Micro Manual Eng

Handbook of Microbiological Culture MediaDehydrated Culture Media ■ Culture Media Ingredients ■ Additives ■ Supplements ■ Reagents

■ Edition No. 10 ■

TABLE OF CONTENTS

Scharlau Microbiology Introduction I

Scharlau Microbiology Quality Policy II

Safety (Regulations, Hazard Categories, Risk and Safety Phrases, Disposal) V

Guidelines for correct manipulation in Microbiology XIII

Dehydrated Culture Media 1-176

Culture Media Ingredients 177-204

Additives 205-213

Supplements 215-229

Reagents 231-244

Index of products in this Manual 245-251

I

Introduction

It is a pleasure to introduce to you this edition no. 8 of our Handbook of Microbiological Culture Media, revised and extended, in a new and more attractive format.

The range of products has been increased, reaffirming the Scharlau Microbiology brand as an attractive alternative to other manufacturers of culture media for Microbiology. You will find new products, according to the standards of the European Pharmacopoeia, USP and other official organisms.In this edition, we have separated additives, reagents and selective supplements into different chapters, mak-ing it easier to locate each product. We have also included additional safety information and R+S sentences, following the EU guidelines 2001/59/CE.

The company

In 1979, Dr. José Sancho Valls, Professor at the Uni-versity of Barcelona, decided to start manufacturing dehydrated culture media. A partnership was formed with a local importer of research chemicals and the products sold on the local market under ADSA=MICRO brand name. In 1992 the company was integrated into the Scharlau group. Thus we look back to more than 25 years of experience in the manufacture of culture media.

At present, a wide rage of products is offered under the Scharlau brand, from standard laboratory chemicals (inorganic salts, acids, buffers, volumetric solution, solvents, etc.), to high purity specialized solvents for HPLC and environmental analysis, up to culture media for microbiology.The technical supervision remains in the hands of Pro-fessor Sancho. Through his wide scientific knowledge, we can offer good technical support.

Contact Scharlau

Don’t hesitate. Contact Scharlau for any requirement of culture media for microbiology.The international presence of Scharlau Microbiology grows day by day. A wider network of distributors and a larger portfolio help to better penetrate the markets.Scharlau Chemie, S.A., which was born 53 years ago to manufacture exclusively organic chemicals for research laboratories, has been increasing its products and proc-esses to become a global manufacturer of laboratory chemicals and culture media for microbiology.

We stock over 700 different microbiological references in our new 4.000 m2 warehouse in Sentmenat (Barcelona).

Scharlau Microbiology is part of the Scharlau Science Group

If you work in Spain please contact:

SCHARLAB, S.L.Tel.: 902 20 18 98Fax: 900 50 29 35E-mail: [email protected]: Gato Pérez, 33. Pol. Ind. Mas d’en Cisa 08181 Sentmenat, Barcelona, Spain

Customers outside of Spain please contact:

SCHARLAU CHEMIE, S.A.Tel.: +34 93 715 18 11Fax: +34 93 715 31 75E-mail: [email protected]: Ctra. de Polinyà a Sentmenat, Km. 8,208181 Sentmenat, Barcelona, Spain

Or ask for the address of our distributors in any of the following countries:

ALBANIAANGOLAARGENTINAAUSTRALIAAUSTRIA BAHRAINBANGLADESHBARBADOSBELGIUMBENINBOLIVIABOSNIA & HERZEGOVINABOTSWANABRUNEIBULGARIACHADCHILECHINA P.R.COLOMBIACOSTA RICAIVORY COASTCROATIACUBACYPRUSCZECH REPUBLICDENMARKDOMINICAN REPUBLICECUADOREGYPTEL SALVADORERITREAESTONIAETHIOPIAFINLANDFRANCEFRENCH POLYNESIAGERMANYGHANAGREECE

GUINEA BISSAUHAITIHONG-KONGHUNGARYICELANDINDIAINDONESIAIRAN IRAQIRELANDISRAELITALYJORDANKENYAKOREAKUWAITLATVIALEBANONLIBYALITHUANIAMADAGASCARMALAYSIAMALTAMAURITANIAMAURITIUSMOLDOVAMOROCCOMOZAMBIQUEMYANMARNEW CALEDONIANEW ZEALANDNIGERIANORWAYPAKISTANPANAMAPERUPHILIPPINESPOLANDPORTUGAL

QATARREUNION ISLANDROMANIARUSSIARWANDASERBIA & MONTENEGROSAUDI ARABIASENEGALSINGAPORESLOVAKIASLOVENIASOUTH AFRICASRI-LANKASUDANSULTANATE OF OMANSWEDENSWITZERLANDSYRIATAIWANTANZANIATHAILANDTHE NETHERLANDSTOGOTONGATRINIDAD & TOBAGOTUNISIATURKEYUGANDAUKRAINEUNITED ARAB EMIRATESVENEZUELAVIETNAMYEMENZAMBIAZIMBAWE

II

Scharlau Microbiology Quality Policy

www.scharlau.com

Please use our website for the latest news and MSDS: www.scharlau.com.Our website has been redesigned recently to offer you a much more modern and intuitive interface.COA come with each bottle, which is why we don´t pub-lish them on the web.

Environmental care

One of Scharlau Chemie’s target regarding environmen-tal issues was achieved in 2003, when we received ISO 14001 approval. Scharlau Chemie has always designed its processes to minimize environmental impacts on the rural area where it is located for about 30 years.

The factory

The microbiological division of Scharlau Chemie, Schar-lau Microbiology, manufactures a wide range of culture media, reagents, stains, supplements and additives.

25 years experience in the production of dehydrated culture media allows us to develop fast new formulations to keep up with the latest developments in microbiology.

New production facilities have been inaugurated in the year 2002, thus enabling us to further improve our proc-esses and expand production capacity. The new factory has been designed following the most latest directives to assure the maximum quality of the final product.

More than 700 references are available from Scharlau Microbiology, ready to be delivered from our new 4000 m2 warehouse in Sentmenat, Barcelona. The strategic situation, within less than 1000 km from most European capitals, makes it possible to quickly restock our distribu-tors. Barcelona, being one of the busiest ports in Europe, boasts excellent world wide shipping connections.

As a contribution to the packaging residue minimization targets, Scharlau has replaced all its packing materials to avoid the use of expanded polystyrene, which is dif-ficult to recycle and expensive to dispose of. All internal and external elements of our packaging are made from recycled cardboard and are fully biodegradable and recyclable.

III


Quality as standard

Our commitment to quality has always been very important to keep our customers trust.

Scharlau Microbiology purchases raw materials and transforms them into microbiological culture media, using various processes and strict analytical controls. Our target is the production of products of the highest possible quality.To achieve this, we have always worked under internal quality standards, even before ISO regulations were adopted. This fact not only allows us to manufacture products of the highest quality, but also to maintain our quality at a constant level.

The quality concept has evolved and nowadays, qual-ity is not only understood as an attribute of the product, but a feature which has to be present in everything done in the company. It is useless to have an excellent product, if delivery times or technical assistance are not adequate. In Scharlau Microbiology we work under these premises, injecting quality into all our services, from the develop-ment of a product designed to cover real customer requirements, to our after sales assistance.

Since 1997 Scharlau Microbiology is ISO 9002 accred-ited through a German TUV.

Certificate of Analysis

Every pack is sent together with its Certificate of Analysis, which guarantees the quality of our products.Together with the Certificate of Analysis we supply a sec-ond certificate to assure absence of Bovine Spongiform Encephalopathy and Foot-and-Mouth Disease in our raw materials of animal origin.

Each certificate includes the following information:Physical-chemical specifications: 1. Of the medium before reconstitution:

ColourAppearanceParticle size / homogenization

2. Of the reconstituted medium ready to use:ClarityPrecipitatesGelifying or solidifying strength

Growth controlResults obtained with control strains under optimal conditions.

IV

We have changed the design of our label to make it more useful and modern.Product name and directions appear now in several languages with medium specification and formula in English. Our labels are printed according to CE regula-tions for dangerous goods.

Expiry date and batch numberGood Laboratory Practices (GLP) which are followed by many of our customers, indicate that all products in the laboratory must be labelled showing batch number, expiry date and other additional data (date of reception, opening date, opened by...)Our labels contain batch number and expiry date and also a specific area is reserved for the user to write down additional data.

Quality in our labels


V

Safety Regulations

Material Safety Data Sheets

MSDS include physical and chemical data, handling rec-ommendations, toxicological information and considera-tions relative to the environment, disposal, storage and transport of each product.In some cases, MSDS are delivered with the products but it is better to get them in advance. We edit MSDS of our products on CD-ROM and they are also available from our web:www.scharlau.com > MSDS

Storage and Transport

Storage and transportation of dangerous goods is sub-jected to special regulations due to the specific risks they involve. All parts handling dangerous goods must adopt the appropriate safety measures to avoid damages.

Safe storageDangerous goods must be stored under special safety conditions to avoid health or environmental injuries. It is always important to keep in mind chemical in compatibili-ties and to separate the products to avoid its mixture in case of accident.Every substance can be classified into a group of sub-stances having similar hazards, that can be stored together. The same security measures will be applied to all the substances belonging to the same classification. If a substance has several hazard degrees, it should be classified as per its highest one: Explosive > Oxidising > Flammable > Toxic > Corrosive > Harmful

There are three basic criteria that should be kept in mind to guarantee a safe storage of chemicals in the laboratories:• Stock of dangerous products must be limited to the minimum quantity needed. As a general rule, purchases should never exceed the quantity needed for one year, and preferably should cover a period of between 3 and 6 months.• Products must be stored in groups depending on its chemical compatibility, as shown below:

• Some products, that are specially dangerous, should be isolated from the rest.Carcinogenic or high toxicity substances should be stored in specific cabinets conveniently marked and closed by key.

Special attention must be paid to the safe storage of flammable liquids, due to the big amounts that are usually needed in the laboratories. They should be stored in security cabinets and security drums.

Storage regulations of dangerous goods indicate minimum distances that should be kept between different classes and special storage conditions for each class. Our new warehouse was built following this and other EC regulations regarding safety and risk prevention.

EXPLOSIVES OXIDIZING FLAMMABLE TOXIC CORROSIVES HARMFUL EXPLOSIVES YES NO NO NO NO NO OXIDIZING NO YES NO NO NO NO FLAMMABLE NO NO YES NO YES* YES TOXIC NO NO NO YES YES YES CORROSIVES NO NO YES* YES YES YES HARMFUL NO NO YES YES YES YES *COULD BE STORED TOGETHER IF CORROSIVE PRODUCTS ARE CONTAINED IN NON FRAGILE BOTTLES

VI

Safety. Regulations

Safe transportationDangerous goods can be transported outside manufac-turing sites only if they are packed, labelled and deliv-ered in safe conditions.

• Packaging must be officially approved to contain dan-gerous goods. Strict tests are performed to evaluate physical resistance to break.

• Packaging must be labelled according to specific regulations for transport of dangerous goods.

• Goods must be accompanied by several documents describing its hazard and how to proceed in case of damage during loading, transporting or downloading operations.

Dangerous goods cannot be transported by unauthor-ized transport means. There are different international regulations that are applied to the transport by road, air or sea. The following classes apply to all means of transport:

Transport regulations

By road / railwayADR (Accord européen rélatif au transport international des merchandises Dangereuses par Route)

This European agreement on the international trans-port of dangerous goods by road changed last July 1st 2001 and all data in this catalogue have been updated according to this new version. The former ADR 1999 can be used until the end of 2002, but, from January 1st 2003, only ADR 2001 will be applied.

RID (Reglement International concernant le transport des marchandises Dangereuses par chemin de fer)

Regulates the international transport of dangerous goods by railway. The new ADR2001 includes all requirements described in RID and could be used instead.

CLASS DESCRIPTION 1 Explosive substances 2 Gases 3 Flammable liquids 4.1 Flammable solids 4.2 Spontaneously flammable solids 4.3 Substances that develop flammable gases in contact with water 5.1 Substances that promote combustion (oxidants) 5.2 Organic peroxides 6.1 Toxic substances 6.2 Substances that induce vomiting or infection 7 Radioactive substances 8 Corrosive substances 9 Various hazardous substances

By airIATA (International Air Transport Association) / ICAO (International Civil Aviation Organization)

IATA regulations for transport of dangerous goods in-clude all requirements from ICAO as well as additional technical instructions. All data in this catalogue refer to the 43rd edition of “Dangerous goods regulation” from IATA. You will also find in this catalogue the packing instruc-tions for passenger aircraft (PAX) and cargo aircraft (CAO). The “Packing Instruction” indicates the condi-tions for the packaging, as well as the quantity author-ized on board the aircraft. If transport of the substance is forbidden, an “F” is printed instead of the PAX or CAO data.The packages that comply PAX conditions, can be always transported on cargo aircraft.IATA issues an ID number that is an identification number for hazardous goods. It is only used in cases where there is no UN number.

By seaIMDG (International Maritime Code for Dangerous Goods) has been drawn by the IMO (In-ternational Maritime Organiza-tion of the UN) and is the regu-lation applied to any transport of dangerous goods by see.

Other transport / security data included in this catalogue:UN number The Committee of Experts of the United Nations (UN) issues recommendations for all freight forwarders on the assessment

of hazardous goods for transport purposes. They are numbered consecutively. These UN numbers are given to each product so that everyone knows how to handle it, no matter if it is going to be sent by road, air or sea.In case there is no UN number, IATA gives an ID number for air transport of hazardous goods.

EC index number (EC = European Community)This number is issued by the EC commission for the classification, packaging and marking of dangerous sub-stances. The marking with hazard symbols, risk warn-ings (R phrases) and safety precautions (S phrases), as used in this catalogue, is linked with this number.

VII

Safety. Hazard categories

E: EXPLOSIVEAre considered all products and preparatio-ns, if they can explode through ignition or if they are more sensitive than dinitrobenzene towards blows and friction.Precautions: Avoid impact, knocks, friction, sparks, fire and heat.

O: OXIDISINGAre considered all products and preparatio-ns, which not being flammable, can produce and enhance fires in contact with flammable products.Precautions: Avoid all contact with flamma-ble substances. Risk of ignition! The substan-ce promotes fires once started and impedes fire fighting.

F+: EXTREMELY FLAMMABLEAre considered all products and preparatio-ns, with a flash point below 0°C and a boiling point of 35°C or below.Precautions: Keep away from naked flames, sparks and sources of heat.

F: FLAMMABLEAre considered all products and preparatio-ns, if:a) They heat up and finally start burning in contact with air at normal temperature without any external energy supplyb) They can start burning in solid condition ater short contact with a source of ignition and continue burning after the source has been taken away,c) They have a flash point below 21°C in liquid condition,d) They form in gaseous condition an explosi-ve mixture with air under normal pressure,e) They create in contact with water or wet air highly flammable gases.Precautions: Keep away from naked flames, sparks and sources of heat.

T+: VERY TOXICare considered all products and preparations, if they lead to death or to acute or cronical health injuries when inhaled, swallowed or absorbed through the skin in very small quantities.Precautions: All contact with the human body must be avoided, as severe or even lethal damage to health cannot be excluded. Particular attention is drawn to the carcinoge-nic, teratogenic or mutagenic risks associated with certain substances.

T: TOXIC Are considered all products and preparations, if they lead to death or to acute or cronical health injuries when inhaled, swallowed or absorbed through the skin in small quantities.Precautions: All contact with the human body must be avoided, as severe or even lethal damage to health cannot be excluded. Particular attention is drawn to the carcino-genic, teratogenic or mutagenic risks associ-ated with certain substances.

Xn: HARMFUL Are considered all products and prepara-tions, that if inhaled, swallowed or absorbed through the skin can cause death or serious acute or chronic effects.Precautions: Avoid contact with the human body, including the inhalation of vapours. Injury to health is possible with improper use. With some substances, carcinogenic, tera-togenic or mutagenic action cannot be fully excluded, as well as possible sensitization.

C: CORROSIVE Are considered all products and preparations, if they can destroy skin by contact.Precautions: Take special measures to protect the eyes, skin and clothes. Do not inhale vapours.

Xi: IRRITANTAre considered all products and prepara-tions, if they can produce irritation in a short, prolonged or repetitive contact with the skin or the respiratory tract.Precautions: Avoid contact with eyes and skin, do not inhale vapours.

N: DANGEROUS FOR THE ENVIRONMENTfor the Environment can be considered those products and substances, which can have detrimental effects upon the ecosystem of water, soil or air, climate, fauna, flora or microorganisms in such a way, that they con-stitute an immediate or future danger for the environment.Precautions: According to the kind of haz-ard do not dispose to wastewater, soil or environment. Observe special disposal regu-lations!

VIII

R: Risk Phrases

1 Explosive when dry.2 Risk of explosion by shock, friction, fire or other sources of ignition.3 Extreme risk of explosion by shock, friction, fire or other sources of ignition.4 Forms very sensitive explosive metallic compounds.5 Heating may cause an explosion.6 Explosive with or without contact with air.7 May cause fire.8 Contact with combustible material may cause fire.9 Explosive when mixed with combustible material.10 Flammable.11 Highly flammable.12 Extremely flammable.13 Extremely flammable liquefied gas.14 Reacts violently with water.15 Contact with water liberates extremely flammable gases.16 Explosive when mixed with oxidising substances.17 Spontaneously flammable in air.18 In use, may form flammable/explosive vapour-air mixture.19 May form explosive peroxides.20 Harmful by inhalation.21 Harmful in contact with skin.22 Harmful if swallowed.23 Toxic by inhalation.24 Toxic in contact with skin.25 Toxic if swallowed.26 Very toxic by inhalation.27 Very toxic in contact with skin.28 Very toxic if swallowed.29 Contact with water liberates toxic gas.30 Can become highly flammable in use.31 Contact with acids liberates toxic gas.32 Contact with acids liberates very toxic gas.33 Danger of cumulative effects.34 Causes burns.35 Causes severe burns.36 Irritating to eyes.37 Irritating to respiratory system.38 Irritating to skin.39 Danger of very serious irreversible effects40 Limited evidence of a carcinogenic effect.41 Risk of serious damage to eyes.42 May cause sensitisation by inhalation.43 May cause sensitisation by skin contact.44 Risk of explosion if heated under confinement.45 May cause cancer.46 May cause heritable genetic damage.47 May cause birth defects.48 Danger of serious damage to health by prolonged exposure.49 May cause cancer by inhalation.50 Very toxic to aquatic organisms.51 Toxic to aquatic organisms.52 Harmful to aquatic organisms.53 May cause long-term adverse effects in the aquatic environment.54 Toxic to flora.55 Toxic to fauna.56 Toxic to soil organisms.57 Toxic to bees.

58 May cause long-term adverse eflects in the environment.59 Dangerous for the ozone layer.60 May impair fertility.61 May cause harm to the unborn child.62 Possible risk of impaired fertility.63 Possible risks of harm to the unborn child.64 May cause harm to breastfed babies65 Harmful: may cause lung damage if swallowed.67 Vapours may cause drowsiness and dizziness.66 Repeated esposure may cause skin dryness or cracking.68 Possible risk of irreversible effects.

Combination of particulars risks

14/15 Reacts violently with water, liberating extremely flammable gases.15/29 Contact with water liberates toxic, extremely flammable gas.20/21 Harmful by inhalation and in contact with skin.20/21/22 Harmful by inhalation, in contact with skin and if swallowed.20/22 Harmful by inhalation and if swallowed.21/22 Harmful in contact with skin and if swallowed.23/24 Toxic by inhalation and in contact with skin.23/24/25 Toxic by inhalation, in contact with skin and if swallowed.23/25 Toxic by inhalation and if swallowed.24/25 Toxic in contact with skin and if swallowed.26/27 Very toxic by inhalation and in contact with skin.26/27/28 Very toxic by inhalation, in contact with skin and if swallowed.26/28 Very toxic by inhalation and if swallowed.27/28 Very toxic in contact with skin and if swallowed36/37 Irritating to eyes and respiratory system.36/37/38 Irritating to eyes, respiratory system and skin.36/38 Irritating to eyes and skin37/38 Irritating to respiratory system and skin39/23 Toxic: danger of very serious irreversible effects through inhalation.39/23/24 Toxic: danger of very serious irreversible effects through inhalation and in contact with skin.39/23/24/25 Toxic: danger of very serious irreversible effects through inhalation, in contact with skin and if swallowed.39/23/25 Toxic danger of very serious irreversible effects through inhalation and if swallowed.39/24 Toxic: danger of very serious irreversible effects in contact with skin.39/24/25 Toxic: danger of very serious irreversible effects in contact with skin and if swallowed.39/25 Toxic: danger of very serious irreversible effects if swallowed.39/26 Very toxic: danger of very serious irreversible effects through inhalation.39/26/27 Very toxic: danger of very serious irreversible effects through inhalation and in contact with skin.39/26/27/28 Very toxic: danger ol very serious irreversible effects through inhalation, in contact with skin and if swallowed.39/26/28 Very toxic: danger of very serious irreversible effects through inhalation and if swallowed.39/27 Very toxic: danger of very serious irreversible effects in contact with skin.

Safety. Risk and Safety Phrases

IX

39/27/28 Very toxic: danger of very serious irreversible effects in contact with skin and if swallowed.39/28 Very toxic: danger of very serious irreversible effects if swallowed.40/20 Harmful: possible risk of irreversible effects through inhalation.40/20/21 Harmful: possible risk of irreversible effects through inhalation and in contact with skin.40/20/21/22 Harmful: possible risk of irreversible effects through inhalation, in contact with skin and if swallowed.40/20/22 Harmful: possible risk of irreversible effects through inhalation and if swallowed.40/21 Harmful: possible risk of irreversible effects in contact with skin.40/21/22 Harmful: possible risk of irreversible effects in contact with skin and if swallowed.40/22 Harmful possible risk of irreversible effects if swallowed.42/43 May cause sensitisation by inhalation and skin contact.48/20 Harmful: danger of serious damage to health by prolonged exposure.48/20/21 Harmful: danger of serious damage to health by prolonged exposure through inhalation and in contact with skin.48/20/21/22 Harmful: danger of serious damage to health by prolonged exposure through inhalation, in contact with skin and if swallowed.48/20/22 Harmful: danger of serious damage to health by prolonged exposure through inhalation and if swallowed.48/21 Harmful: danger of serious damage to health by prolonged exposure in contact with skin.48/21/22 Harmful: danger of serious damage to health by prolonged exposure in contact with skin and if swallowed.48/22 Harmful: danger of serious damage to health by prolonged exposure if swallowed.48/23 Toxic: danger of serious damage to health by prolonged exposure through inhalation.48/23/24 Toxic: danger of serious damage to health by prolonged exposure through inhalation and in contact with skin.48/23/24/25 Toxic: danger of serious damage to health by prolonged exposure through inhalation, in contact with skin and if swallowed.48/23/25 Toxic: danger of serious damage to health by prolonged exposure through inhalation and if swallowed.48/24 Toxic: danger of serious damage to health by prolonged exposure in contact with skin.48/24/25 Toxic: danger of serious damage to health by prolonged exposure in contact with skin and if swallowed.48/25 Toxic: danger of serious damage to health by prolonged exposure if swallowed.50/53 Very toxic to aquatic organisms, may cause long- term adverse effects in the aquatic environment.52/53 Harmful to acquatic organisms, may cause long- term adverse effects in the acquatic environment.51/53 Toxic to acquatic organisms, may cause long-term effects in the aquatic environment.

68/20 Harmful: possible risk of irreversible effects through inhalation.68/21 Harmful: possible risk of irreversible effects in contact with skin.68/22 Harmful: possible risk of irreversible effects if swallowed.68/20/21 Harmful: possible risk of irreversible effects through inhalation and in contact with skin.68/20/22 Harmful: possible risk of irreversible effects through inhalation and if swallowed.68/21/22 Harmful: possible risk of irreversible effects in contact with skin and if swallowed.68/20/21/22 Harmful: possible risk of irreversible effects through inhalation, in contact with skin and if swallowed.

S: Safety phrases

1 Keep locked up.2 Keep out of reach of children.3 Keep in a cool place.4 Keep away from living quarters.5 Keep contents under _ (appropriate liquid to be specified by the manufacturer)5.3 Keep contents under paraffin oil.6 Keep under (inert gas to be specified by the manufacturer)7 Keep container tightly closed.8 Keep container dry.9 Keep container in a well ventilated place.12 Do not keep the container sealed.13 Keep away from food, drink and animal feeding stuffs.14 Keep away from _ (incompatible materials to be indicated by the manufacturer)14.1 Keep away from alkalis.14.2 Keep away from oxidizing and acidic substances as well as heavy metal compounds.14.9 Keep away from flammable organic substances.15 Keep away from heat.16 Keep away from sources of ignition - No Smoking.17 Keep away from combustible material.18 Handle and open container with care.20 When using do not eat or drink.21 When using do not smoke.22 Do not breathe dust.23 Do not breathe gas/fumes/vapour/sray (appropiate wording to be specified by the manufacturer).23.2 Do not breathe vapour.24 Avoid contact with skin.25 Avoid contact with eyes.26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.27 Take off immediately all contaminated clothing.28 After contact with skin, wash immediately with plenty of _ (to be specified by the manufacturer).28.1 After contact with skin, wash immediately with plenty of water.28.2 After contact with skin, wash immediately with soap and water.28.3 After contact with skin, wash immediately with soap and water, if possible also with polyethylene glycol 400.28.6 After contact with skin, wash immediately with polyethylene glycol 400 (then rinse with plenty of water).


X


29 Do not empty into drains.30 Never add water to this product.33 Take precautionary measures against static discharges.34 Avoid shock and friction.35 This material and its container must be disposed of in a safe way.36 Wear suitable protective clothing.37 Wear suitable gloves.38 In case of insufficient ventilation, wear suitable respiratory equipment.39 Wear eye/face protection.40 To clean the floor and all objects contaminated by this material use _ (to be specified by the manufacturer).41 In case of fire and/or explosion do not breathe fumes.42 During fumigation/spraying wear suitable respiratory equipment (appropriate wording to be specified)43 In case of fire, use _ (indicate in the space the precise type of fire-lighting equipment. If water increases the risk add - Never use water)43.1 In case of fire, use water.43.3 In case of fire, use powder extinguisher. Never use water.43.6 In case of fire, use sand. Never use water.43.7 In case of fire, use metal-fire powder. Never use water.43.8 In case of fire, use sand, carbon dioxide or powder extinguisher. Never use water.44 If you feel unwell, seek medical advice (show the label where possible)45 In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible)46 If swallowed seek medical advice immediately and show this container or label47 Keep at temperature not exceeding _ ºC (to be specified by the manufacturer)48 Keep wetted with _ (appropriate material to be specified by the manufacturer)49 Keep only in the original container.50 Do not mix with _ (to be specified by the manufacturer)50.1 Do not mix with acids.51 Use only in well ventilated areas.52 Not recommended for interior use on large surface areas.53 Avoid exposure - obtain special instruction before use.54 Obtain the consent of pollution control authorities before discharging to wastewater treatment plants.55 Treat using the best available techniques before discharge into drains or the aquatic environment.56 Dispose of this material and its containes at hazardous or special waste collection point.57 Use appropriate containment to avoid environmental contamination.58 To be disposed of as hazardous waste.59 Refer to manufacturer/supplier for information on recovery/recycling60 This material and its container must be disposed of as hazardous waste.

61 Avoid release to the environment.Refer to special instructions/Safety data sheets.62 If swallowed, do not induce vomiting: seek medical advice immediately and show this container or label.63 In case of accident by inhalation: remove casualty to fresh air and keep at rest.64 If swallowed, rinse mouth with water (only if the person is conscious).

Combination of safety precautions

1/2 Keep locked up and out of reach of children.3/7 Keep container tightly closed in a cool place.3/7/9 Keep container tightly closed, in a cool well ventilated place.3/9 Keep in a cool well ventilated place3/9/14 Keep in a cool, well ventilated place away from _ (incompatible materials to be indicated by the manufacturer)3/9/14.1 Keep in a cool, well ventilated place away from alkalis.3/9/14/49 Keep only in the original container in a cool, well- ventilated place away from ... (incompatible materials to be indicated by the manufacturer).3/9/49 Keep only in the original container in a cool, well- ventilated place.3/14 Keep in a cool place away from ... (incompatible materials to be indicated by the manufacturer).7/8 Keep container tightly closed and dry.7/9 Keep container tightly closed and in a well- ventilated place.20/21 When using do not eat, drink or smoke.24/25 Avoid contact with skin and eyes.27/28 After contact with skin, take off immediately all contaminated clothing, and wash immediately with plenty of ... (to be specified by the manufacturer).29/35 Do not empty into drains; dispose of this material and its container in a safe way.36/37 Wear suitable protective clothing and gloves.36/37/39 Wear suitable protective clothing, gloves and eye/ face protection.36/39 Wear suitable protective clothing and eye/face protection.37/39 Wear suitable gloves and eye/face protection.47/49 Keep only in the original container at temperature not exceeding _ ºC (to be specified by the manufacturer).

XI

Disposal of Laboratory Waste

Avoid contamination of the waterElimination of residue through the drain is strictly forbid-den. You have to bear in mind, that many chemicals shall not be reduced by the waste water plants and shall contaminate the environment.

Residue in the laboratoryWe highly recommend naming a person in charge of the residue of your company. This person should be in-formed of the laws and regulations regulating the waste of residue in your own country.In order to hand over the residue to a treatment plant, it is necessary to organize the collection and storage previously.

Collection of residue in the laboratoryThe material of the residue containers has to comply with the following conditions:1. One has to be able to close the containers hermeti-

cally and the material has to be resistant to the con-tents. Plastic containers should not form toxic fumes when disposed of through burning.

2. Generally, plastic containers are being used.3. Corrosive products should be collected in metal

drums with inner plastic lining, like our Combi drum (Ref. 055-0C0025).

4. Inflammable or oxidizing products should be collect-ed in metal or plastic drums resistant to the solvent.

5. For products that produce gases or vapours, special containers with a security valve are required, in order to avoid the danger of explosions.

If the collection containers are handed over to a trans-port agency, they have to carry the UN number that approves them for transport by road.

Safety. Disposal

Hazardous chemicals In this handbook risk phrases (R-phrases), safety phrases (S- phrases) and hazard symbols are added in accordance with the EU Directive (67/548) and (83/467). The absence of R- and/or S-phrases or hazard symbols does not mean that those substances are harmless. The normal safety precautions for handling chemicals should always be observed.

General Waste - Disposal proceduresThe disposal methods outlined below are intended as a guideline for the technician. We do not assume respon-sibility of their use. Careful consideration must be given to the chemical and physical properties of the substance. In addition, local laws and regulations may preclude the use of these methods which were primarily designed for the quantities we list. Observe all Federal, State and local laws concerning health and pollution. All procedures require the use of goggles or face shield, gloves and apron. Gas masks or self- contained breath-ing units may also be required.

1. Neutralize the bases with diluted sulphuric acid, then flush neutral solution down the drain with an abun-dance of water.

2. Treat the aldehydes or peracids with an excess of sodium bisulfite solution. Use starch iodide paper to ensure complete reduction of any peracid. Flush the resulting solution down the drain with an abundance of water.

3. The compound should be burned. Ideally, all hydro-carbons and related solvents should be burned in an approved presence of sodium carbonate and cal-cium hydroxide (slaked lime). The solid or the liquid absorbed on vermiculite should be wrapped in paper and burned in an approved incinerator.

4. Bury in an approved chemical and hazardous-waste sand fill site, well away from streams, rivers or wells.

5. Azides and azo-compounds should be treated with dilute ceric ammonium nitrate solution with cool-ing. Solutions may be discarded upon completion of the reaction. Mixtures should be separated and the organic portion burned.

6. Treat with an alcohol such as ethanol or butanol in an appropriate solvent. The chemical reaction may be vigorous and/or exotermic. Provisions must be made for venting of large volumes of highly flammable hydrogen and/or hydrocarbon gases. Neutralize the solution with aqueous acid. Filter off any solid residue for disposal as hazardous waste. Burn residual organic materials in an incinerator equipped with an afterburner and scrubber.

XII

7. Neutralize the base with dry sodium bisulfate add water slowly and flush the neutral solution down the drain with an excess of water.

8. Carefully mix the acidic compound with dry sodium bicarbonate. Dilute slowly and flush down the drain with an excess of water.

9. Oxidize the mercaptan or nitrite with a weak aqueous solution (up to 15%) of sodium or calcium hypoclorite. Vigorous stirring may be required to ensure comple-tion of the reaction. Neutralize the resulting mixture, then discard down the drain with an excess of water.

10. Flush down the drain with an excess of water. 11. Bury in a site approved for disposal of poisonous or

hazardous wastes. 12. Refer to our Material Safety Data Sheet before han-

dling or disposing.

Caution regarding hazardous componentsSome media contain components that are toxic or carci-nogenic. Appropriate safety precautions must be taken when using media with such components.

Basic fuchsin and acid fuchsin are carcinogens and caution must be used in handling media with these com-pounds to avoid dangerous exposure that could lead to the development of malignancies . Thalium salts, sodium azide, sodium biselenite and cyanide are among the toxic components found in some media. These com-pounds are poisonous and steps must be taken to avoid ingestion, inhalation, and skin contact. Azides also react with many metals, especially copper, to form explosive metal-azides. The disposal of azides must avoid con-tact with copper or achieve sufficient dilution to avoid the formation of such hazardous explosive compounds. Media with sulphur containing compounds may result in the formation of hydrogen sulphide which is a toxic gas. Care must be used to ensure proper ventilation. Media with human blood or human blood components must be handled with great caution to avoid exposure to hu-man immunodeficiency virus and other pathogens that contaminate some blood supplies. Proper handling and disposal procedures must be followed with blood-con-taining as well as other media that are used to cultivate microorganisms.

Safety. Disposal

XIII

Guidelines for correct manipulation in Microbiology

Following instructions provide the correct guidelines for the manipulations in microbiology. Every microbiological laboratory is responsible for their proper implementation.

A. Personal techniquesA.1. Hygiene is the basic element and need for the safe

manipulation.A.2. All manipulated microorganisms must be presumed

potentially pathogenic, regardless of their nature. The following procedures or techniques must therefore be avoided : oral pipetting, application of cosmetics ,eating, drinking or smoking in the laboratory. Wrappings and labels in the labortory should be self-adhesive.

A.3. Manipulation of the microorganisms should be conducted in such a way that will not provoke any environmental contamination or hazard. For example, sprays should always be avoided and pipettes must be submerged immediately in the disinfectant after use.

A.4. Manipulators (technicians) must be aware of the risks and should know how to prevent them.

A.5. Manipulators must make sure that both, the user and the equipment itself are perfectly safe from any potential risk.

B. Equipment in generalB.1. A standard equipment for the sterilization of any

potentially hazardous material must be available (autoclave, jars with disinfectant for the pipettes, dry heat ovens etc).

B.2. Protective clothes must be used exclusively in the working area, and should never be worn while in public areas.

B.3.Toilets and other facilities must be available close to the working areas, and separate from those of use to the general public.

B.4. Any device which may probably produce the sprays must be always handled in microbiological secu-rity cabins like Laminar Air Flow .

B.5. In case of any accident, all necessary substances and equipments , either to minimize or neutralize the risks must be readily available.

C. Basic characteristics of the laboratoryC.1. The laboratory or plant should not be located in a

passage or corridor so that any outside person would always remain away from it.

C.2. It must be equiped with an appropiate ventillation system, mechanically regulated if possible, so as to create a drift of flowing air from the areas of low risk to the contaminated areas, and then go-ing outside, through the HEPA filters.

C.3. The laboratory must be built of easily washable, waterproof and disinfectant-resistant surfaces.

D. Administrative proceduresD.1. Systems to confirm and assure the effectiveness of

the biological security code carried out by quali-fied personnel.

D.2. Training programmes regarding the security during or at work, adapted to the level required by every maninpulator / technician.

D.3. Adequate medical surveillance and facilities .D.4. Procedures for the verification and maintenance

of the ventilation system and the equipment in general.

D.5. Emergency procedures and systems for use in case of any accident.

D.6. Strategies to restrain the freak entries and exits, to maintain the security standards of laboratory and the plant.

D.7. Procedures for the transportation, postage and reception of the biological material (postage regulations and related things).

D.8. Training of personnel to act as security officers.D.9. Systems to eliminate any potentially hazardous

residues.

ReferencesReport of Committee of Enquiry into Smallpox (1974) Outbreak in March and April 1973. Cmnd.5626, Her Majesty’s Stationery Office.Report of the Working Party on the Laboratory Use of Dangerous Pathogens. (The Godber Report) (1975). Cmnd. 6054, Her Majesty’s Stationery Office. Report of the Working Party on the Experimental Manip-ulation of the Genetic Composition of Micro-organisms. (1975) Cmnd. 5880, Her Majesty’s Stationery Office.PUBLIC HEALTH LABORATORY SERVICE (1980) Safety Precautions-Notes for Guidance. Colindale, MILLER, B.M. (1987) Laboratory Safety: Principles and Practices. ASM. Whasington, D.C.HARTREE, E., U. BROTH (1977) Safety in Biological Laboratories. Biochem. Soc. Spec. Pub. nº5. The Bio-chemical Society. LondonCOLLINS, C.H., E.G. HARTLEY, R. PILSWORT (1976) The Prevention of Laboratory Adquired Infection. PHLS Monograph nº6. Her Magesty Stationery Office.SMITH, J.A. (1996) Laboratory Safety in Clinical Microbi-ology. Cumitech nº29. ASM. Whasington, D.C.WHO (1983) Laboratory Biosafety Manual. Geneva.

The appearance and quality of the prepared medium usually depends on the method used for rehydration and storage. It is therefore very important to follow the recommendations stated below.

XIV

container. Use a container with a capacity of approxi-mately 2 to 3 times more the volume of the medium, to allow plenty of space for manipulation.Sometimes, It is necessary to boil the medium for more than 1 minute. In such cases, the volume of evaporated water must be restored.

Media not containing the agar or any solidifying agents (broths and Nutrient Solutions) tend to dissolve eas-ily and rapidly in preheated water, however sometimes boiling is necessary to attain the complete dissolution. These media have a tendency to form a concentrated syrup or suspension which settles at the bottom of the container. This thick suspension usually remains at the bottom, risking the destruction of the medium contents by hydrolysis, caramelization and pH drift when the heat is applied. Therefore, thorough mixing should be as-sured before heating.

Media containing solidifying agents (Agars and Fluid Media) must be preheated before sterilization. Media containing agar must be brought to the boiling, since agar is insoluble in water below 98-100° C. It is useful to let the agar soak in preheated water for 5 to 10 minutes

before heating, in order to al-low it to swell and to secure its solubility and uniform distribu-tion and dissolution.

A highly recommended procedure can be practised by removing the rehydrated medium from the source of heat when the mixture begins to boil, letting it stand for a while and then quickly bring-ing it to the boiling again. This may be repeated 2 or 3 times with constant agitation. The complete dissolution of the agar will be indicated by the absence of granules in the container.Dissolution of culture media directly during sterilization in the autoclave is a frequent and totally incorrect practice. It alters the medium’s quali-ties and frequently causes the uneven dissolution of the agar into layers with different concentration gradients, pH drifts and browning.

Nowadays, heating the rehy-drated medium in a microwave oven in order to melt the agar


RehydrationDirections for the individual reconstitution are detailed on the container of every media and must be strictly followed.

The water used for reconstitution purposes must be distilled or deionized, of «pharmaceutical grade». The solution or suspension must be completely homogene-ous and the exposure to heat if necessary, should be minimal.

In any case, it is always helpful to heat the water previ-ously to 50-60°C, to help dissolve the media. For the preparation of the culture media the most recom-mended technique is the following:Add necessary amount of powder to half the volume of water needed. The water should have already been warmed to 50-60° C. Stir the mixture thoroughly until the powder is dissolved. Use rest of the water to dissolve the powder sticking to the walls or sides of the container and continue stirring and heating if necessary. Heat must be applied directly and smoothly. Constant agitation will prevent the components from sticking to the walls of the

XV

or dissolve the medium components is a very common procedure. In this case also, it is recommended to let the agar-medium to soak for some time before using the microwave and use the containers suitable for the volume of medium to be prepared. Using a microwave with 800 W for 4 minutes are enough to achieve the total dissolution or melting of agar. However, it must be noted that since this is a static procedure, a concentration gra-dient will appear which will create a stratification. Thus, it may be necessary to shake the rehydrated medium vigorously to homogenize the solidifying agent before using or sterilizing it in the autoclave, in the same way as if it were an autosterile medium. Microwave heating can never be adopted as a substitute for autoclave sterilization.

Dehydrated culture media usually maintain their features if reconstituted properly. Nevertheless, verification is encouraged since some of them (colour, pH, etc) may slightly change subject to the conditions of reconstitution and the type of water used.

These recommendations are particularly important when referring to fluid media, since such media contain small amounts of solidifying agent and are therefore very deli-cate. Cooling becomes as important as heating, as any violent process may induce the formation of flakes and clouds. A slow and smooth cooling process is recom-mended.

Media containing phosphate buffers together with glu-cose or other carbohydrates may darken if overheated as a concentrated solution. Precipitates may also appear when using poor quality water.

Special attention must be paid to all media containing agar at a pH under 5, since it hydrolyzes and looses the ability to form gels. In those cases, unnecessary heating must be avoided.

SterilizationThe indications given on every container must be fol-lowed, while bearing in mind that they refer to quantities of up to 1 litre. As for bigger volumes, autoclaving and heat penetration conditions of the medium will have to be taken into consideration.

In all the cases autoclaving must be monitored regu-larly by the manometers and thermometers, as well as the even distribution of heat. Nowadays this can be done mechanically (thermocouples), chemically (ther-mal indicators) or biologically (thermoresistent spores). Considering that overheating is one of the main causes of culture media alteration, it is only natural to prevent, whenever possible, the treatment of large quantities of media and the prolonged exposure to heat. For large autoclaves it is recommended to preheat before the me-

dium is introduced in the autoclave , and to let the tem-perature drop to 70-80°C before removing the medium so as to avoid severe temperature fluctuations. Although the use of cold water to cool media is widespread, it is not recommended for media containing agar since it causes flakes and cloud formation.


XVI

Containers used for sterilization purposes must have a large head space for air to allow foaming. If screw-cap containers are used,the closures must be a half turned or screwed to allow inner and outer pressure balancing.All containers should be chemically inert to prevent al-teration of the pH of contents. Borosilicate glass contain-ers are highly recommended.Media containing carbohydrates darken a little after heat sterilization, therefore, sterilization should be carried out at a temperature below 120°C if possible.Should autoclave not be available, sterilization can be done in a pressure vessel (at 100°C for 30 minutes). In such case it is useful to resterilize the medium during three days, if the medium allows the treatment.Some media containing selective substances do not need sterilization. The preparation process is quicker since they are ready for use after dissolution. Since these media are usually not very stable and preparation is brief and easy, the exact quantities required should be prepared.

Additions to the medium (if any) after sterilization must be done aseptically, that is, additives should have been previously sterilized. Additives are generally thermolabi-les if not they would be part of the dehydrated medium’s composition. It is therefore necessary to allow the medi-um to cool to 50-60°C before their addition, so as not to harm the additive while permitting its correct distribution. Once the addition is over, reheating must be avoided.Distribution into final containers must be completed before sterilizing, to avoid any manipulation. Since it is rarely possible to do so, manipulation should be done in safety cabinets instead. The sterile cabinets or rooms are not be exposed to any radiations and so, no any kind of highly active reagent could affect the medium’s components. Excessive steam condensation can be eluded by distributing the medium at temperatures close to solidification, ranging from 45 to 50°C.

Storage of dehydrated mediaDehydrated culture media should be stored safely, pro-tected from humidity or moisture, light and heat, which are the most frequent causes of their alteration.SCHARLAU media are supplied in opaque, waterproof, screw-cap plastic bottles, with an internal relief which eliminates the need for an intermediate seal. Never-theless a hermetic seal is assured by accurately fitting the cap and keeping both its sides and the border of its mouth clean.

It is important to open the bottles in dry atmosphere and close them immediately after their use. Refrigera-tion is not necessary even if low temperatures prolong the medium’s effectivity. The nature of the containers of SCHARLAU make culture media are suitable for prolonged storage in cool, dry places. If the media are moist they become stiff and hard, lose their properties

and can even allow direct growth of microorganisms in them. Under such circumstances they must not be used. Although they usually have a dry and powdery appear-ance, the media may occasionally appear moist, oily but never stiffened, without there being any alteration in their composition (as e.g.in MRS, Tributyrin Agar, etc).In any case, storage of dehydrated media is not indefi-nite. It is not recommended to store large quantities but to keep small amounts in stock, so as to maintain a rotation of ready prepared dehydrated media. Register-ing the reception dates on the containers will avoid the accumulation of older media.

Storage of ready prepared culture mediaAlthough it is best to prepare the media as you required, it is common to store them ready prepared and sterilized in order to spare preparation time.Under these circumstances the biggest drawback is the dehydration and every precaution taken to avoid it will prolong the medium’s useful life, which is usually be-tween 4 and 6 weeks. We therefore recommend the use of hermetically sealed screw-capped containers (either bottles or tubes) instead of those plugged with cotton wool. A moderate refrigeration (4°C) usually prolongs the life of the medium but it should also be noted that a refrigerated environment is very dry and consequently renders dehydration. In some cases, as regards in thioglycolate media and almost all media recommended for anaerobes, they are best stored at room temperature for the less penetration of air. Direct light must always be avoided, particulary when it is specifically indicated on the label of the medium.Solid media for the plates are best stored in their original containers than in the poured plates. Nevertheless, poured plates may also be prepared and stored if the indications below are followed:a) Refrigerate the media immediately after solidification

while incubating «controls» to check the sterility of the batch.

b) If the media are to be stored for a longer period, plates should be individually sealed with a waterproof seal and, if possible, stored in single containers.

c) If storage is to last more than a few days, the plates should be wrapped in plastic bags to avoid dehydra-tion of the media.

Since condensation water will inevitably appear, we sug-gest to store the poured plates inverted. Contamination with condensation water can be avoided by pouring the molten medium on the plates as cold as possible.Media which have been stored in refrigeration should be allowed to warm up gradually. It is recommended to keep them at room temperature for a few hours before inoculation, allowing misted containers to clear and avoiding the delayed initiation of growth (or eventually its total failure).


XVII


Stored ready prepared media which may involve a high level of dehydration should never be used. Dehydra-tion is indicated by excess condensation of water or by a decrease in volume of liquid media; an increase of concentration or precipitation of fluid and liquid media; or retraction, cracking and precipitation of solid media. Plates with very dry surfaces or those which appear to have changed colour should not be used either.

Remelting solid mediaAlthough most solid culture media can be stored ready prepared and sterilized, and can be remelted and poured into plates whenever necessary, this procedure should not be applied to media which do not need sterilization or those with a pH 5 or less, since it would alter their properties.Remelting is done in a boiling water bath or by fluent steam autoclaving for 30 minutes. Do not apply direct heat. However, it must be emphasized that remelted media are prone to precipitation and darkening if held in a molten state for more than 1 hour and at temperatures ranging from 45 to 65°C. Such media may also undergo nutritional impairment to the desired microorganisms. It is therefore recommended to avoid remelting of solid media whenever possible.

One of the quickest and most recommended remelt-ing methods for culture media is the use of microwave ovens. In this case all the precautions exposed must be carefully observed and practised in the media which are labile and consequently radiation and minimum intensity should be dosed for the shortest time intervals.A usual and erroneous practice consists of imposing strong radiation intensities for short periods, which re-sults in partial remelting, sudden boiling and overflowing of the medium and an overall alteration of its properties.

Differentiation of culture mediaSCHARLAU names for culture media are based on their final consistency or appearance. Thus, a medium is named as:Agar: Solid media with an agar content of 1% or more.Fluid Medium (FM): Semisolid media with an agar con-

tent of less than 1%.Broth: Liquid culture media with undefined organic com-

ponents (peptones, organ and tissue extracts, etc...)

Nutrient Solution: Culture media with defined chemical composition.

Selective Medium: Medium that allows the growth of a biotype or a few biotypes between those that are present in the inocule.

Differential Medium: Medium that allows to distinguish or differentiate between similar culture types.

Enrichment Medium: Medium that aids the growth of a defined biotype over the rest in the inocule.

General Medium: Medium that supports the growth of a very wide range of microorganisms without special nutrient needs.

Membrane Filter (MF): Media used when the Mem-brane Filter Technique is employed.

Membrane Filter MediaUtilization of the Membrane filter technquies has offered some undoubted advantages to the Microbiology which has certain specific problems. Membrane filtration allows to examine big samples (in volume of liquid) with very low microorganisms concentration or load, to separate the microorganisms from the culture medium and even the exchange of the microorganisms between two media without affecting their growth. Moreover, the MPN technique is more precise in these media and also, the membrane with the colonial growth may be stored and filed.The membrane filtration system is accepted by most pharmacopeias as an alternative or the single method to examine antimicrobial or with strong inhibitors sub-stances, since a proper wash of the filter remove all the substances that interfere with the microbial growth.On other hand, the wide range of filters and qualities available allows the examination of any product, since you may find membranes resistant to almost all the dis-solvents.

TechniqueEssentially, the technique consists of filtering the sample through a filter with a suitable porosity (0,22 microns for bacteria and 0,45 microns for fungi) with pressure or suction, in such a way that the microorganisms are retained on the membrane.If the fluid that has been filtered contains inhibitors, wash the membrane several times with rinse liquid to remove them. Membrane is removed aseptically and it is taken and placed on the culture medium.For sterility controls, the membrane is incubated directly in the classical media. For these tests refer: Thioglyco-late Broth (Ref. 2-186), Thioglicolate USP Fluid Medium (Ref. 3-187), Tryptone Soy Broth (Ref. 2-200), Sabour-aud USP Broth (Ref. 2-165).

Should an enumeration of colonies be desired, incubate the filter on a solid medium or on a pad soaked in liquid medium, but beware that the lower surface touches the culture medium and that there are no air bubbles or air gap in between.Usually, general media may be used for these tech-niques. However, there are several culture media spe-cifically developed for these techniques, specially in the water Microbiology.

Dehydrated Culture Media

2

Culture media manufacture process flow chart

3

Acetamide Medium

Ref. 03-428

SpecificationLiquid medium for the enrichment and confirmative test of presence of Pseudomonas aeruginosa in water acc. EN 12780:2002 and ISO 16266 standard.

Formula (in g/L)Acetamide ............................................2,0000Magnesium sulfate, anhydrous ............0,2000Sodium chloride ....................................0,2000Ferrous sulfate .....................................0,0005Monopotassium phosphate ..................1,0000Sodium molybdate ................................0,0050Final pH 7,0 ± 0,5

DirectionsDissolve 3,4 g of powder in 1 L of distilled water. Heat only if necessary. Sterilize in the autoclave at 121°C for 15 minutes. Prepared medium may be opalescent and with precipitate. The prepared medium remain active for 3 months if it is stored in the dark in a cool place.

DescriptionThis nutrient solution has acetamide as the unique carbon and nitrogen source, and it allows the growth of only those microorganisms that are able to use aceta-mide. In water and in almost all the food materials, these microorganisms are the non fermenting gramnegatives, thus Pseudomonas aeruginosa is the only one that can liberate ammonia by deaminating acetamide.

Some authors suggest the use of this nutrient solution as a previous enrichment medium before the isolation me-dium, especially if you are studing very polluted samples with companion flora.

TechniqueMedium is inoculated with a couple of loops from the culture or inoculum to be assayed and is incubated at 32-35°C for 24-48 hours before proceeding for the isola-tion medium.

To confirm Pseudomonas aeruginosa, inoculate a loop of culture inoculum in Asparagine Broth (Ref. 02-271) and incubate at 35-37°C for 24 hours. After this period, pour 1 or 2 drops of Nessler’s Reagent (Ref. 06-084) on to the culture and observe for ammonia production: a change in colour to yellow indicates ammonia production and thus the presence of Pseudomonas aeruginosa .

ReferencesEN 12780 Standard (2002) Water Quality-Detection and enumeration of Ps.aeruginosa by membrane filtration. CEN. Brussels.DIN Standard 3841. Deutsche Einheitsverfahren zür Wasser, Abwasser und Schlammuntersuchung Mikro-biologische Verfahren: Nachweiss von Pseudomonas aeruginosa (K8).KELLY, N.M., C.T. KEANZ (1983) Acetamide Broth for Isolation of Pseudomonas aeruginosa from patients with cystic fibrosis. J. Clin. Microbiol. 17:159-163.ISO 16266 (2006). Water Quality. Detection and enu-meration of Pseudomonas aeruginosa. Method by membrane filtration.

Ref. 03-428 Acetamide Medium. Left: control; center: Pseudomonas aeruginosa ATCC 27853; right: Pseu-

domonas aeruginosa ATCC 9027.

R-40S-36/37

Xn

4

Actinomycete Media

Actinomycete Agar

Ref. 01-003

SpecificationSolid culture medium for actinomycete maintenance and propagation according to Ajello et al. formulation.

Formula (in g/L)Meat peptone .........................................10,00Casein peptone ........................................4,00Heart extract ...........................................10,00Yeast extract .............................................5,00Dextrose ...................................................5,00Potassium phosphate .............................15,00Sodium chloride ........................................5,00Starch .......................................................1,00Ammonium sulfate ....................................1,00Cysteine ...................................................1,00Magnesium sulfate ...................................0,20Calcium chloride .......................................0,01Agar ........................................................20,00Final pH 7,0 ± 0,2

DirectionsSuspend 77 g of powder into 1 L distilled water and let it soak. Bring to boiling with constant stirring and distribute into suitable containers. Sterilize by autoclaving at 121°C for 15 minutes.

Actinomycete Broth

Ref. 02-003

SpecificationLiquid culture medium, able to form a fluid medium, for the pathogenic actinomycete maintenance and propaga-tion, according to Ajello et al. formulation.

Formula (in g/L)Meat peptone .........................................10,00Casein peptone ........................................4,00Heart extract ...........................................10,00Yeast extract .............................................5,00Dextrose ...................................................5,00Potassium phosphate .............................15,00Sodium chloride ........................................5,00Starch .......................................................1,00Ammonium sulfate ....................................1,00Cysteine ...................................................1,00Magnesium sulfate ...................................0,20Calcium chloride .......................................0,01Final pH 7,0 ± 0,2

DirectionsDissolve 57 g of powder into1 L of distilled water. Heat if necessary. Distribute into suitable containers and steri-lize by autoclaving at 121°C for 15 minutes.Should a fluid medium be required, incorporate 7 g of Agar and follow the directions for the solid medium. After the sterilization, cool it quickly but gently to avoid agar flocculation.

DescriptionBasically Pine and Watson formulated this medium for the maintenance of pathogenic actinomycytes which was then further modified by Ajello et al. Those media do not possess any inhibitory agents, and so are not recom-mended for the isolation, but their slight reducing power and their high nutritive capacity make them suitable for facultative anaerobes also.

TechniqueIf growth conditions require strict anaerobiosis, the incu-bation must be done in special flasks, but if the condi-tions are only moderate or not strict anaerobic, a good anaerobiosis can be obtained by sealing the flasks with Sealing Anaerobic Agar (Ref. 01-174) or with the classic alkaline pyrogallate caps.

Best results are obtained using an Anaerobic Jar with a CO

2 mixture generator.

The use of a liquid medium allows freezing of the cul-tures after their growth and the use also as a massive production system for cellular preparations. In the fluid format, the Actinomycete Broth reaches a high reduction potential allowing an easy growth to strict anaerobics.

There is only one difference between solid and broth formulation which is the addition of agar 20,0 g/L to the solid medium as a solidifying agent, providing the suf-ficient strength to support the surface growth.

ReferencesAJELLO, GEORG, KAPLAN & KAUFFMAN (1963) CDC Lab Manual for Medical Mycology, Publication nº994 US Govt. Prmtmg. Office. Washington.LENNETTE, SPAULDING & TROULANT (1974) Manual of Clinical Microbiology. ASM 2d. Ed. Washington.PINE, L., A.HOWELL, S.J.WATSON (1960) Studies on the morphological characters of Actynomyces bovis. J. Gen. Microbiol. 23:403-424.ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press. London.

5

AFP Agar (Aspergillus flavus/parasiticus Agar)

Ref. 01-405

SpecificationSelective solid medium for the isolation and enumeration of Aspergillus flavus and Aspergillus parasiticus.

Formula (in g/L)Peptone ................................................10,000Yeast extract .........................................20,000Ammoniun Iron Citrate ..........................0,500Dichloran ................................................0,002Chloramphenicol ....................................0,100Agar ......................................................15,000Final pH 6,3 ± 0,2

DirectionsSuspend 45,6 g of powder in 1 L of distilled water and bring to the boil. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

DescriptionAFP Agar is a modification of the original medium devel-oped by King, Hocking & Pitt for the enumeration and isolation of moulds in foods. The inclusion of ammonium iron citrate and the high temperature (30ºC) of incubation enhances the quickly typical pigmentation in Aspergillus flavus and A. parasiticus.

The elimination of glucose from the formula, with the low value of pH and the presence of chloramphenicol inhibits the bacterial growth and the dichloran repress the spreading of the fungal colonies.

Technique The sample is inoculate on the surface of the AFP Agar ad the plates are incubated at 30ºC for 42-48 hours. Count the fungal colonies that shows the typical yellow-orange pigmentation on the reverse. Express the results as “Aspergillus flavus/parasiticus colonies per g or mL of sample”.

In this conditions Aspergillus orizae can produce a very similar pigmentation than induce error in the counting. Also, Aspergillus niger can be present but its pigment is pale yellow and never turns to orange and after 48 hours of incubation begin to produce the typical black conidi-ophora.

ReferencesKING D.A. A.D. HOCKING & J.I. PITT (1979) Dichloran-rose bengal médium fot enumeration and isolation of moulds from foods. Appl. Environm. Microbiol 37:959-964VANDERZANT, C. & D.F. SPLITTSTOESSER (1992) Compendium of methods for the microbiological exami-nation of foods. APHA. Washington.ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press. London

R-45S-45-53

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Ref. 01-405 AFP Agar. Left: control; right: Aspergillus ssp.

6

Algae Media

Algae Agar

Ref. 01-007

SpecificationSolid culture medium for the isolation and cultivation of algae from soil, water and waste water.

Formula (in g/L)Sodium nitrate ........................................1,000Dipotassium phosphate ..........................0,250Magnesium sulfate .................................0,513Ammonium chloride ................................0,050Calcium chloride .....................................0,058Ferrous chloride .....................................0,003Agar ......................................................15,000Final pH 7,0 ± 0,2

DirectionsSuspend 17 g of powder into 1 L of distilled water and let it soak . Bring to boiling with constant stirring. Distribute into appropriate containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionThe balanced nutrient medium composition provide all necessary nutrients for the good growth of the algae , but it does not support the good growth of fungi and bacteria which can not grow well or have difficulties in the medium.It is also suitable for algicide testing, however it is essen-tially recommended for the algae-pattern maintenance and cultivation or for the isolation of water contaminants.

TechniqueFor the maintenance of algal strains it is recommended to incubate at room temperature, under a suitable light source (natural, fluorescent tube or incandescent lamp) until a good growth is obtained (within one to two weeks).In these conditions, and without gel dehydration, cultures can be maintained up to two months.

Algae Broth

Ref. 02-007

SpecificationNutritive solution for algae and cyanobacteria, suitable for water algicide biotesting.

Formula (in g/L)Sodium nitrate ........................................1,000Dipotassium phosphate ..........................0,250Magnesium sulfate .................................0,513Ammonium chloride ................................0,050Calcium chloride .....................................0,058Ferrous chloride .....................................0,003Final pH 7,0 ± 0,2

DirectionsDissolve 1,87 g of powder in 1 L of distilled water and distribute into suitable containers. Sterilize in the auto-clave at 121°C for 15 minutes.

DescriptionThis liquid medium is suitable for algae and cyanobac-teria cultivation, and it is especially adapted for inocule preparation and algicide biotesting, as per Fitzgerald’s technique.Due to the less content of energy source, fungi and bacteria do not grow or grow badly unlike algae, which obtain all the necessary nutrients, except the energy source.

TechniqueFitzgerald’s procedure for the algicide ability verification of chemical products is:

a) Inoculum preparationPrepare the Algae Broth and distribute 20 mL. each in the 50 mL capacity conical flasks. Sterilize and keep them cool until usage.Regularly, one of the conical flasks is inoculated with a couple of loops from Chlorella emersonii culture from slanted Agar (Ref 1-007) and it is incubated at room temperature until good growth is observed.This culture can be used as biotest inoculum, but for only up to 30 days.

b) Biotest1- SamplesPrepare one litre of pure distilled water and 1 litre of distilled water containing the inhibitor. Add 120 mg of Sodium nitrate and 2,5 g of Di-potassium phosphate to each sample.2- Technique of the TestPrepare a double series of 50 mL capacity conical flasks and add 5, 12.5 and 25 mL respectively of water algicidal mixture, and then refill with pure water to get 25 mL in each conical flask.Add only 25 mL of pure water in one or two conical flasks to use them for control purposes.All the conical flasks are inoculated with the same vol-ume of inoculum, the necessary amount to get an algae concentration about 300.000 cells/mL in each flask. As a practice (not exactly) this concentration produces a slight greenish tinge. If necessary, adjust the inoculum by counting or photocolorimetry.

7

Algae Media

Incubate the inoculated flasks at room temperature under an homogeneous and standardized light (i.e. 20 W fluorescent light).Countings of all the flasks is carried out daily with a glob-ule-cell count (Thoma, Neubauer type or similar).The test is said to be over when the control conical flasks have an average concentration greater than 5x106 cells/mL, comparing the other flasks to them.3-InterpretationInhibitor concentration in the flasks with equal growth to the control is considered non toxic or ineffective. If algal concentration is maintained or remains same as at the starting of experiment, it is considered algistatic. Concentrations that have reduced starting population are considered as algicidic, with the different effectivity ratio depending on the amount of loss.

ReferencesCLESCERI, L., A.E. GREENBERG, A.D. EATON (1998) Standard Methods for Examination of Water and Waste-water. APHA-AWWA-WEF. Washington, D.C.ALLEN, (1952) Arch. Microbiol. 17:34FITZGERALD (1962) Water and Sewage Works. 109:361.

Antibiotic Media

While performing the antibiotic assays, the only meth-odology accepted universally is the microbiological methodology. National pharmacopeia provides different directives to adapt to new compounds. Nowadays, the European Pharmacopeia (1996), following a path to standardize all the countries in the EU, provides several rules and recommendations but it does not impose strict criteria, since they could interfere with the local legisla-tion. Instead of this, the USP 25/NF 20 (2002) incorpo-rates more concrete guidelines about the antibiotics that are accepted for human consumption and their assay methodology. However, many times the USP refers to the US/FDA, which is the more detailed publication about the antibiotics assay.Kirshbaun and Arret, in 1959, published a report with all the variations of this methodology. The success of their first attempt, and the discovery of new antibiotics made the same authors revise their work and , in 1967, they published a new report detailing 57 official methods, and in 1971 last edition they detailed 83 official methods and 10 non official methods. The XIX edition of the USP con-sider this last 1971 edition as its information source.The present article simply pretends to resume the most important characteristics of this methodology, following the guidelines of the current pharmacopeia. There are no detailed directions to perform the assay because it is considered that this is going to be read by microbiol-ogists who are familiar with the basic techniques.If you are interested in this subject, we would ask you to read

the bibliography mentioned at the end of this publication, especially 21CFR for the details about the preparation of samples and the extraction of antibiotics for each phar-maceutical preparation.The reference substances used in the assays are sub-stances whose activity has been precisely determined with references to the corresponding international stand-ard or international reference preparation.

Microorganisms

Strains used in the antibiotics assay as well as the preparation methods and inocula are resumed in the Table I. Strains may be obtained from the following and other collections:

ATCC: American Type Culture Collection, 12301 Park-lawn Drive, Rockville, Maryland 20852-176. USA. Fax +1 301-231-5826

CIP/CNCM: Collection de l’Institut Pasteur / Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS CEDEX 15. France. Fax +33 143 06 98 35

CECT: Colección Española de Cultivos Tipo, Dpto Micro-biologia, Fac. Ciencias Biológicas, Dr. Moliner 50, 46100 Burjasot (Valencia) Spain. Fax +34 63 86 43 72

Ref. 01-007 Algae Agar. Chlorella sp.

8

Antibiotic Media

NCTC:National Collection of Type Cultures, PHLS Central Public Health Laboratory, 61 Colindale Avenue, LONDON NW9 5HT England.

NCIB: The National Collections of Industrial and Marine Bacteria Ltd., 23 St Machar Drove, ABERDEEN AB2 1RY, Scotland. U.K. Fax +4402 24 487 658

NCYC: National Collection of Yeast Cultures. Institute of Food Research. Norwich Laboratory. Colney Lane. NORWICH NR4 7UA. U.K.

Preparation methodsMethod #1

Strains to be assayed are mantained in slanted tubes of Antibiotic Medium 1 (Ref. 01-009), subculturing to fresh tubes every week. The growth on a slanted tube, incubated at 32-37° for 24 hours, is washed with 3 mL of Ringer Solution (Ref. 06-073), and the resulting suspension is used to inoculate a Roux flask containing 250 mL of sterile and controlled Antibiotic Medium 1 (Ref. 01-009).For the maintenance of antibiotic resistent strains prepare Antibiotic Medium 1 (Ref. 01-009) with the addition of proper amounts of antibiotic, as described in the Table II. The growth obtained in the Roux flask after a 24 hours incubation at 32-37°C is washed and collected with 50 mL of Ringer Solution (Ref. 06-073) and it is stored in refrigeration (4-6°C)

Method #2Process is the same as the Method #1 but the standardization is performed in the following way:Centrifuge and collect the pellet. Sediment is sus-pended with 50-70 mL of Ringer 1/4 Solution (Ref. 06-073) and heat up to 70°C for 30 minutes. Refrig-erate the spore suspension obtained in this way.

Method #3Proceed as per Method #2 performing the thermal shock before the centrifugation. Wash the spore suspension 3 consecutive times with distilled water (25-50 mL each time) and centrifuge. Reconstitute the final suspension with 50-70 mL of sterile distilled water and store it in refrigeration.

Method #4Let the fungi grow for 6-8 weeks at room temperature (20-25°C) in several 3 L Erlenmeyer flasks, contain-ing each one about 200 mL of Antibiotic Medium 22.After six weeks, test the sporulation regularly and, when it reaches 80%, collect the spores of the aerial micelium with a sterile spatula or any other suitable instrument. The collected spores are suspended in 20 mL of Ringer 1/4 Solution (Ref. 06-073) and store refrigerated.

Table I. Strains (Test organisms) for the Antibiotic Assays# Strain Genus & species Strain and Type culture collection # Method American Collection European Collections A Staphylococcus aureus ATCC 6538P CIP 53.156 NCTC 7447 1 B Staphylococcus aureus ATCC 29737 1 C Staphylococcus aureus ATCC 9144 CIP 53.134 NCIB 6751 1 D Staphylococcus epidermidis ATCC 12228 CIP 68.21 NCIB 8853 1 E Micrococcus luteus ATCC 9341 CIP 53.45 NCTC 8340 1 F Micrococcus luteus (flavus) ATCC 10240 CIP 53.160 NCTC 7743 1 G Enterococcus hirae ATCC 10541 CIP 58.55 NCIB 8192 5 H Bacillus pumilus CIP 76.18 NCTC 8241 2 I Bacillus cereus ATCC 11778 CECT 193 NCTC 10320 3 J Bacillus subtilis ATCC 6633 CIP 52.62 NCTC 10400 2 K Bacillus subtilis CIP 1.83 NCTC 8236 2 L Klebsiella pneumoniae ATCC 10031 CIP 53.153 NCTC 7427 1 M Escherichia coli ATCC 10536 CIP 54.127 NCIB 8879 1 N Escherichia coli ATCC 9637 CIP 2.83 NCIB 8666 1 O Bordetella bronchiseptica ATCC 4617 CIP 53.157 NCTC 8344 1 P Saccharomyces cerevisiae ATCC 9763 CIP 1432.83 NCYC 87 61

Q Saccharomyces cerevisiae ATCC 2601 CECT 1324 NCYC 853 6 R Pseudomonas aeruginosa ATCC 25619 NCIB 10817 1 S Mycobacterium smegmatis ATCC 607 NCTC 7017 7 T Candida tropicalis CIP 1433.83 NCYC 1393 6 1 For candicidine, use method #1

9

Antibiotic Media

Method #5With the help of a loop inoculate a flask containing 100 mL of sterile and controlled Antibiotic Medium 3 (Ref. 02-011). from a slant culture. Incubate for 16-18 hours.

Method #6Follow the directions stated in the Method #1 but in-cubate the slanted subcultures at 30°C for 24 hours, and the Roux flasks at 30°C for 48 hours.

Method #7Organisms are maintained on Antibiotic Medium 36 in slanted tubes, subculturing them each week with an incubation at 37°C for 48 hours.Collect the growth of a recent subculture with 3 mL of Ringer 1/4 Solution (Ref. 06-073) and use it to inoculate a 500 mL Erlenmeyer flask containing 100 mL of Antibiotic Medium 34. Add asseptically to the flask 50 g of sterile glass balls and incubate at 27°C for 5 days with constant stirring (120 rpm and 3,5 cm radius). Keep the suspension in refrigeration.

Inoculum DeterminationFor many years the official US/FDA methods provided specific instructions for the inoculum standardization. Af-ter 1985 the USA Federal Regulations and the USP XIX adopted the criterion suggested by Kirshbaum, Kramer and Gorth that recommends to dilute the microorgan-isms suspension until it provides a 25% transmitance at 580 nm in a 13 mm tube. However, even with this stand-

ardization it is necessary to test every batch of inoculum to determine the exact volume that must be added to each assay medium.If the inoculum suspensions are prepared as described in the above sections, they will be homogeneous enough to determine the volumes that are going to be added, with tests, without any other previous standardization step.Diffusion agar Assay

Prepare a series of flasks with 100 mL of sterilized, melted and cooled Seed Layer Medium (Ref. 01-009) and add to each flask a different volume of micro-organisms suspension, as per the volume pattern suggested in Table IV.Prepare Petri plates with the inoculated medium according to the specificacions of each antibiotic and put a steel or glass cylinder or a disc with the refer-ence concentration of antibiotic. Incubate the plates and measure the inhibition zones. The volume of suspension that produces optimum zones, in defini-tion as well as in diameter, is the one that is adopted for the inoculum. In Table IV the optimum inhibition zones are shown that are supposed to appear in the different assays.

Turbidimetric AssayProceed in the same way as in the previous case but instead of inoculating over the melted medium, inoc-ulate in the flasks containing 100 mL of the suitable liquid medium, using the volumes pattern specified in

Table II. Buffer solutions and diluents # Buffer1 Formula (in g/L of distilled water)

1 Phosphate buffer 1% at pH 6,0±0,05 K2HPO

4 ................................. 2,000 g

KH2PO

4 ................................. 8,000 g

3 Phosphate buffer 0,1M at pH 7,9±0,1 K

2HPO

4 ............................... 16,730 g

KH2PO

4 ................................. 0,523 g

4 Phosphate buffer 0,1M at pH 4,5±0,05 KH

2PO

4 ............................... 13,600 g

6 Phosphate buffer 10% at pH 6,0±0,05 K

2HPO

4 ............................... 20,000 g

KH2PO

4 ............................... 80,000 g

10 Phosphate buffer 0,2M at pH 10,5±0,1 K

2HPO

4 ............................... 35,000 g

KOH 10N ............................... 2,000 mL 16 Phosphate buffer 0,1N at pH 7,0 K

2HPO

4 ............................... 13,600 g

KH2PO

4 ................................. 4,000 g

17 Phosphate-methanol buffer at pH 6 Methanol .............................. 50,000 mL Buffer #1 ........................................ q.s.

18 Sodium carbonate solution 1% Sodium carbonate ............... 10,000 g

1 May require adjustment with phosphoric acid 18 N or KOH 10N before or after sterilization.

10

Antibiotic Media

Table III . With these volumes of inoculated medium continue as if you were performing an assay, using just the highest and lowest concentrations in relation to the pattern. Incubate for 3-4 hours and read the absorbances. With all these data you will be able to establish the right inoculum that provides a better answer between the high and low point, and so adopt it for the assay.

Strain maintenanceAssay strains are maintained by vegetative propaga-tion in slants and in duplicate. One of the duplicates is used only for the next subculture, and the other one is used for all the other operations. Medium, if there is no other directive, is Antibiotic Medium 1 (Ref. 01-009) with antibiotics or other additive if necessary. Details may be obtained in Table II. Nonetheless, other media may be used if considered necessary.

Buffer Solutions and diluents

Composition of the most common buffer solutions and diluents is given in Table II. Dilutions of pure chemical products as well as organic diluents have been om-mited. In anycase, the compounds must be according to the purity standards of the corresponding pharmaco-peia. Water must always be distilled and be of reagent grade. All buffer solutions and diluents have to be sterile. Sometimes a little pH readjustment is necessary after the sterilization.

Culture Media

The composition of the several SCHARLAU Microbiol-ogy Antibiotic Media is given below. Media still have the nomenclature according to the US/FDA, which uses the one described by Grove and Randall (media 1 to 13) and Kirshbaum and Arret (media 18 to 21). The Euro-pean Pharmacopeia names the media with alphabetical letters, and the corresponding Scharlau media are as follows:

Medium A ..............Antibiotic Medium 1 and 11Medium B .......................Antibiotic Medium 10Medium C ........................Antibiotic Medium CMedium D ........................Antibiotic Medium DMedium E ........................ Antibiotic Medium EMedium F........................Antibiotic Medium 19Medium G .......................Antibiotic Medium 35

Note: Medium D is very similar to Antibiotic Medium 3 but with the addition of 2 g/L of potassium nitrate in it.

SCHARLAU media described following are prepared with the ingredients specified in the USP 23/NF 18, Eur. Pharm. 3 and 21 CFR for these components, providing an absolute reproducubility and reliability.

The components of the culture media accomplishes the specifications of the several pharmacopeia, however the technician is responsible for the end use of dehydrated culture media or any other variation in it, as long as they still have the same characteristics described for the above media. pH of the medium must be checked when it is completely reconstituted and at 25°C.

Antibiotic Medium 1(Eur. Phar. Antibiotic Medium Aat pH 6,6)

Ref. 01-009

SpecificationAntibiotic Medium 1 or Seed Layer is used for the anti-biotic assays by the diffusion method in agar, that may be performed in several ways (cylinder, punched-hole or paper disc method).

Formula (in g/L)Peptone .....................................................6,0Casein Peptone ..........................................4,0Yeast extract ...............................................3,0Beef extract ................................................1,5Dextrose .....................................................1,0Agar ..........................................................15,0Final pH 6,6 ± 0,2

DirectionsAdd 30,5 g of medium to 1 L of distilled water. Heat to the boiling and dispense into suitable containers. Steri-lize in the autoclave at 121°C for 15 minutes.

11

Antibiotic Media

Antibiotic Medium 2

Ref. 01-010

SpecificationAntibiotic Medium 2 or Basal Layer is used for the anti-biotic assays by the diffusion method in agar, that may be performed in several ways (cylinder, punched-hole or paper disc method).

Formula (in g/L)Peptone .....................................................6,0Yeast extract ...............................................3,0Meat extract ................................................1,5Agar ..........................................................15,0Final pH 6,5 ± 0,2


Antibiotic Medium 3

Ref. 02-011

SpecificationAntibiotic medium 3 or Antibiotic Assay Broth may be used in the inoculum preparation, serial dilutions or turbi-dimetric antibiotic assays.

Formula (in g/L)Peptone ...................................................5,00Yeast extract .............................................1,50Meat extract ..............................................1,50Sodium chloride ........................................3,50Dextrose ...................................................1,00Monopotassium phosphate ......................1,32Dipotassium phosphate ............................3,68Final pH 7 ± 0,05


Antibiotic Medium 4

Ref. 01-012

SpecificationAntibiotic Medium 4 is used in the antibiotic assays by the diffusion method in agar, which may be performed in several ways (cylinder, punched-hole or paper disc method)

Formula (in g/L)Peptone .....................................................6,0Yeast extract ...............................................3,0Meat extract ................................................1,5Dextrose .....................................................1,0Agar ..........................................................15,0Final pH 6,5 ± 0,1


Antibiotic Medium 5

Ref. 01-013

SpecificationAntibiotic Medium 5 is used in the antibiotic assays by the diffusion method in agar, which may be performed in several ways (cylinder, punched-hole or paper disc method).



Antibiotic Medium 8

Ref. 01-014




12

Antibiotic Media

Antibiotic Medium 9

Ref. 01-015


Formula (in g/L)Casein peptone ........................................17,0Soy peptone ...............................................3,0Sodium chloride ..........................................5,0Dipotassium phosphate ..............................2,5Dextrose .....................................................2,5Agar ..........................................................20,0Final pH 7,2 ± 0,2

DirectionsAdd 50 g of medium to 1 L of distilled water. Heat to the boiling and dispense into suitable containers. Sterilize in the autoclave at 121°C for 15 minutes.

Antibiotic Medium 10 (Eur. Phar. Antibiotic Medium B)

Ref. 01-016


Formula (in g/L)Casein peptone ........................................17,0Soy peptone ...............................................3,0Sodium chloride .........................................5,0Dipotassium phosphate ..............................2,5Dextrose .....................................................2,5Agar ..........................................................12,0Final pH 7,2 ± 0,2

DirectionsAdd 42 g of medium to 1 L of distilled water. After disso-lution, add 10 mL of Polysorbate 80. Heat to the boiling and dispense into suitable containers. Sterilize in the autoclave at 121°C for 15 minutes.

Antibiotic Medium 11(Eur. Phar. Antibiotic Medium A at pH 8,0)

Ref. 01-017


Formula (in g/L)Peptone .....................................................6,0Casein peptone ..........................................4,0Yeast extract ...............................................3,0Meat extract ................................................1,5Dextrose .....................................................1,0Agar ..........................................................15,0Final pH 8,0± 0,2


Antibiotic Medium 13

Ref. 02-544

SpecificationAntibiotic Medium 13 is used in the turbidimetric antibi-otic assays.

Formula (in g/L)Peptone ....................................................10,0Dextrose ...................................................20,0Final pH 5,5 ± 0,2


Antibiotic Medium 19(Eur. Phar. Antibiotic Medium F)

Ref. 01-434


13

Antibiotic Media

Formula (in g/L)Peptone .....................................................9,4Yeast extract ...............................................4,7Beef extract ................................................2,4Sodium chloride ........................................10,0Dextrose ...................................................10,0Agar ..........................................................23,5Final pH 6,0 ± 0,1



Ref. 01-069

This medium is sold by Scharlay Microbiology under the name Sporulating AK Agar.


Formula (in g/L)Peptone .................................................6,000Casein peptone ......................................4,000Yeast extract ...........................................3,000Meat extract ............................................1,500Dextrose .................................................1,000MnSO

4.H

20 .............................................0,003

Agar ......................................................15,000Final pH 6,5 ± 0,2


Antibiotic Medium 34 Base

Ref. 02-545

SpecificationAntibiotic Medium 34 is used in the turbidimetric antibi-otic assays.

Formula (in g/L)Peptone ....................................................10,0Meat extract ..............................................10,0Sodium chloride ..........................................3,0Final pH 7,0 ± 0,1

DirectionsAdd 33 g of medium to 1 L of distilled water.Add 10 mL of glycerol. Heat to the boiling and dispense into suitable containers. Sterilize in the autoclave at 121°C for 15 minutes.

Antibiotic Medium 35 Base (Eur. Phar. Antibiotic Medium G)

Ref. 01-545


Formula (in g/L)Peptone ....................................................10,0Meat extract ..............................................10,0Sodium chloride ..........................................3,0Agar ..........................................................15,0Final pH 7,0 ± 0,1

DirectionsAdd 38 g of medium to 1 L of distilled water containing 10 mL glycerol. Heat to the boiling and dispense into suitable containers. Sterilize in the autoclave at 121°C for 15 minutes.


Ref. 01-200

Sharlau Microbiology sold this medium as Tryptic Soy Agar (TSA).


Formula (in g/L)Casein peptone ........................................15,0Soy peptone ...............................................5,0Sodium chloride ..........................................5,0Agar ..........................................................15,0Final pH 7,3 ± 0,1

DirectionsAdd 40 g of medium to 1 L of distilled water.Heat to the boiling and dispense into suitable containers. Sterilize in the autoclave at 121°C for 15 minutes.

14

Antibiotic Media


Ref. 02-547

SpecificationAntibiotic Medium 39 may be used in the inoculum preparation, serial dilutions or turbidimetric antibiotic assays.

Formula (in g/L)Peptone ...................................................5,00Yeast extract .............................................1,50Meat extract ..............................................1,50Sodium chloride ........................................3,50Dextrose ...................................................1,00Monopotassium phosphate ......................1,32Dipotassium phosphate ............................3,68Final pH 7,9 ± 0,1



Ref. 01-546

SpecificationAntibiotic Medium 40 is used in the antibiotic assays by the diffusion method in agar, which may be performed in several ways (cylinder, punched-hole or paper disc metho

Formula (in g/L)Polypeptone ...............................................5,0Dextrose ...................................................10,0Yeast extract .............................................20,0Polysorbate 80 ...........................................0,1Monopotassium phosphate ........................2,0Agar ..........................................................12,0Final pH 6,8 ± 0,1



Ref. 02-548

SpecificationAntibiotic Medium 41 is used in the antibiotic assays by the turbidimetric method.

Formula (in g/L)Casein Peptone .......................................9,00Yeast extract .............................................5,00Sodium citrate ........................................10,00Dextrose .................................................20,00Monopotassium phosphate ......................1,00Dipotassium phosphate ............................1,00Final pH 6,8 ± 0,1

DirectionsAdd 46g of medium to 1 L of distilled water. Heat to the boiling and dispense into suitable containers. Sterilize in the autoclave at 121°C for 15 minutes.

Antibiotic Medium C (Eur. Phar. Antibiotic Medium C)

Ref. 02-601

SpecificationAntibiotic Medium C may be used for the inoculum prepa-ration, serial dilutions or turbidimetric antibiotic assays.

Formula (in g/L)Peptone ...................................................6,00Yeast extract .............................................3,00Beef extract ..............................................1,50Sodium chloride ........................................3,50Dextrose ...................................................1,00Monopotassium phosphate ......................1,32Dipotassium phosphate ............................3,68Final pH 7,0 ± 0,2

DirectionsAdd 20 g of medium to 1 L of distilled water. Heat to the boiling and dispense into suitable containers. Sterilize in the autoclave at 121°C for 15 minutes. If pH 8,0 is desired, adjust with NaOH1N.

Antibiotic Medium D (Eur. Phar. Antibiotic Medium D)

Ref. 02-549

SpecificationAntibiotic Medium D may be used for the inoculum prepa-ration, serial dilutions or turbidimetric antibiotic assays.

Formula (in g/L)Heart extract .............................................1,50Yeast extract .............................................1,50Casein peptone ........................................5,00Sodium chloride ........................................3,50Dextrose ...................................................1,00Monopotassium phosphate ......................1,32Dipotassium phosphate ............................3,68Potassium nitrate .....................................2,00Final pH 7,0 ± 0,2

15

Antibiotic Media


Antibiotic Medium E (Eur. Phar. Antibiotic Medium E)

Ref. 01-430

SpecificationAntibiotic Medium E is used in the antibiotic assays by the diffusion method in agar, which may be performed in several ways (cylinder, punched-hole or paper disc method).

Formula (in g/L)Peptone ...................................................5,00Meat extract ..............................................3,00Disodium phosphate ...............................26,90Agar ........................................................12,00Final pH 7,9 ± 0,2

DirectionsAdd 46,9 g of medium to 1 L of distilled water. Heat to the boiling and dispense into containers. Sterilize in the autoclave at 121°C for 15 minutes. Precipitates ap-pearing after sterilization do not interfere in its normal operation.

Table III. Specifications for the antibiotic master standard solutions

Drying cond. Initial solvent Master soln Days (cool) Final solvent

Amikacin 6 Distilled water 1,0 mg 15 Distilled waterAmphotericin B1 1 Dimethylsulfoxide 1,0 mg 1 10Bacitracin 1 1 100,0 ui 7 1Bleomycin 7 16 2,0 ui 15 16Carbenicillin None 1 1,0 mg 15 1Cephalothin 1 1 1,0 mg 5 1Cycloserine 1 Distilled water 1,0 mg 30 1Chloramphenicol None Ethanol 1,0 mg 30 1Chlortetracycline None HCl 0,01N 1,0 mg 4 4Cloxacillin None 1 1,0 mg 7 1Colistimethate Na 1 Distilled water 1,0 mg 1 6Colistin 1 Distilled water 1,0 mg 15 6Demeclocycline None HCl 0,01N 1,0 mg 5 4Dihydrostreptomycin 5 3 1,0 mg 30 3Doxycycline None HCl 0,01N 1,0 mg 5 Distilled waterErythromycin 1 Methanol 1,0 mg 14 3Framicetine 3 Distilled water 1,0 mg 7 3Gentamicin 3 3 1,0 mg 30 3Gramicidin 1 Ethanol 1,0 mg 30 EthanolKanamycin SO

4 None 3 1,0 mg 30 3

Methacycline None HCl 0,01 N 1,0 mg 15 4Methylmicin None 3 1,0 mg 7 3Nafcillin None 1 1,0 mg 2 1Natamycin 4 Dimethylsulfoxide 1,0 mg 1 10Neomycin 1 3 1,0 mg 15 3Novobiocin 5 Absolute Ethanol 1,0 mg 5 6Nystatin 4 Dimethylformamide 1000,0 ui 1 6Oxytetracycline None HCl 0,1N 1,0 mg 4 4Paromomycin 1 3 1,0 mg 21 3Penicillin G None 1 1000,0 ui 2 1Polymyxin B 1 Distilled water 10000,0 ui 15 6Rifampin None Methanol 1,0 mg 1 1Rolitetracycline None Distilled water 1,0 mg 1 Distilled waterSisomycin None 3 1,0 mg 15 3Spectinomycin 6 Distilled water 1,0 mg 30 Distilled waterSpiramycin 1 Methanol 1,0 mg 7 3Streptomycin 1 3 1,0 mg 30 3Tetracycline None HCl 0,1N 1,0 mg 1 4Ticarcillin None 1 1,0 mg 1 1Thiostreptone None Dimethylsulfoxide 1,0 ui 1 DimethylsulfoxideTobramycin 3 Distilled water 1,0 mg 15 16Troleandomycin None Isopropanol 1,0 mg 1 Distilled waterVancomycin 1 Distilled water 0,4 mg 7 4

1 Use amber glass or work under red light

16

Antibiotic Media

Reference Standard

Each antibiotic must be assayed with a proper reference standard or master standard pattern. Patterns may be obtained from the USP NF Convention Inc. or other organisms, such as:European Pharmacopeia Secretariat, B.P. 907, F-67029 Strasbourg. CEDEX 1. Fax +33 388 41 20 36. e-mail:[email protected] Reference Standards, 12601 Twinbrook Park-

way, Rockville. Md. 20852, USABritish Pharmacopoeia Comission Laboratory, Govern-

ment Buildings, Block 2, Honeypot Lane, Stan-more, Middlesex, HA7 1A4. England.

However, you may use domestic or working standards if they have been compared to a reference pattern. In Table III you may find details about the reference stand-ard: dessication conditions, initial diluent to obtain the mother solution, activity period,etc...Following are described the dessication methods for the standards. US/FDA numeration have been maintained.

US/FDA Method #1In vacuum oven, dry the pattern or master reference standard at 60°C for 3 hours, at pressure 5 mm Hg.




US/FDA Method #6In vacuum oven, dry the pattern or master reference standard at 45°C for 3 hours, at pressure 5 mm Hg .

US/FDA Method #7In vacuum oven, dry the pattern or master reference standard at 35°C for 4 hours, at pressure 5 mm Hg .

Diffusion Method in Agar (Plate assay)

Once we decide the assay conditions (tabulated in Table IV), we can prepare the plates as per the following method .

Plate preparation..Pour basal medium, melted and cooled to 50°C, into each plate, and let it solidify horizontally. Using 10 cm. diameter plates, suggested volumes are 21 mL for each basal layer and 4 mL for the seed layer. Nonetheless, in the case of amphotericin and nystatin a single seed layer of 8 mL is used, without any basal layer. Another exception to this rule is bleomycin, for which a 10 mL of basal layer and a 6 mL of seed layer is used. In practise, when the standard 9 cm diameter plastic plates are used it is advisable to use the volumes of about 15 mL for the basal layer and 4 mL for the seed layer, in order to avoid the contact between the cylinders and the plate cover.While the basal layer solidifies, adjust the temperature for the medium of seed layer to 45-48°C, and inocu-late with the suggested (or tested) volume of inoculum suspension. Homogenize by gentle stirring and carefully pour the medium over the basal layer in 4 mL volumes, except for the cases mentioned above. Be very careful in this operation, since the final results may be affected if the layer is not very homogeneous, and forms bubbles or clots. Once the agar solidifies, place the cylinders or make the holes. Put or make 6 cylinders or holes per plate, centered in the plate at 60° angle from each other.

Reference pattern preparation and standard curve Master pattern is prepared according to the specifica-tions in Table IV, starting from the stock solution and using the adequate diluent until the desired concentra-tion is obtained. Reference level is always the medium concentration among the 5 concentrations used to obtain the standard curve (see Table IV).

Assay techniqueTo obtain the curve, 12 plates are used, three for each level, except for the reference standard, which is in-cluded in all. In each batch of three plates, 3 alternate holes or cylinders are filled with the reference concentra-tion and the other three are filled with the corresponding concentration. By proceeding in this way we can obtain 30 inhibition halos or inhibition zones corresponding to the reference concentration and 9 halos for each one of the other assay standards.Three plates, with 6 holes or cylinders, are used in each test. Holes or cylinders are filled alternatively with the reference level and the diluted sample. Plates are incubated at appropriate temperature (see Table IV) for 16-18 hours. After this period, inhibition halo readings are performed using the proper measuring instrument.

17

3a+2b+c-eA = 5

3e+2d+c-aB = 5

Antibiotic Media

Potency calculationTo extrapolate from the standard curve, use the average of the 36 diameters of the reference level and the sev-eral batches of 9 halos obtained in each assay level. The average of the 36 halos of reference is the correction value of the standard curve, and thus we can achieve each point as the function of the average value of the reference values of the corresponding standard. Values obtained in this way are plotted over a semiloga-rithmic graph paper, drawing the antibiotic concentration (in mcg/mL) on X axis, and the halo or zone diameters on the Y axis. Standard curve is drawn by linking the five points or by tracing a straight line between the high and low point, which are calculated using the following statistical formulae:

where:A: Halo diameter, calculated for the highest standard

concentration.B: Halo diameter, calculated for the lowest pattern con-

centration.c: Reference halo diameter, average of the 36 halos

of the pattern plates.a,b,d,e: Corrected average values of the other pattern

assay levels,from the highest to the lowest order.

To estimate the potency of the test sample, calculate average of the inhibition halo diameters and the refer-ence and sample levels of the three plates, and perform the corrections related to the correction point. Corrected value of the problem is projected on the standard curve and thus the theoritical concentration is obtained. Theo-ritical concentraion multiplied by the dilution factor gives the real concentration of antibiotic in the sample.

Turbidimetric assay

Reference pattern preparation and standard curveOnce the assay conditions are determined as per Table III, prepare the pattern according to the specifications in Table V. At the time of assay experiment, take an aliq-uote of mother solution and prepare appropriate dilutions to obtain the desired concentrations. Reference level is the medium concentration between the five proposed for the standard curve.

Amphotericin B P 19 1,00 - 19 30 0,640 0,800 1,000 1,250 1,560 16-18Bacitracin F 1 0,30 9 10 32-35 0,640 0,800 1,000 1,250 1,560 16-18Bleomycin S 36 1,00 35 35 32-35 0,010 0,020 0,040 0,080 0,160 18-20Carbenicillin R 1 0,50 9 10 37 12,800 16,000 20,000 25,000 31,200 16-18Cephalothin A 1 0,10 2 1 32-35 0,640 0,800 1,000 1,250 1,560 14-16Cloxacillin A 1 0,10 2 1 32-35 3,200 4,000 5,000 6,250 7,810 18-20Colistimethate sodium O 1 0,10 9 10 37 0,640 0,800 1,000 1,250 1,560 13-15Colistin O 1 0,10 9 10 37 0,640 0,800 1,000 1,250 1,560 15-17Dihydrostreptomycin J 1 0,05 5 5 37 0,640 0,800 1,000 1,250 1,560 14-16Doxycyclin I 1 0,40 8 8 30 0,640 0,800 1,000 1,250 1,560 18-20Erythromycin E 1 1,50 11 11 32-35 0,640 0,800 1,000 1,250 1,560 17-19Framicetin J,H 1 0,40 1 1 35-37 0,640 0,800 1,000 1,250 1,560 14-16Gentamicin D 1 0,03 11 11 37 0,640 0,800 1,000 1,250 1,560 15-17Nafcillin A 1 0,30 2 1 32-35 1,280 1,600 2,000 2,500 3,120 14-16Natamycin P 19 1,00 19 19 28-30 3,200 4,000 5,000 6,250 7,810 17-19Neomycin A,H,J 1 0,40 11 11 32-35 6,400 8,000 10,000 12,500 15,600 16-18Netilmycin D 1 0,25 11 11 32-35 0,064 0,080 0,100 0,125 0,156 14-16Novobiocin D 1 4,00 2 1 35 0,320 0,400 0,500 0,625 0,781 16-18Nystatin Q 19 1,00 - 19 30 12,800 16,000 20,000 25,000 31,200 16-18Paromomycin D 1 2,00 11 11 37 0,640 0,800 1,000 1,250 1,560 19-21Penicillin G A 1 1,00 2 1 32-35 0,640 0,800 1,000 1,250 1,560 17-19Polymyxin O 1 0,10 9 10 37 6,400 8,000 10,000 12,500 15,600 15-17Rifamycin J 1 0,10 2 2 30 3,200 4,000 5,000 6,250 7,810 15-17Sisomycin D 1 0,03 11 11 32-35 0,064 0,080 0,100 0,125 0,156 15-17Spiramycin J 1 0,20 1 1 35-37 0,640 0,800 1,000 1,250 1,560 15-17Ticarcillin B 1 0,40 8 8 32-35 3,200 4,000 5,000 6,250 7,810 17-19Vancomycin J 1 0,20 8 8 37 6,400 8,000 10,000 12,500 15,600 16-18

Assay strain

(Table I)

Maintenance Medium(A.M. nº)

Inoculum(mL/100 mL

medium)Seed strain

Incubation Temperat.

(°C) Ref.

Final concentrations: assay levels for the standard curve in IU/mL or in mcg/mL Reference

halo(mm)

Basal strain

Medium

Antibiotic

Table IV. Agar diffusion test specifications

18

3a+2b+c-eA = 5

3e+2d+c-aB = 5

Antibiotic Media

Assay TechniqueThe assay is performed in the following way:

Prepare 3 tubes and put 1 mL (or 0,1 mL if the antibiotic is gramicidin or tyrothricin) of every assay level of stand-ard and sample. Thus, 15 tubes will be employed for the pattern and 3 tubes for each problem.Add asseptically 9 mL of inoculated assay broth to each tube, and put them into a boiling water bath for 2-4 hours. The exact incubation time is determined by observing the growth inside the tube, taking the standard tube as the reference.Tubes are removed from the boiling water bath and 0,5 mL of 12% formaldehyde is added in each tube in order to stop the growth. Growth in the form of turbidity is measured by a Spectrophotometer at the wavelength 530 nm. The zero is fixed by the device and 100% trans-mission is provided by a blank containing only the sterile medium and formaldehyde at the same proportion or concentration as that of the assay.

Potency calculationStatistical values corresponding to the highest and lowest concentrations are determined according to the following formulae:

where:A: Absorbance value, calculated for the highest

value of the pattern curve.B: Absorbance value, calculated for the lowest value of

the pattern curve.a,b,c,d,e: Average values for each value in the triplicate

standard curve, from the highest to the lowest concentration.

Potency estimation is performed by taking the average value of each assay level in the pattern curve over a semilogarithmic paper. Draw the absorbance over the logarithmic scale and concentration over the abscissa. If the calculated values are used, the result is a straight line, but a curve may be drawn by linking the five points. The absorbance average value of the test sample is pro-jected over the standard curve, obtaining in this way the theoritical concentration which, multiplied by the dilution factor, gives the real potency of the test sample.

Cross Contamination with Penicillin

To perform this assay all the instrumentation and even the environment must be free of penicillin. Plates are prepared following the general method, using 10 mL of Antibiotic Medium 1 (Ref. 01-009) in the basal layer and 4 mL of Antibiotic Medium 4 in seed layer and inoculating the C strain of microorganism (see Table I).

Amikacin A 0,50 3 32 6,000 8,000 10,000 12,500 15,600Candicidin P 0,20 13 29 0,030 0,042 0,060 0,084 0,120Capreomycin L 0,05 3 37 64,000 80,000 100,000 125,000 156,000Chloramphenicol M 0,70 3 32 2,000 2,240 2,500 2,800 3,120Chlortetracycline A 0,10 3 32 0,038 0,048 0,060 0,075 0,094Cycloserine A 0,40 3 32 32,000 40,000 50,000 62,500 78,100Demeclocycline A 0,10 3 32 0,064 0,080 0,100 0,125 0,156Dihydrostreptomycin L 0,10 3 37 24,000 26,800 30,000 33,500 37,500Doxycyclin A 0,10 3 32 0,064 0,080 0,100 0,125 0,156Gramicidin G 1,00 3 37 0,028 0,034 0,040 0,048 0,057Kanamycin B 0,20 3 32 8,000 8,900 10,000 11,200 12,500Methacycline A 0,10 3 32 0,038 0,048 0,060 0,075 0,094Neomycin L 2,00 39 37 0,640 0,800 1,000 1,250 1,560Oxytetracycline A 0,10 3 32 0,160 0,200 0,250 0,312 0,390Rolitetracycline A 0,10 3 32 0,160 0,200 0,250 0,312 0,390Spectinomycin M 0,10 3 32 24,000 26,800 30,000 33,500 37,500Streptomycin L 0,10 3 37 24,000 26,800 30,000 33,500 37,500Tetracycline A 0,10 3 32 0,160 0,200 0,250 0,312 0,390Thiostreptone G 0,20 3 37 0,400 0,600 0,800 1,000 1,250Tobramycin A 0,15 41 32 2,000 2,236 2,500 2,795 3,125Troleandomycin L 0,10 3 32 16,000 20,000 25,000 31,200 39,000

Antibiotic

Table V. Turbidimetric assay specifications

Inoculum(mL/100 mL

medium)

Assay strain(Table I)

Assay medium

Incubation tempera-

ture(°C)

Final concentrations: assay levels for the pattern curve in IU/mL or mcg/mL

Ref.

19

Antibiotic Media

Reference standard is prepared according to the stand-ard for penicillin, proposed in Table II, but taking the mother solution of 100 units and the final concentrations at 0,0005;0,0125; 0,0050; 0,100 and 0,200 penicillin units. Reference is the one with final concentration 0,05 units (see Table III).

Sample is prepared by dissolving 1 g in 18 mL of distilled water. From this initial solution, take 9 mL into a separat-ing funnel, and add 20 mL of amyl acetate. Add then 1 mL of Solution #11 (see Table II) and stir it vigorously. Let it rest and once the two layers are separated, take the aqueous layer to another separating funnel. Check the pH of the solution and if it is higher than 3, readjust to 2,5 with HCl. Extract again with amyl acetate and discard the aqueous layer.Mix the two parts of amyl acetate, and wash them with 10 mL of Solution #2 (see Table II). Discard the aqueous layer. Extract penicillin from the amyl acetate with 10 mL of phosphate buffer pH 6 (Solution #1, Table II). Penicillin presence is determined in this solution.Use 15 plates to perform the assay: three for each standard curve assay, except for the reference stand-ard, which is present in all of them. Place 6 holes or cylinders in each plate and fill them alternatively with the reference solution and the corresponding assay level. In this way, you will obtain 45 inhibition halos for the refer-ence level and 9 halos more for each one of the other levels.A part of sample (2-5 mL) is treated with 0,1 mL of peni-cillinase and incubated at 37°C for 1 hour. Use 3 plates for each sample, and fill 2 holes or cylinders in each one

with the pattern reference level. Fill another 2 holes or cylinders in each plate with the untreated sample, and the last 2 holes or cylinders in each plate with the treated sample.Plates are incubated at 30°C for overnight. Presence of inhibition halos in the untreated sample, and the absence of holes in the treated sample are a sign of penicillin contamination. Should a qualitative estimation of penicillin be desired, measure the halos and proceed as in the normal penicillin assay.

Special lab requirements

The instruments used in all these assays must be washed carefully before and after each test, in order to remove all the traces of antibiotic.To sterilize lab commodities, cover them well and use a hot air oven at 200-220°C for 2 hours. Volumetric flasks , pipets, ... must be washed carefully.

Figure 1. Holes, discs or cylinders at 90° Figure 2. Holes, discs or cylinders at 60°

Plate external Ø 9 mm Cylinder ext. Ø 6 mmCylinder int. Ø 8 mm

20

Antibiotic Media

Scharlau Chemie may supply all the necessary instru-ments to perform antibiotic assays, which is detailed below:

Volumetric materialIf it is possible, use guaranteed, checked and class A glass.

Cylinders Cylinders must be made of stainless steel. Exterior diameter: 8 mm, interior diameter: 6 mm, height: 8-10 mm.

HolesHoles should be made with a suitable punching instrument of the cylinder dimension.

DiscsUse normalized paper filter discs, 6 mm diameter.

PlatesUse plastic or glass plates. If glass plates are em-ployed, they must be washed and sterilized.

TubesAll the tubes used in one assay must be equal and uniform in their dimensions.

Colourimeter / SpectrophotometerIt must be able to read at 530 nm, have its own zero adjustment and 100% transmission can be deter-mined with a sterile medium solution at the same conditions of assay.

ReferencesARRET, B. DIANA, P. JOHNSON y A. KIRSHBAUM (1971) Outline of Details for Microbiological Assays of Antibiotics: Second Revision. J. PHARM, Sci. 60,11,1689-1694.EUROPEAN PHARMACOPEIA (1997) 3rd Ed. 2.7 Bio-logical Assays. 2.7.2 Microbiological Assay of Antibiotics. Council of Europe. Strasbourg.SANCHO, GUINEA, PARES. (1980) Microbiología Analítica Básica. Ed. JIMS. Barcelona, U.S. PHARMACOPEIA XIX (1975) Antibiotic Assays.U.S./F.D.A.: 21 CFR (1976 and following) 436.100 and following.U.S. PHARMACOPEIA XX/National Formulary XV (1980) Antibiotic Assays.U.S. PHARMACOPEIA 23/National Formulary 18 (1995) Biological Tests and Assays. {81} Antibiotics Microbial Assays.U.S. PHARMACOPEIA 25/National Formulary 20 (2002) Biological Tests and Assays. {81} Antibiotics Microbial Assays.EUROPEAN PHARMACOPEIA, Supplement (2002), 4th Ed.,Council of Europe, Strasbourg,

21

APT Media

APT Agar

Ref. 01-026

SpecificationSolid medium for general purposes, especially designed for the cultivation of the heterofermentative lactic acid bacteria that causes meat greening.

Formula (in g/L)Casein Peptone ....................................12,500Yeast Extract ..........................................7,500Sodium Chloride .....................................5,000Dipotassium Phosphate .........................5,000Trisodium Citrate ....................................5,000Dextrose ...............................................10,000Magnesium Sulfate .................................0,800Manganous Chloride ..............................0,140Iron Sulfate .............................................0,040Thiamine HCl ..........................................0,001Polysorbate 80 .......................................0,200Agar ......................................................15,000Final pH 6,7 ± 0,2

DirectionsSuspend 61,2 g of powder into 1 L of distilled water and let it soak . Heat to boiling with constant stirring. Distrib-ute in suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

APT Broth

Ref. 02-026

SpecificationLiquid version of the medium with the same name. It is especially designed for the cultivation of lactic acid bacteria.

Formula (in g/L)Casein Peptone ....................................12,500Yeast Extract ..........................................7,500Sodium Chloride .....................................5,000Dipotassium Phosphate .........................5,000Trisodium Citrate ....................................5,000Dextrose ...............................................10,000Magnesium Sulfate .................................0,800Manganous Chloride ..............................0,140Iron Sulfate .............................................0,040Thiamine HCl ..........................................0,001Polysorbate 80 .......................................0,200Final pH 6,7 ± 0,2

DirectionsDissolve 46,2 g of powder into 1 L of distilled water, heating up slightly if necessary. Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionThese two general purpose media (APT= All Purpose with Tween), originally formulated by Evans and Niven, have been used successfully for the isolation and culti-vation of lactic acid bacteria that alter the food quality and composition (especially meat) and require a high thiamine level. For this reason, ignoring what peptone and yeast extract could provide, the medium has been complemented with an extra amount of thiamine. Both versions, solid and liquid, have demonstrated their efficacy for detection of lactobacilli that produce meat greening. Moreover, if media are supplemented with 5% fruit juices, as APHA states, they are converted into irreplaceable prospection media for any kind of food biomodifier.However, without the inclusion of any inhibitory agent in the formulation signifies that the media have no selective ability, and thus they can support the growth of almost all the microbial types.

TechniqueUsual detection technique for the bacteria those causes the greening is as mentioned below:Products to be examined are crushed carefully in Tryp-tone Water (Ref. 03-156), and with the same diluent a dilution bank is prepared. From each dilution, APT Agar plates are inoculated in mass and in triplicate, and they are incubated at 32°C for 48 hours. After the incubation period, colonies are counted using usual technique, and different types are selected. Every type is inoculated in APT Broth, and is incubated at 32°C for 24 hours or more. From these pure cultures, streak on Frankfurt sausages slices, and incubate those slices and also the one without the culture as a Control, in a humid room or atmosphere to verify its greening capacity. Final identification is done by morphological and biochemical characteristics.

ReferencesEVANS, J.B. & C.F. NIVEN (1951) Nutrition of the het-erofermentative lactobacilli that cause greening of cured meat products J.Bact. 62:599DEIBEL, R.H, J.B. EVANS & C.F. NIVEN (1957) Micro-biological assay for the thiamin using Lactobacillus viridescens. J. Bact. 74:818-821DOWNES, F.P., K. ITO (2002) Compendium of methods for the microbiological examination of food. 4rd. ed. APHA. Washington.

22

Asparagine Broth

Ref. 02-271

SpecificationLiquid medium for the presumptive assay and enumera-tion of Pseudomonas aeruginosa in bottled water by MPN method.

Formula (in g/L)Asparagine .................................................2,0Dipotassium phosphate ..............................1,0Monopotassium phosphate ......................10,0Magnesium sulfate .....................................0,5Final pH 7,0 ± 0,2

DirectionsDissolve 13,5 g of powder in 1 L of distilled water con-taining 8 mL of glycerol. Sterilize by filtration and dis-tribute in tubes (10 mL/tube). To obtain broth of double strength, dissolve 27 g of powder in 1 L of distilled water containing 16 mL of glycerol.

DescriptionAsparagine medium is recommended for the microbio-logical analysis of bottled water. This is an excellent enrichment medium for Pseudomonas aeruginosa, since it is composed of a mineral base and the only carbon source is asparagine. It may also be used in the multiple tube technique in microbiological analysis of recreational waters and as a presumptive test medium for the differ-entiation of non fermentative gramnegative bacteria. TechniqueSome standards suggests the viable enumeration by MPN method with 5 tubes per series, inoculating 10 mL, 1 mL and 0,1 mL. All the tubes are incubated at 37°C for 48 hours. Growth, with or without pigmentation, is estimated as a presumptive evidence of presence of Pseudomonas aeruginosa. Enumeration is carried out with MPN tables for 5 tubes (See MPN chapter in this book).Confirmation is performed subculturing a loop of each tube in Acetamide Medium (Ref. 03-428).

ReferencesPASCUAL ANDERSON, M.R. (1992) Microbiología Alimentaria. Diaz de Santos. Madrid.

23

Azide Dextrose Media

Azide Dextrose Broth

Ref. 02-343

SpecificationLiquid and selective medium for the detection of strepto-cocci in water, waste water and milk.

Formula (in g/L)Meat extract ................................................4,5Tryptose ....................................................15,0Dextrose .....................................................7,5Sodium chloride ..........................................7,5Sodium azide ..............................................0,2Final pH 7,2 ± 0,2

DirectionsDissolve 35 g of powder in 1 L of distilled water, heat if necessary. Distribute into tubes and sterilize in the auto-clave at 121°C for 15 minutes. To make double strength broth, dissolve 70 g of powder in 1 L of distilled water.

DescriptionAzide Dextrose Broth is formulated according to the “Standard Methods for the Examination of Water and Wastewater”.Azide concentration in the medium restrains the growth of gramnegative bacteria and lets a good growth of en-terococci. This medium composition is very similar to the Azide Dextrose Broth acc. to Rothe (Ref. 02-027), but without phosphate buffering. Therefore, it is advisable to use this medium with samples with a high salts concen-tration, to avoid any precipitation in the medium.

Azide Dextrose Broth acc. to Rothe (Rothe Azide Broth)

Ref. 02-027

SpecificationMedium for the detection and enumeration of entero-cocci in water.

Formula (in g/L)Meat peptone ...........................................10,0Casein peptone ........................................10,0Dextrose .....................................................5,0Sodium chloride ..........................................5,0Dipotassium hydrogen phosphate .............. 2,7Potassium dihydrogen phosphate .............. 2,7 Sodium azide ..............................................0,2Final pH 7.0 ± 0,2

DirectionsDissolve 35,6 g in 1 L of distilled water. Heat if neces-sary to help dissolution. Divide into 10 mL portions and pour into tubes. Sterilize by autoclaving at 121°C for 15 minutes. As for the double strength medium, dissolve 71,2 g/L and proceed as indicated above.

DescriptionThe Azide Dextrose Broth acc. to Rothe has been widely used since 1948 for the detection of fecal streptococci. It usually provides higher positive results than other similar media. Its efficiency is due to the Sodium azide, which is both selective for enterococci and inhibitor of the ac-companying flora through interference of the electron transport chain.This medium is also used for the primary enrichment of food samples, particularly frozen vegetables.

TechniqueStreptometry in WaterAdd 10 mL of water to be examined to each one of three tubes containing 10 mL of double strength medium.Add 1 mL of sample to another three tubes containing 10 mL of medium each, of single strength. Then add 0.1 mL of water to each one of three tubes containing 10 mL of medium of single strength. Incubate at 37° C and examine after 24 hours and 48 hours. All tubes which show turbidity due to growth will be considered as PRE-SUMPTIVLY POSITIVE and will have to be confirmed on EVA Broth (Ref.2-028). All tubes which result positive on this second testing should be considered for the count of the Most Probable Number (MPN).When considering other type of samples, dilute them in Ringer solution 1/4 or peptone water and then inoculate the tubes as done before. In case of the highly contaminated samples, dilutions should be done before inoculation.

ReferencesAPHA-AWWA-WPCF (1980) Standard Methods for the Examination of Water and Wastewater. 15th. Ed. APHA Inc., Washington, D.C.CLESCERI, L., A.E. GREENBERG, A.E. EATON (1998) Standard Methods for the Examination of Water and Wastewater. APHA-AWWA-WEA. Washington.GUINEA, SANCHO y PARÉS. (1979) Análisis Microbiológico de Aguas: Aspectos Aplicados. Ed. Omega,Barcelona,.ROTHE (1948) Illinois State Health Department.DOWNES, F.C. & K.ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food. 4th ed. APHA. Washington.

R-22-32-52/53S-7-46-61

Xn

R-22-32-52/53S-7-46-61

Xn

24

Bacillus cereus Media

Bacillus cereus Agar (MYP Agar)

Ref. 01-262

SpecificationSelective solid medium, according to Mossel, for the isolation and identification of Bacillus cereus from food samples, acc. ISO 7932 (2003) and ISO 21871:2006.

Formula (in g/L)Peptone ................................................10,000Mannitol ................................................10,000Sodium chloride ....................................10,000Meat extract ............................................1,000Phenol red ..............................................0,025Agar ......................................................15,000Final pH 7,2 ± 0,2

DirectionsSuspend 46 g of powder in 900 mL of distilled water. Let it soak and bring to boil. Sterilize in the autoclave at 121°C for 15 minutes. Let it cool to 50°C and then add 100 mL of Egg’s Yolk Sterile Emulsion (Ref. 06-016) and 100 mg/L of Polymyxin (Ref. 06-021CASE). Homogenize well and distribute into plates. Do not reheat or remelt the complete medium.

DescriptionMossel’s formulation is developed to detect and enu-merate B.cereus in any kind of food, since it lets a good differentiation and selection of these microorganisms. Polymyxin addition inhibits most of accompanying bacteria, but it does not affect the growth of B.cereus.This bacteria do not ferment mannitol and thus there is no change in the indicator around the colonies. On other hand, due to lecithinase activity of B.cereus it produces a halo or zone of white precipitate around the colonies.A count of B.cereus over 100.000 cells/g of the food sample is considered to be hazardous, since the ac-cumulated phosphoril-choline may cause intoxication symptoms in children. For this reason, besides common isolation and identification, a viable enumeration must be performed to evaluate the real population of cells.

TechniqueAccording to the authors, dehydrated or dry samples must follow a recovery process in the following way: 20 g of sample is mixed with 90 mL of Tryptone Water (Ref. 03-156) for a minimum period of 1 hour, at room tem-perature. Afterwards, add 90 mL more of Tryptone Water and homogenize it. A dilution of 1:10 should be obtained. If necessary, a decimal dilution bank using Tryptone wa-ter as the diluent can be prepared. With a Drigalsky loop (Ref. 5-010), spread aliquotes of 0,1 mL over the surface of the agar plates and let the agar medium absorb those aliquots. Incubate the plates at 30°C for 18-24 hours to allow spore germination before giving definite results, which are referred as B. cereus colonies per gram of sample.Suspected colonies show the following appearance: irregular borders, pink colour and even purple in the center, and with a halo of white precipitate. Colonies with yellow halos must be discarded surely.

Sometimes,the confusion with other colonies of gram-positive bacilli is possible, and hence to identity this, confirmation has to be performed verifying the glucose fermentation, gelatin degradation and nitrate reduction, which are positive tests for Bacillus cereus.

ReferencesATLAS, R.M. & L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press. Londres.CORRY, J.E.L., G.D.W. CURTIS & R.M. BAIRD. (2003) Handbook of Culture Media for Food Microbiology. Elsevier Sci. B.V. Amsterdam. The Netherlands.DOWNES, F.P. & K. ITO (2001) Compendium of methods for the microbiological examination of foods. 4th ed. APHA. Washington DC. USA.FIL-IDF 181:1998 Provisional Int. Standard. Dried Milk Products. Enumeration of Bacillus cereus.- Most probable number techniqueISO 7932 Standard (2004) 3rd ed. Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of presumptive Bacillus cereus. Colony count technique at 30ºC.ISO 21871 Standard (2006) Microbiology of food and animal feeding stuffs.- Horizontal method for the determination of low numbers of presumptive Bacillus cereus.- Most probable number technique and detection method.MOSSEL, D.A.A., KOOPMAN. M.J. y JONGERIUS, E. (1967) Enumeration of Bacillus cereus in foods. Appl. Microbiol. 15:650-653.PASCUAL ANDERSON, Mª.Rª (1992) Microbiología Alimentaria. Díaz de Santos, S.A. Madrid.

Bacillus cereus Selective Agar (PEMBA)

Ref. 01-487

SpecificationSelective solid medium for the enumeration of Bacillus cereus in food, according to ISO 21871.

Formula (in g/L)Peptone ....................................................1,00Mannitol ..................................................10,00Sodium chloride ........................................2,00Magnesium sulfate ...................................0,20Disodium phosphate .................................2,50Potassium phosphate ...............................0,25Brom thymol blue .....................................0,12Sodium pyruvate ....................................10,00Agar ........................................................14,00Final pH 7,2 ± 0,2

DirectionsSuspend 40 g of powder in 950 mL of distilled water. Let it soak and bring to the boiling. Distribute into suitable containers and sterilize by autoclaving at 121ºC for 15 minutes. Let it cool to 50ºC and then add 50 mL/L of Egg’s Yolk Sterile Emulsion (Ref. 06-016) and Polymyxin B Sulfate (Ref. 06-021CASE) to reach a 100 U/mL con-centration. Homogenize and pour into plates.

25

Bacillus cereus Media

DescriptionBacillus Cereus Selective Agar is formulated according to the Food Analysis Nordic Committee (NMLK) stand-ards. This standard uses this medium and Blood Agar Base (Ref. 01-352) simultaneously for the detection and enumeration of B. cereus in any type of food. This me-dium can also be used to confirm doubtful colonies, and in this last case Polymyxin addition may be ommitted.

TechniqueNMLK proposes the simultaneous use of Bacillus cereus Selective Agar and Blood Agar Base (Ref. 01-352). Both media are inoculated by surface streaking with 0,1 mL aliquotes which are spread with a Drigalsky loop. Both series of plates are incubated at 30ºC for 24 hours.Typical B.cereus colonies over Blood Agar are big, irreg-ular, dirty white or grey-like colour with a hemolysis halo surrounding them. In B.cereus Selective Agar, colonies are blue, surrounded by a clear zone of egg yolk diges-tion (lecithinase positive).If there is an equal amount of typical or similar colonies in both the media, later confirmation is not necessary.

ReferencesISO 21871 Standard (2006) Microbiology of food and animal feeding stuffs.- Horizontal method for the

determination of low numbers of presumptive Bacillus cereus.- Most probable number technique and detection method.

Baird Parker Agar Base (Eur. Phar. Medium O)

Ref. 01-030

SpecificationSolid and selective culture medium for the screening of staphylococci from a variety of samples, acc. to pharma-copeias and ISO standard.

Formula (in g/L)Tryptone ...................................................10,0Sodium pyruvate ......................................10,0Glycine .....................................................12,0Meat extract ................................................5,0Lithium chloride ..........................................5,0Yeast extract ...............................................1,0Agar .........................................................17,0Final pH 7,0 ± 0,2

DirectionsSuspend 60 g in 950 mL of distilled water. Allow it to soak and then bring to the boil with constant stirring. Sterilize by autoclaving at 121° C for 15 minutes. Cool to 50° C and add 50 mL of Egg’s Yolk Tellurite Sterile Emul-sion (Ref. 06-026). Homogenize and distribute into plates. Once prepared, the medium must not be reheated nor sterilized again.

DescriptionThe Baird Parker Agar Base is specially recommended for the detection and enumeration of staphylococci in food and other material, since it allows a good differen-tiation of coagulase-positive strains. The growth of the

accompanying bacteria is usually suppressed by the high concentration in lithium, glycine and pyruvate. Lithium and glycine enhances the growth of staphylococci. Even if its high selectivity does not affect staphylococci it may occasionally permit the growth of some Bacillus species, yeast and very rarely, Proteus. The growth of Proteus species can be suppressed by adding 50 mg/l of sul-phamethazine.The presence of tellurite and egg’s yolk, which must always be added to the medium after sterilization, allows the differentiation of presumptly pathogenic staphylococ-cal colonies. A high correlation has been found between the coagulase test and the presence of clearing zones of lypolysis in this medium, which is due to the staphylo-coccal lecithinase. On the other hand, studies show that almost 100% of coagulase-positive staphylococci are capable of reducing tellurite, which produces black colo-nies, whereas other staphylococci can not always do so.When using sterile reagents other than SCHARLAU MICROBIOLOGY, the complete medium may be ob-tained by adding 50 mL sterile egg’s yolk and 10 mL of 1% potassium tellurite solution. Plates should be used on the same day of preparation or within 48 hours, to avoid the loss of definition in the precipitated zones . The basal medium, without the yolk or the tellurite, is perfect-ly stable and therefore can be repeatedly melted.

TechniqueThe inoculation is done by spreading 0,5 mL of sample over each plate with a Drigalski loop (Ref. 5-010). After 18-24 hours of incubation at 35° C, select the colonies

Control

Bacillus cereus ATCC 10876

Ref. 01-262 Bacillus cereus Agar

26

which are black, shiny and convex with regular margins surrounded by a zone of clearing. These can be pre-sumptly identified as coagulase-positive Staphylococcus aureus.Colonial appearance after 24 h. at 35°C:Staphylococcus aureus: Black, shiny, convex, regular

margins, 1.0-1.5 mm diameter, surrounded by a clearing zone of lipolysis 2-5 mm in width. May produce wide opaque precipitate zones extending into the cleared medium after 48 hours.

Other species of Staphylococcus: Black, usually dully, with regular margins. Sometimes they are brown with zones of clearing but these present wide opaque zones.

Micrococcus spp: Brown, very small and without clear-ing.

Bacillus spp: Various shades of brown, big. May produce clearing after 48 hours.

Yeasts: White, big and smooth.The egg’s yolk emulsion can be prepared by mixing a fresh egg’s yolk with an equivalent quantity (w/v) of saline solution. Sterilize by filtration and aseptically add to the medium. This reagents´s reference, already steri-lized, is SCHARLAU 6-016.

The potassium tellurite solution is prepared by dissolv-ing 3,5 g potassium tellurite in 100 mL distilled water. Sterilize by filtration. This sterile reagent’s SCHARLAU MICROBIOLOGY reference is 6-011.Although these solutions can be mixed to be added to the Baird Parker Agar Base forming the commonly known Egg Yolk Tellurite Sterile Emulsion (Ref. 06-026), they are also stable as the separate supplement and can be used in many other culture media.

ReferencesATLAS R.M. & L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press. LondresBAIRD-PARKER, A.C. (1962) An improved diagnostic and selective médium for isolating coagulase-positive staphylococci. J. Appl. Bact. 25:12.DOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Foods. 4rd ed. APHA. Washington. USAEUROPEAN PHARMACOPOEIA (2007) 5ª ed. Suppl. 5.6 § 2.6.13 Microbiological examination of non-sterile products. EDQM Council of Europe. Strasbourg.ISO 5944:2001 Standard. Milk and Milk based products – Detection of coagulase positive staphylococci – MPN Technique. Geneva.ISO 6888-1:1999 Standard.Microbiology of food and animal feeding stuffs – Horizontal method for the enu-meration of coagulase-positive staphylococci – Part 1 Technique using Baird-Parker Agar médium. Geneva.ISO 22718:2006 Standard. Cosmetics – Detection of Staphylococcus aureus.

FIL-IDF 60:2001 Standard. Latí et produits à base de lait – Détection des staphylocoques à coagulase positive – Technique du nombre le plus probable. Brussels.USP 29 – NF 25 (2006) <61> Microbial Limit Tests. US Phamacopoeial Conv. Inc. Rockville. Md, USAZANGERL, P. & H. ASPERGER. (2003) Media used in the detection and enumeration of Staphylococcus aureus in Handbook of Culture Media for Food Microbiol-ogy Corry et als. Eds. Elsevier Sci. BV Ámsterdam

Bile Esculin Agar

Ref. 01-265

SpecificationSolid culture medium for the identification of probiotic streptococci in food samples.

Formula (in g/L)Meat extract ................................................3,0Peptone ......................................................5,0Bile ..........................................................40,0Ferric citrate ................................................0,5Esculin ........................................................1,0Agar ..........................................................15,0Final pH 7,0 ± 0,2

DirectionsSuspend 64,5 g of powder in 1 L of distilled water and let it soak. Heat to boiling and distribute into containers. Sterilize in the autoclave at 121°C for 15 minutes.

Bile Esculin Media

DescriptionThis medium formulation is based on the modification by Facklam and Moody of the original formulation by Swan to verify the esculin hydrolysing capacity of streptococci and their resistance to bile salts which inhibit gram posi-tive bacteria.In fact, this medium can substitute KAA Confirmative Agar (Ref. 01-263), but it has not the same selectivity. Hence, it is used just as a substrate to verify the two as-says simultaneously in the biochemical tests that identify enterococci.

TechniqueAssay is performed by inoculating the surface of a slant with the pure culture that is going to be verified. After the 24 hours incubation at 35°C, it might produce translucid colonies, surrounded by black halos or zones, due to esculin hydrolysis. Resistance to bile salts is indicated by the growth.

ReferencesLEUCHNER, R.G.K., J.BEW, K.J.DOMIG, & W.KNEIFEL. (2002) A collaborative study of a method for enumeration of probiotic enterococci in animal feed J. Appl. Microbiol. 93:781-786

Baird Parker Agar Base (Eur. Phar. Medium O)

27

DEIBEL, R.H., HARTMAN, P.A. (1976) The enterococci, Compendium of Methods for the Microbiological Exami-nation of Food. APHA.FACKLAM, R.R., MOODY, M.D. (1970) Presumptive identification of group D streptococci: The bile-esculin test. Appl. Microbiol. 20:245.PASCUAL ANDERSON, MªRª. (1992) Microbiología Alimentaria. Diaz de Santos, S.A.,Madrid,SPAIN.SWAN,A. (1954), The use of bile-esculin medium and Maxted’s technique of Lancefield grouping in the identifi-cation of enterococci. J.Clin.Pathol. 7:160.FDA (1998) Bacterilogical Analitical Manual. 8th ed. Rev. A. APHA International. Gaitherburg, VAATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Prss. London.DOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological examination of Foods. APHA. Washington

Bile Esculin Azide Agar

Ref. 01-592

SpecificationSolid medium for the enterococci confirmative test in water by the membrane filtration method according to ISO 7899-2.

Formula (in g/L)Tryptone .................................................17,00Peptone ....................................................3,00Yeast extract .............................................5,00Bile .........................................................10,00Sodium chloride .......................................5,00Esculin ......................................................1,00Ammonium-ferric citrate ...........................0,50Sodium azide ............................................0,15Agar ........................................................15,00Final pH 7,2 ± 0,2

DirectionsSuspend 56.6 g of powder in 1 L distilled water and bring to the boil. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes. Cool to 50-60ºC and pour plates to 3-5 mm thickness. These plates can be stocked at 2-8ºC until two weeks.

DescriptionThe Bile Esculin Azide Medium is a modification of the classical Bile Esculin proposed by Isenberg, Goldberg and Sampson in 1970, with a reduction in the amount of bile and the addition of the sodium azide. Brodsky and Schieman shown that this medium, also know as Pfizer Enterococci Selective gave best results with the filtration technique.The actual formulation is according the ISO Standard 7899-2:2000 for the second step in the confirmation and enumeration of enterococci in water by the membrane filtration method. The colonies previously selected in the Slanetz-Bartley Agar (Ref. 01-579 + 6-023) must be confirmed by a short incubation on the Bile Esculin Azide Medium that permits the verification of the esculin hydrolysis in a selective environment.

TechniqueAfter an incubation of 24-48 hours on Slanetz Bartley Agar (Ref. 01-579 + 6-023), the membrane filter that show typical colonies is transferred, with sterile forceps and upright position, to a pre-warmed plate of Bile Esculin Azide Agar. After two hours of incubation at 44 ± 0,5ºC the membrane filter is inspected. All the typical col-onies that show a brown to black colour in the surround-ing medium are considered as positives and enumerate as intestinal enterococci.A heterogeneous distribution of the colonies or the pres-ence of abundant and different micro organisms can interfere with the differentiation of positive colonies.

References ISO Standard 7899-2 (2000) Water Quality. Detection and enumeration of intestinal enterococci. Part 2: Mem-brane filtration method.ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press Boca Raton. Fla.ISENBERG, H.D., D. GOLDBERG & J. SAMPSON (1970) Laboratory studies with a selective enterococcus medium. Appl. Microbiol. 20:433BRODSKY M.H. & D.A. SCHIEMANN (1976) Evalua-tion of Pfizer Selective Enterococcus and KF media for recovery of fecal streptococci from water by membrane filtration. Appl. Environ. Microbiol. 31:695-699

Bile Esculin Media

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Ref. 01-592 Bile Esculin Azide Agar. Entero-coccus faecalis ATCC 29212

28

Blood Media

Blood Agar Base

Ref. 01-352

SpecificationGeneral purpose medium for the isolation and cultivation of microorganisms.

Formula (in g/L)Meat extract ..............................................10,0Tryptone ...................................................10,0Sodium chloride ..........................................5,0Agar ..........................................................15,0Final pH 7,3 ± 0,2

DirectionsSuspend 40 g of powder in 950 mL. of distilled water and let it soak. Heat to the boiling and distribute into contain-ers. Sterilize by autoclaving at 121°C for 15 minutes. Let it cool to 45-50°C and then add defibrinated blood in a proportion about 7% or the desired enrichment.

DescriptionBlood Agar Base may be used for the cultivation of non fastidious microorganisms, since it has a balanced nutri-ent base . For the fastidious microorganisms, it is advisable to add special enrichments, such as ascitic liquid, egg yolk, etc..This medium, with the addition of blood, is very suitable for studies in hemolytic activity, but for the isolation of pathogens Blood Agar Base Columbia type (Ref. 01-034) is more suitable.

ReferencesATLAS, R.M. and L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, London.

Blood Agar Base No. 2

Ref. 01-505

SpecificationImproved base with high nutrient properties especially adapted for the growth of very fastidious microorganisms, acc. ISO 7932 (2003).

Formula (in g/L)Proteose peptone .....................................15,0Liver extract ................................................2,5Yeast extract ...............................................5,0Sodium chloride ..........................................5,0Agar ..........................................................15,0Final pH 7,0 ± 0,2

DirectionsSuspend 42,5 g in 950 mL of distilled water and bring to boil. Distribute into flasks and sterilize by autoclaving at 121ºC for 15 minutes. Cool to 45-50ºC and aseptically add 7% of sterile defibrinated blood. Mix gently and pour into plates.Note: Blood and medium should be mixed in a big flask to assure proper blood oxidation and mixing.

DescriptionBlood Agar Base No. 2 allows a maximum recovery of weak organisms without altering or interfering in their hemolytic reactions. Compared to other Blood Agar bases, this one shows an equal or higher stimulatory growth ability, however it specially helps the formation of pigment in the chromogenic bacteria.

ReferencesATLAS, R.M. and L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, London.CASMAN, E. (1947) A noninfusion blood agar base for neisseriae, pneumococci and streptococci. Am. J. Clin Path. 17:281-289FDA (1998) Bacterilogical Analitical Manual. 8th ed. Rev. A. APHA International. Gaitherburg, VADOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological examination of Foods. APHA. WashingtonISO 7932 Standard (2003). Microbiology of food and animal feeding stuffs. Horizontal Methods for the enu-meration of presumptive Bacillus cereus. Colony count tecnique at 30°C.

Blood Agar Base (Columbia)

Ref. 01-034

SpecificationMedium especially rich in peptone, appropriate for blood addition or to prepare Chocolate Agar.

Formula (in g/L)Casein Peptone ........................................12,0Meat peptone ........................................... 11,0Starch .........................................................1,5Sodium Chloride .........................................5,0Agar ..........................................................15,0Final pH 7,3 ± 0,2

DirectionsAdd 44,5 g of powder to 950 mL of distilled water and bring it to the boil. Distribute into suitable containers and sterilize at 121°C for 15 minutes. To obtain Blood Agar cool it to 45-50°C and aseptically add sterile defibrinated blood at 5% proportion.

DescriptionBlood Agar Base contains an equilibrated mixture of meat and casein peptones, being suitable for preparing selective and as diagnostic media with the addition of

29

Blood Media

blood or inhibitors. As it is presented, without additions, it is also an excellent general culture medium.Generally, Blood Agar base contains a casein peptone, that aids big size colonies formation, or a meat petone, that provides a well defined hemolysis halos or zones. Blood Agar Base is prepared according to the Columbia University formulation, and meets the two conditions mentioned above.

TechniqueSome applications for this base are:Base Agar without either enrichment and inhibitors:

This medium supports growth of normal micro-organisms as enterobacteriaand othyers more strengh as Pasteurella, Brucella and Clostridium perfringens.

Clostridium Selective Base Agar: Should a selective clostridium medium be desired, add 240 mg/L Sodium Azide and 180 mg/l Neomycin before the sterilization, or alternatively the contents of SC Inhibitor container (Ref. 06-012CASE).

Blood Agar: Aseptically add to the sterile medium 5% sterile defibrinated sheep blood and cool it to 45°C. This way, medium is enriched and allows the determination of typical hemolytic reactions necessary for the identification of enterococci, staphylococci and other microorganisms.

Selective Gram-positive cocci Blood Agar: Simulta-neously at the time blood addition, also add 10 mg/L of colistine and 15 mg/L of Nalidíxic Acid, or, the contents of a CP Inhibitor container (Ref. 06-013CASE), to obtain an excellent selective medium for gram-positive cocci.

Note: Some authors recommend Blood Agar Base as the maintenance medium for Campylobacter.

ReferencesCASMAN, E. (1947) A non infusion blood agar base for neiseriae, pneumococci and streptococci. Am. J. Clin. Path. 17:281-289.ATLAS, R.M. and L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, London.

Blood Azide Agar Base

Ref. 01-242

SpecificationSelective basal medium for staphylococci and strepto-cocci isolation. With blood added can be used for the determination of haemolytic reactions.

Formula (in g/L)Tryptose ..................................................10,00Meat extract ..............................................3,00Sodium chloride ........................................5,00Sodium azide ............................................0,20Agar ........................................................15,00Final pH 7,2 ± 0,2

DirectionsSuspend 33 g of powder in 1 L of distilled water and bring to the boil. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes. Cool to 45-50ºC and aseptically add 5% of defibrinated blood. Pour in sterile plates.

DescriptionThis medium is a nutrient base (Tryptose and Meat extract) with a suitable osmotic value (Sodium chlo-ride) that includes a selective agent (Sodium azide) to suppressing the growth of gram-negative bacteria. The addition of 5% defibrinated blood supplies growth factors for the fastidious microorganisms and is used for the determination of the haemolytic patterns.

TechniqueThe plates are inoculated by the surface striking and stabbing the agar several times to deposit inoculum be-neath the agar surface, to show the hemolytic reaction of both oxygen-stable and oxygen-labile streptolysins. After an incubation of 18-24 and 48 ours in a suitable environ-ment the hemolytic reactions are displayed: Alpha-(α)-haemolysis is the reduction of haemo-

globin to met-haemoglobin that sows a greenish decolourisation of the medium surrounding the colony.

Beta-(β)-haemolysis is the total lysis of erythro-cytes that produce a clear zone surrounding the colony.

Gamma-(γ)-haemolysis means no haemolysis and there is no change in the medium

Alpha-prime-(α’)-haemolysis is a small zone of complete haemolysis that is surrounded by area of partial lysis around the colony.

In the haemolysis studies must be in mind that the haemolytic reactions are affected by several conditions: atmosphere (aerobic, anaerobic or CO

2 enriched) of in-

cubation, the composition of the culture media (presence of sugars or growth factors) and the source of the blood (horse rabbit, sheep, human…)

ReferencesATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press. LondonISENBERG, H.D. (1992) Clinical microbiology proce-dures handbook. Vol. I ASM. Washington DC. PACKER, R.A (1943) The use of sodium azide as an inhibition substance of gram-negative bacteria. J. Infect. Dis. 67:113

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30

RUOFF, K.L. (1995) Streptococcus p. 299-305, in P.R. Murray et al. (ed.) Manual of Clinical Microbiology. 6th ed. ASM Washington DC.

Blood Tryptose Agar Base

Ref. 01-551

SpecificationSolid highly nutrient medium developed as Base for Blood Agar for the isolation and cultivation of fastidious microorganisms.

Formula (in g/L)Tryptose .................................................10,00Meat extract ..............................................3,00Sodium chloride ........................................5,00Agar ........................................................15,00Final pH 7,3 ± 0,2

DirectionsSuspend 33 g of powder in 1 L of distilled water and let it soak. Bring to the boiling and distribute into suitable containers. Sterilize autoclaving at 121°C for 15 minutes. Let it cool to 45-50ºC and then add 5-10% defibrinated blood or the suitable enrichment. Homogenize and pour plates.

DescriptionCasman proposed Blood Tryptose Agar Base in 1947 as alternative medium without tissue infusion components. Their original formulations include dextrose that inter-feres with haemolytic reactions and it is omitted in the present formulation.This medium, with the addition of blood, is very suitable for studies in haemolytic activity, but to isolate pathogens Blood Agar Base Columbia type (Ref. 1-034) is more suitable. This medium support the growth of a wide vari-ety of fastidious microorganisms but several species of Streptococcus and Neisseria require the addition of 1 g/L of Yeast Extract for the optimal growth.

TechniqueThe plates are inoculate by the surface striking and stab-bing the agar several times to deposit inoculum beneath the agar surface, to show the hemolytic reaction of both oxygen-stable and oxygen-labile streptolysins. After an incubation of 18-24 and 48 ours in a suitable environ-ment the hemolytic reactions are displayed: Alpha- (α)-haemolysis is the reduction of haemo-

globin to met-haemoglobin that sows a greenish decolourisation of the medium surrounding the colony.

Beta- (β)-haemolysis is the total lysis of erythro-cytes that produce a clear zone surrounding the colony.

Gamma- (γ)-haemolysis means no haemolysis and there is no change in the medium

Alpha-prime- (α’)-haemolysis is a small zone of complete haemolysis that is surrounded by area of partial lysis around the colony.

In the haemolysis studies must be in mind that the haemolytic reactions are affected by the environmental conditions (aerobic, anaerobic or CO

2 enriched) of incu-

bation, the composition of the culture media (presence of sugars or growth factors) and the source of the blood (horse rabbit, sheep, human…)

ReferencesATLAS, R.M. and L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press. London.BALLOWS, A. & W.J. HAUSLER (1981) Diagnostic Pro-cedures for Bacterial, Mycotic and Parasitic Infections 6th Ed. APHA Washington D.C.CASMAN, E.P. (1947) A non-infusion blood agar base for neisseriae, pneumococci and streptococci. Am. J. Clin. Pathol. 17:281-289HARMON, S.M. et al. (1998) FDA Bacteriological Analytical Manual. 8th ed. AOAC International. Gaithers-burg. ISENBERG, H.D. (1992) Clinical Microbiology Proce-dures Handbook. Vol. I ASM. Washington DC.

Blood Media

31

BPRM Broth Base

Ref. 02-409

SpecificationBase medium to cultivate Bacteroides fragilis and phage recovery from the environmental samples according to ISO Standard 10705-4.

Formula (in g/L)Peptone ..................................................10,00Tryptone .................................................10,00Yeast extract .............................................2,00Sodium chloride ........................................5,00Glucose ....................................................1,80Magnesium sulfate ...................................0,12L-Cysteine HCl .........................................0,50Final pH 6,8 ± 0,2

DirectionsDissolve 29,42 g of powder in 1 L of distilled water. Add 1 mL of a CaCl

2 5% solution. Distribute in flasks and

sterilize in the autoclave at 121°C for 15 minutes. Cool to 40°C and add 1 mL/L of 1% sterile soution of hemin prepared in 0,02% NaOH. Mix well. Just before the utilization add 25mL/L of a sterile solution of 10,6% (w/v) NaCO

3. Adjust the pH to 7,0 with HCl.

DescriptionBPRM (Bacteroides Phage Recovery Medium) was formulated according the specifications by the Microbi-ology Department of the Barcelona University. Tartera and cols. verified its efficiency in the Bacteroides fragilis phage recovery used as human fecal pollution indica-tors in environmental samples. Initially Kanamycin (100 mg/L) and Vancomycin (7,5 mg/L) were added to the medium to prevent the growth of unwanted accompany-ing bacteria, but later the Vancomycin was replaced by Nalidixic acid (100 mg/L).BPRM Basal Broth can be used in double strengh and, with the addition of adequate amounts of agar, in the preparation of solid or semisolid media.

ReferencesTARTERA, C., R. ARAUJO, T. MICHEL and JOFRE (1992) Culture and decontamination methods affecting enumeration of phages infecting Bacteroides fragilis in sewage. Appl. Environm. Microbiol. 58:8:2670-2673.ISO Standard 10705-4 (2001) Water Quality-Detection and enumeration of bacterigophages. Part 4: Enumera-tion of bacteriophages infecting Bacteroides fragilis.

32

Brain Heart Infusion Agar (BHI Agar)

Ref. 01-599

SpecificationGeneral purpose solid medium for various fastidious pathogenic microorganisms.

Formula (in g/L)Brain Extract .............................................12,5Heart Extract ..............................................5,0Proteose peptone .....................................10,0Sodium chloride ..........................................5,0Di-sodium phosphate .................................2,5Dextrose .....................................................2,0Agar ..........................................................15,0Final pH 7,4 ± 0,2

DirectionsSuspend 52 g of powder in 1 L of distilled water and bring to the boil . Distribute in tubes or flasks and steri-lize by autoclaving at 121°C for 15 minutes.

Brain Heart Infusion Broth (BHI Broth)

Ref. 02-599

SpecificationLiquid culture medium for general purpose according to ISO 5944.

Formula (in g/L)Brain extract .............................................12,5Heart extract ...............................................5,0Proteose peptone .....................................10,0Dextrose .....................................................2,0Sodium chloride ..........................................5,0Di-sodium phosphate .................................2,5Final pH 7,4 ± 0,2

DirectionsDissolve 37 g of powder in 1 L of distilled water, heating up if necessary. Distribute into containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionBrain Heart Infusion is used for the cultivation of fastidi-ous bacteria (streptococci, pnemococci, meningococci, etc.) and also is recommended for the cultivation of pathogenic fungi.Growth of the accompanying bacterial flora can be almost completely suppresed by adding 20 I.U. penicillin and 40 µg streptomycin per mL culture medium.

If this medium is to be used for the selective isolation of fastidious fungi (especially of Histoplasma capsulatum and Blastomyces) add 10% sterile defibrinated blood and from mixinfected samples add also 0,05 µg cy-cloheximide/mL and 0,5 µg choramphenicol/mL.This medium is not suitable for obtaining the character-istic hemolytic reactions even after addition of the blood because of its glucose contents.

ReferencesROSENOW, E.C. (1919) Studies on elective localization. J. Dent. Research. 1:205-209HAYDEN R.L. (1923) Elective localization in the eye of bacteria from infected teeth. Arch. Int. Med. 32:828-849HOWELL(1948) The efficiency of methods for the isola-tion of Histoplasma capsulatum Public. Health. Reports 63, 173, 178.CONANT (1950) Diagnostic Procedures and Reagents 3rd Ed., APHA, Inc., New York, 452.DIN Standard 10163. Bestimmung koagulase positiver Staphylokokken.APHA-AWWA-AWPC (1998) Standard methods for the examination of Water and Wastewater. 20th ed. Wash-ington.FDA (1998) Bacterilogical Analitical Manual. 8th ed. Rev. A. APHA International. Gaitherburg, VAATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Prss. London.DOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological examination of Foods. APHA. WashingtonISO 5944 Standard (2001) Milk and milk based products: Detection of coagulase-positive staphylococci - MPN Technique.

Brain Heart Infusion Media (BHI Media)

33

Brilliant Green Agar (BGA)(Eur. Phar. Agar Medium L)

Ref. 01-203

SpecificationMedium for Salmonella isolation, according to the Euro-pean Pharmacopoeia

Formula (in g/L)Meat peptone .......................................5,0000Casein peptone ....................................5,0000Sodium chloride ....................................5,0000Yeast extract .........................................3,0000Lactose ...............................................10,0000Sucrose ..............................................10,0000Phenol Red ...........................................0,0800Brilliant Green .......................................0,0125Agar ....................................................15,0000Final pH 7,0 ± 0,2

DirectionsSuspend 53 g of powder in 1 L of distilled water and heat to boiling with constant stirring. Dispense into containers and sterilize at 121°C for 15 minutes.

DescriptionBGA is a differential selective medium, able to detect the presence of enteropathogenic bacteria in different samples. This medium is a modification to Kauffman’s original formulation, and it complies with the ISO, HMO, Eur. Phar., USP and APHA specifications. Since it has a high brilliant green concentration, it inhibits notably the growth of most bacteria, except Salmonella. However, S. typhi and S. paratyphi are also inhibited. Therefore, when their presence or Shigella is suspected, it is recommended to use other media in parallel, as Deoxycholate Lactose Agar (Ref. 01-057), MacConkey Agar (Ref. 01-118), Salmonella Shigella Agar (Ref. 01-171), Xylose Lysine Deoxycholate Agar (Ref. 01-211 or 1-552) or Endo Agar Base (Ref. 01-589) which are less inhibitory.

Presence of lactose and sucrose allows a good dif-ferentiation between Salmonella, which produce pink or colourless colonies with a red halo or zone, and the companion flora, which produce smaller and green yel-lowish colonies with yellow halo, due to acid created by lactose and/or sucrose fermentation.Osborn and Stokes suggested the addition of 0,08 g/L of sulfadiacine or 1 g/L of sulfapyridin in order to make this medium more selective for Salmonella and provide suit-able qualities to this medium to perform the examination of food and eggs and their derivatives.

ReferencesU.S.PHARMACOPOEIA (2002) 25 ed. Chap. <61> “Mi-crobial Limit Tests”. USP/NF Conv.Inc. Rockville.MDKAUFFMAN, F. (1935) Weitere Erfahrungen mit der kombinierten Anreicherungsverfahren für Salmonellaba-zillen Z. Hyg. Infekt. Krhn, 117; 26-32EUROPEAN PHARMACOPOEIA,Supplement 4.2 (2002), 4th Ed., Council of Europe,Strasbourg, France

Brilliant Green Bile 2% Broth

Ref. 02-041

SpecificationLiquid medium for the detection of coliforms in water, as recommended by APHA and ISO 4831 and 9308-1 Standards.

Formula (in g/L)Bile .......................................................20,000Lactose .................................................10,000Peptone ................................................10,000Brilliant Green .........................................0,013Final pH 7,2 ± 0,2

DirectionsDissolve 40 g of powder in 1 L of distilled water and bring to the boil. Distribute into containers containing Durham tubes and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionBrilliant Green Bile 2% Broth has been widely used as a medium for the assay of presumptive colimetries in food, milk and water, through the Most Probable Number Technique. This broth offers some advantages over other similar broths as its balanced composition of bile and Brilliant Green effectively suppresses the growth of gram-positive bacteria, even that of the more fastidious Clostridium perfringens.It is recommended by the APHA for colimetries in water, milk and food.British and Australian methodology use the broth as an intermediate stage between presumptive and confirma-tive colimetry, as it was an enrichment at 32°C. Other authors suggest it as an optimal base for the Eijkman testing of gas production at 44°C, for the identification of E. coli.This medium can be used as Presumptive broth for E.coli (by fluorescent reaction) if before sterilization MUG (Ref. 06-102CASE) is added.

ReferencesAPHA (1985) Standard Methods for the Examination of Water and Wastewater, 16th ed. Washington.VANDERZANT, C., SPLITTSTOESSER, D.F.(1992) Compendium of Methods for the Microbiological Exami-nation of Food. 3rd. Ed. APHA. Washington.

Brilliant Green Media

34

DOWNES F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food. 4rd. Ed. APHA. Washington.FDA (1998). Bacteriological Analitical Manual.8th ed. Rev. A AOAC International, Gaithersburg MD.. PASCUAL ANDERSON, MªRª (1992) Microbiologia Alimentaria. Diaz de Santos, S.A.,Madrid.ISO 4831 Standard (1991) General guidance for the enumeration of coliforms. MPN technique.ISO 9308-1. Standard (1990) Water Quality-Detection and enumeration of coliforms, thermotolerants coliforms and presumptive E.coli. MPN Method.

Brilliant Green Modified Agar

Ref. 01-309

SpecificationSolid culture medium for selective isolation of Salmonel-lae in food (except S. typhi) according ISO 6579, 6340, 6785 and IDF 93 Standards.

Formula (in g/L)Peptone ................................................10,000Meat extract ............................................5,000Yeast extract ...........................................3,000Lactose .................................................10,000Sucrose ................................................10,000Disodium phosphate ...............................1,000Sodium phosphate .................................0,600Phenol red ..............................................0,090Brilliant green .........................................0,005Agar ......................................................15,000Final pH 6,9 ± 0,2

DirectionsSuspend 54,5 g of powder in 1 L of distilled water. Let it soak and heat up to boiling with constant stirring. Distrib-ute in plates. Do not autoclave.

DescriptionIn this modification of the classical medium for Salmo-nellae, the concentration of brilliant green has been reduced to obtain a less inhibitory medium. At the same time, the nutrient basis has been enriched to enhance the recovery of those microorganisms that are weakened during the food production process.

This formulation was subsequently adopted by the ISO and DIN official method for detecting Salmonellae in meat.

TechniqueA previous enrichment in Tetrathionate Base Broth (Ref.2-033) is recommended. Inoculate on the surface of this plate medium in order to get separate colonies. Incubate at 35-37°C for a 18-24 hours period.Salmonella colonies (except S.typhi) are red, pinkish or white, but they are always surrounded by a red halo or zone, which shows the non lactose or sucrose fermen-tation. Colonies of lactose and/or sucrose fermenting bacteria produce yellow-green colonies surrounded by a yellow halo. Sometimes, Proteus or Pseudomonas may appear, and they produce red pointed colonies.In very polluted samples, it is recommended to include 1 g/L of sodium sulfacetamide and 250 mg/L of sodium mandelate.

ReferencesDIN. 10181 Mikrobiologische Milchuntersuchung. Nach-weis von Salmonellen. Referenzverfahren. DIN 10160. Untersuchung von fleisch und fleischerzeng-nissen. Nachweis von Salmonellen. ReferenzverfahrenISO Standard 6579 Meat and meat Product. Detection of Salmonellae. Reference Method. (1993) PASCUAL ANDERSON MªRª (1992) Microbiología Ali-mentaria. Diaz de Santos, S.A.Madrid,.ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc.,Boca Raton, Fla.ISO 6785 Standard (2001) Milk and milk products - De-tection of Salmonella spp.FIL-IDF Standard 93 (2001) Milk and milk based prod-ucts - Detection of Salmonella spp.ISO 6340 Standard (1995) Water Quality - Detection of Salmonella spp.

Brilliant Green Media

Ref. 02-041 Brilliant Green Bile 2% Broth. Left: control; center: E.coli ATCC 25922; right: Salmonella typhimu-

rium ATCC 14028

Ref. 01-203 Brilliant Green Agar.

Control

Salmonella typhimurium ATCC 14028

E. coli ATCC 25922

35

Bromcresol Purple Base Broth

Ref. 02-031

SpecificationLiquid culture medium with indicator to carry out sugar fermentation studies.

Formula (in g/L)Gelatin peptone ......................................10,00Sodium chloride ........................................5,00Bromocresol purple ..................................0,02Final pH 6,8 ± 0,2

DirectionsDissolve 15 g of powder in 1 litre of distilled water. Add substrate to assay in the desired concentration and distribute into containers provided with Durham’s tubes. Sterilize in the autoclave at 121°C for 10 minutes. Heat up the autoclave before putting in the tubes to avoid sugar caramelization.Addition of some kind of sugars may require a pH adjust-ment.To study the fermentation of some sugars like Glucose (Ref. 06-048), Lactose (Ref. 06-051), Maltose (Ref. 06-052), Mannitol (Ref. 06-050) and Sucrose (Ref. 06-049) it is advisable to add 10 g/L of each one.

DescriptionBromcresol Purple Base Broth is the liquid version suit-able to determine gas production (by Durham´s tubes) by enterobacteria which are sensitive to phenol red.In the SCHARLAU formulation, meat extract has been omitted as it was found unnecessary and it also provid-ed low concentrations of fermentable sugars that could change or give erroneous results.The bacteria when ferment the carbohydrates, changes the medium colour to yellow due to Bromcresol purple pH indicator, and if they produce gas, it is retained in the Durham’s tube.

ReferencesATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc.,Boca Raton, Fla.

Brucella Agar

Ref. 01-042

SpecificationSolid medium for the cultivation of Brucella and other fastidious microorganisms

Formula (in g/L)Peptone of casein .....................................10,0Meat Peptone ...........................................10,0Yeast Extract ..............................................2,0D(+) Glucose .............................................1,0Sodium chloride ..........................................5,0Sodium bisulphite ......................................0,1Agar ..........................................................15,0Final pH 7,0 ± 0,2

DirectionsSuspend 43 g of powder in 1 L of distilled water and bring to the boil. Distribute into suitable containers and sterilize in autoclave at 121ºC for 15 minutes. If a high selectivity medium is wanted, add 2 flasks of Brucella Selective Supplement (Ref. 06-025CASE). When Ethyl Violet 1,25 mg/L is added to the culture medium, the growth of biotype 2 of Brucella abortus is inhibited.Some microbiologists promote this medium as suitable for Campylobacter maintenance.

Brucella Media

Brucella Broth

Ref. 02-042

SpecificationLiquid culture medium for Brucella and other fastidious microorganisms

Formula (in g/L)Peptone of casein .....................................10,0Meat Peptone ...........................................10,0Yeast Extract ..............................................2,0D(+) Glucose .............................................1,0Sodium chloride ..........................................5,0Sodium bisulphite ......................................0,1Final pH 7,0 ± 0,2

DirectionsDissolve 28 g of powder in 1 L of distilled water, heat-ing if necessary. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes. If a highly selectivity is wanted, add aseptically after autoclave, Brucella Selective Supplement (Ref. 06-025CASE). When Ethyl Violet 1,25 mg/L is added to the culture medium, the growth of biotype 2 of Brucella abortus is inhibited.

36

DescriptionThe Brucella Media are prepared from composition of the APHA’s Albimi Broth used for isolation of Brucella species and the only difference between Broth and Agar is the solidifying agent. Both media are suitable for the isolation and cultivation of a lot of fastidious microorgan-isms including Streptococcus, Neisseria and Campylo-bacter, but they became selective with the addition of antibiotics like polymyxin or bacitracin or chemical inhibi-tors like cycloheximide and ethyl violet. With some dyes (fuchsin and thionin) the media became differential. See the suitable reference for the technique in every case.

Caution Brucella species are classified as Biosafety Level 3 pathogens. All manipulations with live cultures and antigens must be confined to a Class II Biological Safety Cabinet. Follow proper established laboratory proce-dures in handling and disposing of infectious materials.

ReferencesALTON, G.G, L.M. JONES & D.E. PIETZ (1976) Las técnicas de laboratorio en la Brucelosis, 21 ed. Mono-graph nº.55 FAO/WHO. Geneve.CRUICKSHANK.(1965) Medical Microbiology. 11th ed. E.S. Livingstone. Edimburgo.ISENBERG H.D. (1992) Clinical Microbiology Proce-dures Handbook. ASM. Washington D.C.MacFADDIN J.D. (1985) Media for Isolation-cultivation-identification-maintenance of medical bacteria. William & Wilkins Baltimore MD.VANDERZANT, C & D.F. SPLITTSTOESSER (1992) Compendium of methods for the microbiological exami-nation of food 3rd Ed. APHA. Washington D.C.

Bryant & Burkey Media

Brucella Media

Bryant & Burkey Lactate Broth(BB-Lactate Broth)

Ref. 02-421

SpecificationLiquid medium for the enumeration of spores of lactate-fermenting clostridia in milk and dairy products

Formula (in g/L)Peptone ..............................................15,0000Meat extract ..........................................7,5000Yeast extract .........................................5,0000L-Cysteine HCl .....................................0,5000Resarzurine ..........................................0,0025Sodium acetate ....................................5,0000Sodium lactate ......................................5,0000Final pH 6,2 ± 0,2

DirectionsDissolve 38 g of powder in 1 L of distilled water, heating up only if necessary. Distribute in suitable containers for every procedure and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionBB medium is used to enumerate, by MPN technique, the spores of gasogenic clostridia that are the produc-ers of the swelling and rancidness of cheese in the dairy industry (late blowing or butyric swelling). In normal conditions of use,the medium allows the growth of other microorganims also which are not directly related to the cheese alteration, e.g. Cl. butyricum or Cl. sporogenes, besides the main responsible organism Cl. tyrobutyri-cum, in presence of enough acetate, Clostridium tyrobu-tyricum ferments lactate, producing acetic and butyric acids, CO

2 and hydrogen.

TechniqueRecommended technique is to enumerate the spores by the MPN technique with fresh made medium, covered with a vaspar layer of 2 mL that acts as a cap that as-sures a low redox potential and at the same time retains gas produced in the reaction.Sample must be previously decontaminated by heat-ing up for 10 minutes at 75°C in order to destroy all the vegetative forms and only leaving the alive spores.Incubation is performed for 7 days at 37°C. Tubes will be declared positive if they show a clear gas produc-tion. Despite the fact that results are often expressed as butyric spores/mL, the limitations of the method suggest that they should be expressed as lactate fermenting clostridial spores.

37

Bryant & Burkey Modified Fluid Medium

Ref. 03-557

SpecificationFluid medium for the enumeration of spores of lactate-fermenting clostridia in dairy products

Formula (in g/L)Tryptose ..............................................15,0000Meat Extract .........................................7,5000Yeast Extract ........................................5,0000Cystine HCl...........................................0,6000Resazurine ...........................................0,0025Sodium lactate .....................................3,0000Sodium acetate ....................................5,0000Sodium thioglycollate ...........................0,2000Agar ......................................................0,7500Final pH 5,9 ± 0,2

DirectionsSuspend 37 g of powder in 1 L of distilled water and bring to the boil. Distribute in suitable containers for every procedure and sterilize in autoclave at 121ºC for 15 minutes.

DescriptionThis modification of the Bryant & Burkey Lactate Broth (Ref. 02-421) add a little amount of agar that makes the medium more thick to be used in greater volumes

maintaining its anaerobic condition by the thioglycollate added. Also the amount of lactate is reduced because the density of the medium retains easily the gas bubbles produced.

TechniqueSamples are processed by established procedures in every product.

ReferencesBERGÈRE, J. L. & S. SIVELA (1989) Detection and enu-meration of clostridial spores related to cheese quality. Classical and new method. FIL-IDF Bull. 51:18-23BRYANT M.P. & L.A. BURKEY (1956) The characteris-tics of lactate-fermenting spore-forming anaerobes from silage. J. Bacteriol. 71:43-46ROSENBERGER, K.F. (1951) The development of meth-ods for the study of obligate anaerobes in silage. Proc. Soc. Appl. Bacteriol. 14:161-164

Ref. 02-494

SpecificationDiluent for the homogenization of samples according to the European Pharmacopeia and ISO 21149.

Formula (in g/L)Peptone ....................................................1,00Sodium chloride ........................................4,30Disodium phosphate .................................7,23Potassium phosphate ...............................3,56Final pH 7,0 ± 0,2

DirectionsDissolve 16 g of powder in 1 L of distilled water, heating up if necessary. Add 1 to 10 mL of Polysorbate 80 (Ref. 06-088) or Polysorbate 20 depending on the type of food to be diluted. Homogenize and distribute into containers. Sterilize by autoclaving at 121ºC for 15 minutes.

Buffered Peptone Water Eur Phar (Buffered Sodium Chloride-Peptone Solution pH 7.0)

DescriptionThis solution is recommended by European Pharmaco-poeia to dilute the sample for microbiological examina-tion. Depending on the amount of fat in the sample to examine it is the technician’s decision regarding the kind and quantity of emulsifying agent to be used.

ReferencesEUROPEAN PHARMACOPOEIA (2007) 5ª ed. Suppl. 5.6 § 2.6.13 Microbiological examination of non-sterile products. EDQM Council of Europe. Strasbourg.ISO 21149:2006 Cosmetics – Enumeration and detec-tion of aerobic mesophilic bacteria.

Bryant & Burkey Media

38

Ref. 02-539

SpecificationLiquid medium to dilute and neutralize samples of phar-maceutical, cosmetic, raw material or end-products for the purpose of enumeration

Formula (in g/L)Casein peptone .....................................20,00Soya lecithin ............................................5,00Final pH 7,3 ± 0,2

DirectionsDissolve 25 g of powder in 960 mL of distilled water pre-warmed at 50ºC . Add 40 mL of polysorbate 20, homog-enize and distribute in suitable containers. Sterilize in autoclave at 121ºC for 15 minutes.

Casein Lecithin Polysorbate Broth Base

DescriptionThis medium is produced according the formulation of the U.S. Pharmacopoeia. In the Section <61> “Microbial Limit Tests” is proposed as alternative system to neutral-ize preservatives and didsinfectants before to proceed with the enumeration process, specially by the mem-brane filtration method.

ReferencesUS PHARMACOPOEIA (2002) <61> Microbial Limits Tests. 25th ed. US Pharmacopoeial Conv. Inc. Rockville. MD

Ref. 01-569

SpecificationSolid medium, acc. Standard Methods, for the detection and enumeration of proteolytic organisms in dairy prod-ucts

Formula (in g/L)Tryptone ...................................................5,00Yeast extract .............................................2,50Dextrose ...................................................1,00tri-Sodium citrate ......................................4,41Sodium caseinate ...................................10,00Calcium chloride .......................................4,40Agar ........................................................15,00Final pH 7,0 ± 0,2


DescriptionCaseinate Agar is preferred to other media like Skimmed Milk Agar for the detection of proteolytic organisms because its better recovery of stressed cells and the absence of false positives.

Caseinate Agar

Technique Pour the caseinate agar into plates so that the medium is 2 mm thick (e.g. 12 mL for a 9-cm plate). After the me-dium is hardened, plate 0,1 mL quantities of the sample or diluted sample on the agar surface and spread evenly with a sterile Drigalski rod. To ensure absorption of the sample allow inoculated plates to dry for 15 minutes, and incubate them for 48 to 72 hours at 32±1ºC or for 72 hours at 21±1ºC. Colonies surrounded by a white or off-white zone of casein precipitate are proteolytic. Highly proteolytic bacteria will also produce a clear inner zone.Report results as “proteolytic count per gram or mL”. Designate the medium used and the incubation condi-tions.

References MARTLEY, F.G., S.R. JAYASHANKAR & R.C. LAW-RENCE. (1070) An improved agar medium for the detec-tion of proteolytic organisms in total bacterial counts. J. Appl. Bacteriol. 33:363-370FRANK, J.F. G.L CHRISTEN & L.B. BULLERMAN (1992) Test for groups of Microorganisms. In R. T. Marshall (ed) Standard Methods for the examination of Dairy Products. APHA. Washington D.C.

39

Cetrimide Agar (Pseudomonas Selective Agar)(Eur. Phar. Agar Medium N)

Ref. 01-160

SpecificationSolid culture medium for selective isolation of Pseu-domonas aeruginosa acc. to ISO 22717.

Formula (in g/L)Gelatin peptone ........................................20,0Magnesium chloride ...................................1,4Potassium sulfate .....................................10,0Cetyltrimethyl-Ammonium Bromide .......... 0,3Agar ..........................................................15,0Final pH 7,2 ± 0,2

DirectionsSuspend 46,7 g of powder in 1 L of distilled water and add 10 mL of Glycerol. Bring to the boil and distribute into suitable containers. Sterilize at 121°C for 15 min-utes.

DescriptionThe Cetrimide Agar is based on the enormous resistance of Ps. aeruginosa strains to the Quaternary Ammonium Compounds (QAC’s). With regard to the Cetyltrimethyl-Ammonium Bromide there has been growth at 1 g/L concentrations, but in such cases it has been very poor and slow.An inhibitor concentration of 0,3-0,5 g/L does not seem to affect the viability of the pyogenic species. Neverthe-less, it does inhibit the rest of the fastidious accompa-nying bacteria, both gram-positive and gram-negative, as well as other species of Pseudomonas which may develop at lower inhibitory concentrations.

Although Ps. aeruginosa prevails over any other fastidi-ous bacteria after a 48 hour incubation at 35°C, it is recommended to first isolate at 42°C with an incubation of 48 hours. By this method, almost complete inhibition of other microorganisms is obtained.

ReferencesLOWBURY,E.J.L. & A.G. COLLINS (1955) The use of a new cetrimide product in a selective medium for Pseu-domonas aeruginosa J. Clin. Path. 8.47BROWN, V.I. & J.L. LOWBURY (1965) Use of an im-proved Cetrimide Agar Medium and of culture methods for Pseudomonas aeruginosa. J. Clin. Path. 18.752FDA (1998) Bacteriological Analytical Manual. 8th ed. Rev. A. AOAC International. Gaitherburg. VA.ATLAS, R.M. and L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press Inc.,Boca Raton,Fla.EUROPEAN PHARMACOPOEIA,(2002) Supplement4.2 4th ed., 2.6.13 Microbiological examination of non-sterile products. Council of Europe,Strasbourg,.U.S. PHARMACOPOEIA (2002) 25 ed. <61> Microbial limit tests. Us Pharmacopoeial Conv. Inc. Rockville, MDISO 22717:2006 Cosmetics – Detection of Pseu-domonas aeruginosa.

control

Pseudomonas aeruginosa ATCC 27853

R-52/53S-61

N

40

Chapman-Stone Agar

Ref. 01-052

SpecificationSolid and differential medium with a high selective ability, for the isolation of staphylococci from food.

Formula (in g/L)Peptone ....................................................10,0Yeast extract ...............................................2,0Gelatin ......................................................30,0D-Mannitol ................................................10,0Sodium chloride ........................................55,0Ammonium sulfate ....................................75,0Dipotassium phosphate ..............................5,0Agar ..........................................................15,0Final pH 7,0 ± 0,2

DirectionsSuspend 202 g of powder in 1 L of distilled water and heat up in boiling water bath until the total dissolution of gelatin. Bring to the boiling. Distribute in tubes or flasks and sterilize at 121°C for 15 minutes. Pour into plates immediately. Avoid overheating.

DescriptionChapman-Stone medium is according to a modification of the classical 110 medium for Staphylococcus. The main modification consists the inclusion of ammonium sulfate, that allows the direct reading or observation of gelatin hidrolysis, instead of adding reagents to the plate medium. Another modification is the reduction in the amount of sodium chloride. The saline content is selec-tive itself, but it is reinforced by the ammonium sulfate, which is also selective. Finally, lactose has been omitted , making the difference between the colonies that fer-

ment mannitol (biqger size) and the colonies that do not ferment mannitol (smaller size) greater, since the latter must grow solely depending on peptone as the nutrient source.

TechniqueMaterial under test is inoculated on the surface to pro-duce separated colonies, and is incubated at 30°C for a 48 hours period. After this time, examine and select colonies on the basis of these criteria:White or non pigmented colonies are discarded, even if they show a gelatin liquefaction halo (coagulase nega-tive).Golden yellow pigmented colonies, surrounded by a clear zone of gelatin hydrolysis (positive Stone’s reac-tion) are selected to verify mannitol fermentation and further, coagulase and hemolysis.

Mannitol fermentation is checked by adding a few drops of Bromcresol purple indicator (aq. sol. 0,04%) over the colonies. Any colour change indicates positive mannitol fermentation.Coagulase production and hemolysis tests are per-formed in later subcultures on suitable media.Chapman-Stone medium composition is very selective, and so, medium can be employed without real steriliza-tion. However, it is always advisable to sterilize it and keep one control before use.

ReferencesCHAPMAN (1948).An improved Stone Medium for the isolation and testing of food-poisoning staphylococci Food Research 13:100-105ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc.London

Ref. 01-053 Chapman TTC Agar.

control E. coli ATCC 25922 Salmonella typhimurium ATCC 14028

E. coli ATCC 25922(MF technique)

Salmonella typhimurium ATCC 14028(MF technique)

41

Ref. 01-053

SpecificationMedium for the detection of coliforms by membrane fil-tration technique in water analysis acc. ISO 9308-1:2000 standard.

Formula (in g/L)Meat peptone .........................................10,00Meat extract ..............................................5,00Lactose ..................................................20,00Yeast extract .............................................6,00Bromothymol blue ...................................0,05Sodium heptadecyl sulfate .......................0,10Agar ........................................................15,00Final pH 7,2 ± 0,2

DirectionsSuspend 56,2 g in 1 L of distilled water and bring to the boil. Distribute into suitable containers and sterilize by autoclaving at 121°C for 15 minutes. Cool to 45-50°C. Add 2-3 mL/L of filter sterile 1% aqueous 2,3,5-triphe-nyltetrazolium chloride (TTC) (Ref. 06-023) and pour plates. Do not reheat.

DescriptionThis medium is formulated for the presumptive iden-tification of coliforms in drinking water, by membrane filtration technique.Due to the instability the triphenyltetrazolium is provided in a separate container, sterilized and ready to use.Poured plates can be stored refrigerated for up to 8 days without losing their effectiveness. They should not be used if any dehydration or drying signs appear.

TechniqueWhile using the membrane filter technique for the pre-sumptive identification of coliforms in water, it should be kept in mind that to every type of water corresponds a minimum volume to be filtered and depends on the type of water. Dilute with sterile phosphate buffer if necessary to obtain the number of colonies on the membrane which are easy to count.

Chapman TTC Agar (Tergitol® 7 Agar)

For every water sample two volumes or double quantity must be filtered over different membranes and incubated on Tergitol 7 Agar at 35°C and 44°C respectively. After 48 hours the typical colonies have the appearance shown in the table.Most coliform can not grow on this medium when incu-bated at 44°C, except E. coli which forms a characteris-tic appearance.Results are always expressed per 100 mL sample in-cluding any applied dilutions. Estimation is done by tak-ing typical colonies which have grown at 35°C as fecal coliform, together with those grown at 44°C as E.coli. Nevertheless, according to the legislation and despite the medium’s selectivity, results can only be considered as presumptive and all coliform colonies have to be con-firmed by following the criteria stated below: Typical appearance in EMB Agar (Ref.1-068) or Endo Agar Base (Ref.1-589); characteristic reactions in Kligler Iron Agar (Ref.1-103). For the confirmation of faecal E. coli, verification of it is: a motile,gram-negative bacillus and lactose fermentator with acid and gas production, which gives negative results on the citrate test and indole production positive.

ReferencesATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc. London.CHAPMAN G.H. (1951). A culture medium for detect-ing and confirming E. coli in ten hours. Am. J. Publ. Hlth 41:1381-1386.DOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Foods. 3rd ed. APHA.Washington.GUINEA, SANCHO,PARES (1979). Análisis Microbiológ-ico de Aguas. Ed. Omega. Barcelona.ISO 9309-1:2000 Standard. Water Quality. Detection and enumeration of Escherichia coli and coliform bacteria.- Part 1: Membrane filtration method.SPECK, M (Ed.) (1982) Compendium of Methods for the Microbiological Examination of Foods. 2nd. Ed. APHA.Washington.

42

Chloramphenicol Glucose Media

Chloramphenicol Glucose Agar(CGA) (Yeast Extract DextroseChloramphenicol Agar acc. FIL-IDF)

Ref. 01-366

SpecificationSolid and selective medium for the isolation and enu-meration of fungi in milk and dairy products according ISO 7954.

Formula (in g/L)Dextrose ...................................................20,0Yeast extract ...............................................5,0Chloramphenicol ........................................0,1Agar ..........................................................15,0Final pH 6,6 ± 0,2

DirectionsSuspend 40 g of powder in 1 L of distilled water and let it soak. Bring to the boil and distribute into containers. Sterilize in the autoclave at 121°C for 15 minutes.

DescriptionThis is the medium recommended by the Federation In-ternational Laitrere - International Dairy Federation (FIL-IDF) for the isolation and enumeration of fungi (moulds and yeast) in milk and dairy products. This medium has also been adopted by the DIN and ISO standard.

Medium relies its selectivity on the bactericidal action of Chloramphenicol which, due to its thermostabity, may be sterilized with the complete medium in the autoclave. Moreover, pH may be adjusted near to neutrality, and this fact allows the medium to be remelted several times without affecting its stability, selectivity and efficacy. Remeltings and overheatings may make the medium darker.

TechniqueGenerally the mass seed method or poured plate method is used to inoculate the medium, and an incuba-tion at 22-25°C for 4 to 5 days is carried out.

Chloramphenicol Glucose Broth(CGB) (Yeast Extract Dextrose Chloramphenicol Broth acc. FIL-IDF)

Ref. 02-366

SpecificationLiquid and selective medium for the enumeration of fungi in milk and dairy products using the MPN technique.

Formula (in g/L)Dextrose ...................................................20,0Yeast extract ...............................................5,0Chloramphenicol ........................................0,1Final pH 6,6 ± 0,2

DirectionsDissolve 25 g of powder in 1 L of distilled water. Distrib-ute in tubes and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionThis is the liquid version of the medium with the same name (Ref. 01-366) recommended by the Federation International Laitrere - International Dairy Federation (FIL-IDF) for the enumeration of fungi (moulds and yeasts in liquid products).Medium is specially adapted to enumerate yeast and, eventually, moulds by the Most Probable Number tech-nique (MPN).

ReferencesFIL-IDF 94B Standard (1991). Enumeration of yeast and moulds. Colony Count Technique at 25 °C.ISO 7954 Standard (1987) General guidance for enu-meration of yeast and moulds - Colony count at 25ºC.DIN Standard 10186. Mikrobiologische Milch Untersu-chung. Bestimmung der Anzahl von Hefen und Schim-melpilzen. Referenzverfahren.

R-45S-53-45

T

R-45S-53-45

T

43

TRef. 01-558

SpecificationSelective differential solid medium for the detection and enumeration of streptococci in dairy products.

Formula in g/LTryptone .................................................10,00Yeast extract ...........................................10,00Sodium citrate ........................................20,00Sodium azide ............................................0,40Tetrazolium blue .......................................0,10Agar ........................................................15,00Final pH 7,0 ± 0,2


DescriptionCitrate Azide Agar is the streptococci detection medium preferred in dairy microbiology over the other media like KF-Streptococcal Agar (Ref. 01-294) used in food micro-biology because its high selectivity and security of use.

Citrate Azide Agar

TechniquePour the melted medium cooled to 45-50ºC over the sample in a petri dish. Let it solidify and dose another 5-7 sterile volume of medium on the surface to facilitate the micro-aerofilic environment. The plates are incubated at 37ºC for 48-72 hours and then the blue colonies are counted. Express the results as “streptococci per g or mL of sample”

ReferencesMARSHALL, R.T. (1992) Standard Methods for the Examination of Dairy Products. 16th Ed. APHA. Washing-ton. DCHARTMANN P.A, R.H. DEIBEL, & L.M. SIEVERDING (1992) Enterococci. en Vanderzant & Splittstoesser: Compendium of methods for the microbiological exami-nation of foods. 3rd Ed. APHA. Washington DC.EFTHYMIOU C.J., P. BACCASH, V.J. LABOMBARDI, & D.S. EPSTEIN (1974) Improved isolation and differentia-tion of enterococci in cheese. Appl. Microbiol. 28:417-422SARASWAT, D.S. G.W. REINBOLD & W.S. CLARK (1963) Selection of a medium for the isolation and enu-meration of enterococci in dairy products. J. Milk Food Technol. 26:114-118

R-45-22-32-52/53S-53-45-7-61

44

CLED Agar (Brolacin Agar)

Ref. 01-047

SpecificationCystine,lactose,electrolyte deficient medium, recom-mended for the isolation and identification of urinary pathogenic bacteria.

Formula (in g/L)Peptone ................................................. 4,000 Trypsic Peptone ..................................... 4,000Meat extract ........................................... 3,000Lactose .................................................10,000L-Cystine ............................................... 0,128Brom Thymol Blue ................................. 0,020Agar ......................................................15,000Final pH 7,4 ± 0,2

DirectionsAdd 36 g of powder to 1 L of distilled water and heat to boiling. Sterilize by autoclaving at 121°C for 15 minutes.

DescriptionThis general purpose medium has been recommended for bacteriological urine analysis. Current formulation is a modification of the original one reported by Sandys, that achieves an excellent colony differentiation without inhibitors. This fact, and also the careful selection of nu-tritive components, makes this medium a substrate able to support growth of most urinary pathogenic bacteria.Presence of lactose as a fermentable sugar allows clas-sic differentiation and, at the same time, lack of electro-lytes suppresses swarming waves on the members of the Proteus species and sometimes growth of Shigella sp. also.The characteristics of colonies that grow on C.L.E.D. Agar (after 18 hours of incubation):Escherichia coli: Yellowish colonies, opaque, with core,

1,25 mm diameter. Non fermentative strains give blue colonies.

Klebsiella sp.: Very mucuous colonies of variable colour, from yellow to blue-white.

Salmonella sp.: Plain and blue colonies.Enterococcus faecalis: Yellow colonies. 0,5 mm diam-

eter.Staphylococcus aureus : Convex yellow colonies. 0,75

mm diameter.

Staphylococci coagulase(-ve): White or light yellow colonies, with halo and the same size as of Enterococci.

Proteus sp.: Blue, translucent and smaller than E.coli.Pseudomonas aeruginosa: Plain, matt and wrinkled

colonies. Green colour and irregular boundary.Corynebacteria: Pointed and grey coloniesLactobacilli: Matt colonies, like Corynebacteria.

ReferencesSANDYS, G.H. (1960) A new method of preventive swarming of Proteus sp. J.Med.Lab.Tech. 17:224MACKEY J.P. and G.H.SANDYS (1966) Diagnosis of urinary tract infections. Brit. Med. J. 1.173

control

Proteus mirabilis ATCC 10975

45

Ref. 01-609

SpecificationsSelective solid medium for the detection of Pseu-domonas aeruginosa according the EN 12780-2002 and ISO 16266 Standard.

Formula (in g/L)Gelatine Peptone ....................................16,00Casein Peptone ......................................10,00Potassium sulphate ................................10,00Magnesium chloride .................................1,40Cetrimide® ................................................0,20Agar ........................................................15,00Final pH 7,1 ± 0,2

DirectionsAdd 52,6 g of powder to 1 L of distilled water with 10 mL of glycerol. Heat to complete solution and sterilize in autoclave at 121ºC for 15 min. Cool to 45-50ºC and add to 500 mL of medium a flask of the Nalidixic Acid Selec-tive Supplement (Ref. 06-124CASE). Homogenise and pour plates.Do not maintain the complete medium melted for more than 4 hours. Do not re-melt. The finished plates can be used without lose its efficiency for one month if they are refrigerated and in a dark place.

DescriptionThe CN Selective Medium for Pseudomonas was pro-gressively developed from the basic media of King, Ward and Raney for the production of pigments. Browne and Lowbury add the cetrimide as selective agent and Goto and Enomoto improves efficiency by adding nalidixic acid. The presence of both inhibitors eliminates the con-taminant microbiota from heavily polluted specimens and was adopted by the CEN (Centre Europeen de Normali-sation) in its EN Standard 12780 for the detection of Ps. aeruginosa by filtering membrane in water.

TechniqueA volume of the sample is filtered thorough a filtering membrane of 0,45 µm pore and the membrane is depos-ited on the surface of the CN medium. The plates are incubated at 36±2ºC for a period of 44±4 h with a partial examination at 22±2 h.All the colonies producing a green or blue (pyocianin) pigmentation in this period must be considered as Pseu-domonas aeruginosa without any other confirmation.All the colonies that produces fluorescence under the Wood’s light (without pyocianin production) era consid-ered presumptive Ps. aeruginosa and must be confir-mate on Acetamide Medium (Ref. 03-428).All the colonies producing a brown-reddish pigment and no produces fluorescence nor pyocianine are considered presumptive Ps. aeruginosa and must be confirmed by the oxidase test and typical growth on Acetamide Me-dium (Ref. 03-428) and King B Agar (Ref. 01-029)

ReferencesKING, E.O., M.K. WARD & E.E. RANEY (1954) Two simple media for the demonstration of pyocianin and fluorescein. J. Lab. Clin. Med. 44:301BROWN, V.L. & E.J.L. LOWBURY (1965) Use of an im-proved Cetrimide Agar Medium and of culture methods for Ps. aeruginosa. J., Clin. Pathol. 18:752GOTO S. & S. ENOMOTO (1970) Nalidixic acid cetrim-ide agar. A new selective plating medium for the selec-tive isolation of Ps. aeruginosa. Jpn. J. Microbiol. 14:65ROBIN, T. & J.M. JANDA (1984) Enhanced recovery of Ps. aruginosa from diverse clinical specimens on a new selective agar. Diag. Microbiol. Infect Dis. 2:207EN STANDARD 12780 (2002) Water Quality. Detection and enumeration of Ps. aeruginosa by membrane filtra-tion. Brussels.ISO 16266:2006 Standard. Water Quality – Detection and enumeration of Pseudomonas aeruginosa. Method by membrane filtration.

CN Selective Agar Base for Pseudomonas

control Pseudomonas aeruginosa ATCC 27853

R-52/53S-61

N

46

Cosmetic Diluent of Beerens

Ref. 02-257

SpecificationDiluent to neutralize preservative systems in common examination of cosmetic products.

Formula (in g/L)Lecithine ...................................................3,00Sodium thiosulfate ....................................5,00L-Histidine HCl .........................................1,00Peptone ....................................................1,00Sodium chloride ........................................8,50Dipotassium phosphate ............................1,00Final pH 7,0 ± 0,2

DirectionsDissolve 19,5 g of powder in 1 L of distilled water con-taining 30 mL of polysorbate 80.(Ref. 06-080). Distribute into suitable containers and sterilize by autoclaving at 121°C for 15 minutes.Let it cool to 50°C and shake gently to redissolve polys-orbate.

DescriptionCosmetic diluent of Beerens has all the necessary compounds to neutralize most of the chemical agents included in cosmetic products to maintain and preserve it free of microorganisms.It complies with the EU recommendation that states that before any microbiological examination, a treatment to remove all the growth inhibitor systems in the cosmetics must be performed.However, this standard also declares that later dilutions have to be performed in less aggressive media, that may be considered as an enrichment and revitalization system, and it suggests Letheen Broth (Ref. 02-236) or Letheen Modified Broth (Ref. 02-237).

ReferencesBEERENS, H., RAMONS, C., LEMAIRE, D. (1976). Rev. Inst. Past. Lyon. 9:127.BRIGIDI, P., MATTEUZZI, D. (1982) II Farmaco Ed. Pr. 37:8:260. Commission del Communautes Europeennes, Groupe Special. Methodes de Controle Microbiologique des Produits Cosmetiques: Limites Numeriques Appli-cables au controle Officiel de la Qualité Microbiologique des Produits Cosmetiques. XI/405A, ISPRA, 1976.

Ref. 01-513

SpecificationSolid medium for the enumeration and isolation of Clostridium perfringens in water acc. the European Directive 12767/97

Formula (in g/L)Tryptose ..................................................30,00Yeast extract ...........................................20,00Sucrose ....................................................5,00L-Cisteine HCl ..........................................1,00Magnesium sulfate.7H

2O ..........................0,10

Bromocresol purple ..................................0,04Agar ........................................................15,00Final pH 7,6 ± 0,2

DirectionsSuspend 71 g of powder in 1 L of distilled water and bring to the boil. Distributein suitable containers and sterilize in autoclave at 121ºC for 15 minutes. Cool to 45-50ºC and aseptically add 2 flasks of Selective Sup-plement m-CP (Ref. 06-125CASE) with the following composition:D-Cicloserine .............................................400 mg/LPolymyxin B sulphate ..................................25 mg/LIndoxil-b-D-Glucoside ..................................60 mg/LFenolftalein diphosphate ...........................100 mg/LIron (III) Chloride .........................................90 mg/L

m-CP Agar Base

Homogenize avoiding bubble formation and pour in plates.

DescriptionThe m-CP Agar Base is a solid medium for counting and isolating vegetative cells and spores of Clostridium perfringens by the membrane filtration method. Its use is compulsory in determining the quality of water for human consumption in European Union by Directive 12767 (12-07-1997) of the European Council.

Technique A suitable volume of water is filtered through a mem-brane filter of 47 mm diameter and 0,45 mm pore. Put the membrane on the surface of a plate of m-CP medium freshly prepared and incubate in an anaerobic atmos-phere at 44±1ºC for 21±3 hours. Expose the growth obtained to amonium hydroxide vapours for 20-30 sec-onds. Count as Clostridium perfringens all the opaque yellow colonies that turn to pink or red after the ammo-nium hydroxide exposure. Express the results as cfu/mL.

ReferencesEuropean Council (1998) Directive 98/83/CE on the quality of the water destined to the human consuption. EC Bull. 11-03-1998.

47

Cystine-Tryptone Fluid Medium (CTA Medium)

Ref. 03-045

SpecificationBasic medium for strain maintenance or sugar fermenta-tion assays. Its fluidity makes it suitable to observe the motility.

Formula (in g/L)L-Cystine HCl .........................................0,500Casein peptone ....................................20,000Sodium chloride ......................................5,000Sodium sulfite .........................................0,500Phenol red ..............................................0,017Agar ........................................................2,500Final pH 7,3 ± 0,2

DirectionsSuspend 28,5 g of powder in 1 L of distilled water and heat to boiling. Dispense and sterilize at 121°C for 15 minutes. Addition of certain sugars may require a pH readjustment.

DescriptionCystine and Tryptone fluid medium is appropiate for the propagation and maintenance of bacterial strains, even the fastidious ones, without additives.The lack of fermentative sugars allows the long survival of microorganims, and sometimes it has been used for studies on severe microaerials fermentation, due to the fact that these microorganisms do not grow on current basal media. Medium has its maximum efficacy when freshly prepared, but it can be stored for long periods of time, taking care to avoid its dehydration. To achieve this, screw caps or hermetic sealing are strongly recom-mended. In any case, if medium has been stored for a long time, it is better to put it in a boiling water bath for a few minutes before using it.

TechniqueOn this medium, we can maintain by deep stab a lot of fastidious microorganisms like Neisseria, Enterococcus, Pasteurella, Shigella and in most cases without needing any additives or CO

2. Even some light-sensitive anaero-

bic microorganisms can grow without special conditions though in reduced atmopheres they give ideal growth on this medium.In the few cases as per the nutritive requirements of the microorganisms like serum or ascitic liquid, which can be added in an aseptic way to the molten medium, when cooled to 45-50°C.Microorganism motility can be easily detected by growth in the stab, and helped by the medium fluidity.Medium, with added sugars, may be used to study sugar fermentation with microorganisms that do not grow on phenol red classical media because of the additional nutritional requirements. Acidification can be easily ob-served with the change in colour of phenol red indicator.

ReferencesVERA, H.D. (1948) A simple medium for identification and maintenance of the gonococcus and other bacteria. J.Bact. 55:531ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc.

m-CP Agar Base

control Clostridium perfringens before the exposure

to Ammonia vapours (AM0257)

Clostridium perfringensafter the exposure

to Ammonia vapours (AM0257)

48

Czapek-Dox Media

Czapek-Dox Agar

Ref. 01-051

SpecificationSemisynthetic solid medium for the cultivation of fungi, with sodium nitrate as the only nitrogen source.

Formula (in g/L)Sucrose ..................................................30,00Sodium nitrate ..........................................2,00Magnesium glycerophosphate ................. 0,50Potassium sulfate .....................................0,35Potassium chloride ...................................0,50Ferrous sulfate .........................................0,01Agar ........................................................15,00Final pH 6,8 ± 0,2

DirectionsSuspend 48,5 g of powder in 1 L of distilled water and heat to boiling. Dispense and sterilize by autoclaving at 121°C for 15 minutes. Should a lower pH be desired, acidify the medium with sterile lactic acid after cooling to 45°C.

Czapek-Dox Broth

Ref. 02-051

SpecificationSemisynthetic liquid culture medium with sodium nitrate as the only nitrogen source.

Formula (in g/L)Sucrose ..................................................30,00Sodium nitrate ..........................................2,00Potassium chloride ...................................0,50Magnesium glycerophosphate ................. 0,50Ferrous Sulfate ........................................0,01Potassium sulfate .....................................0,35Final pH 6,8 ± 0,2

DirectionsDissolve 33,5 g of powder in 1 L of distilled water. Dis-pense in suitable containers and sterilize in the auto-clave at 121°C for 15 minutes. Do not overheat

DescriptionCzapek-Dox medium, both liquid and solid version, is a general cultivation medium of defined chemical composi-tion, where the sole nitrogen source is sodium nitrate, and the carbon source is sucrose. It has been employed successfully in the isolation and cultivation of soil micro-organisms especially fungi.

Its original version had a pH near neutrality, but it can be rendered selective medium for the fungi by adding, (after sterilization and before solidification), 10 mL of sterile

lactic acid 10% solution, producing a pH drop to 3,5. In this medium, Warcup adds 5 g/L of Yeast extract (Ref. 07-079) and an antibiotic mixture (Streptomycin 30 mg/L and Tetracycline 2 mg/L) to achieve total efficacy and selectivity. In any case, bacterial growth is very poor.

Czapek-Dox medium has been adopted for the morpho-logical studies of soil fungi, and it aids chlamydospora formation by Candida albicans in shorter periods (Daw-son, 1962). Temperatures and times of incubation al-ways varies, and they can go from 1 to 5 weeks at room temperature. Usually, it is 8-15 days at 15°C. In case of Candida, 28°C for 48 hours seems to be optimum ; for Penicillium, 22°C and nonetheless, Aspergillus grows better at 30-32°C.

ReferencesCZAPEK, F. (1903) Untersuchung uber die sticstoff-gewinnungund einweissbildung der Pflanze.Beitr. Chem. Physiol. Pathol. 1:540DOX, A.W. (1910) The intracellular enzymes of Penicil-liumand Aspergillus with special references to those of P. camemberti. US Dept Agr. Bur. Animal Ind. Bull. 120:70APHA-AWWA-WPCF (1992) Standard Methods for the examination of Water and Wastewater. 18th ed. APHA. Washington.RAPER, K.B. & D.J.FENELL (1965) The genus Aspergil-lus. William & Wilkins Co. Baltimore.THOM,C & M.B. CHURCH (1926 The aspergilli. William & Wilkins Co. Baltimore. WARCUP, J.H. (1950) The soil-plate method for isolation of fungi from soil. Natur 166:117-118

Fusarium sp.

49

Ref. 01-610

SpecificationSolid culture medium for the neutralization and testing of antiseptics and disinfectants acc. ISO 22717 and 22718 standards.

Formula (in g/L)Tryptone ...................................................5,00Yeast extract .............................................2,50Dextrose .................................................10,00Lecithin .....................................................7,00Sodium thiosulphate .................................6,00Sodium sulphite ........................................2,50Sodium thioglycollate ...............................1,00Polysorbate 80 .........................................5,00Bromcresol purple ....................................0,02Agar ........................................................15,00Final pH 7,6 ± 0,2

DirectionsSuspend 54 g of powder in 1 L of distilled water and bring to the boil. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes. The ap-pearance of precipitates is normal and do not interferes the results.

DescriptionDey & Engley developed this medium in 1983 to recov-ery chemically damaged staphylococci, At the present its use is generalized for testing by the contact method (RODAC Plates) the efficiency of antiseptics and disin-fectants on impervious surfaces. The present formulation incorporate neutralizing substances for almost all the active products used as antiseptics and disinfectants. Lecithin neutralizes quaternary ammonium compounds (QAC’s); Polysorbate acts on phenolics and formalin; thioglycollate neutralizes the organic-mercurial com-pounds; thiosulfate-sulfite inactive halogen-compounds and lecithin + polysorbate neutralizes ethanol and other alcoholic compounds.

D/E Neutralizing Agar

TechniqueWhen the RODAC (Replicate Organisms Detection and Counting) plates are filled in the laboratory be careful with the meniscus of the agar: It should rise above the rim of the plate to give a slightly convex surface to make a proper contact with the surface to be sampled.For sampling remove the cover of the RODAC plate and carefully press the agar surface to the surface being sampled. Make certain that the entire agar meniscus contacts the surface. Replace the cover and incubate in an inverted position under the time and temperature conditions for the microorganisms in question. Express the results as “colonies per RODAC plate” or “Colonies per cm2”

ReferencesDEY, B.P. & F.B. ENGLEY (1983) Methodology for recovery of chemically treated Staphilococcus aureus with neutralizing medium. Appl. Environm. Microbiol. 453:1533-1537HICKEY, P.J., C.E. BECKELHEIMER, & T. PARROW (1992) Microbiological tests for equipment, containers, water and air. In R.T. Marshall (Ed.) Standard Methods for the examination of Dairy Products 16th ed. APHA Washington.EVANCHO, G.M., W.H. SVEUM, LL. J. MOBERG & J.F. FRANK (2001) Microbiological Monitoring of the Food Processing Environment. In DOWNES & ITO (Eds) Compendium of Methods for the Microbiological Exami-nation of Foods. 4th ed. APHA. Washington DC.ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-biological Culture Media. CRC Press. Boca Ratón, Fla.ISO 22717:2006 Standard. Cosmetics – Detection of Pseudomonas aeruginosa.ISO 22718:2006 Standard. Cosmetics – Detection of Staphylococcus aureus.

R-43S-24-37

Xi

50

Decarboxylase Lysine Broth acc. to Taylor

Ref. 02-336

SpecificationLiquid medium to differentiate enteric bacteria in the L-Lysine decarboxylation assays according to ISO 6785, 21567 and IDF 93 Standards

Formula (in g/L)Yeast extract ...........................................3,000D(+)Glucose ...........................................1,000Bromcresol purple ..................................0,016L-Lysine ..................................................5,000Final pH 6,8 ± 0,2

DirectionsDissolve 9 g of powder in 1 L of distilled water. Distribute in thin tubes in an amount of 2 or 5 mL per tube. Steri-lize in the autoclave at 121°C for 10 minutes.

DescriptionThe capacity to decarboxylate some aminoacids has been widely employed to classify Enterobacteriaceae.Taylor’s formulation, including lysine, has been recently included in several standards for the identification of Sal-monella. This modification shows an improved perform-ance, in comparison to Falkow’s formulation.

TechniqueIt is advisable to use a vaseline seal to avoid spontane-ous oxidation. The use of glucose in anaerobic condi-tions produces an acidification of the medium, the indica-tor will then turn to yellow if there is growth. The use of the aminoacid will acidify the medium again, and then it turns to grey and finally to violet, thereby showing the positive reaction. The observations of these biochemi-cal tests are performed after an incubation period of 24 hours at 37°C.

ReferencesDOWNES, F.P. & K. ITO (2001) Compendium of meth-ods for the microbiological examination of foods. APHA. Washington. DC.TAYLOR, W.I. (1961) Isolation of Salmonellae from Food Supplies. V Determination of the Method Choice for Enumeration of Salmonella. App. Microbiol. 9, 487-490.ISO 6785 Standard (2001) Milk and milk products - De-tection of Salmonella spp. FIL-IDF 93 Standard (2001) Detection of Salmonella spp.ISO 21567:2004 Food and feeding stuffs – Horizontal method for the detection of Shigella ssp.

Ref. 01-057 Deoxycholate Lactose Agar

control

Salmonella typhimurium ATCC 14028 Escherichia coli ATCC 25922

51

Deoxycholate Media

Deoxycholate Citrate Agar(Eur. Phar. Medium J)

Ref. 01-056

SpecificationDifferential and moderately selective plating medium for enteric pathogenic bacteria, according the European Pharmacopoeia.

Formula (in g/L)Peptone .................................................10,00Meat extract ............................................10,00Lactose ...................................................10,00Ferric Citrate .............................................1,00Sodium Citrate ........................................20,00Sodium deoxycholate .............................. 5,00Neutral red ...............................................0,02Agar ........................................................15,00Final pH 7,3 ± 0,2

DirectionsSuspend 71 g of powder in 1 L of distilled water and bring to the boil. Immediatelly pour into plates. Plates may be used at once or refrigerated for a few days. Do not autoclave or overheat.

Description The European Pharmacopoeia formulation is one of these modifications to rhr original medium developed by Leifson in 1935. The inhibition of gram-positive microor-ganisms is due primarily to its content of sodium deoxy-cholate, although the two citrate compounds also are active inhibitors. The lactose achieves differentiation of enteric bacilli. Organisms that ferment lactose produce acid that, in presence of neutral red indicator, results in the formation of red colonies. Lactose non-fermenting produces colourless colonies. The black centres due to the ferric sulphide depot detect the production of SH2.

TechniqueInoculate the specimen as soon as possible directly onto surface of medium. Incubate the plates at 35 ± 2ºC for 18-24 hours. Plates can be incubated for an additional 24 hours if no lactose-fermenting are observed.Typical colonial morphology on Deoxycholate Citrate Agar is as follows:Escherichia coli: Large, flat, rose-redEnterobacter / Klesiella: Large, mucoid, pale with pink

centre.Proteus: Large, colourless to tan.Salmonella: Large, colourless to tan.Shigella: Colourless to pinkPseudomonas: Irregular, colourless to brownGram-positive bacteria: No growth to slight growth

ReferencesLEIFSON, E. (1935) New culture media based on so-dium deoxycholate for the isolation of intestinal patògens and for the enumeration of colon bacilli in milk and water. J. Path.. Bact. 40:581-599.HYNES, M. (1942) The isolation of intestinal pathogens by selective media. J. Path. Bact. 54:193-207EUROPEAN PHARMACOPOEIA (2002) 4th ed. Suppl. 4.2 . § 2.6.13 Test for specified micro-organisms. Council of Europe. Strasbourg.MAC FADDIN, J.F. (1985) Media for isolation-cultivation-identification-maintenance of medical bacteria. William & Wilkins, Baltimore, MD.ATLAS, R.M. & L.C. PARKS (1991) Handbook of Micro-biological Media. CRC Press London.

Deoxycholate Lactose Agar

Ref. 01-057

SpecificacionDifferential solid medium for the isolation of enterobacte-ria according to APHA.

Formula (in g/L)Peptone ..................................................10,00Lactose ...................................................10,00 Sodium chloride ........................................5,00 Sodium citrate ..........................................2,00 Sodium deoxycholate ...............................0,50 Neutral red ................................................0,03Agar ........................................................15,00Final pH 7,1 ± 0,2

DirectionsSuspend 42,5 g of powder in 1 L of distilled water and heat to the boil. Do not autoclave and pour into sterile petri plates. The medium loses its efficiency if overheat-ed and so avoid autoclaving and remelting.

DescriptionThe Deoxycholate-Lactose Agar is very close to the Deoxycholate Agar, differing only in the deoxycholate amount and in its reduced inhibitory power. The present formulation is made according to the recommendation of APHA and AOAC.

ReferencesGREENBERG, A.E., L.S. CLESCERI & A.D. EATON (1995) Standard Methods for the examination of Water and Wastwater. 19th ed. APHA-AWWA-WEF. Washing-ton D.C.SPECK, M.L (1984) Compemdium of methods for the microbiological examination of food.2nd ed. APHA. Washington D.C. ATLAS, R.M. and L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Boca Raton, Fla.

52

Dextrose Media

Dextrose Agar

Ref. 01-089

SpecificationGeneral purpose solid culture medium.

Formula (in g/L)Meat peptone .............................................5,0Casein peptone ..........................................5,0Meat extract ................................................3,0D(+)Glucose .............................................10,0Sodium chloride ..........................................5,0Agar ..........................................................15,0Final pH 6,9 ± 0,2

DirectionsSuspend 43 g of powder in 1 L of distilled water and heat to boiling . Sterilize by autoclaving at 121°C for 15 minutes. Should the acid pH is required, add sterile tartaric acid solution when the medium is at 45°C. Do not reheat.

DescriptionThis solid culture medium is suitable for many objectives and it supports growth of most non fastidious microor-ganisms. Adding 5% sterile defibrinated blood to this me-dium, makes it an excellent culture medium which satisfy nearly all kinds of nutritive needs, even for meningococci and pneumococci, however due to its high glucose con-tent it is not suitable for hemolytic reaction studies.This formulation is also in accordance with the one suggested for the study of frozen food, and also for fruit juices. In this latter case, it is suggested to use it in duplicate, and one of the samples must be acidified in order to facilitate the growth of moulds and yeast in such a selective way.Acidification can be easily achievevd by adding 7-8 mL of 10% sterile tartaric acid to the sterile, and cooled me-dium at 45°C,producing a pH drop to 3,5 ± 0,2. In these conditions, do not remelt the medium, as then agar tends to get hydrolysed and do not solidify again.

ReferencesAPHA (1958) Recommended Methods for the Microbio-logical Examination of Food. APHA. Inc, New York.SPECK, M. L. (1984) Compendium of Methods for the Microbiological Examination of Food. 2nd ed. APHA. Washington.VANDERZANT & SPLITTSTOESSER (1992) Compen-dium of Methods for the Microbiological Examination of Food. 3rd ed. APHA. Washington.

Dextrose Broth

Ref. 02-089

SpecificationLiquid culture medium for general purposes.

Formula (in g/L)Casein peptone ........................................10,0D(+)Glucose ...............................................5,0Sodium chloride ..........................................5,0Final pH 7,2 ± 0,2

DirectionsDissolve 20 g of powder in 1 L of distilled water, heating up if necessary. Dispense into containers and sterilize by autoclaving at 121°C for 10 minutes.

DescriptionThis version of Dextrose Broth is formulated without meat extract in order to make dextrose the single carbo-hydrate in the medium. It is a liquid culture medium for the faster growth, since most microorganisms can use glucose as their energy source, but it has one draw-back that it is not buffered, and the strong acidification produced by fermentation may damage its maintenance. Hence, though it is used many times in blood cultures, it has been replaced by other, more buffered media.Due to the relatively high proportion of glucose, it is advisable to use it freshly prepared, and with short sterilization period, as otherwise toxic furfurales may be developed.On the other hand, its simple formulation makes it the best medium for checking gas production from glucose if Durham’s tubes are used, as it has no indicator that could interfere with it.

ReferencesAPHA (1958) Recommended Methods for the Microbio-logical Examination of Food. APHA. Inc, New York.DOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food.4th ed. APHA. Washington.MacFADDIN, J.D. (1985) Media for isolation-cultivation-identification-maintenance of medical bacteria. William & Wilkins Co. Baltimore

Dextrose Purple Broth

Ref. 02-416

SpecificationLiquid medium for E.coli detection in water, according to German legislation (DEV).

53

Dextrose Media

Formula (in g/L)Peptone ..................................................10,00Meat extract ..............................................3,00Sodium chloride ........................................5,00Dextrose .................................................10,00Bromocresol purple ..................................0,02Final pH 7,1 ± 0,2

DirectionsDissolve 28 g of powder in 1 L of distilled water. Dis-tribute into tubes with Durham’s tubes and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionMedium can be used for any assay of degradation of sucrose. It has been adopted by the German Federal Government for detecting E.coli in water, according to Eijkman’s test, which is based on the production of acid and gas (yellow change of indicator) from sucrose after an incubation of 20 hours at 44°C (±0,5).

ReferencesDIN Normative (Standards) 38411 (1991) Teil 6 (Juni 1991): Mikrobiologische Verfahren (Gruppe K): Nach-weis von Escherichia coli und coliformen keimen (K6).DEUTSCHE EINHEITSVERFAHREN zur Wasser-, Abwasser- Und Schlammuntersuchung. VCH Verlags-gesellschaft D-6940 Weinheim.VERORDNUNG über Trinkwasser und über wasser fur Lebensmittelbetreibe vom 12/12/1990. Bundesgesetzbl. Teil I 2613-2629 (1990)

Dextrose Tryptone PB Agar

Ref. 01-556

SpecificationSolid medium for the cultivating the “flat-sour” food spoil-ing microorganisms

Formula (in g/L)Tryptone .................................................10,00Dextrose ...................................................5,00Bromocresol Purple ..................................0,04Agar ........................................................15,00Final pH 6,9 ± 0,2

DirectionsSuspend 30 g of powder in 1 L of distilled water and bring to the boil. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

DescriptionThis medium was adopted in 1930 by the National Can-ners Association for the detection of the microorganisms causing the “flat-sour” spoilage in the canned foods.Latter it was used for the detection and enumeration of all the micro-organisms related with acid spoilage

of foods, like Bacillus coagulans (causing the typical “flat-sour”) other Bacillus and Sporolactobacillus and the thermophilic Bacillus stearothermophilus.

Technique The samples or its dilutions are inoculated in the me-dium, melted and cooled to 50ºC. Then are poured in petri dishes and incubated for 72 h at 32ºC (mesophiles) or for 48 hours at 55ºC (thermophiles). After incubation the acid-producing colonies can be easily enumerated because they show a yellow zone that contrast with the purple medium.

ReferencesNATIONAL CANNERS ASSOCIATION (1968) Labora-tory Manual for food canners and Processors. Vol. 1. NCA. WashingtonDOWNES, F.P. & K. ITO (2001) Compendium of meth-ods for the Microbiological Examination of Foods. 4th ed. APHA. Washington.HORWITZ, W. (2000) Official Methods of Analysis. AOAC International, Gaithersburg. MD.

Dextrose Tryptone PB Broth

Ref. 02-556

SpecificationLiquid medium for the microbiological examination of canned foods

Formula (in g/L)Tryptone .................................................10,00Dextrose ...................................................5,00Bromocresol Purple ..................................0,04Final pH 6,9 ± 0,2

DirectionsDissolve 15 g of powder in 1 L of distilled water, heating if necessary. Distribute in suitable containers and steri-lize in autoclave at 121ºC for 15 minutes.

DescriptionBaumgartner and Hersom proposed this medium, in 1956 for the microbial examination of canned foods and at the present it is recommended for all the medium and low acidity (pH 4,5) canned or heat-processed foods. The microbial growth is supported by the peptone and the pH indicator that turns from purple to yellow detects the acid-producers from glucose.

TechniqueVolumes of 10-20 mL of medium in duplicate are inocu-lated with 1-2 g or mL of sample to detect the aerobic microorganisms. For the anaerobic ones it is convenient inoculate other series of tubes with a more reducer me-dium like Liver Broth (Ref. 02-098). The incubation must be at 35 and 55ºC for both series.

54

Dextrose Media

ReferencesBAUMGARTNER, J.G. & A.C. HERSOM (1956) Canned Foods. 4th ed. Churchill Ltd. LondonDOWNES F.P. & K. Ito (2001) Compendium of methods for the Microbiological Examination of Foods. 4th ed. APHA. Washington. D.C.

Dichloran Glycerin Selective Agar (DG18 Agar)

Ref. 01-485

SpecificationSolid differential and low water activity medium for the determination of xerophilic fungi from low moisture food.

Formula (in g/L)Peptone .................................................5,000Dextrose ...............................................10,000Monopotassium phosphate ....................1,000Magnesium sulfate .................................0,500Dichloran ................................................0,002Chloramphenicol ....................................0,100Agar ......................................................15,000Final pH 5,6 ± 0,2

DirectionsSuspend 31,7 g of powder in 820 mL of distilled water and heat to the boiling. Add 180 mL of glycerin. Distrib-ute into suitable containers and sterilize by autoclaving at 121ºC for 15 minutes.

DescriptionAmong the culture media for xerophilic fungi, those that have played a more successful role are the ones which include any agent that restrains the continuous growth of zygomicete fungal colonies. Dichloran (Dichloreben-zalkonium cloride) and Rose bengal are two of those inhibitors. DG18 Agar medium is according to the formulation pro-posed by Hocking & Pitt in 1980, and it includes Dichlo-ran which limits the size of fungal colonies more ef-ficiently than Rose bengal. Chloramphenicol inhibits the bacterial growth very well and its thermostability allows it to be included in the medium before sterilization.

The inclusion of 18% (w/v) of Glycerine makes the medium with a water activity (aw) of 0,995 without caus-ing any problems that generally appear when this water activity is reached with sodium chloride or sugar.

TechniqueIt is advisable to inoculate in mass and thus a surface inoculation is recommended. This seed may be by surface streaking as well as swab spreading or by Dri-gaslky loop. Never use inoculum more than 0,1 mL.According to the standardized technique, plates must be incubated at 22-25ºC, with partial readings after 3 and 5 days, and a definitive readings after 7-8 days. Results are expressed in cfu xerophiles/g or mL of food sample.

ReferencesHOCKING, A.D., J.I. PITT (1980) Dichloran-glycerol me-dium for enumeration of xerophilic funghi from low-mois-ture food. Appl. Environm. Microbiol. 39:488-492PITT, J.I., A.D. HOCKING, D.R. GLENN (1983) An improved medium for the detection of Aspergillus flavus and A. parasiticus. J. Appl. Bacteriol. 54:109-114.PITT, J.I., A.D. HOCKING (1985) Fungi and Food Spoil-age. Academic Press: SydneySAMSON, R.A., E.S. HOEKSTRA (2001) Introduction to the Food Borne fungi. 6th. Ed. Centralbureau voor Schimmelcultures: Baarn.

R-45S-53-45

T

55

Differential Reinforced Clostridial Medium (DRCM)

Ref. 02-410

SpecificationLiquid medium for the enumeration of clostridia in food samples and other products by MPN technique.

Formula (in g/L)Peptone ................................................10,000Meat extract ............................................8,000Yeast extract ...........................................1,000Starch .....................................................1,000Glucose ..................................................1,000L-Cysteine HCl .......................................0,500Sodium acetate ......................................5,000Sodium bisulfite ......................................0,500Ferric-ammonium citrate .........................0,500Resazurine .............................................0,002Final pH 7,0 ± 0,2

DirectionsDissolve 27,5 g of powder in 1 L of distilled water. Bring to the boil, distribute in tubes and sterilize in the auto-clave at 121°C for 15 minutes.

DescriptionThis medium is a modification by Freame and Fitzpatrick of the Gibb’s classic medium, to easily detect the pres-ence of sulfite reducing clostridia. The modification is, mainly, an addition of sodium bisulfite and ferric citrate, that make colonies black and thus more conspicu-ously visible. The current version of this medium has no agar in order to facilitate the blackness of the medium. Resazurine,the redox indicator allows the verification

of anaerobiosis in the medium in the same assay. L-Cysteine acts as reducing agent in this medium.

TechniqueSample to be examined is distributed in tubes as per the MPN technique, and is covered with paraffin or vaseline oil to help the anaerobiosis. The bank of tubes is kept in a boiling water bath at 75°C for 30 minutes to remove all the dissolved oxygen and vegetative cells. Then, incubate at 30°C up to 7 days before concluding any negative results.Generally, the spores of sulfate reducing clostridia germinate between the second and fourth day, and the medium turns black, in which case the test is positive.The medium can be rendered selective by the addition of 70 IU/mL of Polymyxin sulftate.Prepared tubes without the inoculation may be stored up to 2 weeks provided the resazurine band does not show an excessive oxidation (more than a 1/3 part of the column).

ReferencesGIBBS, B.M., FREAME, B. (1965) Methods for the re-covery Clostridia from food. J.Appl. Bact. 36:23-33FREAME, B., FITZPATRICK, B.W.F. (1972) The use of DRCM for the isolation and enumeration of Clostridia from Food. In Isolation of Anaerobics. Ed. Shapton AND Board. Academic Press. London.DIN Standard 38411 (1991) Teil 6 (Juni 1991): Mikro-biologische Verfahren (Gruppe K): Nachweis von Es-cherichia coli und coliformen keimen (K6).

56

DNAse Agar

Ref. 01-346

SpecificationSolid culture medium for the determination of the deox-yribonuclease activity of microorganisms, especially of staphylococci and Serratia sp.

Formula (in g/L)Tryptose ..................................................20,00DNA .........................................................2,00Sodium chloride ........................................5,00Agar ........................................................15,00Final pH 7,3 ± 0,2

DirectionsSuspend 42 g of powder in 1 L of distilled water and heat to the boiling with constant stirring. Distribute into suit-able flasks and sterilize in the autoclave at 121°C for 15 minutes. Cool it to 50°C and pour it into the plates.

DescriptionJeffries, Holtman and Guse (1957) incorporated DNA into a general medium with agar to study bacterial and fungal DNAse production. Microorganisms that are able to produce DNAse, break DNA, reducing it to nucleotide fragments.This reaction is observed by the appearance of a clear zone surrounding the growth, the rest of the plate re-maining turbid. The process is as follows: Hydrochloric acid reacts with DNA producing white precipitates that make the medium turbid, although it does not react with nucleotide fragments.The same authors also observed that there is a correla-tion between coagulase production and DNase activity, thus DNAse Medium may be used as a laboratory test to diagnose pathogenic staphylococci.

Mannitol fermentation may be simultaneously deter-mined if 10 g of mannitol and 0,025 g of phenol red are added to 1 L of DNAse Agar, before sterilization. Positive results in both tests will determine with more certainty that the microorganism is a pathogenic Staphylococcus aureus.This medium is also useful to identify Serratia marces-cens in clinical specimens, since it is a DNAse producer. Davis (1967) modified the medium by adding toluidine blue and crystal violet, and stated that gramnegative DNAse producing bacilli that grew on this medium may be accepted as Serratia species.

TehniqueDNAse Agar plates are inoculated with the microorgan-ism to be studied by thick streak or inoculating at the bottom and are incubated at 35-37°C for a 18-24 hours period.To read, flood the plates with 1N chlorhydric acid and observe if there are any clear or transparent zones sur-rounding the streak. If the plate becomes totally turbid without any clear zone then the test is Negative however if any clear zone developes around the growth, then the test is accepted as Positive.

ReferencesJEFFRIES, C.D., D.F. HOLTMAN y D.G. GUSE (1957). Rapid Method for Determining the Activity of Microorgan-isms on NucleicAcids. J. Bacteriol. 73:590-591DISALVO, J.W. (1958). Desoxyrribonuclease and Coagulase Activity of Micrococci. Med. Tech. Bull. U.S. Armed Forces. Med. J. 9:191SMITH, P.B., G.A. HANCOCK & D.L. RHODEN (1969) Improved médium for detecting deoxyrribonuclease-pro-ducing bacteria. Appl. Microbiol. 18:991-993

control

Serratia marcescens ATCC 13880 Staphylococcus aureus ATCC 25923

57

EC Broth

Ref. 02-060

SpecificationSelective medium for the detection of enterobacteria, in water and foodstuff according to ISO 9308-2 and 7251 standards.

Formula (in g/L)Peptone ....................................................20,0Bile Salt n.3 ................................................1,5Lactose .......................................................5,0Dipotassium phosphate ..............................4,0Potassium dihydrogen phosphate .............. 1,5Sodium chloride ..........................................5,0Final pH 6,9 ± 0,2

DirectionsDissolve 37 g of powder in 1 L of distilled water. Distrib-ute into tubes or containers with inverted Durham tubes (for gas production). Sterilize at 121°C for 15 minutes.

DescriptionEC Broth is a buffered medium containing lactose. It is in the range of selective broths for Enterobacteriaceae. Its efficiency or selectivity is based on bile salts’ inhibitory effect on other microorganisms.This broth may be used for routine testing of water and food, either alone or by using the Most Probable Number method of enumeration.The type of sample will determine how precise the results are. If the incubation is at 35-37°C for 48 hours, gas formation may be interpreted as presumptive evi-

dence of coliform bacteria. Later confirmation will have to be done using any of the classical methods.Should the incubation take place at 44,5°C, gas forma-tion could be interpreted as a confirmation of the pres-ence of Escherichia coli. Nevertheless, it must be taken into account that the validity of this test is highly limited by technical variations. A maximum incubation time of 24 hours in a water bath with a very precise temperature regulation, is therefore recommended. When using the samples more than 10 mL, the medium must be reconstituted at a concentration equivalent to that specified on the directions, once the sample is added.

ReferencesAPHA-AWWA-WEF (1998). Standard Methods for the Examination of Water and Wastewater, 20th ed. APHA, Inc. Washington D.C., U.S.A.PERRY and HAJNA, (1943). Am. J. Pub. Hlth. 33:550.DOWNES, F.P. & K. ITO (2001) Compendium of meth-ods for the microbiological examination of foods. 4th ed. APHA. Washigton DCATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc. London.ISO Standard 9308-2 (1990) Water Quality Detection and enumeration of coliform organisms, thermotoler-ant coliform organisms and pressumptive E. coli - MPN method.ISO Standard 7251 (1993). Microbiology General Guidance for enumeration of presumptive E. coli. M.P.N. Technique.

E. coli Direct Agar (ECD Agar)

Ref. 01-484

SpecificationSolid culture medium for the detection of coliforms and E.coli in water and food.

Formula (in g/L)Tryptone .................................................20,00Yeast extract .............................................5,00Bile salts ...................................................1,50Disodium phosphate .................................5,00Monopotassium phosphate ......................1,50Sodium chloride ........................................5,00Agar ........................................................15,00Final pH 7,2 ± 0,2

DirectionsSuspend 53 g of powder in 1 L of distilled water and add 5 g/L of carbohydrate. Heat to the boiling. Distribute into suitable containers and sterilize by autoclaving at 121ºC for 15 minutes. Cool to 50ºC and add 2 flasks/L of MUG Supplement (Ref. 06-102CASE)

DescriptionThis medium is formulated according to the Swiss Stand-ards for the detection of coliforms in the food. Although the medium is complete by itself, it is recommended to add 5 g/L of glucose or lactose in order to make the colonial growth more evident.Direct detection is achieved by adding to the preparation the fluorescent agent (MUG, Ref. 06-102). After incuba-tion of the sample dilution or filter membrane, E.coli colonies show a light blue fluorescence when examined under UV light.

ReferencesANONYMOUS. Schweizerisches Lebensmittelbuch. 5th. Ed. Chap. 56A.ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press. Boca Raton. Fla.

58

EE Broth (Eur. Phar. Enrichment Broth Medium E)

Ref. 02-064

SpecificationLiquid culture medium for the enrichment of enterobacte-ria from food samples according to ISO 8523 standard.

Formula (in g/L)Gelatin peptone ....................................10,000Dextrose .................................................5,000Ox bile ..................................................20,000Di-sodium phosphate .............................6,450Monopotassium phosphate ....................2,000Brilliant green .........................................0,015Final pH 7,2 ± 0,2

DirectionsSuspend 43,5 g of powder in 1 L of distilled water and heat at 100°C for 30 min. and cool immediately.

DescriptionAs the name suggests, this medium is for the enrichment of enterobacteria, and is a modification by Mossel(1963) of the classic Brilliant Green Bile 2% Broth (Ref. 02-041). Substitution of lactose by glucose makes it more suitable for enteric bacteria detection, whether gas or non-gas-producer, in food and different samples.

Usual recommended technique is as follows: sample to be studied is added to sterile broth at a proportion of 10%. After strong homogenization, the mixture is incu-bated for a period of18-20 hours at 35-37°C. Afterwards, subcultures are performed on a solid media appropiate for the selective enterobacteria isolation. For this step, Violet Red Bile Agar (Ref. 01-164) is specially recom-mended, though there are also the MacConkey (Ref. 01-118), Deoxycholate or Brilliant green based media.From the suspected colonies on this media, identification can be performed following the common methodology.

ReferencesMOSSEL, VISSER and CORNELISSEN (1963) The ex-amination of foods for Enterobacteriaceae using a test of the type generally adopted for the detection of salmonel-lae J. Appl.Bact. 26:444-452PASCUAL ANDERSON, MªRª. (1992) Microbiología Alimentaria. Díaz de Santos, S.A. Madrid,.EUROPEAN PHARMACOPOEIA,Supplement 4.2 (2002),2.6.13 Test for specified micro-organisms 4th ed.,Council of Europe, Strasbourg.ISO 8523 Standard (1991) General guidance for the detection of Enterobacteriaceae with pre-enrichment.

Elliker Broth

Ref. 02-288

SpecificationLiquid culture medium for the enrichment of lactobacilli and streptococci in the dairy industry.

Formula (in g/L)Casein peptone ........................................20,0Yeast extract ...............................................5,0Gelatin ........................................................2,5Dextrose .....................................................5,0Lactose .......................................................5,0Sucrose ......................................................5,0Sodium chloride ..........................................4,0Sodium acetate ..........................................1,5Ascorbic acid ..............................................0,5Final pH 6,8 ± 0,2

DirectionsDissolve 48,5 g of powder in 1 L of distilled water, heat-ing up to 50°C to totally dissolve gelatin. Distribute into suitable containers, and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionMedium was formulated by Elliker, Anderson and Han-nesson to cultivate lactobacilli and streptococci from milk and other dairy products. Sodium acetate, in this me-dium at this concentration, gives two advantages: on one hand it restrains gramnegative bacteria growth, and on the other hand, it enhances lactic bacteria growth. The latter produce massive growth due to the high amount of sugars.

TechniqueReadings are performed after 3-5 days of incubation at 37°C. Should a solid medium be desired, add 15 g/L of Agar Bacteriological (Ref. 07-004).

ReferencesELLIKER, P.R., ANDERSON, A.W., HANNESSON, G. (1956) An Agar Culture Medium for Lactic Acid Strepto-cocci and Lactobacilli. J. Dairy Sci. 39:1611.HAUSLER, W.J. (1976) Standard Methods for the Examination of Dairy Products, 14 Ed. (APHA). Wash-ington, D.C.MARSHALL, R.T. (1992) Standard Methods for the Examination of Dairy Products, 16 Ed. (APHA). Wash-ington, D.C

59

Endo Media

Endo Agar Base

Ref. 01-589

SpecificationSolid selective medium for the detection of coliform and other enteric organisms, in milk and water, according to the APHA specifications.

Formula (in g/L)Peptone ....................................................10,0Lactose .....................................................10,0Sodium sulfite .............................................2,5Di-potassium hydrogen phosphate ............. 3,5Agar ..........................................................15,0Final pH 7,2 ± 0,2

DirectionsSuspend 41 g of powder in 1 L of distilled water. Bring to the boil and add 2 vials of Basic Fuchsin 250 Supple-ment (Ref. 06-607CASE). Homogenize and distribute into suitable containers. Sterilize at 121°C for 15 min-utes.Cool to 45-50°C, homogenize and pour into plates. Medium must appear slightly pinkish. If colour is very in-tense red, it can be decolourised by adding a few drops of a sterile solution of sodium sulfite 10% before pouring it into the plates. Medium must be freshly prepared for the use, and must not be used when it is red.

DescriptionEndo Agar is used to confirm the detection of and to count coliform bacteria following presumptive test of drinking water, as well as for the detection and isolation of coliforms and fecal coliforms from milk, dairy products and other food.Inoculate the plates by the streak-plate method and incubate for 24 hours at 37°C.Colonies of coliform bacilli, which ferment lactose, are pink to rose red, with or without green metallic sheen: marked reddening of the medium may occur. Colonies of other enteric bacilli, including Salmonella and of non lactose fermentors are about the same colour as the medium, being almost colourless to faint pink.On exposure to oxygen, the plated medium gradually becomes red due to the oxidation of sulfite and can thus no longer be used. It can only be kept for a few days even if it is stored in the dark and at refrigerator tem-perature.

ReferencesAPHA /AWWA/WEF, (1985). Standard Methods for the Examination of Water and Wastewater, 15th ed., Inc. Washington D.C., U.S.A.APHA (1967). Standard Methods for the Examination of Dairy Products, 12th ed. , APHA Inc. Washington D.C., U.S.A.ENDO, S (1904), Über ein Verfahren Zum Nachweis von typhusbazillen. Zbl BaKt. Hyg. Abt. I, Orig, 35:109MARSHALL, R.T. (1992) Standard Methods for the Examination of Dairy Products, 16th ed. , APHA Inc. Washington D.C., U.S.A.

Endo Dev Agar Base

Ref. 01-606

SpecificationSelective agar for the isolation and differentiation of E.coli in water, according to the german legislation.

Formula (in g/L)Meat extract ............................................10,00Peptone ..................................................10,00Lactose ...................................................10,00Sodium chloride ........................................5,00Sodium sulfite ...........................................2,50Agar ........................................................20,00Final pH 7,3 ± 0,2

DirectionsSuspend 57,5 g of powder in 1 L of distilled water and bring to the boil. Add 2 vials of Basic Fuchsin 250 Sup-plement (Ref. 06-607CASE). Homogenize and distribute into suitable containers. Sterilize in the autoclave at 121°C for 15 minutes. Cool it to 45-50°C, homogenize and pour into plates. In these conditions, medium must appear slightly pink-ish. If colour is very intense red, it can be decolourised by adding a few drops of a sterile solution of sodium sulfite 10% before pouring it into the plates. Medium must be freshly prepared for the use, and must not be used when it is red.

DescriptionMedium is a modification over the classical ENDO (Ref. 01-589), according to the German legislation, to obtain a better detection of damaged coliforms. Since the buffer system is removed in this medium, this formulation includes a more rich nutrient base and sodium chloride to restore the osmotic balance. Agar concentration has been increased to keep the strengh of gel after the water sample is incorporated.On this medium, E.coli colonies appear red, with metallic sheen, meanwhile Klebsiella and Enterobacter only take on the red colour. Colonies of other enteric bacteria are colourless.

TechniqueDEV standards recommends this medium to incubate membrane filters used in the coliform detection and in the re-seed on the surface of suspicious colonies for their confirmation and isolation. However, the agar strength allows the incorporation of the sample to be as-sayed in the medium mass, without any loss of consist-ency.For times and temperatures of incubation, it is recom-mended to follow the standard for every purpose, or fol-low the technician’s criteria. In any case, an incubation at 25-30°C for 24-48 hours usually provides good results.

60

Endo Media

ReferencesDEUTSCHE EINHEITSVERFAHREN zur Wasser-, Abwasser- Und Schlammuntersuchung. VCH Verlags-gesellschaft D-6940 Weinheim.BUNDESGESUNDHEITSAMT: Amtliche Sammlung von Untersuchungverfahren nach. 35 LMBG Beuth Verlag Berlin Köln.DIN 38411: Teil 6 (Juni 1991): Mikrobiologische Ver-fahren (Gruppe K): Nachweis von Escherichia coli und coliformen keimen (K6).

Endo LES Agar Base

Ref. 01-604

SpecificationMedium for detecting and enumerating coliforms in water and food, by the membrane filtration method.

Formula (in g/L)Peptone ..................................................15,00Yeast extract .............................................1,20Lactose .....................................................9,40Sodium chloride ........................................3,70Dipotassium phosphate ............................3,30Potassium phosphate ...............................1,00Sodium deoxycholate ...............................0,10Sodium laurylsulfate .................................0,05Sodium sulfite ...........................................1,60Agar ........................................................15,00Final pH 7,2 ± 0,2

DirectionsSuspend 50,3 g of powder in 1 L of distilled water and bring to the boil. Add 4 vials of Basic Fuchsin 200 Sup-plement (Ref. 06-617CASE). Homogenize and distribute in sterile plates. Do not autoclave. Final medium is intense red in colour.

DescriptionMedium is a modification of the classical Endo’s me-dium, reformulated to use it with the membrane filtration technique and a previous enrichment in Tryptose Lauryl Sulfate Broth (Ref. 02-108) is always helpful and recom-mended for achieving in more growths and more brilliant colonies.

TechniqueMembrane(s) that have filtered the sample are incu-bated at 35°C for 2-3 hours over a pad with Tryptose Lauryl Sulfate Broth (Ref. 02-108), and after this they are transferred to a plate with Endo LES Agar. Incubate at 35°C for 24 hours. Coliform colonies appear red with a characteristic metallic sheen.

ReferencesAPHA-AWWA-WEF (1985) Standard Methods for the Examination of Water and Wastewater. 16th. Ed. Wash-ington DC.ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press Inc.,London

Endo MF Broth Base

Ref. 02-605

SpecificationLiquid medium especially formulated for the incubation of membrane filters over the absorbent pads.

Formula (in g/L)Casein peptone ......................................10,00Meat peptone .........................................10,00Yeast extract .............................................1,50Lactose ...................................................12,50Sodium chloride ....................................... 5,00Potassium phosphate .............................. 5,75Sodium lauryl-sulfate ............................... 0,05Sodium deoxycholate .............................. 0,10Sodium sulfite .......................................... 2,10Final pH 7,2 ± 0,2

DirectionsSuspend 47g of powder in 1 L of distilled water. Heat to boiling. Add 4 vials of Basic Fuchsin 250 Supplement (Ref. 06-607CASE) and homogenize. Optimum results are obtained using the media on the same day it is pre-pared. Do not sterilize in the autoclave.

DescriptionEndo Broth for membrane filtration has the suggested composition according to “Standard Methods” for detec-tion and enumeration of coliforms by the membrane filtration method.Basically, it is the classical Endo medium, but selectivity has been improved by adding lauryl-sulfate and sodium deoxycholate and also the sodium sulfite and basic fuchsin. The mixture of peptones and yeast extract pro-vide the medium with a nutritive substrate by which most of the enteric bacteria can have the better recovery and the colonies of coliforms take on a characteristic metal-lic sheen. So,the members of Enterobacter, Citrobacter and Klebsiella types, after 20-22 hours of incubation, take a metallic green-blue sheen, but not so intense as Escherichia coli.

TechniqueEndo Broth for membrane filtration may be used directly or soaked in a pad as just by solidifying it with agar. In some cases, it seems that a better results are obtained if double-step method is used. That is, a short incuba-tion of 2-3 hours on Tryptose Lauryl Sulfate Broth (Ref.

61

Endo Media

02-108) and a later incubation of 20-22 hours over Endo Broth at 35°C (see Endo LES Agar Base, Ref. 01-604).The procedure suggested by APHA is filtration of mini-mal amounts of water (50 mL), but in any case, mem-branes with more than 400 colonies must be rejected for enumeration. In the direct method, as in the double-step method, lactose fermenting colonies show a characteris-tic sheen, and they have to be considered as presump-tive coliforms, meanwhile the lactose non fermenting colonies are colouress and transparent.

Ref. 01-068

SpecificationSelective differential medium for the isolation of coliforms from water acc. ISO 21150 Standard.

Formula (in g/L)Peptone ................................................10,000Lactose .................................................10,000Dipotassium Hydrogen phosphate ......... 2,000Yellowish Eosin ......................................0,400Methylene Blue .......................................0,065Agar ......................................................15,000Final pH 7,1 ± 0,2

DirectionsAdd 37,5 g to 1 L of distilled water. Bring to the boil and distribute in suitable containers. Sterilize in the autoclave at 121°C for 15 minutes.

DescriptionA very versatile medium originally developed for the differentiation of E. coli and Enterobacter aerogenes. It has also proved very effective in the rapid identification of Candida albicans and presents a high correlation with the coagulase test for staphylococci.

It has been repeatedly recommended for the detec-tion, enumeration and differentiation of members of the coliform bacteria.

TechniqueThe Weld method for the identification of Candida albi-cans uses this medium with Chlortetracycline (100 mg/l) in a 10% CO

2 environment. The method’s effectivity has

been tested with a variety of samples, such as sputum, oral secretions, faeces, nails and vaginal secretions, all of which provide definite results within 24-48 hours. sta-phylococci are also easily identified, particularly coagu-lase-positive strains. These have a very characteristic appearance: small colourless colonies with a central red nucleus.Nevertheless, the medium’s prevailing application is in the differentiation of E. coli and E. aerogenes. The medium should be sterilized once distributed into tubes containing 20 mL of product each, and then be refrigerated. Melt in a boiling water bath before use and stir until it acquires a dark purple colour. Pour a tube into each sterile plate and allow to solidify. It is advisable to dry the medium’s surface before use, leaving the plate open but inverted on a heater.For each doubtful lactose broth tube,inoculate one plate by streaking , and incubate for 24 to 48 hours at 37°C. Examine afterwards.

Eosin Methylene Blue Agar (EMB Agar)

ReferencesAPHA-AWWA-WEF (1995) Standard Methods for the Examination of Water and Wastewater, 19th ed. APHA Washington. D.C.FIFIELD, C.W. & C.P. SCHAUFUS (1958) Improved membrane filter medium for the detection of coliform organisms.J. Amer.Water Works Assoc. 50:193

Salmonella typhimurium ATCC 14028 Escherichia coli ATCC 25922

control

62

Eosin Methylene Blue Agar (EMB Agar)

Escherichia coli and Citrobacter form flat colonies of 2-3 mm in diameter and are dark violet in colour with a black centre which produces a distinctive green metallic glow when light is reflected on it.

Enterobacter and Klebsiella form convex colonies which are twice as big as the very smooth E. coli , have no metallic glow and are pink in colour with a dark blue centre. Non-lactose fermenting organ-isms produce colourless colonies.

Candida albicans colonies incubated in a CO2 atmos-

phere have a very peculiar cotton-like appear-ance which distinguishes them from other Candida species that produce classical yeast like colonies.

ReferencesCLESCERI, L.S., A.E. GREENBERG & A.D. EATON. (1998) Standard Methods for the Examination of Water and Wastewater. 20th edition. APHA-AWWA-WEF. Washington D. C.HOLT-HARRIS, J. E. y TEAGUE O.A. (1916) A New Cul-ture Medium for the Isolation of Bacillus typhosus from Stools J. Infect. Dis. 18:596-600.ISO 21150:2006 Cosmetics – Detection of Escherichia coli. LEVINE, M (1918) Diferentation of E. coli and A. aero-genes on simplified Eosin-ethylene Blue Agar. J. Infect. Dis. 23:43-47.MENOLASINO, N.I., GRIEVES B. Y PAYNE P. (1960) Isolation and Identification of Coagulase Positive Sta-phylococci on Levine’s Eosin-Methylene Blue Agar. J. Lab. Clin. Med. 56(6) 908-910.WELD, J. (1953) Candida albicans: Rapid Identification in Cultures made directly from Human materials Arch. Dermat. Syph. 67(5):473-478.WINDLE TAYLOR, E. (1958)The Examination of Water and Water Supplies. Churchill Ltd. 7th ed. Londres.USP 29 – NF 24 (2006) 2nd Suppl. <61> Microbial Tests. USP Con. Inc. Rockville, MD, USA

Ethyl Violet Azide Broth (EVA Broth)

Ref. 02-028

SpecificationMedium for the confirmation of enterococci in water.

Formula (in g/L)Meat Peptone .....................................10,0000Casein Peptone ..................................10,0000Dextrose ...............................................5,0000Sodium chloride ....................................5,0000Monopotasium phosphate ....................2,7000Dipotassium phosphate ........................2,7000Sodium Azide .......................................0,3000Ethyl Violet ............................................0,0005Final pH 6,8 ± 0,2

DirectionsDissolve 35,6 g of powder in 1L of distilled water, heating up slightly if necessary. Distribute in tubes or flasks and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionEVA Broth is a highly selective medium for some kinds of enterococci, and it has been adopted by many Official Organisations, National and International.Medium´s high selectivity is due to the presence of sodium azide and ethyl violet, as they inhibit other ac-companying bacteria, blocking their respiratory chains, leaving enterococci unaffected. In general, this medium is always used as a confirmation medium in the sec-ond stage, recommending an inoculum from a suitable medium such as Rothe Azide Broth (Ref. 02-027) to be inoculated in this medium.

TechniqueEach of the EVA Broth tubes is inoculated with one or two loops from a presumed positive Rothe Azide Broth (Ref. 02-027) flask, and are incubated for a 24-48 hours period at 37°C. Enterococcus presence is noted by the turbidity in the medium.Occasionally a slight turbidity may appear accompanied by ample violet sediment at the bottom of the tube.Commonly, growth confirmation in this medium is considered enough to state Enterococcus presence. However, confirmative identification must be carried out by isolation in solid media and classification in one of the four faecal enterococci species: Enterococcus faecalis, Enterococcus faecium, Enterococcus bovis and Entero-coccus equinum.

ReferencesLITSKY, MALLMAN and FIFIELD (1953) Amer.J.Publ.Hlth 43:873APHA-AWWA-WPCF (1995) Standard Methods for the Examination of Water and Wastewater. 19th. ed. APHA Washington.SPECK, APHA/Intersociety (1976). Compendium of methods for the microbiological examination of food. Washington.GUINEA, SANCHO and PARES (1979). Análisis micro-biologicos de aguas: aspectos aplicados. Ed. Omega, Barcelona.

R-22-32-52/53S-7-46-61

Xn

63

Ref. 02-532

SpecificationLiquid medium used in routine antibacterial testing of antiseptics and disinfectants.

Formula (in g/L)Meat peptone .........................................10,00Meat Extract .............................................5,00Sodium chloride ........................................5,00Final pH 6,9 ± 0,2

DirectionsDissolve 20 g of powder in 1 L distilled water, heating if necessary. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

Description This medium is produced according the formulation specified in U.S. Food ad Drug Administration (FDA), Association of Official Analytical Chemists (AOAC) and American Association of Textile Chemists and Colour-ists (AATCC) procedures for the testing of antiseptics

FDA Broth (AATCC Bacteriostasis Broth)

and disinfectants for antibacterial activities. It has the formula required by the AOAC for “Nutrient Broth” used in Phenol Coefficient and others determinations. The AATCC uses this medium in the procedures for the de-tection of antibacterial activity of fabrics and in the pro-duction of the solid medium for this type of evaluations.

ReferencesAATCC (1985) 1986 Technical Manual of the AATCC Vol. 61. Research Triangle Park. N.C.MACFADDIN J.F. (1985) Media for Isolation Cultivation-Identification-Maintenance of Medical Bacteria. William & Wilkins. BaltimoreATLAS R.M, & LC PARKS (1993) Handbook of Microbio-logical Media. CRC Press. Boca Raton Fla.HORWITZ, W. (2000) Official Methods of Analysis. 17th. Ed. AOAC International. Gaithersbourg, M.D.

Fecal Coliforms Media (FC Media)

Fecal Coliforms Agar (FC Agar)

Ref. 01-287

SpecificationSolid, selective and differential medium for coliform enu-meration by membrane filter technique.

Formula (in g/L)Tryptose ....................................................10,0Yeast extract ...............................................3,0Proteose peptone .......................................5,0Bile salts # 3 ...............................................1,5Sodium chloride ..........................................5,0Lactose .....................................................12,5Aniline blue .................................................0,1Agar ..........................................................15,0Final pH 7,4 ± 0,2

DirectionsSuspend 52,1 g of powder in 1 L of distilled water and heat to boiling. Add two vials of Rosolic Acid Selective Supplement (Ref. 06-085CASE). Mix and pour into ster-ile Petri plates. Do not autoclave nor overheat. Use freshly prepared medium.

Fecal Coliforms Broth (FC Broth)

Ref. 02-287

SpecificationLiquid, selective and differential medium for coliform enumeration by membrane filter technique.

Formula (in g/L)Tryptose ....................................................10,0Yeast extract ...............................................3,0Proteose peptone .......................................5,0Bile salts # 3 ...............................................1,5Sodium chloride ..........................................5,0Lactose .....................................................12,5Aniline blue .................................................0,1Final pH 7,4 ± 0,2

DirectionsSuspend 37,1 g of powder in 1 L of distilled water and heat to boiling. Add two vials of Rosolic Acid Selective Supplement (Ref. 06-085CASE). Do not autoclave nor overheat. Use freshly prepared medium.

64

DescriptionFC Agar and Broth are formulated according to Geldre-ich et al., to detect the faecal coliforms in polluted water. The bile salts included in these media make these media selective for enterobacteria, and also selective for coliforms due to the high temperature of incubation: 44,5°C±0,5°C.Freshly prepared medium has a red-garnet colour. Fae-cal coliform colonies are greenish-blue, and the medium also turns to this colour. In case of other bacteria, when they grow, show pinkish colonies, and then the medium turns to fluorescent red.

TechniqueEssentially, the technique consists of filtering the test sample to be examined through a membrane filter of suitable porocity (0,22-0,45 µm), assisting the filtration by pressure or suction, so that the microorganisms are retained on the membrane. Remove the membrane carefully and aseptically and take it to the culture me-dium.Put the membrane over the agar,if using the solid me-dium, or over the impregnated pad if using the liquid ver-sion. Cover the Petri plates and incubate them at 37°C for 24 hours. After incubation, proceed with the counting

of coliforms. Should a total E.coli selectivity be desired, incubate at 44,5°C.

ReferencesGELDREICH, E.E, H.F.CLARK, C.B. HUFF and L.C.BEST (1965) Fecal-Coliform-organism medium for the membrane filter technique. J. Am. Water Works As-soc, 57:208-214APHA-AWWA-WPCF (1995) Standard Method for the examination of water and wastewater. 19th ed. APHA Washington DC.

Fecal Coliforms Media (FC Media)

Ref. 03-593

SpecificationSemi-solid culture media for the motility test perform-ance.

Formula (in g/L)Gelatin ....................................................52,00Tryptose ..................................................10,00Heart extract ...........................................10,00Sodium chloride ........................................5,00Agar ..........................................................5,00Final pH 7,2 ± 0,2

DirectionsSuspend 82 g of powder in 1 L distilled water, heating until total solution. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

DescriptionIn this medium, the presence of diffuse growth away from the line or spot of inoculation evidences motil-ity. Non-motile organisms growth only along the line of inoculation. In the medium all the components supplies nutrients and the little amount of agar introduces enough strength to maintain solidity even at incubation tempera-tures, therefore, it is adaptable to use in both tubes and

GE Motility Medium

plates for motility studies, improving the original formula-tion by Jordan et al.

TechniqueAfter sterilization, cool the tubes by placing in cool water bath up to the depth of the medium (in a 16x160 mm tube 8 cm depth or 15 mL of medium). For plates, cool flasks of medium to 50ºC and pour into sterile petri dishes to a dept of 5 mm or more and allow solidifying.From an overnight culture spot the inoculum on the surface or stab just below the medium surface. If tubes are used, inoculate by depth stab inoculation. Incubation must be suitable in time and temperature to the suspect-ed organism being tested. Periodically examine tubes or plates for growth and signs of motility at last for 7 days.

ReferencesATLAS, R.M. & L.C. PARKS. (1993) Handbook of Microbiological Media. CRC Press. LondonMACFADDIN, J.F. (1985) Media for isolation-cultivation-identification-maintenance of medical bacteria. William & Wilkins, Baltimore MDD’AMATO, R.F. & K.M. TOMFOHRDE (1981) Influence of media on temperature-dependant motility test for Yersinia enterocolitica. J. Clin Microbiol. 14:347-348JORDAN, E.O. M.E CALDWELL & D. REITER (1934) Bacterial motility. J. Bacteriol. 27:165-173.

Escherichia coli ATCC 25922


Without Rosolic Acid

With Rosolic Acid

65

Giolitti-Cantoni Broth

Ref. 02-230

SpecificationLiquid medium for the recovery and enumeration of low numbers of coagulase-positive staphylococci in foods acc. to the ISO 5944:2001, IDF 60:2001 and EN-ISO 6888-3:2003 Standards

Formula (in g/L)Tryptone ...................................................10,0Meat extract ................................................5,0Yeast extract ...............................................5,0Lithium chloride ..........................................5,0D-Mannitol ................................................20,0 Sodium chloride ..........................................5,0Glycine .......................................................1,2Sodium pyruvate ........................................3,0Polysorbate 80 ...........................................1,0Final pH 6,9 ± 0,2

DirectionsDissolve 55,2 g of powder in 1 L of distilled water. The medium can be prepared at single strength or double strength using double quantity of powder Distribute into tubes dispensing 10 mL/tube (Single strength) or 20 mL/tube (double strength). Sterilize by autoclaving at 121°C for 15 minutes. Cool and add 1% Potassium Tel-lurite Sterile Solution (Ref. 06-089) using 0,1 mL/tube for single strength and 0,2 mL/tube for double strength.

DescriptionThis medium for the selective enrichment of staphyloco-cci was formulated by Giolitti and Cantoni in 1966.

The growth of staphylococci is promoted by pyruvate, glycine and above all by a high concentration of man-nitol. Addition of Polysorbate 80 is necessary for the suc-cessful recovery of Staphylococcus aureus (Chopin et al., 1985). Competitive microbiota is inhibited by lithium chloride and potassium tellurite. Anaerobic growth condi-tions increase the selectivity of the medium. Generally, growth of staphylococci can be recognized by a blacken-ing or black precipitates in the culture medium due to reduction of tellurite to metallic tellurium.

The prepared basal culture medium can be stored for about 1-2 weeks in the refrigerator. The ready-to-use medium must be used the same day of preparation. It is advisable that the stored basal medium be degasified at the moment of use by heating for 15 minutes at 100°C, cool rapidly and add sterile potassium tellurite solution.

TechniqueRefer to the standard protocol for specific products (Food and animal feeding stuffs EN-ISO 6888-3:2003; Milk and milk based products ISO 5944:2001 and FIL-IDF 60:2001). As a general technique the following is suggested:

Use food macerates or decimal dilutions and inoculate 1 mL to single strength medium. To lower the detection limit 10 mL of the test sample (liquid products) or of the

first dilution (other products) may be inoculated in double strength medium. MPN procedures need at least three tubes for at least three dilution steps. If no anaerobic jar is available, overlay with a layer of sterilized vaseline (Ref. 6-077) or vaspar. Incubate anaerobically for 24-48 h at 37°C.

After 24 hours, subculture any tubes showing blackening or black precipitate by streaking onto Baird-Parker Agar (Ref. 01-030). Incubate the remainder of the tubes for a further 24 h and subculture all tubes showing growth (irrespective of blackening) to Baird-Parker Agar.

When determining the bacterial count by the MPN method, all tubes showing growth are considered as presumptive positive for staphylococci and they are confirmed only if they produce a positive result in the coagulase test.

ReferencesCHOPIN, A. et altri (1985) ICMSF Methods Studies XV. Comparison of four media and methods for enumerat-ing Staphylococcus aureus in powdered milk. J. Food Protect. 48:21-27 EN-ISO 6888-3 Standard (2003) Microbiology of food and animal feeding stuffs. Horizontal method for the enu-meration of coagulase positive staphylococci (Staphylo-coccus aureus and other species). Part 3: Detection and MPN technique for low numbers.FIL-IDF (2001) Milk and milk based Products. Detection of coagulase-positive staphylococci. MPN technique. Standard 60:2001. Brussels. GIOLITTI, G. A. CANTONI, C (1966) A medium for the isolation of staphylococci from foodtuffs. J.Appl. Bact. 29, 395-398.HARRIGAN, WF. a. McCANCE, M.E. (1976) Labora-tory Methods in Food and Dairy Microbiology. Academic Press. London.ISO 5944 Standard (2001) Milk and milk based Prod-ucts. Detection of coagulase-positive staphylococci. MPN technique.

Left: control; right: Staphylococcus aureus ATCC 25923

66

Glucose Bromcresol Purple Agar

Ref. 01-502

SpecificationSolid medium for the confirmation of enterobacteria in diverse samples according to ISO 4702 and 8523 standards.

Formula (in g/L)Tryptone ...............................................10,000Yeast extract ...........................................1,500Dextrose ...............................................10,000Sodium chloride ......................................5,000Bromcresol purple ..................................0,015Agar ......................................................15,000Final pH 7,0 ± 0,2

DirectionsSuspend 41,5 g of powder in 1 L of distilled water and bring to the boil. Distribute into containers and sterilize by autoclaving at 121ºC for 15 minutes.

DescriptionThis medium is used to confirm enterobacteria by their ability to ferment glucose. Glucose fermentation can be detected by the production of acid that change the colour of the medium to yellow.

ReferencesISO 4702 Standard (1993) Microbiology General Guid-ance for the enumeration of entrobacteriaceae without ressucitation. MPN technique and colony count tech-nique.ISO 8523 Standard (1991) Microbiology General Guid-ance for the detection of Enterobacteriaceae with pre-enrichment.ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press. Boca Raton. Fla.

Glutamate Starch Pseudomonas Agar (GSP Agar)

Ref. 01-092

SpecificationSolid, semiselective and differential solid medium for the isolation of Pseudomonas and Aeromonas from very contaminated samples.

Formula (in g/L)Sodium L(+) glutamate ...........................10,00Soluble starch .........................................20,00Monopotassium phosphate ......................2,00Magnesium sulfate ...................................0,50Phenol red ................................................0,36Agar ........................................................15,00Final pH 7,2 ± 0,2

DirectionsSuspend 48 g of powder in 1 L of distilled water and bring to the boil. Dispense in tubes or flasks and sterilize by autoclaving at 121°C for 15 minutes. Cool to 50°C and add 100.000 I.U. of sodium G penicillin and 0,01g of pimaricine per litre. Pour into sterile plates.

DescriptionThis formulation is according to Kielwein’s modification to Korth’s medium, improving the latter because it sup-ports growth of almost all types of Pseudomonas and Aeromonas.Selectivity is achieved due to the antibiotics (penicillin and pimaricine) and glutamate, which is hard to metabo-lize by gram-negative bacteria. Differentiation between Pseudomonas and Aeromonas is based on the utiliza-

tion of starch, which is degraded by Aeromonas with acid production which causes change in colour of phenol red to yellow. Pseudomonas also grows on this medium but do not degrade starch and there is no acid produc-tion and thus their colonies remain blue-violet.

TechniqueAntibiotics are added to this medium after sterilization and cooling to 50°C, in such a way that final concentra-tions in the medium are 100 u/mL of penicillin and 10 mcg/mL of piramicine (it may be replaced by amphoter-icin or nystatin).After solidification in plates, medium may be used by surface inoculation or by leaving for the membrane filters. Aeromonas colonies turn to yellow, and Pseu-domonas ones do not.The incubation is performed at room temperature (20-25°C) for 3 days. Sometimes, enterobacteria may also grow but very slowly with pinpoint colonies.

ReferencesKORTH, H. (1963) Ein Nährboden zur Züchtung von Pseudomonaden. Zbl.Backt.Parasit. Hyg. Abt. 190:225STANIER, R., N. PALLERONI, M. DOUDOROFF. (1966) The aerobic pseudomonads: A taxonomic study. J. Gen. Microbiol. 42:159-271KIELWEIN, G. (1971) Die Isolierung und Differenzierung von Pseudomonaden aus Lebensmitteln. Arch G. Leb-ensmillelhyg. 22:29-37.

67

Gram Negative Broth (GN Broth)

Ref. 02-093

SpecificationLiquid culture medium for enteric bacteria according Hajna’s formulation.

Formula (in g/L)Peptone ....................................................20,0Dextrose .....................................................1,0D-Mannitol ..................................................2,0Sodium citrate ............................................5,0Sodium deoxycholate .................................0,5Di-potassium phosphate .............................4,0Monopotassium phosphate ........................1,5Sodium chloride ..........................................5,0Final pH 7,0 ± 0,2

DirectionsDissolve 39 g of powder in 1 L of distilled water. Dis-pense in tubes or flasks and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionGN Broth (Gram Negative Broth) is an enrichment and selective medium for enterobacteria, with a strong inhibi-tory action against gram-positive because of its high

content of citrate and deoxycholate. On the other hand, mannitol restrains the growth of Proteus and facilitates the proliferation of Salmonella and Shigella.The medium is strongly recommended for primary enrichment, 14-16 first hours, before going to selective media such as EMB (Ref. 01-068) or MacConkey (Ref. 01-118). Its author, Hajna, declares an extraordinary selectivity of the medium, whatever may the origin of the sample, if everything is kept in a transport medium upto the inoculation.

ReferencesHAJNA, A.A. (1955) A new enrichment medium for gram-negative organisms of the intestinal group Pub.Hlth.Lab 13:83EDWARDS and EWING (1973). Identification of Entero-bactericeae. Burgess Pub.Co. Minneapolis.VANDERZANT & SPLITTSTOESSER (1992). Compen-dium of Methods for the Microbiological Examination of Food. 3rd. Ed. APHA. Washington.DOWNES, F.P. & K.ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food. 4th ed. APHA. Washington.

Ref. 01-298

Specification Selective solid medium for the enumeration of molds in cosmetic products.

Formula (in g/L)Tryptone ...................................................2,50Proteose peptone .....................................2,50Yeast Extract ............................................5,00Dextrose .................................................20,00Disodium phosphate .................................3,50Monopotasium phosphate ........................3,40Ammonium chloride ..................................1,40Magnesium sulphate ................................0,06Sodium carbonate ....................................1,00Chloramphenicol ......................................0,10Agar ........................................................15,00Final pH 7,0 ± 0,2

DirectionsSuspend 54,6 g of powder in 1 L of distilled water and bring to boil. Add 20 mL of polysorbate 80 and homog-enize. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

HC Agar Base

DescriptionThe HC Agar was developed by Mead & O’Neill in 1986 to attain reliable enumeration of moulds in cosmetic products in short time. The nutrient basis of the medium s the dextrose with the peptones and yeast extract that supplies the energy, nitrogen and vitamins and growth factors. The inorganic ions are given by ammonium chlo-ride and magnesium sulphate and both phosphates acts buffering the medium. Sodium carbonate and polysorb-ate are detoxifiers and neutralising preservatives and others toxic substances. The selectivity against bacteria is due to the chloramphenicol.

TechniqueSuitable sample is inoculated into surface of me-dium plates per duplicate and incubate aerobically at 27,5±0,5ºC for 72 hours. Count colonies of moulds from duplicate plates and record average count of mould count per g or mL of sample.

ReferencesMEAD, C. & J. O’NEILL (1986) A three-day mould as-say for cosmetics and toiletries. J. Soc. Cosmet. Chem. 37:49-57

R-45S-53-45

T

68

Ref. 02-564

Specification Liquid medium for fastidious microorganisms cultivation

Formula (in g/L)Heart Extract ..........................................10,00Tryptose ..................................................10,00Sodium Chloride .......................................5,00Final pH 7,4 ± 0,2


Heart Extract Broth

DescriptionThe Heart Extract Broth is a very old and classical gen-eral purpose medium that can be used for the cultivation of fastidious microorganisms. Its components supply a very rich nutritive base which supplemented with suitable additives can be used in all laboratory purposes, from Blood Agar Base until Base for carbohydrate fermenta-tion studies.

ReferencesHUNTOON, F.M. (1918) “Hormone” Medium. A simple medium employable as a substitute for serum medium. J. of infect. Dis. 23:169-72ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press Boca Ratón Fl.MAC FADDIN, J.F. (1985) Media for Isolation-Cultiva-tion-Identification-Maintenance of Medical Bacteria. William & Wilkins. Baltimore.FDA (1998) Bacteriological Analytical Manual. 8th ed. Rev.A. AOAC International. Gaithersburg. MD.

Hektoen Enteric Agar

Ref. 01-216

SpecificationSolid, selective and differential culture medium for isola-tion of pathogenic enterobacteria from very contami-nated samples acc. ISO 21567.

Formula (in g/L)Peptone ..................................................12,00Yeast extract .............................................3,00Bile salts ...................................................9,00Lactose ...................................................12,00Sucrose ..................................................12,00Salicin .......................................................2,00Sodium chloride ........................................5,00Sodium thiosulfate ....................................5,00Ammonium ferric citrate............................1,50Acid fuchsin ..............................................0,10Bromothymol blue ....................................0,06Agar ........................................................15,00Final pH 7,7 ± 0,2

DirectionsSuspend 77 g of powder in 1 litre of distilled water and let it soak. Heat up by constant stirring until boiling. Cool to 55-60°C and pour into sterile plates. Do not autoclave. This medium is very thermolabile and thus overheating should be avoided.

DescriptionThis culture medium, originally developed by King and Metzger, has a high nutrient content like peptones, fermentable sugars and combination of indicators which makes this medium less toxic. All these characteristics and the bile salts makes it a very selective and effective medium.

TechniqueIn order to avoid the spreading of Proteus, it is neces-sary that the agar surface be perfectly dry at the moment of inoculation. Inoculation must be carried out by surface streaking, directly from rectal swabs or faeces dilutions. If colonies are well separated after 18 hours of incuba-tion, first characteristic appearances or colony charac-ters may be observed, as then those colonies become more prominent after a longer period:Shigella spp., Proteus inconstans: Raised colonies,

humid, green colour.Salmonella sp.: Green-blue colonies, with or without

black core.Pseudomonas spp.: Irregular colonies, plain, green or

brown.Companion and non pathogenic bacteria : Salmon colour

colonies.

ReferencesATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-biological Media CRC Press. BocaRaton. Fla. USAHORWITZ, W. (2000). Official Methods of Analysis of the AOAC Internacional 17th ed. Gaithersburg Md. USA DOWNES, F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Foods.4th ed. APHA. Washington DC. USA.FORBES, B.A., D.F SAHM & A.S. WEISSFELD (Eds) (1998) Bailey & Scott’s Diagnostic Microbiology 10th ed. Mosby. St Louis, Mo. USAMURRAY, P.R., E.J. BARON, J.H. JORGENSEN, M.A. PFALLER & R.H. YOLKEN (Eds) (2003) Manual of Clini-cal Mcrobiology 8th ed. ASM Press. Washington DC, USAKING S.y METZGER W. Y. (1968). A new plating method for the isolation of the enteric pathogens. Appl. Microbiol. 16:577.US FDA (1998) Bacteriological Analytical Manual 8th ed. AOAC Internacional. Gaithersburg, Md. USA.ISO 21567. Standard (2004) . Horizontal method for the detection of Shigella ssp.

69

Ref. 01-593

SpecificationSolid medium for the enumeration of heterotrophic mi-cro-organisms from water by filter membrane technique

Formula (in g/L)Peptone .................................................20,00Gelatine ..................................................25,00Agar ........................................................15,00Final pH 7,1 ± 0,2

DirectionsSuspend 60 g of powder in 1 L of distilled water and bring to the boil. Add 10 mL of glycerol and homogenize. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

DescriptionThe membrane-Heterotrophic Plate Count Agar was developed in 1979 by Taylor and Geldreich as an adap-tation to the membrane filtration method of the Standard Plate Count Medium and it is also know as m-SPC. In this medium peptone supplies all he nutrients and gelatine overcomes the trouble of liquefaction and over-growth of the colonies. The m-HCP is recommended by Standard Methods for Examination of Water and Waste-water in the filtration technique applied to the greats volumes of water with a low microbial population.

m-HPC Agar

TechniqueSamplingRefer to the Section 9060 of the Standard Methods or the ISO 5667 Standard. In any case the time between the sampling and analysis must exceed 8 hours (6 for transfer and 2 for analysis). The samples can be refriger-ated but never chilled. After 24 hours refrigeration the samples must be rejected.ProcedureA suitable volume to obtain 20-200 colonies on the 47 mm filter must be filtered. Grilled filter of 0,45 µm pore are preferred. The funnel and the filter are washed three times with 20-30 mL of sterile water. The filter is placed on the surface of a m-HPC Agar plate and incubated for 48 h. at 35±2ºC. Express the results as “Cfu/unity of volume filtered”

ReferencesTAYLOR, R.H & E.E. GELDREICH (1979) A new mem-brane filter procedure for bacterial counts in potable wa-ter and swimming pool samples. J. Amer. Water Works Assoc. 71:402-405EATON, A.D., .S. CLESCERI & A.E. GREENBERG (Eds) (1995) Standard Methods for the Examination of Water and Wastewater. 19th Ed. APHA Washington DC.

Indole Nitrite Fluid Medium

Ref. 03-101

SpecificationGeneral purpose medium for indole production tests and nitrite detection.

Formula (in g/L)Casein peptone ........................................20,0Di-sodium phosphate .................................2,0Dextrose .....................................................1,0Potassium nitrate ........................................1,0Agar ............................................................1,0Final pH 7,2 ± 0,2

DirectionsSuspend 25 g of powder in 1 L of distilled water. Heat to boiling and dispense in tubes. Sterilize at 121°C for 15 minutes. If tubes are kept in refrigerator, heat them up in boiling water bath for 2 minutes before using them.

DescriptionThe extraordinary richness of this medium, due to high quality and quantity of peptone, allows its usage as a general medium. Even the fastidious microorganisms

such as Lactobacillus or Clostridium grow well on this medium, and indole and nitrite tests can be carried out.Adding 2 g/L of Bacteriological Agar to this medium can make it suitable for motility test. Indole production can be observed with Kovacs’ Reagent (Ref. 06-018) with or without previous extraction with chloroform, and in an-other tube, nitrate assay can be carried out with Nitrate reagents (Ref. 06-003 and Ref. 06-004).This medium is not the most suitable one for indole production detection in enterobacteria, because some times there are some interferences. For that assay, SIM Medium (Ref.3-176) or Nitrate Broth (Ref. 02-138) are more suitable. Nonetheless, Indole Nitrite Broth is the medium of choice for nitrate reduction assays or denitrification assays with any other bacterial biotype especially the grampositive ones .

ReferencesVANDERZANT & SPLITTSTOESSER (1992). Compen-dium of Methods for the Microbiological Examination of Food.3rd. Ed. APHA. Washington.

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SpecificationSolid media for the verification of antimicrobial inhibi-tor substances in food and packaging material.

Assay Agar at pH 6,0

Ref. 01-437

Formula (in g/L)Casein peptone ........................................3,45Meat peptone ...........................................3,45Sodium chloride ........................................5,10Agar ........................................................15,00Final pH 6,0 ± 0,1

DirectionsSuspend 27 g of powder in 1 L of distilled water and heat to the boil. Distribute in the suitable containers and steri-lize in the autoclave at 121°C for 15 minutes. Check the pH and adjust it if necessary. Cool to 50°C and inoculate with the spore suspension of Bacillus subtilis to obtain a confluent culture. Homogenize well and pour the medium into sterile plates, (15 mL per plate). Once the agar is solidified, put the plates into the refrigerator until their use.


Ref. 01-438

Formula (in g/L)Casein peptone ........................................3,45Meat peptone ...........................................3,45Sodium chloride ........................................5,10Trisodium phosphate ................................2,40Agar ........................................................15,00Final pH 8,0 ± 0,1

DirectionsSuspend 29,4 g of powder in 1 L of distilled water and heat to the boiling. Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes. Check the pH and adjust it if necessary. Cool to 50°C. Inoculate a part of medium with the spore suspension of Bacillus subtilis to obtain a confluent culture. Inoculate the other part of medium with a cell suspension of Micrococcus luteus ATCC 9341. Homogenize well and pour the medium into sterile plates, (15 mL per plate). Once the agar is solidified, put all the plates into the refrigerator until their use.


Ref. 01-439

Formula (in g/L)Casein peptone ........................................3,60Meat peptone ...........................................3,60Sodium chloride ........................................5,00Trisodium phosphate ................................0,80Agar ........................................................15,00Final pH 7,2 ± 0,1

DirectionsSuspend 27 g of powder in 1 L of distilled water and heat to the boiling. Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes. Check the pH and adjust it if necessary. Cool to 50°C and add 50 mcg/L of Trimetoprim. Inoculate with the spore sus-pension of Bacillus subtilis to obtain a confluent culture. Homogenize well and pour the medium into sterile plates, 15 mL per plate. Once the agar is solidified, put the plates into the refrigerator until their use.All the plates must be sealed with an adhesive and water-proof tape, named, and packed in plastic bags before storing them into the refrigerator until their use. If they are stored at 3-6°C, plates may be thus stored up to 3 weeks. Do not freeze the plates, and never put them back in the refrigerator once they have reached 10°C.

TechniqueAssay may be performed with 2 or 4 plates. If two plates are used, one must be at pH 6.0 and the other one at pH 8.0, and both must be inoculated with Bacillus subtilis. If four plates are used, include another plate at pH 7,2, inoculated with Bacillus subtilis too, and another plate at pH 8,0 inoculated by Micrococcus luteus.If the sample is solid, use a punch to extract 8 mm x 2 mm diameter cylinders. If the sample is liquid, use anti-biogram assay discs. Sample pieces or discs are placed over the plates surface, 2 per plate, on all the plates. As a control, put discs alternatively with the samples in each plate, impregnated with the following standard substances:Assay Medium at pH 6,0: Penicillin 0,01 IU/discAssay Medium at pH 8,0: Streptomycin 0,5 mcg/discAssay Medium at pH 7,2: Sulfamidin 0,5 mcg/disc

Plates are incubated at 30°C for 18-24 hours, except those inoculated with M. luteus, which are incubated at 37°C.After the incubation period, read the inhibition halos (zones of inhibition), but do not consider the diameter of the discs or cylinders.Assay is considered reliable when control discs provide ø12 mm halos, and then samples with average diameter greater than ø4 mm are considered positive (with inhibi-tor presence), and samples with ø1-4 mm halos must be considered doubtful.

Inhibitory Substances Test Media

71

ReferencesBAUR, E. (1975) Untersuchungen von Fleisch und Wurstwaren mit dem Hemmstoff im Rahmen das Tierärt-zlichen Lebensmittelüberwachung. Fleischwirtschaft 55:843-845.BOGAERTS, R., F. WOLF (1980) Eine standardisierte Methode sum Nachweis von Rückständen antibakterial wirksauer substanzen in fischen Fleich. Fleischwirtschaft 60: 667-675DEUTSCHES FLEICHBESCHANGESETZ: Aus-führungsbestimmungen A über die Untersuchung und Gesundheitspolizeiliche Behandlung der Schlachtiere und des Fleisches bei Schlachtungen im Inland. Anlage 1 zu § 20 Abs. 4: Vorschriften über die Bakteriologische Fleischuntersuchung.

Inhibitory Substances Test Media

Iron Sulfite Agar

Ref. 01-328

SpecificationMedium for detection and identification of sulfite reduc-ing clostridia.

Formula (in g/L)Tryptone ...................................................10,0Sodium sulfite .............................................0,5Ferric citrate ................................................0,5Agar ..........................................................15,0Final pH 7,1 ± 0,2

DirectionsDissolve 26 g of powder in 1 L of distilled water, heating up to boiling with constantl stirring. Distribute in suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionAlthough the medium was originally described by Wilson Blair, it remained unused because it was not safe. There have been many modifications, and the one by Tanner in 1944 for the National Canners Association of America was the more lasting. However, it was also modified because it was demonstrated that clostridia were highly inhibited at concentrations of sulfite over 0,1%, this be-ing the reason why the Wilson Blair formulation was un-

suitable for clostridia. The actual formulation is according to the one widely experimented by several authors, and provides a relatively low false negative results.Most of clostridia have sulfite reductases in their cito-plasm, but they are unable to expel them to the exte-rior. So, when H

2S is produced from sulfite, the colony

becomes dark due to the formation of precipitates of iron sulfide from citrate, which is less toxica than the alum described by Wilson and Blair.Note that there are many gram negative bacteria able to reduce sulfite with iron sulfide production in this medium, but in these cases the enzymes are extracellular and all the medium becomes dark, rendering their enumeration impossible.

TechniqueTo enumerate sulfite reducing clostridia, vegetative cells have to be eliminated by heating up the sample to 80°C for 10 minutes. Sample is cooled under water and mixed aseptically with an equal volume of sterile, double concentrated, melted and cooled to 60°C medium. Let it solidify and incubate at 37°C for 48 hours, with visual ex-amination after 24 hours. Enumerate the black colonies, which clearly contrast with the medium, and express the result as spores of sulfite reducing clostridium per volume of sample.Tubes with blackish medium, without discrete colonies, must be rejected and the assay has to be restarted heat-

72

ing up the sample at the same temperature but for ad-ditional 5 minutes more than the previous heating time.Should thermophilic species are desired to be enumer-ated, incubate at 55°C.If the assay is performed by membrane filtration, it is advisable to put the membrane over a layer of medium solidified in the plate, and then cover it with another layer of medium (20 mL) melted and cooled to 60°C.

ReferencesTANNER, F.W. (1944) The Microbiology of Food. 2 Ed. Garrad Press U.S.A.BUTTON, A.W.J. (1959) A note on the enumeration of thermophilic sulfate-reducing bacteria. J. Appl. Bact., 22(2) 278-280.SCARR, M.P. (1959) Selective Media Used in the Micro-biological Examination of Sugar Products. J. Sci. Food Agri., 678-681.

Iron Sulfite Agar

Kanamycin Esculin Azide Agar(KAA Agar)

Ref. 01-263

SpecificationSolid medium for confirmative detection and isolation of Lancefield’s group D streptococci in food samples, ac-cording to Mossel et al.

Formula (in g/L)Tryptone .................................................20,00Yeast extract .............................................5,00Sodium chloride ........................................5,00Disodium citrate ........................................1,00Esculin ......................................................1,00Ferric-Ammonium citrate ..........................0,50Sodium azide ............................................0,15Kanamycin sulfate ....................................0,02Agar ........................................................15,00Final pH 7,0 ± 0,2

DirectionsSuspend 48 g of powder in 1 L of distilled water and let it soak. Heat to boiling and distribute into suitable contain-ers. Sterilize in the autoclave at 121°C for 15 minutes.

Kanamycin Esculin Azide Broth(KAA Broth)

Ref. 02-263

SpecificationLiquid medium for the presumptive detection of Lance-field’s group D streptococci in food samples, according to Mossel et al.

Formula (in g/L)Tryptone .................................................20,00Yeast extract .............................................5,00Sodium chloride ........................................5,00Disodium citrate ........................................1,00Esculin ......................................................1,00Ferric-Ammonium citrate ..........................0,50Sodium azide ............................................0,15Kanamycin sulfate ....................................0,02Final pH 7,0 ± 0,2

DirectionsDissolve 33 g of powder in 1 L of distilled water. Distrib-ute in suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

Kanamycin Esculin Azide Media (KAA Media)

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Left and center: Clostridium perfringens ATCC 13124; right: control.

73

Kanamycin Esculin Azide Media (KAA Media)

DescriptionKAA Presumptive Broth and Confirmative Agar are the two media that the Spanish National Center for Food and Nutrition (CeNAN) recommend to detect, enumerate and isolate Lancefield’s group D streptococci in samples of food and beverages like: bottled water, fresh, refriger-ated, frozen or mashed meat, fish and molluscs, soft drinks, pastries, spices and semiconserves. Kanamycin and sodium azide are the selective inhibitory com-pounds.

TechniquePrepare tubes with 9 mL of broth, and Petri plates with the agar. Make a decimal dilution bank from the sample in duplicate, and inoculate 1 mL fractions in the tubes. Incubate at 37°C for 24 hours.

Presumptive presence of streptococci is indicated by the development of a blackish-brown colour and the loss of fluorescence behind Wood’s light. These tubes are con-sidered as positive, and then inoculate 0,1 mL aliquotes from them over the surface of plates with Confirmative Agar, spreading with a Drigalsky loop (Ref. 5-010). Incubate those plates, in inverted position, at 37°C for 24 hours. Colonies that appear surrounded by a black halo are considered as group D streptoccoci, and are isolated to confirm them biochemically and morphologically with the following tests: microscopical examination, catalase assay (that should be negative) in an azideless medium, growth at 45°C and resistance to a high saline concen-tration (6,5% of NaCl in BHI Broth (Ref. 02-102). Finally, they have to grow in Bile Esculin Agar (Ref. 01-265) with a similar appearance of the colonies in the Confirmative Agar. Nonetheless, there are some exceptions to this rule, i.e. Streptococcus equinus and S.bovis do not grow

in the hypersaline broth, and therefore, definitive identifi-cation has to be performed by serological methods.

This methodology does not allow the enumeration of cells from the original sample, and as this is a neces-sary data, it is recommended to use the Most Probable Number (MPN) technique with the presumptive broth, doing it at double strength if necessary. For bottled water, soft drinks and molluscs, CeNAN suggest the fol-lowing technique:Prepare broth tubes at normal concentration and at double strength. Using a sterile pipet, inoculate five broth tubes of double strength with 10 mL of sample. Inoculate five tubes of normal concentration with 1 mL of sample and five tubes of normal concentration with 0,1 mL of sample. Homogenize them well and incubate at 37°C for 48 hours. Tubes that show a blackish-brown colour after the incubation period, are considered positive. Note down the results and carry out the counting using the MPN tables.

ReferencesGUINEA, J., SANCHO, J., PARES, R. (1979). Análisis Microbiológico de Aguas. Ed. Omega, Barcelona,.MOSSEL, D.A.A., P.G.M. BUKER, J. ELDERING (1978) Streptokokken der Lancefield Gruppe D in Lebensmitteln und Trinkwasser. Arch F. Lebensmittelhyg. 29:121-127PASCUAL ANDERSON, MªRª (1992) Microbiología Alimentaria. Diaz de Santos, S.A. Madrid.VANDERZANT & SPLITTSTOESSER (1992). Compen-dium of Methods for the Microbiological Examination of Food.3rd. Ed. APHA. Washington.

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Kenner Fecal Media (KF Media)

Kenner Fecal Agar (KF Agar)

Ref. 01-294

SpecificationSolid and selective medium for enterococci enumeration and detection.

Formula (in g/L)Proteose peptone .................................10,000Yeast extract .........................................10,000Sodium chloride ......................................5,000Sodium glycerophosphate ....................10,000Maltose .................................................20,000Lactose ...................................................1,000Sodium azide ..........................................0,400Bromocresol purple ................................0,015Agar ......................................................20,000Final pH 7,2 ± 0,2

DirectionsSuspend 76,4 g of powder in 1 L of distilled water and heat to boiling by constant stirring. If it has to be used immediately, it may not to be sterilized. Otherwise, sterilize in the autoclave in small volumes, at 121°C and for 10 minutes maximum. In both the cases, let it cool to 50°C and add 10 mL/L of TTC Sterile Solution 1% (Ref. 06-023). Homogenize well and distribute in sterile plates.Note: Non homogeneous appearance is normal, and it

does not affect the medium´s quality and efficacy.

Kenner Fecal Broth (KF Broth)

Ref. 02-294

SpecificationLiquid and selective medium for enterococci enumera-tion by the MPN or membrane filter methods.

Formula (in g/L)Proteose peptone .................................10,000Yeast extract .........................................10,000Sodium chloride ......................................5,000Sodium glycerophosphate ....................10,000Maltose .................................................20,000Lactose ...................................................1,000Sodium azide ..........................................0,400Bromocresol purple ................................0,015Final pH 7,2 ± 0,2

DirectionsSuspend 56,4 g of powder in 1 L of distilled water. If it has to be used immediately, it may not to be sterilized, just heat to boiling for one or two minutes, with constant stirring. Otherwise, sterilize in the autoclave in small volumes, at 121°C and 10 minutes maximum. In both cases, let it cool to 50°C and add 10 mL/L of TTC Sterile Solution 1% (Ref. 06-023). Homogenize well and distrib-ute in sterile tubes.Note: Non homogeneous appearance of medium is nor-

mal, and it does not affect the medium´s quality and efficacy.

DescriptionKenner, Clark and Kabler (1960,1961) discovered that KF media were excellent for detecting enterococci in pol-luted water. Carbohydrates in this medium viz. lactose and maltose, are utilised by most of enterococci, produc-ing a big amount of acid and making the indicator turn from violet to yellow. streptococci that do not belong to D group may also grow in the medium, but they do not produce enough acid to change the indicator colour. Other microorganisms, are strongly inhibited by sodium azide. enterococci reduce TTC to formazan and so their colonies are red coloured.

TechniqueMedia can be used by following several techniques depending on the sample. When a marginal contami-nation is thought, then often it is performed using the membrane filter or MPN method. On the contrary, if a high population of enterococci is suspected, it is more advisable to make a plate count.

To use the membrane filter technique, incubate at 37°C for 48 hours on the surface of KF Agar or on an absorb-ent pad impregnated in KF Broth.To use MPN technique, directly inoculate samples, of up to 1 mL to tubes with 10 mL of KF Broth in normal con-centration, and for larger samples, to tubes with 10 mL of KF Broth in double strength.If the sample is suspected of being highly contaminated, prepare a decimal dilution bank and inoculate on the surface 0,1 mL with a Drigalsky Loop (Ref. 5-010) or, if desired, inoculate by stabbing 1 mL. Anyway, incubation should be carried out at 37°C and for a 48 hours period.After the incubation, readings are performed by observ-ing the indicator colour turning from violet to yellow, and on the solid medium, colonies will be also pink or red coloured.

It is very important to maintain the pH of the medium over 7,0, or otherwise, false results may appear. Steri-lization longer than the specified period could result in caramelization and thereby a decrease in the pH.

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ReferencesKENNER, B.A., CLARK, H.F., KABLER, P.W. (1961) Fecal streptococcci I. Cultivation and Enumeration of streptococci in Surface Waters. Appl. Microbiol. 9:15.KENNER, B.A., CLARK, H.F., KABLER, P.W. (1960) Fecal streptococci. II. Quantification of streptococci in faeces. Am. I. Publ. Health, 50:1553.VANDERZANT & SPLITTSTOESSER (1992). Compen-dium of Methods for the Microbiological Examination of Food.3rd. Ed. APHA. Washington.APHA-AWWA-WEF (1998) Standard Methods for the Examination of Water an WasteWater. 20th. Ed. Wash-ington.

Kenner Fecal Media (KF Media)

King Media

King A Agar

Ref. 01-001

SpecificationSolid Medium to enhance the pyocyanine production by Pseudomonas aeruginosa acc. ISO 16266, 22717 standards.

Formula (in g/L)Peptone ....................................................20,0Magnesium Chloride ..................................1,4Potassium Sulfate ....................................10,0Agar ..........................................................15,0Final pH 7,2 ± 0,2

DirectionsSuspend 46,4 g of powder in 1 L of distilled water with glycerol 10 mL and let it soak . Heat with constant stirring until it boils. Distribute into suitable containers and steri-lize by autoclaving at 121°C for 15 minutes. If tubes are used, let them solidify with short slant and good butt.

DescriptionThis A medium was formulated by King, Ward and Raney in 1954 to enhance the pyocyanine production by Pseudomonas aeruginosa. The blue pigment Pyocyanine diffuses into the culture medium and its production varies depending on the strains of Pseudomonas aeruginosa and on the growth conditions.

Sometimes, although this medium enhances especially blue pigment production, it is possible that green (piover-dine) or brown (piomelanine) pigments also appear and mask the pyocyanine. Anyway, fluorescence and other pseudomonas pigments can be noticed on other more suitable media, like King B Agar (1-029).

TechniqueSlanted tubes or Petri dishes are inoculated by surface inoculation by streaking and are then incubated at 30-32°C for 4-5 days. Petri plates usage has the disadvan-tage of the dehydration of the medium during incubation. Therefore, it is better to use slanted tubes being careful for the aeration by loosening the screw caps or replacing them with cotton or aluminium caps.

In the recently isolated pathogenic strains from the pathological material, pigment production is often shown early i.e. after 24-48 hours of incubation, however if the material is non pathogenic or if they come from water, food or soil,then the pigmentation can be later or delayed.

When pigment has not got the usual blue colour, it is due to the production of two or more coloured substances. At this time, and if it is not confirmed on other culture medium, it is recommended to confirm by extraction: on the culture slant, 0,5-1 mL chloroform is added, and it is shaken for a few minutes until the pyocyanine is diffused, which makes the solvent blue. After that, chloroform is acidified with a few drops of HCl, obtaining a rapid change in colour from blue to red,the fact that confirms the presence of pyocyanine.

ReferencesKING E.O., M. WARD y D.E. RANEY (1954) Two simple media for the demonstration of pyocyanin and fluores-cein. J.Lab.Clin.Med. 44:301-307US Pharmacopeia (2002) 25th ed. <61> Microbial Limit Test. US Pharmacopoeial Conv. Inc. Rockville MD. ISO 16266:2006 Standard. Water Quality.– Detection and enumeration of Pseudomonas aeruginosa. Method by membrane filtrationISO 22717:2006 Standard. Cosmetics – Detection of Pseudomonas aeruginosa.

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King Media

King B Agar (F Agar)

Ref. 01-029

SpecificationCulture media for enhancing the fluorescein production by Pseudomonas species.acc. EN 12780:2002 and ISO 16266, 22717 standards.

Formula (in g/L)Meat peptone ...........................................10,0Casein peptone ........................................10,0Dipotassium phosphate ..............................1,5Magnesium sulfate .....................................1,5Agar ..........................................................15,0Final pH 7,2 ± 0,2

DirectionsSuspend 38 g of powder in 1L of distilled water with 10 mL of glycerol and let it soak . Heat to boiling and distribute in suitable containers. Sterilize in the autoclave at 121°C for 15 minutes. Cool by solidifying in slanted position with a long slant.

DescriptionF medium was formulated by King, Ward and Raney in 1954 to enhance green fluorescent pigment (pio-verdine) production by Pseudomonas fluorescens and Ps.aeruginosa, in which pyocyanine production is restricted.Green-yellowish pigments, soluble and from fluorescent type, define the Pseudomonas group I according to the 9th. edition of Bergey’s Manual of Systematic Bacteriol-ogy, and therefore, detection of its productive capacity is critical.

TechniqueSlanted tubes are inoculated with Pseudomonas strains and incubated at 30-31°C for a 2-4 days period. If after this time a green-yellowish colour does not appear on the medium, the tubes should be kept under observa-tion at room temperature for a period of 6-20 days more before the culture can be rejected as negative. It can be observed that Pseudomonas aeruginosa and Pseu-domonas putida strains coming from water, soil or food, produce pigments in a very slow way.Pioverdine is not soluble in chloroform, so the confirma-tion of presence is usually done by characteristic fluo-rescence verification under Wood’s light, comparing the dubious tube to another uninoculated F medium , which is considered as the control.

ReferencesKING, E.O., M.WARD and D.E. RANEY (1954) Two simple media for the demonstration of pyocyanin and fluorescein J.Lab.Clin.Med. 44:30-307LENNETTE, E.H., E.H. SPAULDING and J.P. TROUANT (1974) Manual of Clinical Microbiology. 2nd. Ed. ASM. Washington.US PHARMACOPOEIA (2002) <61> Microbial Limit Tests. 25th ed. US Phamacopoeial Conv. Inc. Rockville. MD.DIN Standard 38411Teil 6 (Juni 1991): Mikrobiologische Verfahren (Gruppe K): Nachweis von Escherichia coli und coliformen keimen (K6).PALLERONI, N. (1984) The genus Pseudomonas, in Bergey’s Manual of Systematic Bacteriology.EUROPEAN STANDARD EN 12780:2002. Water quality. Detection and enumeration of Pseudomonas aeruginosa by membrane filtration. CEN. Brussels.ISO 16266:2006 Standard. Water Quality.– Detection and enumeration of Pseudomonas aeruginosa. Method by membrane filtrationISO 22717:2006 Standard. Cosmetics – Detection of Pseudomonas aeruginosa.

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Kligler Iron Agar (KIA)

Ref. 01-103

SpecificationSolid and differential medium for primary identification of enterobacteria based on the fermentation of two sugars and the hydrogen sulphide production according ISO Standard 6340.

Formula (in g/L)Meat extract ..............................................3,00Yeast extract .............................................3,00Peptone ..................................................20,00Lactose ...................................................10,00Sodium chloride ........................................5,00Dextrose ...................................................1,00Ammonium iron citrate .............................0,50Sodium tiosulfate ......................................0,50Phenol red ................................................0,03Agar ........................................................15,00Final pH 7,4 ± 0,2

DirectionsAdd 58 g of powder to 1 L of distilled water and heat to the boiling. Distribute in tubes and sterilize in the auto-clave at 121°C for 15 minutes. Let it solidify with short slant and plenty of butt.

DescriptionKligler Agar is a differential medium that has all the characteristics of the 2-Sugar Russell Agar and the Lead Acetate Medium for H

2S detection. In this medium,

lactose fermentation and hydrogen sulphide production can be detected, so it allows a presumptive diagnostic of most enterobacteria. Glucose fermentation is shown by acid production, which makes the indicator turn from red to yellow, but since there is little sugar (dextrose), acid production is very limited and then a reoxidation of the indicator is produced on the surface of the medium, and the indicator remains red. Otherwise, when lactose is fermented, the large amount of acid produced avoids reoxidation and then all the medium turns to yellow.Hydrogen sulphide production is indicated by the me-dium turning black, due to the reaction of H

2S (liberated

from tiosulfate) with the Fe ions from ammonium iron citrate.

TechniqueKligler Iron agar is used in slanted tubes with short slant and plenty of butt, that are inoculated on the surface as much as in stab. Inoculum must be copious, it has to come from a solid medium, because otherwise, readings may be delayed (up to 2-3 days more). Normal incubation is 18 hours at 37°C.It is recommended to use tubes with caps that allow ventilation, like cotton caps, cellullose caps or cap-o-test. Should screw caps be used, do not tighten them because otherwise they can hinder the reoxidation of the indicator.Kligler medium provides excellent results if it is used freshly prepared, however if it has been prepared before a few days then, it is advisable to remelt it and solidify it again to obtain more precision.When H

2 S production is more, it may make the read-

ings difficult, and hence the early readings are strongly recommended. Anyway, one can obtain more precise readings if Three Sugar Iron Agar (Ref. 01-192) is used, since this one has the sucrose that allows a greater dif-ferentiation between members of Proteus, Salmonella and Shigella types.

ReferencesKLIGLER (1918) Modification of Culture Media used in the Isolation and Differentiation of Typhoid, Dyesentery and allied Bacilli. J. Exper. Med. 28:319-332KLIGLER (1917) A simple medium for the differentiation of members of typhoid-paratyphoid groups. Am. J. Pub. Hlth 7:1042-1044VANDERZANT & SPLITTSTOESSER (1992) Compen-dium of Methods for the Microbiological Examination of Food. 3rd. Ed. APHA. Washington.DOWNES, F.P. & K.ITO (2001) Compendium of Methods for the Microbiological Examination of Food. 4th. ed. APHA. Washington.

Left: control; center: Escherichia coli ATCC 25922; right: Salmonella typhimu-rium ATCC 14028.

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Kligler Iron Agar (KIA)

Typical reactions of enterobacteria on Kligler Iron Agar

79

Lactose Broth(Eur. Phar. Broth Medium D)

Ref. 02-105

SpecificationMedium for the pre-enrichment and the detection of enterobacteria and coliforms in milk and water according ISO 9308-2 and 21150 standards.

Formula (in g/L)Peptone ......................................................5,0Meat extract ................................................3,0Lactose .......................................................5,0Final pH 6.9 ± 0,2

DirectionsAdd 13 g of powder to 1 L of distilled water, or in the quantity required for the desired concentration. Dissolve it and distribute into containers fitted with Durham tubes. Sterilize by autoclaving at 121°C for 15 minutes. Avoid any further reheating.

DescriptionLactose Broth is a classical medium for use in the presumptive testing for coliforms and for the enrichment of Salmonella. This formulation is as per the standards recommended by APHA, AWWA, USP-NF and Euro-pean Pharmacopoeia.It is commonly used with Durham fermentation tubes for the examination of gas formation. If the volume of sam-ple to inoculate is important, reconstitute the medium at a concentration such, as to remain normal once the sample has been added to it.Although it is not the original formulation, this broth pro-vides excellent results in Eijkman assays of gas produc-tion at 45°C, which is a characteristic of Escherichia coli.While preparing this medium it is important to avoid overheating and to distribute it into tubes before sterili-zation.

ReferencesFDA (1998) Bacteriological Analitical Manual 8th ed. Rev A. AOAC International. Gaithersburg. Va. USA..VANDERZANT & SPLITTSTOESSER (1992) Compen-dium of Methods for the Microbiological Examination of Foods. 3rd. Ed. APHA. Washington.DOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Foods. 4th ed. APHA. Washington.EUROPEAN PHARMACOPOEIA (2005) 5th ed. §2.6.13 Test for specified micro-organisms. EDQM. Council of Europe. Strasbourg.ISO 9308-2 Standard. (1990) Water Quality – Detection and enumeration of coliform organisms, thermotolerant coliform and presumptive E. coli – MPN technique.ISO 21150:2006 Standard. Cosmetics – Detection of Escherichia coli

US PHARMACOPOEIA (2005) <61> Microbial limit test. US Pharmacopoeial Conv. Inc. Rockville. Md USAAPHA-AWWA-WPCF (1998) Standard methods for the examination of water and wastewater. 20th ed. APHA Washington

Lactose Peptone Broth

Ref. 02-414

SpecificationLiquid medium for the enrichment and enumeration of coliforms in water.

Formula (in g/L)Casein peptone ......................................17,00Soy peptone .............................................3,00Lactose ...................................................10,00Sodium chloride ........................................5,00Bromocresol purple ..................................0,02Final pH 7,2 ± 0,2

DirectionsDissolve 35 g of powder (or 70 g if double concentrated medium is desired) in 1 L of distilled water. Distribute in tubes provided with Durham’s tubes and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionThis medium is according to the German standards for quality control of water.

TechniqueGerman standards suggests the use of MPN technique with 0,1, 1 and 10 mL of sample and an incubation at 36±1°C for 44±4 hours. Tubes that change to yellow and eventual gas production/accumulation in Durham´s tubes are considered positive.

ReferencesDEUTSCHE EINHEITSVERFAHREN zur Wasser-, Abwasser- Und Schlammuntersuchung. Mikrobiologie Verfahren (Gruppe K) Nachweiss vom E. coli und colifor-men keimen.VERORDNÜNG über Trinkwasser und Wasser für Leb-ensmittelbetriebe vom 12- Dezember-1990- Bundesges-etzblatt; Teil I, 2613-2629 (1990)MURRAY, PR, EJ BARON, MA PFALLER, FC TENO-VER & RH YOLKEN (eds) (1995) Manual of Clinical Microbiology, 6th ed. ASM Washington.MaCFADDIN J.A. (1985) Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria. William& Wilkins.Baltimore.

Lactose Media

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Lactose Purple Modified Broth

Ref. 02-417

SpecificationLiquid medium for coliforms and E. coli identification.

Formula (in g/L)Meat peptone .........................................10,00Meat extract ..............................................3,00Sodium chloride ........................................5,00Lactose ...................................................10,00Bromocresol purple ..................................0,02Final pH 7,2 ± 0,2

DirectionsDissolve 28 g of powder in 1 L of distilled water. Distrib-ute in tubes provided with Durham’s tubes. Sterilize in the autoclave at 121°C for 15 minutes.

DescriptionThis medium is according to the German standards for quality control of water.

TechniqueGerman standards suggests the use of MPN technique with 0,1, 1 and 10 mL of sample and an incubation at 36±1°C for 44±4 hours. Tubes that change to yellow and eventual gas production/accumulation in Durham´s tubes are considered positive.

ReferencesDEUTSCHE EINHEITSVERFAHREN zur Wasser-, Abwasser- Und Schlammuntersuchung. VCM Verlags-gesellschaft, D-6940. Weinhem.VERORDNÜNG über Trinkwasser und Wasser für Leb-ensmittelbetriebe vom 12- Dezember-1990- Bundesges-etzblatt; Teil I, 2613-2629 (1990)

Lactose Sulfite Broth Base(Eur. Phar. Medium R)

Ref. 02-519

SpecificationLiquid medium for the determination of H

2S produc-

tion by Clostridium perfringens according to ISO 7937 standard.

Formula (in g/L)Peptone ..................................................5,000Yeast extract ...........................................2,500Sodium chloride ......................................2,500Lactose .................................................10,000L-Cysteine HCl .......................................0,300Final pH 7,1 ± 0,2

DirectionsDissolve completely 10,1 g of powder in 500 mL of distilled water and sterilize in the autoclave at 121°C for 15 minutes. Cool and aseptically add a flask sodium meta-bisulfite sterile solution (Ref. 06-114CASE) and a flask of sterile solution of ammonium ferric citrate (Ref. 06-113CASE). Mix well and distribute into sterile tubes provided with Durham´s tubes.

DescriptionThis is a simple medium that selects Cl. perfringens among other sulfite reducing clostridia by their ability to produce gas from lactose, at 46°C. It has interferences only with Cl. paraperfringens, however this microorgan-ism is very rare in food samples.

TechniqueAll of the freshly prepared media tubes (or regenereated) are inoculated in duplicate with 1 mL of sample dilution. Sample dilution must be previously kept in boiling water bath, boiling it for 10 minutes. Tubes are incubated in anaerobic conditions at 46°C for a period of 18-24 hours. Cl. perfringens presence is observed by an iron sulfide precipitate appearing in the tubes. It indicates the sulfite reducing activity. Accumulation of gas in the Durham’s tubes is a sign of lactose fermentation.

ReferencesPASCUAL ANDERSON, MªRª (1992) Microbiología Alimentaria. Diaz de Santos, S.A.,Madrid,.EUROPEAN PHARMACOPOEIA (2002) Suppl. 4.2 (2001). Chap. 2.6.13 Tests for specified microo-organ-isms 4th Ed., Council of Europe, Strasbourg.ISO 7937 Standard (2004) Microbiology of food and animal feeding stuff. Horizontal method for enumeration of Cl. perfringens. Colony-count technique.

Lactose Media

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LB Media

Under this generic name there are several formulations included which are derived from the base medium that was originally described by Luria and lately modified by different authors.This nutrient base has become very popular among culture media for the maintenance and propagation of Escherichia coli in the assays about molecular genetics. The nutrient richness and simplicity in their composi-tion allow easy and quick modifications, where the most common are the addition of different antibiotics and inhibitors as well as the variations in the sodium chloride concentration.

The current SCHARLAU formulation program covers the following formulations:

LB Agar

Ref. 01-384

SpecificationSolid medium for general purposes recommended for the molecular genetics studies with E. coli.

Formula (in g/L)Casein peptone ........................................10,0Yeast extract ...............................................5,0Agar ..........................................................15,0Final pH 7,2 ± 0,2

DirectionsSuspend 30 g of powder in 1 L of distilled water and heat to the boil with constant stirring. Distribute into suitable containers and sterilize by autoclaving at 121°C for 15 minutes.

LB Broth

Ref. 02-384

SpecificationLiquid medium for general purposes, recommended for molecular genetics studies with E. coli.

Formula (in g/L)Casein peptone ........................................10,0Yeast extract ...............................................5,0Final pH 7,2 ± 0,2

DirectionsDissolve 15 g of powder in 1 L of distilled water. Distrib-ute into suitable containers and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionFormulation of both the media, solid and liquid, are ac-cording to the Luria and Bartani base, in which sodium chloride has been omitted to help in the variation of the saline concentration with other additives.

LB Agar acc. to Lennox

Ref. 01-406


Formula (in g/L)Casein peptone ........................................10,0Yeast extract ...............................................5,0Sodium chloride ..........................................5,0Agar ..........................................................15,0Final pH 7,0 ± 0,2


LB Broth acc. to Lennox

Ref. 02-406


Formula (in g/L)Casein peptone ........................................10,0Yeast extract ...............................................5,0Sodium chloride ..........................................5,0Final pH 7,0 ± 0,2


DescriptionFormulation of both the media, solid and liquid, are according to the Luria and Bartani base modified by Lennox.

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LB Agar acc. to Miller

Ref. 01-385


Formula (in g/L)Casein peptone ........................................10,0Yeast extract ...............................................5,0Sodium chloride ........................................10,0Agar ..........................................................15,0Final pH 7,2 ± 0,2


LB Broth acc. to Miller

Ref. 02-385


Formula (in g/L)Casein peptone ........................................10,0Yeast extract ...............................................5,0Sodium chloride ........................................10,0Final pH 7,2 ± 0,2


DescriptionFormulation of both the media, solid and liquid, are ac-cording to the Luria and Bartani base modified by Miller, who has increased the sodium chloride concentration.

ReferencesAUSUBEL, F.M., R. BRENT, R.E. KINGSTON, D.D. MOORE, J.G. SEIDMAN, J.A. SMITH & K. STRUHL (1994) Current protocols in molecular biology. Greene Pub. Assoc. Inc. Brooklyn, N.Y.ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc. London.GHERNA, R., P. PIENTA, R. COTE (Eds.) 1992. ATCC Catalogue of Bacteria and Bacteriophages. Media #1065, #1226, #1226, #1235, #1236, #1315, #1364. American Type Culture Collection. Rockville MD. USA.LURIA, S.E. & J.W. BURROUS (1955) Hybridization between Escherichia coli and Shigella. J. Bacteriol. 74:461-476LENNOX, E.S. (1955) Transduction of linked genetic character of the host bacteriophage P1. Virology 1:190-206.MILLER, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y.SAMBROOK, J., E.F. FITSCH & T. MANIATIS (1989) Molecular cloning: A laboratory manual. 2nd ed. Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y.

LB Media

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Letheen Media

Letheen Agar

Ref. 01-236

SpecificationSolid medium for assays of antimicrobial action of qua-ternary ammonium compounds (QAC’s).

Formula (in g/L)Tryptone ...................................................5,00Meat extract ..............................................3,00Dextrose ...................................................1,00Lecithin .....................................................1,00Agar .......................................................15,00Final pH 7,0 ± 0,2

DirectionsSuspend 25 g of powder in 1 L of distilled water with 7 mL of Polysorbate 80 (Ref. 06-088). Let it soak and heat to boiling with constant stirring. Distribute in suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionLetheen Agar is formulated according to AOAC direc-tions, that were taken from the research work of Weber and Black for the assay of bactericidal action of quater-nary ammonium compounds (QAC’s). In fact, the medium is the classical one for the standard-ized counting with the addition of lecithin and polysorb-ate, which act as the neutralizers of the QAC’s.

Letheen Broth

Ref. 02-236

SpecificationLiquid culture medium for the determination of germicidal activity coefficients of cationic detergents.

Formula (in g/L)Peptone ....................................................10,0Meat extract ................................................5,0Lecithin .......................................................0,7Sodium chloride ..........................................5,0Final pH 7,0 ± 0,2

DirectionsDissolve 20,7g of powder in 1 L of distilled water with 5 mL of Polysorbate 80 (Ref. 06-088). Distribute in suitable containers and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionThis is the liquid version of Letheen Agar (Ref. 01-236), recommended by AOAC to verify the germicidal activity coefficients in cationic soaps, although its formulation is not the same.

Letheen Modified Agar

Ref. 01-237

SpecificationSolid medium for the primary screening of microorgan-isms in cosmetics according to the FDA

Formula (in g/L)Casein peptone ........................................10,0Meat peptone ...........................................10,0Meat extract ...............................................3,0Yeast extract ...............................................2,0Dextrose .....................................................1,0Lecithin .......................................................1,0Sodium chloride ..........................................5,0Sodium bisulphite .......................................0,1Agar ..........................................................15,0Final pH 7.0 ± 0,2

DirectionsSuspend 47 g of powder in 1 L of distilled water and add 7 mL of Polysorbate 80 (Ref. 06-088). Allow to soak and heat to the boil with constant agitation. Distribute into final containers and sterilize by autoclav-ing at 121°C for 15 minutes.

Letheen Modified Broth

Ref. 02-237

Specification FDA recommended liquid medium for the primary recov-ery of stressed microorganisms in the microbial exami-nation of cosmetics.

Formula (in g/L)Casein Peptone ........................................15,0Meat Peptone ...........................................10,0Meat extract ................................................5,0Yeast extract ...............................................2,0Sodium chloride ........................................10,0Lecithin .......................................................0,7Sodium bisulfite ..........................................0,1Final pH 7,0 ± 0,2

DirectionsDissolve 43 g of the dry powder in 1 L of distilled water with 5 mL of Polysorbate 80 (Ref. 06-088). Distribute in suitable containers and autoclave at 121°C for 15 minutes.

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DescriptionIn the early 40’s, Weber and Black recommended the use of lecithin and polysorbates to neutralize the anti-microbial action of the quaternary amonium compounds (QAC’s).In 1965 the methodology was accepted by AOAC for the antimicrobial assays and extendes their use to all the cationic tensoactives (detergents).The TAT (Tryptone Azolactin Tween) medium in the Newburger Cosmetic Analysis Manual (2nd. Ed., 1977) is closely similar in formulation and uses to the AOAC recipe. In 1978 the FDA (Bacteriological Analytical Manual, 1978) incorporated it as previous presumptive and enrichment medium for every microbial examination in cosmetics.

The SCHARLAU formulation of the Letheen Media covers the old recipe of Lucas (1977) in the Newburger Manual (Ref.1-236 Letheen Agar and Ref. 02-236 Letheen Broth) and the new recipe (Ref. 01-237 Letheen Modified Agar and Ref. 02-237 Letheen Modified Broth) of the FDA (B.A.M., 1979)

ReferencesASTM Standard E 640-78 (1991) Test method for pre-servatives in water-containing cosmetics. Philadelphia. PAATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc. London.FDA (1998) Bacteriogical Analytical Manual. 8th ed. Revision A. AOAC International. Gaithersburg. MDHORWITZ, W. (2000) Official Methods of Analysis. 17th ed. AOAC International. Gaithersburg. MDLUCAS, J.P. (1977) Microbiological Examination of Cosmetics. Newburguers’ Manual of Cosmetic Analysis. AOACWEBER, G.R. & L.A. BLACK (1948) Relative efficiency of quaternary inhibitors. Soap and Sanit Chem. 24:134-139ISO 21149:2006 Cosmetics – Enumeration and detec-tion of aerobic mesophilic bacteria.ISO 22717:2006 Standard. Cosmetics – Detection of Pseudomonas aeruginosa.ISO 22718:2006 Standard. Cosmetics – Detection of Staphylococcus aureus.

Letheen Media

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Liebermeister & Braveny Agar

Ref. 01-446

SpecificationSolid medium for the selective isolation of ß-hemolytic streptococci from throat samples.

Formula (in g/L)Meat peptone ...........................................1,00Meat extract ..............................................0,60Yeast extract .............................................0,50L(+)Lysine .................................................0,02Sodium chloride ........................................6,00Disodium phosphate .................................2,00Agar ........................................................15,00Final pH 7,2 ± 0,2

DirectionsSuspend 25 g of powder in 930 mL of distilled water and heat to the boil. Distribute into suitable containers and sterilize in an autoclave at 121°C for 15 minutes. Cool to 45°C, then add 70 mL/L of sterile defibrinated lamb’s blood. Homogenize well and pour into sterile plates.

DescriptionDespite its simplicity, this medium has a better yield in the recovery of ß-hemolytic streptococci than the com-monly used Blood Agar.Okamoto et al. and, later Bernheimer and Rodbart demonstrated the strong stimulatory effect of the nucleic acids on the hemolytic properties of streptococci. Lieber-meister and Braveny formulated a medium with insuf-ficient nutrients for the development of normal microor-ganisms but with an increased amount of nucleic acids

in the yeast extract. Moreover, they also included lysine, which has an effect on the hemolysis similar to that of the nucleic acids.The result is that microorganims fail to grow or form only small colonies that have hemolysis zones(ß-hemolysis) of normal or greater than normal size. Viridans strep-tococci (alpha-hemolytic) virtually do not grow, and if hemolysis zones form at all, they are minimal.

TechniquePlates are inoculated by surface seeding and incubated at 37°C for 24-48 hours. After the incubation small colo-nies form which are surrounded by a large, well defined hemolytic zone. Staphylococci and enterococci are almost viridans streptococci.

ReferencesOKAMOTO, H., S. KYODA, R. ITO (1939) Jap. J. Med. Sci. VI Pharmacol. 12:167.BERNHEIMER, A.W., M. RODBART (1948) The effect of nucleic acids and carbohydrates on the formation of streptolysin. J. Exp. Med. 88:149.LIEBERMEISTER, K., J. BRAVENY (1971) Ein Nährsub-strat zur Isolierung von haemolytischen Streptokokken. Z. Med. Mikrobiol. Inmunol. 156:149-153.MILATOVIC, D. (1981) Comparison of five selective media for beta-haemolytic streptococci. J. Clin. Pathol. 34:556-558

86

Lingby Iron Agar Base

Ref. 01-584

SpecificationSolid medium for the isolation and enumeration of heterotrophic H

2S producing bacteria from fish and fish

products.

Formula (in g/L)Tryptone ..................................................20,0Meat extract ................................................3,0Yeast extract ...............................................3,0Ferric citrate ................................................0,3Sodium thiosulfate ......................................0,3Sodium chloride ..........................................5,0Agar ..........................................................15,0Final pH 7,4 ± 0,2

DirectionsSuspend 46,6 g of powder in 1L of distilled water and heat to the boil. Distribute into suitable containers and sterilize by autoclaving at 121ºC for 15 minutes. Cool to 45-50ºC and aseptically add 8 mL/L of a filter sterilized solution of 5% L-Cysteine hydrochloride. Homogenize well and pour into sterile plates.

Description

Lingby Iron Agar Base has been formulated accord-ing to the Food Analysis Nordic Committee standard. This standard states that cysteine addition must be done separately in order to achieve better results in the recovery of the H

2S producing bacteria from fish and fish

products that have not been under heat treatment or with any addition of preservatives.

TechniqueFor the total count of H

2S producing bacteria, the Com-

mittee prescribes the following technique:Transfer 1 mL of sample dilution to sterile Petri plates. Add 10-12 mL of molten medium and cool to 45ºC. Ho-mogenize by shaking it gently. When cooled, add a new layer of medium and incubate at 20±1ºC for 3 days. Select the adequate plates and proceed with total count. H

2S producing bacteria form black colonies.

If incubation temperature is too high or the covering layer is too thin, black colonies loses the colour rapidly.

ReferencesNordisk Metodikkommité för Livsmedil. (1994) UDC. 637.56:576.8.08-No96. 2ntg- Bacteriological examina-tion of fresh and frozen seafood.

Listeria Enrichment Media

ListeriosisListeriosis is the generic name for the whole group of disorders caused by Listeria monocytogenes, which is the only species of the Listeria genus that is important as a human pathogen, howewer occasionally L.seeligeri, L.welshimeri and L.ivanovii have been related with human diseases. In anycase, all the species are patho-genic between the ovin and bovine cattle.Positive diagnosis of listeriosis can be obtained only by the isolation and cultivation of the responsible bacte-ria from blood or cerebrospinnal fluid samples of the affected organism. Faecal samples have a very debat-able value, since actually it is estimated that between 1-10% of human population may be an intestinal carrier of L.monocytogenes. This species of bacteria have been isolated from 37 species of mammals, domestic or wild, as well as from 17 species of birds and some species of fishes and shellfishes.Listeria monocytogenes bears a notorious tolerance to heat, cold and desiccation, and it acts as a real psi-chrotrophic. These facts and the high morbidity when it infects annimals and humans increase its interest as a food-borne pathogen.Its presence has been related with food as raw milk or supposedly pasteurized milk, soft-ripped cheese, ice creams, vegetables, raw meat, cured fermented meat, raw or smoked fish, but it has been isolated also from soil, silages and other environmental samples.

Detection and confirmation ofListeriaFood control requires to culture the samples that use to need 5-7 days before provide definitive results, and although the commercial production of DNA probes to help the diagnosis, they are not available yet.The traditional technique of low temperature (4ºC) en-richment has been substituted for selective enrichments at temperatures near to the optimum growth tempera-ture. Isolation can be also performed over selective and differential media, that most of the cases allow to disregard the special Henry lighting technique, but final identification must be carried out by biochemical and serological methods.The type of sample restrains the technique of detection because the companion flora is very important. If a pop-ulation more than 100 Listeria cells/g is suspected, the sample can be inoculated directly over the solid selective media, but in other cases, an enrichment technique, in one or two stages, must be adopted.

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Specific identificationAll the Listeria species are grampositive, oxidase- and urease-negative. They produce catalase, do not reduce nitrate and hydrolyze esculin. Provide +/+ result in the O/F test for glucose and they also acidify the bottom and surface in TSI Agar. Provide a positive result in the Voges-Proskauer and Phenol Red tests. Differential characteristics are shown in the table above.

ReferencesDONNELLY, C.W., R.E. BRACHETT, S. DOORES, W.H. LEE, J. LOVETT (1992)Listeria, in Compendium of method for the microbiologi-cal examination of foods, Vanderzant & Splittstoesser (eds) APHA. Washington.ADAMS, M.R., M. O. MOSS (1995) Food Microbiology . The Royal Society of Chemistry. Cambridge. U.K. FDA/ Center for Food Safety & Applied Nutrition. (March 1999) Foodborne Pathogenic Microorganisms and Natural Toxins Handbook: Listeria monocytogenes http://vm.cfsan.fda.gov/mow

Following you will find the Scharlau Microbiology produc-tion program in selective and differential media for the enrichment and isolation of Listeria.

Listeria Enrichment Broth Base(UVM)

Ref. 02-472

SpecificationLiquid culture medium for the enrichment of Listeria sp.

Formula (in g/L)Proteose Peptone .....................................5,00Tryptone ...................................................5,00Meat extract ..............................................5,00Yeast extract .............................................5,00Sodium chloride ......................................20,00Esculin ......................................................1,00Disodium phosphate ...............................12,00Dipotassium phosphate ............................1,35Final pH 7,4 ± 0,2

DirectionsDissolve 54,5 g of powder into 1 L of distilled water. Dis-tribute 500 mL in each container and autoclave at 121ºC for 15 minutes. Cool to 50ºC and then aseptically add the suitable selective supplement toeach 500 mL: UVM I for primary enrichment (Ref. 06-106CASE) and UVM II/Fraser for secondary enrichment (Ref. 06-111CASE).Note: Prepared medium (broth+supplement) must be

kept away from light, since it helps the produc-tion of acriflavine-oxidised photocomplexes that repress Listeria growth.

DescriptionThis basal broth for the enrichment of listeria is made ac-cording the AOAC modifications over the medium of the Vermont University (UVM), since it was demonstrated that a slight increase of acriflavine concentration in the secondary enrichment and a strong reduction in the amount of nalidixic acid in all the stages allowed more positive isolations.

TechniquePrimary enrichmentAdd 25 g or 25 mL of sample to 225 mL of primary enrichment broth (Base Broth, Ref. 02-472, and UVM I, Ref. 06-106CASE) and homogenize it all in a stomacher for 2 minutes. Incubate the mixture at 30ºC for 24 hours, but after the first 4 hours take aliquots of 0,2 mL to plates with Oxford Selective Agar (Ref. 01-471) in order to make the isolation.Secondary enrichmentAfter 24 hours of primary enrichment, inoculate the sec-ondary enrichment broth (Base Broth, Ref. 02-472, and UVM II/Fraser, (Ref. 06-111CASE) at the rate of 1:100. Incubate at 30ºC. After 4 and 24 hours take aliquots of 0,2 mL to plates with Oxford Selective Agar (Ref. 01-471) in order to make the isolation.IsolationIsolation is carried out on the Oxford Selective Agar (Ref. 01-471) plates prepared during the primary and second-ary enrichment, after 24-48 of incubation at 30-37ºC. Sometimes it is advisable to alkalinize the inoculum before the seeding, by mixing 1 mL of enrichment broth with 5 mL of 0,5% sterile KOH solution.

88

Listeria Enrichment Broth Baseacc. to Lovett

Ref. 02-498

SpecificationLiquid culture medium for the enrichment of Listeria, ac-cording Lovett et al.

Formula (in g/L)Tryptone .................................................17,00Yeast extract .............................................6,00Soy peptone .............................................3,00Sodium chloride ........................................5,00Dextrose ...................................................2,50Dipotassium phosphate ............................2,50Final pH 7,3 ± 0,2

DirectionsDissolve 36 g of powder into 1 L of distilled water and distribute 500 mL per flask. Sterilize by autoclaving at 121ºC for 15 minutes. Cool to 50ºC and aseptically add to each flask the content of one vial of Listeria Supple-ment for Selective Enrichment acc. to FDA/IDF (Ref. 06-107CASE). Homogenize and distribute into suitable containers.Note: Prepared medium (broth+supplement) must be

kept away from light, since it helps the produc-tion of acriflavine oxidised photocomplexs that repress Listeria growth.

DescriptionThis medium according to Lovett et cols. formulation has been adopted by the FDA for the analysis of food, and it is recommended by the IDF/FIL for the selective enrich-ment of Listeria in milk samples, due to its good results in the recovery of stressed bacteria, with only an enrich-ment stage.

TechniqueMix the sample (25 mL or 25 g) with 225 mL of complete enrichment broth and incubate at 30ºC for 7 days. Make subcultures after 24 hours, 48 hours and 7 days in the following way:Inoculate 0,5 mL of enrichment culture in a solid medium for the Listeria isolation (Oxford Agar Base, Ref. 01-471, or Palcam Agar Base, Ref. 01-470, with their respective selective supplements).Alkalinize 0,5 mL of enrichment culture by mixing with 4,5 mL of 0,5% sterile KOH solution and inoculate on a solid medium for Listeria isolation.

Listeria Enrichment Broth Baseacc. to Fraser

Ref. 02-496

SpecificationLiquid culture medium for the enrichment and detection of Listeria spp. according ISO standards 11290-1 and 11290-2.

Formula (in g/L)Proteose peptone .....................................5,00Tryptone ...................................................5,00Meat extract ..............................................5,00Yeast extract .............................................5,00Sodium chloride ......................................20,00Esculin ......................................................1,00Dissodium phosphate .............................12,00Monopotassium phosphate ......................1,35Lithium chloride ........................................3,00Final pH 7,2 ± 0,2

DirectionsDissolve 57,4 g of powder into 1 L of distilled water. Distribute 500 mL per flask and sterilize in the autoclave at 121ºC for 15 minutes. Cool to 50ºC. Aseptically add the Ferric Ammonium Citrate for Bacteriology (Ref. 06-112CASE) and Listeria Supplement for Secondary Enrichment UVM II/Fraser (Ref. 06-111CASE) to each flask and homogenize well.Note: Prepared medium (broth+supplement) must be

kept away from light, since it helps the production of acriflavine oxidised photocomplex that repress Listeria growth.

DescriptionThis broth base for Listeria enrichment is made accord-ing to the modifications of Fraser and Sparber over the UVM medium, which have been adopted by the USDA-FSIS. The inclusion of lithium chloride inhibits the devel-opment of enterococci which also may hydrolyze esculin in the same way of Listeria. Thus, any darkness in the medium produced by the reaction of esculetin coming from esculin hydrolysis with iron present in the medium can be taken as a presumptive presence of Listeria. Moreover, it seems the ferric citrate helps L. monocy-togenes development.

TechniqueAlthough some authors use the Fraser Broth as the only enrichment, it has been verified than better results are obtained if it is employed as secondary enrichment, ac-cording the following methodology:Inoculate the sample to examine in a primary enrichment broth (UVM I, Ref. 02-472 or Lovett Broth, Ref. 02-498) and incubate for 18-24 hours.


89

Take aliquots of 0,1 mL, inoculate them in tubes with 10 mL of Fraser Broth and incubate for 24-28 hours.Tubes that become dark are considered presumptively positive and must be subcultured over isolation and confirmation solid media, such as Oxford Agar Base (Ref. 01-471) or Palcam Agar Base (Ref. 01-470). Tubes that remain clear are considered negative and can be discarded or kept in incubation for 24 hours more to clear the doubts if any.

Palcam Agar Base

Ref. 01-470

SpecificationSolid, selective and differential medium for the detection, enumeration and isolation of Listeria spp. according ISO standards 11290-1 and 11290-2.

Formula (in g/L)Tryptone .................................................23,00Lithium chloride ......................................15,00Mannitol ..................................................10,00Sodium chloride ........................................5,00Yeast extract .............................................3,00Starch .......................................................1,00Esculin ......................................................0,80Ferric Ammonium citrate...........................0,50Dextrose ...................................................0,50Phenol red ................................................0,08Agar ........................................................13,00Final pH 7,2 ± 0,2

DirectionsSuspend 72 g of powder in 1 L of distilled water and let it soak. Heat to boil and distribute 500 mL per flask. Sterilize by autoclaving at 121ºC for 15 minutes. Cool to 50ºC and aseptically add the Palcam Agar Selective Supplement (Ref. 06-110CASE) to each flask. Mix well and pour into sterile plates.Note: Prepared medium (broth+supplement) must be


DescriptionPalcam Agar is based in the formulation described initial-ly by van Netten et cols., which has a high selectivity and a good colonial differentiation. Selectivity is achieved by the inclusion of lithium chloride, acriflavine, polymixin B and ceftacidine, since they inhibit the growth of almost all the gramnegative bacteria and most of grampositive companion bacteria.Listeria hydrolyze esculin to esculetin, which reacts with ferric ammonium citrate producing a dark precipitate colouring the colonies to green-grey with beige halos. The colonies of enterococci or staphylococci that may overpass the high selectivity of this medium can be easily recognized, since they use mannitol and produce

yellow colonies and halos, contrasting with the cherry red colour of medium.However, when there are many Listeria colonies, the entire medium turns dark, and the differentiation can be interfered. In these cases it is advisable to perform a more diluted inoculation.

TechniqueSeed the Palcam Agar with a growth from a primary enrichment broth (UVM I, Ref. 02-472 or Lovett, Ref. 02-498) or a secondary enrichment broth (UVM II, Ref. 02-472 or Fraser, Ref. 02-496). Incubate in a microaer-ophile atmosphere for 48 hours at 37ºC.In these conditions, Listeria colonies have a size approx. 2 mm diameter, green-grey coloured with black core and halo. Enterococcus and Staphylococcus colonies are bigger, grey with green-brown halo if they do not use mannitol or form yellow colonies with yellow halo if they do. Anyway, suspicious colonies must be confirmed biochemically and serologically.

Oxford Agar Base

Ref. 01-471

SpecificationSolid, selective and differential medium for the detection, enumeration and isolation of Listeria sp. according ISO standards 11290-1 and 11290-2.

Formula (in g/L)Tryptone .................................................10,00Lithium chloride ......................................15,00Proteose peptone ...................................10,00Sodium chloride ........................................5,00Yeast extract .............................................3,00Starch .......................................................1,00Esculin ......................................................1,00Ferric Ammonium citrate...........................0,50Agar ........................................................13,00Final pH 7,2 ± 0,2

DirectionsSuspend 58,5 g of powder in 1 L of distilled water and let it soak. Heat to boil and distribute 500 mL per flask. Sterilize by autoclaving at 121ºC for 15 minutes. Cool to 50ºC and aseptically add the Oxford Agar Selective Sup-plement (Ref. 06-109CASE) to each flask . Mix well and pour into sterile plates.Note: Prepared medium (broth+supplement) must be



R-22-36/38S-26-37/39-46

Xn

90

DescriptionOxford Agar is derivated from the original formulation by Curtis et al, which worked a medium with a high nutritive ability as the Columbia Agar and added inhibitor agents to remove all the undesirable companion bacteria.The current formulation keep the high capacity to sup-port growth and restrain all the gramnegative flora and most of grampositive, including yeast. Thanks to the inhibitors incorporated in the selective supplement: cycloheximide, acriflavine, colystin, phosphomicyn and ceftacidine. These inhibitors in addition to lithium chloride restrain the growth of all other bacteria except Listeria.However, selectivity is not total, but Listeria colonies are easily recognizable since as they hydrolyze esculin. Free esculetin that reacts with the ferric ions and produce a dark precipitate around the colonies, which typically present a grey-blue colour with a very dark core.

TechniqueAlthough the selectivity of the medium is enough to allow the isolation and differentiation by direct surface inocula-tion, a previous dilution of the inoculum is advisable, or even more when the sample is highly polluted.In anycase, most authors prefer one or two previous cultures in any of the primary enrichment media (UVM I, Ref. 02-472 or Lovett, Ref. 02-498) or secondary enrich-ment media (UVM II, Ref. 02-472 or Fraser, Ref. 02-496) before inoculating in Oxford Agar.Incubation is carried out at 37ºC, and after 24 hours typical colonies of Listeria monocytogenes are visible. However, it is recommended to extend incubation for more 20-24 hours in order to evidence the slow grow-ing strains even though this could allow staphylococci or streptococci development, since they would grow weakly.

ReferencesMcCLAIN, D., W.H. LEE (1988) Development of USDA-FSIS Method for isolation of Listeria monocytogenes from raw meat and poultry. JAOAC 71:3:660-664.ATLAS, R.M. (1993) Handbook of Microbiological Media. CRC Press. Boca Raton, Florida.VANDERZANT, C., D.F. SPLITTSTOESSER (1992) Compendium of methods for the microbiological exami-nation of food. APHA, Washington D.C.LOVETT, J., D.W. FRANCIS, J.M. HUNT (1988) Listeria monocytogenes in raw milk; Detection, incidence and pathogenicity. J. Food Protect. 50;188-192LOVETT,J., A.D. HITCHINS (1989) Listeria isolation. FDA Bacteriological Analytical Manual. 6th Ed. Supp. Sept. 1987 (2nd Print):29.01FRASER, J.A., W.H. SPERBER (1988) Rapid detection of Listeria sp. in food and environmental samples by esculin hydrolysis. J. Food Prot. 51:762-765.van NETTEN, P., J.PERALES, A. van de MOODSDUCK, G.D.W. CURTIS, D.A.A. MOSSEL (1989) Liquid and solid selective differential media for the detection and enumeration of Listeria monocytogenes. Int. J. Food Microbiol. 8:299-316.CURTIS, G.D., R.G. MITCHELL, A.F. KING, E.J. GRIF-FIN (1989) A selective differential medium for the isola-tion of Listeria monocytogenes. Letters Appl. Microbiol. 8:95-98ISO 11290 standard (1996) Microbiology of food and animal feeding stuff. Horizontal method for the detec-tion and enumeration of Listeria monocytogenes. Part 1 - Detection method. Part 2 - Enumeration method.


Ref. 02-496 Listeria Enrichment Broth acc. Fraser. Left: control; right: positive.

91

Lysine Media

Lysine Iron Agar (LIA)

Ref. 01-094

SpecificationDifferential medium for Enterobacteria, recommended by Edwars and Ewing for Salmonella identification.

Formula (in g/L)Gelatin peptone ........................................5,00Yeast extract .............................................3,00Dextrose ...................................................1,00L-Lysine ..................................................10,00Ammonium Ferric citrate ..........................0,50Sodium thiosulfate ....................................0,04Bromocresol purple ..................................0,02Agar ........................................................15,00Final pH 6,7 ± 0,2

DirectionsSuspend 34,5 g of powder in 1 L of distilled water and heat to boiling . Dispense in tubes and sterilize by autoclaving at 121°C for 15 minutes. Allow to solidify in slanted position, with copious depth and short slant.

DescriptionLysine and Iron medium has been widely used for the differentiation among different biotypes of Salmonella, above all those corresponding to S. arizona, which, on usual selective isolation in plate media, like MacConkey or Deoxycholate, may give colour or colourless colonies due to the fact that their lactose fermentative capacity is quite variable.If it is considered that these microorganisms, when cultured in tubes with Kligler Iron Agar (Ref. 01-103) or Triple Sugar Iron (Ref. 01-192), produce enough acid to avoid sulfide formation, it is comprehensible that they are sometimes not recognized or overlooked as pathogen, and wrong or negative results are consequently obtained. On the other hand, Salmo-nella is the only genus of enterobacteria that normally decarboxylates lysine and produces important amounts of hydrogen sulfide.LIA works perfectly verifying these two characteristics, and that is the reason why it is used at the same time of use of Kligler Iron Agar (Ref. 01-103) and/or TSI (Ref. 01-192) in the second phase of isolation of pathogenic enterobacteria.

TechniqueFrom some suspicious colonies in the isolation media, and with a needle, inoculate a Kligler’s medium tube, and without recharging the inoculation loop, pass it to LIA by surface streaking and depth stab. Incubate them closed, but at the same time sufficiently ventilated, at 35-37°C for 24 hours.Microorganisms that decarboxylate the lysine, quickly, produce a strong alkalinization in all the medium, that is observed by the indicator turning to purple. Whereas, those that have no lysine decarboxylase activity, acidify the medium at the bottom producing a yellow coloura-tion, meanwhile the surface of the medium remains with the same original colour or with alkaline reaction.Proteus type members are distinguished easily, since, over the yellow bottom, they produce a typical red or orange colour on the surface, due to oxidative deamina-tion of lysine. The microorganisms which produces of hydrogen sulfide blacken the medium because of iron sulfur precipitates.Although the gas production may be observed, generally this medium does not offer optimal conditions for this, and gives very irregular results, even giving total inhibi-tion in some cases.

ReferencesEDWARDS, P.R. and M.A. FIFE (1961) Lysine-Iron Agar in the detection of Arizona cultures. Appl. Microbiol. 99, 478-480.EWING, J. (1982) Edwards and Ewing’s identification of Enterobacteriaceae. 4th. Ed. Elsevier Sci. Pub. Co. Inc. N.Y.McFADDIN, J.F. (1985) Media for the isolation, cultiva-tion, identification and maintenance of medical bacteria. William & Wilkins. BaltimoreATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press,LondonDOWNES, F.P. & K. ITO (2001)) Compendium of Meth-ods for the Microbiological Examination of Food. 4th ed APHA. WashingtonHORWITZ, W. (2000) Official Methods of Analysis. 17th ed. AOAC International. Gaitherburg. MD.MARSHALL R.T. (1992) Methods for the examination of dairy products. APHA. Washington.

92

Lysine Media

93

M-17 Media

M-17 Agar

Ref. 01-245

SpecificationSolid selective medium for Streptococcus thermophilus in the common examination of yoghurt acc. to ISO 7889: 2003 and IDF 117: 2003.

Formula (in g/L)Tryptone ...................................................2,50Meat peptone ...........................................2,50Soya peptone ...........................................5,00Yeast extract .............................................2,50Meat extract ..............................................5,00Sodium glycerophosphate ......................19,00Magnesium sulfate ...................................0,25Ascorbic acid ............................................0,50Lactose .....................................................5,00Agar .......................................................15,00Final pH 7,0 ± 0,2

DirectionsSuspend 57 g of powder in 1 L of distilled water and let it soak. Heat to boiling and dispense into suitable contain-ers. Sterilize by autoclaving at 121°C for 15 minutes. Avoid unnecessary overheating and remelting.

M-17 Broth

Ref. 02-245

SpecificationLiquid selective medium for Streptococcus thermophilus in the common examination of yoghurt.

Formula (in g/L)Tryptone ...................................................2,50Meat peptone ...........................................2,50Soya peptone ...........................................5,00Yeast extract .............................................2,50Meat extract ..............................................5,00Sodium glycerophosphate ......................19,00Magnesium sulfate ...................................0,25Ascorbic acid ............................................0,50Lactose .....................................................5,00Final pH 7,0 ± 0,2

DirectionsSuspend 42 g of powder in 1 L of distilled water and let it soak. Heat if necessary and dispense into suitable con-tainers. Sterilize by autoclaving at 121°C for 15 minutes.

M-17 w/o Lactose Broth

Ref. 02-580

SpecificationLiquid selective medium for Streptococcus thermophilus in the examination of yoghurt and other dairy products.

Formula (in g/L)Tryptone ...................................................2,50Meat peptone ...........................................2,50 Soya peptone ...........................................5,00Yeast extract .............................................2,50Meat extract ..............................................5,00Sodium glycerophosphate ......................19,00Magnesium sulfate ...................................0,25Ascorbic acid ............................................0,50Final pH 7,0 ± 0,2

DirectionsDisolve 37 g of powder in 1 L of distilled water, heating if necessary. Dispense into suitable containers. Sterilize by autoclaving at 121°C for 15 minutes.

DescriptionM-17 Agar was developed by Terzaghi and Sandine for the screening of bacteriophages in streptococci of dairy industry. Afterward, Shankar and Davies demonstrated the efficacy of this medium for selective isolation of Streptococcus thermophilus in yoghurt. Medium com-bines a strong buffer, which aids development of strep-tococci, with a high concentration of glycerophosphate, which inhibits the growth of lactobacilli.

TechniqueSuggested technique to enumerate streptococci is to seed in mass or by stabbing, with agar melted and cooled to 50-55°C, and then to incubate them at 42°C for a 24 hours period. With these conditions, all the colo-nies might be streptococci. Longer incubation periods or lower temperatures may cause morphological changes in the colonies which hinders in the the recognition of the colonies.FIL-IDF has adopted this medium for yoghurt examina-tion, and uses it simultaneously with MRS Agar (Ref. 01-135).Lactobacilli count is performed analogously in MRS Me-dium, at 30°C and in CO

2 enriched atmosphere.

Colonies of lactose positive streptococci are visible after 15 hours, and after 5 days they may reach a diameter of about 3-4 mm, whereas those lactose negatives are 1 mm of diameter. However, longer incubation period may hinder the observations, due to the growth of lactobacilli.Bacteriophages presence is observed by the appear-ance of characteristic plaques over the bacterial growth.

94

ReferencesTERZAGHI, B.E. and SANDINE, W.E. (1975) Improved medium for lactic streptococcacal phages from cheese factories. Appl. Environm. Microbiol 29:807-813SHANKAR, P.A. and DAVIES, F.L. (1977) Selective Technique for yogurt Bacteria Enumeration. J. Soc. Dairy Technol. 30:28.FIL-IDF Standard 146A (1998) Identification of charac-teristic micro-organisma of yoghurt.ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press,LondonDOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food. 4th ed.APHA. Washington.ISO 7889 standard (2003). Yogurt - Enumeration of characteristic microorganisms - Colony- count technique at 37°C.

M-17 Media

95

MacConkey Media

In the production program of SCHARLAU you can find the following formulations of the MacConkey media:

MacConkey Agar ....................................... Ref. 01-118(Eur. Phar. Medium H)MacConkey Broth ...................................... Ref. 02-118MacConkey Modified Broth ...................... Ref. 02-120MacConkey Sorbitol Agar .......................... Ref. 01-541MacConkey w/o Salt Agar ......................... Ref. 01-320MacConkey WHO Broth ........................... Ref. 01-121Mac Conkey G Broth ................................ Ref. 02-611 (Eur Pharm Medium G)

MacConkey Agar (Eur. Phar. Medium H)

Ref. 01-118

SpecificationA selective and differential medium for the detection, isolation and enumeration of coliforms from a variety of samples according European Pharmacopoeia and ISO standard.

Formula (in g/L)Peptone ................................................20,000Lactose .................................................10,000Bile Salts #3 ...........................................1,500Sodium chloride ......................................5,000Neutral red ..............................................0,030Crystal violet ...........................................0,001Agar ......................................................15,000Final pH 7,2 ± 0,2

DirectionsSuspend 51,5 g of powder to 1 L of distilled water. Bring to the boil and sterilize by autoclaving at 121°C for 15 minutes.

MacConkey Broth

Ref. 02-118

SpecificationLiquid medium for the detection and enumeration of coliforms by MPN technique.

Formula (in g/L)Peptone ................................................20,000Lactose .................................................10,000Bile Salts ................................................5,000Sodium chloride ......................................5,000Neutral Red ............................................0,075Final pH 7,4 ± 0,2

DirectionsDissolve 40 g of powder in 1 L of distilled water. Bring to the boil and distribute into suitable containers fitted with Durham tubes. Sterilize by autoclaving at 121°C for 15 minutes.

MacConkey G Broth (Eur. Phar. Medium G)

Ref. 02-611

Specification Liquid medium for the detection and enumeration of coliforms by MPN technique. It is a modification of the classic medium, where neutral red is replaced by a less aggressive indicator, according European Pharmaco-poeia.

Formula (in g/L)Peptone ..................................................20,00Lactose ...................................................10,00Bile Salts ..................................................5,00 Bromocresol purple ..................................0,01Final pH 7,4 ± 0,2

DirectionsDissolve 35 g of powder in 1 L of distilled water. Heat only if necessary to help the dissolution. Distribute into suitable containers fitted with Durham tubes. Sterilize by autoclaving at 121°C for 15 minutes.

MacConkey Modified Broth

Ref. 02-120

Specification Liquid medium for the detection and enumeration of coliforms by MPN technique according ISO standard.

Formula (in g/L)Peptone ..................................................20,00Lactose ...................................................10,00Bile Salts ..................................................5,00Sodium chloride ........................................5,00Bromocresol purple ..................................0,01Final pH 7,4 ± 0,2

DirectionsDissolve 40 g of powder in 1 L of distilled water. Heat only if necessary to help the dissolution. Distribute into suitable containers fitted with Durham tubes. Sterilize by autoclaving at 121°C for 15 minutes.

96

MacConkey Sorbitol Agar

Ref. 01-541

SpecificationSelective and differential solid medium for the detection of Enterohaemorrhagic Escherichia coli (EHEC O157:H7) according ISO standard.

Formula (in g/L)Peptone ................................................20,000Sorbitol .................................................10,000Bile salts .................................................1,500Sodium chloride ......................................5,000Neutral Red ............................................0,030Crystal violet ...........................................0,001Agar ......................................................15,000Final pH 7,1 ± 0,2

DirectionsSuspend 51,5 g of powder into 1 L of distilled water and heat to boiling. Sterilize in the autoclave at 121ºC for 15 minutes. If the medium is used the same day of prepara-tion autoclaving is not necessary but the boiling must be for 3 minutes at least.

DescriptionThe substitution of lactose by sorbitol for the isolation of the enteropathogenic serotypes O111 y O55 of Es-cherichia coli was proposed in 1952 by Rappaport and Hening. The usefulness of the medium was showed by March and Ratman (1986) and Adas (1991) for the de-tection, differentiation and isolation of the enterohaemor-rhagic (EHEC) and the verocytoxin-producing (VTEC) strains of the serotype O17:H7 of E. coli.The only modification on the typical MacConkey Media formulations is the replacement of lactose with sorbitol. The enterohaemorrhagic strains do not use this sub-strate and produce colourless colonies, but the other serotypes can ferment sorbitol and produce red coloniesIn all others aspects, MacConkey Agar with Sorbitol works similarly as the other media in the MacConkey group. Peptone supply the nitrogen and sodium chloride the osmotic environment. Crystal violet and bile salts inhibits the growth of grampositive bacteria. Neutral red acts as the pH indicator.

TechniqueSpread the inoculum on the dry surface of the medium and incubate at 35±2ºC for 24 hours. Usually, the O157:H7 serotype forms colourless colonies and the other strains of E. coli red colonies. The results must be recorded at 24 hours because an extended incubation produce a decreasing colouring in the sorbitol-ferment-

ing colonies and some times there is some colony of the o157:H7 serotype that begins to ferment sorbitol.Some gramnegative bacteria like Pseudomonas, Pro-teus and Klebsiella can growth on the MacConkey Agar with Sorbitol but their colonies are diverse and easy to differentiate from E. coli. Because the failure in the fermentation of sorbitol by some strains on E. coli no-enterotoxigenic and the atypi-cal colony production by some enterohaemorrhagic ones it is recommended the use of some others media in con-comitance with the MacConkey Agar with Sorbitol and to confirm the suspect colonies by serological, biochemical or molecular techniques.

MacConkey w/o Salt Agar

Ref. 01-320

SpecificationA selective and differential medium for the detection, isolation and enumeration of enterobacteria, especially Proteus sp. from the clinical specimens.

Formula (in g/L)Peptone ................................................20,000Lactose .................................................10,000Bile Salts #3 ...........................................1,500Neutral red ..............................................0,075Agar ......................................................15,000Final pH 7,4 ± 0,2

DirectionsAdd 46,5 g of powder to 1 L of distilled water. Bring to the boil and distribute in suitable containers. Esterilize by autoclaving at 121°C for 15 minutes.

MacConkey WHO Agar

Ref. 01-121

SpecificationDifferential medium with moderate selectivity, without crystal violet, according to WHO recommendation, for the isolation of enterobacteria.

Formula (in g/L)Peptone ................................................20,000Lactose .................................................10,000Bile Salts mixture ....................................2,500Sodium chloride ......................................5,000Neutral Red ............................................0,075Agar ......................................................15,000Final pH 7,4 ± 0,2

MacConkey Media

97

DirectionsSuspend 53 g of powder in a litre of distilled water. Bring to the boil, distribute in suitable containers and autoclave at 121°C for 15 minutes.

DescriptionThe MacConkey media are well known and popular enrichment system for coliform bacteria.At the beginning of the last century, MacConkey made the original formulation and included ox bile as inhibitor of the gram-positive bacteria and the litmus as the indi-cator of the acid production from lactose sugar. Lately the litmus has been substituted by phenol red indicator to make interpretations easier and more precise.The media have been adapted to facilitate the coliform detection with the advancement of knowledge of the bacterial physiology. The most significant modification to the original formulation has been the substitution of the ox bile by purified bile salts that improve the selectivity and avoid the inherent turbidity which is due to the fat material of the bile. The effectivity of the inhibition of the bile salts is variable and depends on the relative concen-tration of cholate and taurocholate.Another important modification was the inclusion of supplementary inhibitors such as crystal violet and/or brilliant green, that are the most popular formulations in America, but not in Europe where the lower selectivity is prefered.In the 60’s the toxicity of neutral red on the stressed cells of coliforms was demonstrated, especially on some strains of E. coli, and then the pH indicator was changed to the Bromcresol purple, being less aggressive than the neutral red.However the most extensive use is in the liquid form, nevertheless there are solids formulations with agar, for every modification.At the moment, the several formulations available of the MacConkey media offer a wide range from a low to high selectivity and the lactose positive bacteria grown on this medium form red colonies due to acid production due to the lactose fermentation and thus E. coli colonies can be easily distinguished as they also form a small precipita-tion zone of bile salts around them.Eventually,some enterococci can also grow, but they are easy to distinguish from the coliforms, as they form smaller colonies and the absence of precipitation zone.Medium can be used as Presumptive medium for E.coli (by fluorescent reaction) if before sterilization MUG (Ref. 06-102CASE) is added. In the medium with MUG the E. coli colonies show a light blue fluorescence under the UV illumination. The formulation without salt offers a low electrolyte content that almost suppresses the swarming growth of Proteus.

TechniqueFrom a decimal dilution bank, 1 mL samples are inocu-lated into empty sterile petri dishes in duplicate. Then ,15 mL of molten medium at 45°C is poured into every plate and mixed carefully . After the solidification, a second

layer of another 5 mL of sterile medium is poured into every plate to seal the surfaceand improve enumera-tion of the colonies. For the enumeration, after an incubation of 24 h. at 35°C, select plates with 30-300colonies. The character-istic colonies must be confirmed for coliform identity by gas production from lactose in a broth culture.The MacConkey broth (code 2-118) can be used for the enumeration of coliforms by the MPN technique, select-ing the positive tubes that shows turbidity, colour turned to red purple and with gas production. The same above characteristics, but turning into a yellow color are valid if the MacConkey broth code 2-120 is used.If the MUG medium is used a supplementary reading under UV illumination must be carried out.

ReferencesADAMS, S. (1991) Screening for verotoxin-producing E. coli. Clin Lab. Science 4:1:19-20.ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc. London.CLESCERI, L.S., A.E. GEENBERG & A.D. EATON (1998) Standard Methods for the Examination of Water and Wastewater. 20th ed. APHA-AWWA-WEF. Washing-ton DC. USADOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Foods.4th. ed. APHA. WashingtonEUROPEAN PHARMACOPOEIA (2007) 5th ed. (Supp.5.7) EDQM. Council of Europe. Strasbourg.HITCHINS, A.D., P. FENG, W.D. WATKINS, S.R. RIPEY y C.A. CHANDLER (1998). E. coli and coliform bacteria. Bacteriological Analytical Manual. 8th ed. AOAC Interna-tional. Gaitherburg. MD. USAHORWITZ, W. (2000) Official Methods of Analysis. AOAC Intl. Gaithersburg. MD. USAISO 9308-2:1990 Standard. Water Quality. Detection and enumeration of coliforms, thermotolerant coliforms and presumptive E. coli. MPN Method.ISO 21150:2006 Standard. Cosmetics – Detection of Escherichia coliISO 21567. Standard (2004) . Horizontal method for the detection of Shigella ssp.MARCH, S.B. y S. RATMANN (1986) Sorbitol-McConkey Medium for detection of E. coli O157:H7 associated with hemorrhagic colitis. J. Clin. Microbiol. 23:869-872McCONKEY, A.T. (1905) Lactose-fermenting Bacteria in faeces. J. Hyg 5:333.MURRAY, P.R., E.J. BARON, M.A. PFALLER, F.C. TEN-OVER, y R.H. YOLKEN (Eds) (1995) Manual of Clinical Microbiology 6th ed. A.S.M. Washington D.C. USARAPPAPORT, F. y E. HENING (1952) Media for the iso-lation and differentiation of pathogenic E. coli (serotypes O111 and O55) J. Clin. Pathology 5:361-362USP 29-NF 25. (2006) <61> Microbial Limit Tests US Pharmacopoeial Corp. Inc. Rockville. MD, USAVANDERZANT, C. y D.F. SPLITTOESSER (Eds) (1992) Compendium of methods for the microbiological exami-nation of foods. 3rd ed. A.P.H.A. Washington D.C.

MacConkey Media

98

MacConkey Media

Ref. 02-467

SpecificationLiquid culture medium for the selective enrichment of Pseudomonas aeruginosa in water, according the DIN 38411 standard.

Formula (in g/L)Meat peptone .........................................15,00Meat extract ..............................................9,00Malachite green (Oxalate) ........................0,03Dipotassium phosphate ............................1,10Final pH 7,0 ± 0,2

DirectionsTo prepare concentrated broth: Dissolve 25 g of pow-der in 1 L of distilled water and distribute into suitable containers.To prepare diluted broth: Dissolve 8,3 g of powder in 1 L of distilled water and distribute into suitable containers.In both the cases, sterilize by autoclaving at 121ºC for 15 minutes.

DescriptionHabs and Kirschner first described this medium in 1948 based on the resistance of Pseudomonas aeruginosa to malachite green as a selective factor. Lately, in 1974 Schubert and Blum modified the medium composition and proposed it as an enrichment step of Pseudomonas in very polluted water since malachite green oxalate at the concentration as above formulation inhibited the growth of almost all gramnegative microorganisms but it did not affect Pseudomonas growth.Years after the proposal of Schubert and Blum, the medium was adopted officially as an enrichment medium

by the German StandardizationInstitute (DIN) and other legal bodies for the microbiological analysis of water and food.

TechniqueIf the product to be examined is not restricted to specific standard (as DIN 38411 for water), it is suggested that the final malachite green concentration for enrichment should not exceed 0,01 g/L. Thus the concentrated or diluted broth should be used in function of the size or volume of the sample.Carry out the incubation at 35±2ºC for 24-48 hours. Cultures that show turbidity due to growth should be selected for the later confirmation for the presence of Pseudomonas aeruginosa.

ReferencesBUNDESGESUNDHEITSAMT: Amtliche Sammlung von Untersuchungverfahren nach 35 LMBG Beuth Verlag Berlin Köln.DEUTSCHE EINHEITSVERFAHREN sur Wasser-, Abwasser- und Schlammuntersuchung. VCH Verlags-gesellschaft D-6940 Weinheim.DIN 38411; Teil 6: Mikrobiologische Verfahren (Gruppe K):Nachweis von Pseudomonas aeruginosa (K8)HABS, H., K.H. KIRSCHNER (1943) Der Pyocyaneus Meerschweinchenhautversuch zur Prüfung von Haut desinfektionsmiteln. Z. Hyg. 124:557-578.SCHUBERT, R., U.BLUM (1974) Zur Frage der Eignun der Malachitgrün-Bouillon nach Habs u. Kirschner als Anreicherungsmedium fur Pseudomonas aeruginosa aus dem Wasser. Zbl. Bakt. Hyg. I Orig. B 158:583-587

Malachite Green Broth

VARNAM, A.H. y M.G. EVANS (1991) Foodborne patho-gens. Manson Publishing Ltd. London. U.K.WHO (1963) International Standards for Drinking Wa-ters, 7th ed., Churchill Ltd. LondonWINDLE-TAYLOR, E.(1958) The Examination of Water and Wastewater Supplies, 7th ed. Churchill Ltd. London

99

Malt Extract Media

Media based on malt extract may be considered as gen-eral growth substrates due to their richness and nutrient balance. They are very suitable for the cultivation of fastidious microorganisms. Classically, with acidic pH, they are used for the isolation, cultivation and mainten-ace of moulds and yeast, but with pH near to neutrality, they support bacterial growth of bacteria with special or fastidious nutritional needs.

The SCHARLAU manufacturing program covers all the published formulations, and included in this manual are some of the most commonly used. We propose that the technicians choose the formulation most suited to their requirements, or, alternatively, ask us for more details or possible modifications.

See also YM Media (Refs. 1-219, 2-219) and Wort Agar (Ref. 01-132)

Malt Extract Agar No. 1

Ref. 01-111

SpecificationCulture medium for moulds and yeast.

Formula (in g/L)Malt extract ...............................................13,0Dextrine ......................................................2,5Gelatin peptone ..........................................5,0Agar ..........................................................15,0Final pH 5,5 ± 0,2

DirectionsSuspend 35,5 g of powder in 1 L of distilled water and heat gently with constant stirring until the boiling. Dis-pense in suitable containers and sterilize by autoclaving at 115°C for 15 minutes. Avoid overheating since the low pH of the medium may hydrolize the agar.

DescriptionMalt Extract Agar is a classic culture medium for moulds and yeast. Malt extract has enough sugar (maltose, glu-cose, sucrose) to allow a copious growth, and in extreme cases, additional necessary growth factors are provided by the gelatin peptone.

TechniqueMalt Extract Agar has been widely used in maintenance, isolation and identification of fungi, and it is also pro-posed in several pharmacopiea as a medium for the control of sterility in pharmaceutical products, though it is mostly used for comparative morphological studies. Should more selectivity be desired, you can add a few millilitres of 10% Lactic Acid, or 5% Tartaric Acid, but

then this will make the solidification of agar more difficult. When acidification is below pH 5,0 do not remelt the agar since the solidifying agent will be hydrolized.


Ref. 01-573

SpecificationSolid medium for the isolation and enumeration of fungi.

Formula (in g/L)Malt extract ...............................................30,0Soy peptone ...............................................3,0Agar ..........................................................15,0Final pH 5,6 ± 0,2

DirectionsSuspend 48 g of powder in 1 L of distilled water and let it soak. Bring to the boil. Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionMalt Extract Agar may support the growth of almost all of the fungi very well, because of its balanced composi-tion, and restrains most of the bacteria due to the strong acidity.Should more selection against the bacterial growth be desired, readjust the pH to 3,5 by adding a sterile solu-tion of 10% lactic acid or 5% tartaric acid to the molten medium. After the additions do not reheat the medium.


Ref. 01-574

SpecificationSolid medium for the isolation and enumeration of fungi.

Formula (in g/L)Malt extract ...............................................30,0Mycological peptone ...................................5,0Agar ..........................................................15,0Final pH 5,4 ± 0,2

DirectionsSuspend 50 g of powder in 1 L of distilled water and let it soak. Heat to the boil and distribute into suitable contain-ers. Sterilize in the autoclave at 121°C for 15 minutes.

100

DescriptionThe balanced and rich nutrient composition of the me-dium makes it suitable for morphogenetic and structural studies of fungi. Due to its low pH it restrains the bacte-rial growth to a greater extent, but the total supression can be achieved by adding to the melted medium at 55°C, 20 mL of sterile solution of 10% Lactic acid or 5% tartaric acid, making the pH reduces to 3,5. In these conditions, do not heat the medium to avoid the hydroly-sis of agar.

Malt Extract Broth No. 1

Ref. 02-111

SpecificationLiquid culture medium for the moulds and yeasts.

Formula (in g/L)Malt extract ...............................................13,0Dextrine ......................................................2,5Gelatin peptone ..........................................5,0Final pH 5,5 ± 0,2

DirectionsDissolve 20,5 g of powder in 1 L of distilled water, heat-ing up if necessary. Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionMalt Extract Broth is a classic culture medium for the moulds and yeasts. Malt extract has sufficient sugar (maltose, glucose, sucrose) to allow a copious growth, and in more demanding cases, the necessary growth factors are provided by the gelatin peptone.

Malt Extract Broth No. 2

Ref. 02-491

SpecificationLiquid culture medium for the moulds and yeasts.

Formula (in g/L)Mycological peptone ...................................3,0Malt extract ...............................................17,0Final pH 5,4 ± 0,2

DirectionsDissolve 20 g of powder in 1 L of distilled water, heat-ing up if necessary. Distribute in suitable containers and sterilize by autoclaving at 121ºC for 15 minutes. Do not overheat, since a browning by Maillard reaction can be produced.

DescriptionThis formulation of classic Malt Extract Broth is ac-cording to Reiss’ modification in order to achieve better results for the cultivation of Aspergillus flavus.

TechniqueMalt Extract Broth has been widely used in mainte-nance, isolation and identification of fungi, and it is also proposed in several pharmacopeia as a medium for the control of sterility in pharmaceutical products, though it is mostly used for comparative morphological studies. Should more selectivity be desired, you can add a few millilitres of 10% Lactic Acid, or 5% Tartaric Acid, but this makes the solidification of agar very difficult. When acidi-fication is below pH 5,0, do not remelt the agar since the solidifying agent is hydrolized below pH 5,0.

ReferencesFDA (1998). Bacteriological Analytical Manual. 8th ed. Revision A AOAC International Gaithersburg) MD.DOWNES, F.P & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food. 4th ed. APHA. Washington.ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press,.BALLOWS, HAUSLER, HERMAN, ISENBERG & SHADOMY (eds.) (1991) Manual of Clinical Microbiol-ogy. ASM. Washington.REIS, J. (1972) Ein selektives kulturmedium für der Nachweiss von Aspergillus flavus. Zbl. Bakt. Hyg. I. Abt. Orig. 220:564-566.RAPP, M. (1974) Indikator-Zusätze zur Keimdifferen-zierung auf Würze und Malzextrakt Agar Milchwiss. 29:341-344.

Malt Extract Media

101

Ref. 01-116

SpecificationSelective medium for the isolation of staphylococci according USP and ISO standard.

Formula (in g/L)Meat extract ............................................1,000Casein peptone ......................................5,000Meat peptone .........................................5,000Sodium chloride ....................................75,000D-Mannitol ............................................10,000Phenol red ..............................................0,025Agar ......................................................15,000Final pH 7,4 ± 0,2

DirectionsSuspend 111 g of powder in 1 L of distilled water and bring to the boil. Dispense in tubes or flasks and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionMannitol Salt Agar is a classical medium for the detec-tion and enumeration of staphylococci. It was described by Chapman and has been adopted by many official or-ganisations. Several modifications have been developed from it with more or less similar effectivity.This medium uses the advantage of high tolerance of staphylococci to salinity, to use sodium chloride as a se-lective agent, since only the staphylococci and halophilic enterobacteria are able to grow freely at this concentra-tion of salt employed in this medium while other bacteria are inhibited. It also exploits the correlation between the pathogenic and fermentative capacity of mannitol of staphylococci, to establish a presumptive diagnosis. Mannitol fermentation with an accumulation of acid prod-ucts is shown by the phenol red indicator turning yellow, that produces a yellow halo surrounding the presumptive pathogen colonies, meanwhile the rest of the medium remains orange in colour.

TechniqueA massive surface inoculation and an incubation at 37°C for 36 hours or at 32°C for 3 days is recommended.The typical appearance of the colonies after the correct incubation is as follows: Presumptive pathogenic staphy-lococci (coagulase +) are mannitol positive and are big colonies with a yellow halo. Non-pathogenic Staphyloco-cci (coagulase -) are usually mannitol negative and are small colonies without halo or change in colour.In any case, coagulase presence must be tested by the classical technique, after a pure culture in the liquid me-dium is obtained, in order to establish its true pahogenic potential.

ReferencesATLAS, R.M. & L.C.PARKS (1993) Handbook of Micro-biological Media. CRC Press. BocaRaton. Fla. USACHAPMAN (1945) The significance of sodium chloride in studies of staphylococci. J. Bact 50:201DOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Foods.4th ed. APHA. Washington DC. USAFDA (1995) Bacteriological Analytical Manual. 8th ed. Revision A. AOAC Internacional Inc. Gaithersburg. Md. USAISO 22718:2006 Standard. Cosmetics – Detection of Staphylococcus aureus. USP 29- NF 25(2005) <61>Microbial Limit Tests. US Pharmacopoeial Convention Inc. Rockville. Md. USA

Mannitol Salt Agar (Chapman Agar)

Staphylococcus aureus ATCC 25923

102

Ref. 02-510

SpecificationIsotonic diluent for the maximal recovery of stressed microorganisms according ISO standard.

Formula (in g/L)Peptone ....................................................1,00Sodium chloride ........................................8,50Final pH 7,0 ± 0,2

DirectionsDisolve 9,5 g of powder in 1 L of distilled water and dis-tribute into suitable containers. Sterilize by autoclaving at 121ºC for 15 minutes.

DescriptionThis formulation combines the osmotic pressure of the physiological saline solution with the protective action of the peptone to obtain a good recovery of stressed microorganisms.The sodium chloride assures the isotonic conditions and the low concentration of the peptone does not allow the cellular growth in the short period (2-4 hours) of time required for the preparation of the dilution bank of the sample.

Technique According to the ISO method, the sample is diluted in a ratio 1:10 with the Maximum Recovery Diluent and ho-mogenized by a vortex mixer or stomacher. After a short period (10-15 minutes) of rest, a decimal dilution bank with the same diluent is released following the standard procedures. Plates are inoculated from the different concentration of the dilution bank.

ReferenceISO/DIS 6649 Meat and Meat Products. Detection and Enumeration of Clostridium perfringensISO 21149:2006 Cosmetics – Enumeration and detec-tion of aerobic mesophilic bacteria.ISO 21150:2006 Standard. Cosmetics – Detection of Escherichia coliISO 22717:2006 Standard. Cosmetics – Detection of Pseudomonas aeruginosa.ISO 22718:2006 Standard. Cosmetics – Detection of Staphylococcus aureus.

Maximum Recovery Diluent

103

Ref. 01-223

SpecificationSolid culture medium for the detection of Leuconostoc in fermentation starters of mixed flora.

Formula (in g/L)Peptone ................................................10,000Yeast extract ...........................................5,000Sucrose ..............................................100,000Dextrose .................................................5,000Sodium citrate ........................................1,000Gelatine ..................................................2,500Sodium azide ..........................................0,075Agar ......................................................15,000Final pH 6,0 ± 0,2

DirectionsSuspend 138,5 g of powder in 1 L of distilled water and heat up in boiling water bath at 50-55°C, till the complete liquefaction of the gelatine is obtained. Heat to boiling and dispense into suitable containers. Sterilize in the autoclave at 121°C for 15 minutes. Avoid overheating, since it may affect the solidification.

DescriptionThis differential and selective medium for Leuconostoc was originally described by Mayeux in 1961, and was later modified by the same author to this formulation. This allows a very specific separation of microorganisms in the lactic fermentation starters with mixed flora.Citrate, glucose and gelatine helps for the growth of Leu-conostoc, and the large amount of sucrose allows a co-pious production of dextrane polymer by L.dextranicum.

Sodium azide avoids the growth of undesired flora, and at the same time, hampers the colonial development of lactic streptococci. Other authors state that the addition of little amounts of tetracycline (15 mcg/mL) produces the inhibition of lactic streptococci without affecting Leuconostoc.

TechniqueThe plates are inoculated by surface inoculation, and then incubated at 21°C for 4 days. Most of the strepto-coccal strains, including Streptococcus lactis, S.cremoris and S.diacetilactis do not grow or grow a little after the third day of incubation. In those cases, their colonies are small, opaque and cream or yellow coloured. Leucon-ostoc colonies have a bigger and earlier growth. Leu-conostoc citrovorum form the colonies of 0,5 to 1 mm diameter, which are translucent and iridescent. L. dextranicum form big colonies (1-5 mm), which are transparent and mucosal.

ReferencesMAYEUX and COLMER (1961) J. Bact. 81:1.009.MAYEUX, SANDINE and ELLIKER (1962) J. Dairy Sci. 45:665.McDONOUGH, HARDGROVE and TITSLER (1962) J. Dairy Sci. 45:656.FIL-IDF Standard 149A (1997) Dairy starters of lactic acid bacteria culture. Composition standard.

Mayeux Agar

104

Ref. 01-562

SpecificationSolid medium for the cultivation of anaerobic microor-ganisms

Formula (in g/L)Meat Extract ...........................................10,00Liver Extract ...........................................10,00Dextrose ...................................................0,75Soluble starch ...........................................0,75Sodium sulfite ...........................................1,20Ammonium Iron (III) citrate .......................0,50Agar ........................................................13,00Final pH 7,6 ± 0,2

DirectionsSuspend 36,2 g of powder in 1 L of distilled water and had to boiling with constantly stirring. Distribute in suit-able containers and sterilize in the autoclave at 121ºC for 15 minutes.

DescriptionThe mixture of meat and liver extract is a highly reduc-ing nutrient basis that provides the supply of nitrogen for the growth of anaerobes. The energy source is provided by the dextrose and the starch acts only as a metabolic detoxifier. The sulphite present in the culture medium is

Meat Liver Agar

reduced to H2S by some Clostridium spp and reacts with

the iron from ammonium iron citrate producing a dark precipitate that blackening the medium.

TechniqueThe meat liver agar can be used in petri dishes or in tubes. When the inoculum is in poured plates, the reducing power of the medium permits the growth of the anaerobes. If spreading plates are used the incubation in an anaerobic environment is compulsory, but some times is enough cover the plate with Sealing Anaerobic Agar (Ref. 01-174). Temperature and time of the incubation must be suitable to the sample, but it is recommended more than 48 hours at 35-37ºC

ReferencesATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press Inc. London.CORETTI, C. (1962) Prüfung eines leberpulvers auf Eignung zur Herstellung von Leberbrühe und Leberagar zur Anaerobenzuchtung. Berl. Münch. Tierärztl. Wissch. 75:205.VANDERZANT, C. & D.F. SPLITTSTOESSER (1992) Compendium of methods for the microbial examination of Foods. 3rd Ed. APHA. Washington.

105

Methyl Red Voges Proskauer Media (MRVP Media)

Methyl Red Voges ProskauerBroth (MRVP Broth)(Clarks Lubs Medium)

Ref. 02-207

SpecificationClassic liquid medium for differential tests (Voges Proskauer and Methyl Red) in Enterobacteria according ISO standards 6579 and 6585 and FIL - IDF 93 stand-ard.

Formula (in g/L)Peptone ......................................................7,0Dextrose .....................................................5,0Potassium phosphate .................................5,0Final pH 7,0 ± 0,2

DirectionsDissolve 17 g of powder in 1 L of distilled water, heating up only if necessary. Dispense in tubes and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionThe classical Clark and Lubs medium which is used to perform the tests of Methyl Red and Voges Proskauer, that together with Indole and Citrate tests (IMViC) allow the differentiation within the coliform group of bacteria. The fundamentals of these reactions are as follows:

Methyl Red Test (M.R. test)Among the Enterobacteriaceae, the E.coli biotype fer-ments glucose by the mixed acid pathway, accumulating acid, which reduces the initial pH. It can be detected by the methyl red indicator, which turns yellow above the pH 5,1 and becomes red below pH 4,4.

Voges Proskauer Test (V.P. test)Enterobacteria of Klebsiella-Enterobacter biotype ferment the glucose by the 2-3-butanediol pathway. Although the acids are produced in this way, the neutral or alkaline products are also formed and at the end the reaction is neutral or alkaline. Due to this, the incubation must be extended up to 3 days. After this period, the methyl red reaction is negative.

Nonetheless, Voges Proskauer test is complementary to Methyl Red test in some ways. It shows the 2-3-bu-tanediol and acetoin production, that are substances difficult to find in the mixed acid pathway. It exploits the fact that these two products, in alkaline medium, oxidize themselves to diacetyl, which reacts with guanidine and produces visibly coloured compounds.

Methyl Red Voges ProskauerModified Broth for Bacillus(MRVP Modified Broth)

Ref. 02-572

SpecificationLiquid culture media to perform the Voges-Proskauer test in Bacillus cultures according FIL-IDF 181 standard.

Formula (in g/L)Tryptone ..................................................7,00Dextrose ...................................................5,00Dipotasium phosphate ..............................5,00Sodium chloride ........................................5,00Final pH 7,0 ± 0,2

DirectionsDissolve 22 g of powder in 1 L of distilled water, heating if it is necessary. Distribute in suitable containers (5 mL in test tubes of 16 x 160 mm) and sterilize in autoclave at 121ºC for 15 minutes

Description This medium is produced according the FIL-IDF formula-tion to perform the test of acetil-metil-carbinol production in Bacillus cereus and other species of Bacillus.

Methyl Red Voges ProskauerSaline Broth(MRVP Saline Broth)

Ref. 02-456

SpecificationLiquid culture medium for the Mehyl Red and Voges Proskauer tests.

Formula (in g/L)Peptone ...................................................7,00Dextrose ...................................................5,00Sodium chloride ......................................30,00Dipotassium phosphate ............................5,00Final pH 7,4 ± 0,2

DirectionsDissolve 47 g of powder in 1 L of distilled water, heating up only if necessary. Distribute into suitable containers and sterilize by autoclaving at 121ºC for 15 minutes.

106

DescriptionThis medium is used to perform the tests of Methyl Red and Voges Proskauer, that, together with Indole and Cit-rate tests allow the differentiation within the enteric bac-teria. The fundamentals of these reactions are described in the Methyl-Red Voges-Proskauer Broth (Ref. 02-207)

TechniqueThere are several techniques to carry out these tests. One of them is as follows:The tube with medium is inoculated with the microorgan-ism to be studied and incubated at 30°C for at least 3 days and up to 5 days maximum. Just before reading, culture is separated in two portions, one for each test.

1) Methyl Red Test.Add 4-5 drops of Methyl Red Reagent (Ref. 06-007) to the culture, and shake in order to homogenize. Observe for the colour development in the medium. The test is considered positive if it turns to red and negative if it remains yellow.

Positive (red colouration): E.coli, Edwardsiella, Shigella, Salmonella, Citrobacter, Proteus, Klebsiella ozoe-nae, Klebsiella rhinoscleromatis, Yersinia.

Negative (yellow colouration): Enterobacter, Hafnia, Ser-ratia, Klebsiella pneumoniae.

With Erwinia, this reaction has no significance since it gives variable reactions.

2) Voges Proskauer TestAdd Barrit’s Reagent to the medium (Ref. 06-027) until it gets a milky appearance and then add O’Meara’s Rea-gent (Ref. 06-006) until milky appearance disappears. Shake vigorously.Test is positive if the medium acquires a pink-violet col-our, forming at the top of the tube. If the test is negative, there is no colour formation. Relative amounts of each reagent depend on initial volumes of the medium. Never incubate above 30°C.

Positive (pink-intense red) : Enterobacter, Hafnia, Kleb-siella pneumoniae, Serratia.

Negative (no colour change): Escherichia, Edwardsiella, Citrobacter, Salmonella, Shigella, Yersinia, Kleb-siella ozonae, Klebsiella rhinoscleromatis.

With Proteus and Erwinia types, this reaction has no significance since it gives variable reactions.

Voges Proskauer test may be performed in a quicker way, using very little volumes of medium and massive in-oculum. This allows the readings with short incubations (18-20 hours), and also the readings may be accelerated by gently heating the culture almost to boiling point after adding the reagents. However, erroneous results are more likely by using this method.

Refer to the FIL-IDF Standard for the specific technique of MRVP Modified Broth.

ReferencesVOGES, O, B. PROSKAUER (1898) Beitragzur Ernährungsphysiologic und zur Differentialdiagnose der hämorrhagischen Septicäemie. Z. Hyg. CLARK, W., H. LUBS. (1915) The differentiation of bac-teria of the colon-aerogenes family by the use of indica-tors. J. Inf. Dis. 17:160-173BARRIT, M. (1936) The intensification of the Voges Proskauer reaction by the addition of alpha-naftol. J. Path. Bact. 42:441-452O’MEARA, R. (1931) A simple delicate and rapid methods of detecting the formation of acetylmethyl/car-binol by bacteria fermenting carbohidrats. J. Path. Bact. 34:401-406MOLLÄNDER, R., J. BÖHMANN, B. GREWING (1982) Die Verstörkung der Voges-Proskauer Reaktion durech fumarat. Zbt. Bakt. Hyg. I Alet. Orig. A 252:316-323.SCHWEIZERISCHES LEBENSMITTELBUCH 5th Ed. Ch. 56A. Berna.FDA (1998) Bacteriological Analytical Manual. 8th ed. Rev. A. AOAC International. Gaithersburg. MD.ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc.,London.PASCUAL ANDERSON, MªRª (1992) Microbiología Alimentaria. Diaz de Santos, S.A.,Madrid,.ISO Standard 6579 (2002) Microbiology of Food and ani-mal feeding stuffs- Horizontal method for the detection of Salmonella species.FIL-IDF (1998) International Provisional Standard 181. Dried Milk Products: Enumeration of Bacillus cereus: Most Probable Number Technique.FIL-IDF (2001) Milk and Milk products Detection of Salmonella. ISO 6585 standard (2001) Milk and Milk products - De-tection of Salmonella.

Methyl-red Voges Proskauer Media (MRVP Media)

107

Microbial Content Test Agar (TSA Lecithin Polysorbate)

Ref. 01-613

SpecificationSolid medium for sampling of surfaces of sanitary impor-tance with RODAC plates technique.

Formula (in g/L)Tryptone .................................................15,00Soy peptone .............................................5,00Sodium chloride .......................................5,00Lecithin .....................................................0,70Polysorbate 80 .........................................5,00Agar ........................................................15,00Final pH 7,3 ± 0,2

DirectionsThe dehydrated medium has a characteristic “brown sugar” appearance and may seem moist.Suspend 45,7 g of powder in 1 L of distilled water and let it soak. Bring to the boil and dDistribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

DescriptionThis medium is a modification of the classical TSA for the surface sampling by the RODAC (Replicate Organ-ism Detection and Counting) plate technique. Collection of samples from identical areas (replicate) “before and after” treatment with disinfectant yields data useful in evaluating cleaning procedures in environmental sanita-tion.Lecithin is incorporated to neutralize quaternary ammo-nium compounds and polysorbate 80 is used to neutral-ize phenolic disinfectants, hexachlorophene, formalin and, with lecithin, ethanol.

ReferencesHICKEY, P.J., C.E. BECKELHEIMER, & T. PARROW (1992) Microbiological tests for equipment, containers, water and air. In R.T. Marshall (Ed.) Standard Methods for the examination of Dairy Products 16th ed. APHA Washington.EVANCHO, G.M., W.H. SVEUM, LL. J. MOBERG & J.F. FRANK (2001) Microbiological Monitoring of the Food Processing Environment. In Downes & Ito (Eds) Compen-dium of Methods for the Microbiological Examination of Foods. 4th ed. APHA. Washington DC.ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-biological Culture Media. CRC Press. Boca Ratón, Fla.

108

Ref. 01-514

SpecificationSolid culture medium for the plate count test in dairy products.

Formula (in g/L)Peptone ....................................................5,00Yeast extract .............................................3,00Powdered milk ..........................................1,00Agar ........................................................15,00Final pH 7,2 ± 0,2

DirectionsSuspend 24 g of powder in 1 L of distilled water and let it soak. Bring to the boil and distribute into suitable con-tainers. Sterilize by autoclaving at 121ºC for 15 minutes.

DescriptionMilk Agar is approved by the European Commission and it is formulated according to the recommendations of the European Association of Ice-Cream Producers (EuroGla-ce) for the microbiological examination of ice-creams.

TechniqueSuggested technique is the standardized count on mass-inoculated plates. Inoculum is obtained from a decimal dilution bank of the sample. Once inoculated, the plates should be left undisturbed for 1-3 hours and then incu-bated at 30ºC for 3 days.

ReferencesKLOSE, J. (1968) Harmonisierung des speisesrechtes under EWC-Sübwaren 14:778-780.KLOSE, J. (1968) Entwarf einer Riehlinie zar Aufleichung der Rechtvorschiften fur Speiseeis unden Mitfliedsstaaten der EWG. Sübwaren 14:780-782ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press. Boca Raton. Fla.

Milk Agar

109

Motility Indole Ornithine FluidMedium (MIO)

Ref. 03-422

SpecificationMedium for the demonstration of motility, indole produc-tion and ornithine decarboxylase activity of enterobacte-ria.

Formula (in g/L)Yeast extract .............................................3,00Dextrose ...................................................1,00Gelatin peptone ......................................10,00Casein peptone ......................................10,00L-Ornithine HCl .........................................5,00Bromocresol purple ..................................0,02Agar ..........................................................2,50Final pH 6,6 ± 0,2

DirectionsSuspend 31,5 g of powder in 1 L of distilled water and let it soak. Bring to the boil and distribute in tubes. Sterilize by autoclaving at 121°C for 15 minutes.

TechniqueRemove all the dissolved air in the medium by heating up the tubes in boiling water bath and cooling them upto room temperature. Taking the growth of the primary isolation as the inoculum, inoculate the tubes by a single deep stab. Incubate aerobically at 35±2°C for a 18-24 hours period.Motility can be observed by the diffuse growth at the upper side of the stab, meanwhile the immotile bacteria grow along the stab, producing a clear streak.Ornithine decarboxylation is indicated by the presence of a dark purple colour throughout the tube. Negative reac-tion produces only a single purple band at the top, and the rest of the tube changes to yellow.Indole production is verified after the addition of a few drops of Kovac’s Reagent (Ref. 06-018) (shake gently). The presence of a red ring signifies the positive reaction, and if the colour is yellow, then the reaction is negative.

ReferencesATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press,Boca Raton,Fla.EDERER, G.M. and M. CLARK (1970) Motility-Indol-Or-nithine Medium. Appl. Microbiol. 2:849.FDA (1998) Bacteriological Analytical Manual 8th ed. REvision A. AOAC International. Gaithersburg. MD.

Motility Nitrate Medium

Ref. 03-612

SpecificationMedium for the motility determination and nitrate reduc-tion of clostridia in food products acc. ISO 7937.

Formula (in g/L)Meat extract ................................................3,0Peptone ......................................................5,0Potassium nitrate ........................................1,0Disodiun phosphate ....................................2,5Galactose ...................................................5,0Agar ............................................................5,0Final pH 7,3 ± 0,2

DirectionsSuspend 21,5 g of powder in 1 L of distilled water con-taining 5 mL of glycerol and heat to boiling. Dispense into suitable containers and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionThis semisolid medium has been made according to the rules suggested by the US Food and Drug Administra-tion for the identification of Clostridium perfringens in food.

TechniquePrepared tubes regenerate themselves if kept in boiling water bath for 10 minutes to eliminate the dissolved oxy-gen. Let them get cooled to 70-80°C and then inoculate them by stabbing the centre. Take a black colony grown on TSN Agar (Ref. 01-195) as the inoculum. Incubate the tubes at 37°C for 18-20 hours without sealing nor in anaerobic chamber.In this medium, if the growth is stopped at 5-7mm. from the surface, signifies that there is anaerobiosis. Non-mo-tile is evident as the growth is observed only inside the stab.To verify the nitrate reduction to nitrite, pour a few drops of Nitrate A Solution (Ref. 06-003) and Nitrate B Solution (Ref. 06-004) on the surface of the medium. If a pink or red colour appears, reaction is positive. Clostridium perfringens is an anaerobic , non-motile and reducer of nitrate to nitrite microorganism.

ReferencesFDA (1998) Baceriological Analytical Manual. 8th ed. Revision A. AOAC International. Gaithersburg.ISO 7937 Standard (2004). Microbiology of food and ani-mal feeding stuffs. Horizontal methods for the enumera-tion of Clostridium perfringens. Colony count technique.

Motility Media

110

MRS Agar

Ref. 01-135

SpecificationSolid culture medium for lactobacilli, according to de Man, Rogosa and Sharpe and ISO standards 9332 and 15214.

Formula (in g/L)Peptone Proteose ...................................10,00Meat extract ..............................................8,00Yeast extract .............................................4,00D(+)Glucose ...........................................20,00Sodium acetate ........................................5,00 Triammonium citrate .................................2,00Magnesium sulfate ...................................0,20Manganese sulfate ...................................0,05Dipotassium phosphate ............................2,00Polysorbate 80 .........................................1,00Agar ........................................................14,00Final pH 6,2 ± 0,2

DirectionsSuspend 66 g of powder in 1 L of distilled water. Bring to the boil slowly with gentle stirring until complete dissolu-tion. Dispense into suitable containers and sterilize by autoclaving at 121°C for 15 minutes.

MRS Broth

Ref. 02-135

SpecificationLiquid culture medium for lactobacilli, according to de Man, Rogosa and Sharpe and ISO standards 9332 and 15214.

Formula (in g/L)Peptone Proteose ...................................10,00Meat extract ..............................................8,00Yeast extract .............................................4,00D(+)-Glucose ..........................................20,00Sodium acetate ........................................5,00 Triammonium citrate .................................2,00Magnesium sulfate ...................................0,20 Manganese sulfate ...................................0,05Dipotassium phosphate ............................2,00Polysorbate 80 .........................................1,00Final pH 6,2 ± 0,2

DirectionsSuspend 52 g of powder in 1 L of distilled water. Heat up to complete dissolution and dispense into suitable con-tainers. Sterilize by autoclaving at 121°C for 15 minutes.

DescriptionMRS Agar and Broth are a modification of the previously used media for the cultivation of lactobacilli, all of them based on tomato juice’s nourishing properties. The ad-dition of magnesium, manganese and acetate, together with the Polysorbate, has provided an improved medium for the growth of lactobacilli, including that of very fastidi-ous species such as Lactobacillus brevis and Lactoba-cillus fermenti. On the other hand, the quality of the peptones in addition to the meat and yeast extracts, combine together all the necessary growth factors that make the MRS media one of the best media for the cultivation of lactobacilli.Nevertheless, these media selectivity is low and the con-taminants tend to grow in these media, which signifies a higher selectivity is needed. We therefore suggest the use of subculture in solid medium, on double layer and broth. In many cases,the growth is encouraged by a CO

2

enriched atmosphere in the medium.MRS media is particularly recommended for the enumer-ation and maintenance of lactobacilli either by the MPN technique (in broth) or on the plate by massive inocula-tion, overlaying it with a second layer of molten medium. This technique overcomes the need of a CO

2 enriched

atmosphere.

ReferencesDOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Foods.4th Ed. APHA. Washington DC. USAFIL-IDF Standard 146 (2003) Yoghurt. Identification of characteristic micro-organisms.IFU Method No 5 (1996) Lactic Acid Bacteria Count Pro-cedure. Schweizerischer Obstverband. CH-6302 ZugIFU Method No 7 (1998) Sterility testing of aseptic filled products, commercial sterile products and preserved products. Schweizerischer Obstverband. CH-6302 ZugIFU Method No 9 (1998) Microbiological examination of potential spoilage micro-organisms of tomato products. Schweizerischer Obstverband. CH-6302 ZugISO Standard 9232 (2003) Yoghurt – Identification of characteristic microorganisms (Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus)ISO Standard 15214 (1998) Horizontal method for the enumeration of mesophilic lactic acid bacteria – Colony count technique at 30ºCMAN, J.C. de, ROGOSA, M. y SHARPE, M. Elisabeth (1960) A médium for the cultivation of lactobacilli. J. Appl. Bact.; 23:130.

MRS Media

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Mueller Hinton Media

Mueller Hinton Agar

Ref. 01-136

SpecificationWidely recommended medium for antibiotic and sulfona-mide susceptibility testing, according to the Kirby-Bauer and the Ericsson methods.

Formula (in g/L)Peptone ....................................................17,5Beef infusion solids ....................................2,0Starch .........................................................1,5Agar ..........................................................17,0Final pH 7,3 ± 0,2

DirectionsAdd 38 g of powder to 1 L of distilled water and let it soak. Bring to the boil to dissolve the medium complete-ly. Sterilize by autoclaving at 121°C for 15 minutes.

DescriptionThe Mueller Hinton Agar was originally designed for the primary isolation of meningococci and gonococci. With the addition of blood it becomes an optimal medium for the growth of Neisseria. It is also more effective if reheated and turned into a Chocolate Agar. It should never be remelted or reheated once the blood has been added to it.

Mueller Hinton Broth

Ref. 02-136

SpecificationLiquid version of the agar with the same name, recom-mended for the studies about MIC of antibiotic.

Formula (in g/L)Peptone ....................................................17,5Starch .........................................................1,5Solids of meat infusion ...............................2,0Final pH 7,3 ± 0,2

DirectionsAdd 21 g of powder to 1 L of distilled water and dissolve it completely. Distribute in suitable containers. Sterilize by autoclaving at 121°C for 15 minutes.

DescriptionMueller Hinton Broth is the liquid version of the agar with the same name, and can be used in parallel with the agar when comparative studies are desired as well as when a broth with a high nutritive capacity is required. It is especially suggested for inoculum preparation for sensitivity assays.

In this medium, and in its solid version, presence of the starch is very important, since it acts as a detoxifying agent against the toxic substances if present in the sam-ple and it also acts as the cell regenerator.

TechniqueFor the culture of Neisseria the best results are obtained if incubation is carried out in a humid chamber with a CO

2 enriched atmosphere, if an anaerobic jar is not

available. This environment can be obtained by placing the plates in a hermetically sealed air-tight container, a dessicator for instance, together with a cotton swab soaked in water and a lighted candle end. Once the con-tainer is full the flame consume oxygen and by the time it is extinguished, the atmosphere inside the container has got 5 to 8% CO

2 enrichment.

The Mueller-Hinton Agar has proved to be one of the most efficient medium in the anti-bacterial susceptibil-ity testing. Without the addition of blood it can even be used for sulfonamide sensitivity testing since it is free from most of its antagonists (nucleotides, etc.). If this type of assay is conducted, the zones of inhibition should be examined just after 12-18 hours, before the usual overgrowth occurs, since after 24 hours it tends to interfere with the examination of sulfonamides sensitivity.For this purpose, a small inoculum will help the early formation of zones of inhibition. It should amount to a 100 to 300 times smaller inoculum than that of the cor-responding strain which is used in the antibiotic sensitiv-ity testing. In 1970 the WHO proposed this medium for antibacte-rial sensitivity testing, and it has been widely used since then.Sensitivity testing can be conducted by a variety of techniques, both on solid and liquid media. The most commonly used method in routine work is that derived from Kirby-Bauer and recommended by the American Association of Clinical Pathologists. It provides informa-tion on growth around a disk impregnated with antibacte-rial substance.

The Bauer-Kirby method is more precise and is semi-quantitative by category. It uses the Mueller-Hinton Agar and disks with high antibiotic concentration. The inocu-lum is first standardized with a Mac-Farland nephlom-eter. Then the plate is inoculated with a swab dipped in the standardized suspension, and finally the disks are arranged properly and at the equidistance from each other on the plate and then incubated.

Some authors suggest that the inoculum should be modified by introducing a double layer of mass inoculat-ed medium. This system undoubtedly provides sharper and more defined zones of clearing or inhibition. Plates are incubated at 37°C

112

overnight and then the zones of inhibition are measured. Results are reported in terms of Resistant, Moderately Resistant and Sensitive strains (Table above).

The Ericsson technique, which has been adopted in most European countries has already standardized the culture medium (Mueller-Hinton) and the quantity per plate (25 mL on 9 cm diameter plates). It has also stand-ardized the inoculum concentration.The fresh culture suspension to be examined (incubated for 18 hours in liquid medium) must be diluted enough, so as to ensure the presence of confluent growth on the agar.Suggested Dilutions:Enterobacteria- Pseudomonas: dilution of 1/300.Staphylococcus - Enterococcus: dilution of 1/300.Streptococcus - Haemophilus: dilution of 1/10.The plate is seeded by flooding its surface. The ex-cess inoculum is removed with a sterile pipette and the antibiotic disks are arranged properly on the plate. Allow a pre-diffusion period of 30-60 minutes before incuba-tion so that the antibiotic can slowly diffuse before the growth. After the incubation at 37°C for 12-18 hours, measure the zones of inhibition and refer to the Assay Regression Curves. Results are reported in terms of

Sensitive, Resistant or as Minimum Inhibitory Concentra-tion (MIC).

This latest technique undoubtedly offers more precision and reliability than the previous ones. Nevertheless, the Kirky method, which is semiquantitative, is much more simple and easy to adopt in everyday practice. On the other hand, the Ericsson technique is highly recom-mended for the effectivity and the sensitivity studies. The Mueller-Hinton medium plates can be stored refrig-erated in plastic bags for a month without affecting their results of sensitivity testing. However, they should not be used if the medium shows any dehydration.The Mueller Hinton Agar Scharlau fulfills the WHO re-quirements for the conducting microbial sensitivity tests and the basic characteristics are verified in every batch. Nevertheless some variation in the results between batches can be observed and technicians claims about the origin of these variability. At this point must keep in mind a lot of factors that are a source of variability:1. Since the nutritional requirements of organisms vary,

some strains may be encountered that fail to grow or grow poorly on these media.


113

2. Numerous factors can affect results: inoculum size, rate of growth, medium formulation and pH, length of incubation and incubation environment, disk con-tent and drug diffusion rate, and measurement of endpoints. Therefore, strict adherence to protocol is required to ensure reliable results.

3. Disk diffusion susceptibility testing is limited to rapidly growing organisms. Drug inactivation may result from the prolonged incubation times required by slow growers.

4. Media containing excessive amounts of thymidine or thymine can reverse the inhibitory effects of sulfona-mides and trimethoprim, causing zones of growth inhibition to be smaller or less distinct.

5. Variation in the concentration of divalent cations, primarily calcium and magnesium, affects results of aminoglycoside, tetracycline, and colistin tests with Pseudomonas aeruginosa isolates. A cation content that is too high reduces zones sizes, whereas a cation content that is too low has the opposite effect.

6. When Mueller Hinton Medium is supplemented with blood, the zone of inhibition for oxacillin and methicil-lin may be 2 to 3 mm smaller than those obtained with unsupplemented agar. Conversely, sheep blood may markedly increase the zone diameters of some cephalosporins when they are tested against ente-rococci. Sheep blood may cause indistinct zones or a film of growth within the zones of inhibition around sulfonamide and trimethoprim disks.

7. Mueller Hinton Medium deeper than 4 mm may cause false-resistant results, and agar less than 4 mm deep may be associated with a false-susceptibility report.

8. A pH outside the range of 7,3±0,1 may adversely affect susceptibility test results. If the pH is too low, aminoglycosides and macrolides will appear to lose potency; others may appear to have excessive activ-ity. The opposite effects are possible if the pH is too high.

9. When Mueller Hinton Medium is inoculated, no drop-lets of moisture should be visible on the surface or on the petri dish cover.

10. Mueller Hinton Medium should be inoculated within 15 minutes after the inoculum suspension has been adjusted.

11. The zone of inhibition diameters of some drugs, such as the macrolides, aminoglycosides and tetracy-clines, are significantly altered by CO

2. Plates should

not be incubated in increased CO2 atmosphere.

For further information on the performance of the anti-biotic disk susceptibility test refer to the M2-A6 NCCLS Monograph.


ReferencesBAUER A.L., W.M.M. KIRBY, J.C.SHERRIS & M.TURCK (1966) Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol 45: 493.BARRY, A.L., M.D. COYLE, C. THORNBERRY, E.H. GARLACH & R.W. HAWKINSON (1979) Methods of measuring zones of inhibition with Bauer-Kirby disk sus-ceptibility test. J. Clin. Microbiol. 10:885-889.ERICSSON & SHERRIS (1971) Antibiotic sensitivity testing. Report of an International Collaborative Study. Acta Pathol. Microbiol. Scand Suppl. 217 p: 90.HINDLER, J. (1998) Antimicrobial Susceptibility Testing In Essential Procedures for Clinical Microbiology. ASM Press. Washington D:C.MUNRO, S. (1995) Disk Diffusion Susceptibility Testing. In Clinical Microbiology Procedures Handbook. H.D. Isenberg (Ed) APHA Whasington D.C.MILLER, J.M., C. THORNBERRY & C.N. BAKER (1984) Disk diffusion susceptibility test troubleshooting guide. Lab. Med. 15:183-185. NCCLS Standard M2-A6 (1997) Performance standards for antimicrobial disk susceptibility tests. 6th ed. National Committee for Clinical Laboratory Standards. Vilanova. PA. THORNSBERRY, C., W.G. GAVAN, E.H. GERLACH & J.C. SHERRIS (1977) Cumitech 6. ASM. Washington.WHO (1977) Requeriments for antibiotic susceptibility tests. Technical Report Series No 610. Geneva.WOODS, G.L. & J.A. WASHINGTON (1995) Antibacteri-al susceptibility tests: dilution and disk diffusion methods. In P.R. Murray, E.J. Baron, M.A. Pfaller, P.C. Tenover and R.H. Yolken (Eds.) Manual of Clinical Microbiology. 6th ed. APHA. Washington, D.C.CFR (1972) Rules and Regulations. 37: 20525.NEUMAN, M.A., D.F. SAMM, C. THORNSBERRY, I.E. McGOWAN (1991) New developments in antimicrobial agent susceptibility testing: A practical guide. ASM. Washington, D.C.

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Ref. 01-131

SpecificationSolid culture medium for the maintenance, enumeration and chromogenesis of fungi.

Formula (in g/L)Soy peptone .............................................10,0Dextrose ...................................................10,0Agar ..........................................................17,0Final pH 7,0 ± 0,2

DirectionsSuspend 37 g of powder in 1 L of distilled water and heat to boiling. Dispense in flasks or tubes and sterilize in the autoclave at 121°C for 15 minutes. Should a selective medium by the acidic pH be desired, adjust the pH to 4,0 with a sterile solution of 10% lactic acid, and do not reheat the medium afterwards.

DescriptionMycological Agar is a general medium that provides enough nutrients for the development of most yeasts and moulds.It is employed in plates for the colonial isolation and characterization, as it aids chromogenesis. In the slants it is used for the maintenance of strains, because its low content of glucose yields very slow acid formation.

ReferencesAJELLO, GEORG, KAPLAN and KAUFFMAN (1963) CDC Lab Manual for Medical Mycology. PHS Pub. N° 994, Washington DC.ATLAS, M.R., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, London.VANDERZANT & SPLITTSTOESSER (1992). Compen-dium of Methods for the Microbiological Examination of Food. 3rd. Ed. APHA. Washington.CTFA Microbiological Guidelines (1993) The Cosmetic Toiletry and Fragance Association. Washington DC.

Mycological Agar

115

Ref. 02-512

SpecificationLiquid medium for neutralizing the antimicrobials accord-ing to the European Pharmacopoeia.

Formula (in g/L)Peptone ....................................................1,00L-Histidine HCl .........................................1,00 Lecithin .....................................................3,00Monopotassium phosphate ......................3,60Disodium phosphate .................................7,20Sodium chloride ........................................4,30Final pH 7,0 ± 0,2

DirectionsDissolve 20,1 g of powder in 1 L of distilled water con-taining 30 mL of Polysorbate 80 (Ref. 06-088). Distribute into suitable containers and sterilize by autoclaving at 121ºC for 15 minutes. Cool to 50ºC and homogenize the solution.

DescriptionNeutralizing Fluid is formulated according to the Euro-pean Pharmacopeia formulation for the microbiological examination of non sterile products. Its composition is the same as the general diluting solution for biological assays with the addition of polysorbate and lecithin as non toxic neutralizing agents. However, the European Pharmacopoeia lets the tech-nician to increase the concentration of polysorbate if the original is not enough or to add other agents when thepreservative type to be neutralized is not known. This way, the European Pharmacopeia suggests the compounds shown in the table below, which have to be always aseptically added to the fluid once sterilized and cooled to 50ºC or below.

ReferencesEuropean Pharmacopoeia (2002) 4th ed. Supplement 4.2.2.6.13. Tests for specified microorganisms. Council of Europe.Strasbourg.

Neutralizing Fluid Eur. Phar.

116

Ref. 01-137

SpecificationSolid medium for the isolation and identification of Can-dida sp.

Formula (in g/L)Yeast extract ...............................................1,0Dextrose ...................................................10,0Glycine .....................................................10,0Sodium sulfite .............................................3,0Ammonium Bismuth Citrate ........................5,0Agar ..........................................................15,0Final pH 6,8 ± 0,2

DirectionsSuspend 44 g of powder in 1 L of distilled water and heat to boiling. Dispense in tubes or dishes, stirring the precipitate before pouring. Do not autoclave. Avoid overheating.

DescriptionNickerson Agar is suitable for the isolation and identi-fication of yeast of the Candida type. Medium is made according to the general principles of Bismuth-Sulfite Agar, as inhibitor and differential agent, and completely selective with the high concentration of glycine. This medium is highly inhibitory, and does not allow bacterial growth, however most Candida grow freely and quickly. In some occasions, tiny colonies of the bacteria or highly repressed moulds may appear. Bacterial development may be totally prevented by adding neomycin sulfate to the medium before pouring it into Petri dishes and its concentration in the medium in this case must be around 2 mcg/mL, so that the antibiotic will not affect the devel-opment or appearance of yeast.

The appearance of the colonies in this medium after an incubation of 48-72 hours at 30-35°C is as follows:Candida albicans: Creamy colonies, very convex, circu-

lar with very slight mycelial border and black or dark brown colour. Neither it has metallic sheen nor the diffused pigment, even after 72 hours of incubation.

Candida tropicalis: Acuminated colonies, creamy, irregu-lar and with slight mycelial borders. Dark brown with black centre. After 72 hours of incubation it may take on a metallic sheen and produce a dif-fussed zone of pigment.

Candida krusei: Big and plain colonies, with irregular borders. Brown colour, darker in the centre. A yel-low halo appears around the colony.

Candida parakrusei: Plain colonies, average size, irregu-lar. Dark red centre and light red borders. Yellow mycelial border.

Candida pseudotropicalis: Big and plain colonies, dark red colour. Mycelial border.

Candida stellatoidea: Average size plain colonies, dark brown colour, without mycelial development.

Rhodotorula: Creamy convex colonies, with irregular border and colours ranging from pink to orange.

Moulds in general: Restricted colonial growth and cot-tony appearance.

To maintain these colony characteristics it is important that the medium is freshly prepared and not reheated or overheated.

ReferencesNICKERSON, W.J. (1953) Reduction of inorganic substance by yeast I. Extracellular reduction of sulfite by species of Candida. J.Inf.Dis 93:43.

Nickerson Agar (BiGGY)

117

Ref. 02-138

SpecificationA liquid culture medium, according to ISO 7932 stand-ard, to determine the ability of enterobacteria to reduce the nitrate to nitrites or free nitrogen gas.

Formula (in g/L)Meat extract ................................................3,0Peptone ......................................................5,0Potassium nitrate ........................................1,0Final pH 7,0 ± 0,2

DirectionsDissolve 9 g of powder in 1 L of distilled water, heating up only if necessary to help the dissolution. Distribute into final containers and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionThe Nitrate Broth is prepared according to classical formula for the assay of nitrate reduction by enterobacte-ria, although it can also be used with aerobic bacilli and other bacterial types.

TechniqueInoculate 2-3 tubes of broth with one loop of pure culture and incubate at 37°C, reading after 18-24 hours, 2 days and 5 days in each tube, adding some drops of Nitrate A Reagent (Ref. 06-003) and of Nitrate B Reagent (Ref. 06-004). If the first two readings are negative, it is rec-ommended to investigate with the third one for the pres-ence of nitrate by the method of zinc powder in order to have quick nitrate reduction reaction .

ReferencesDOWNES, F.P. & K. ITO (2001). Compendium of Meth-ods for the Microbiological Examination of Food.4th ed. APHA. Washington.F.D.A. (1998) Bacteriological Analytical Manual 8th ede. Rev. A. AOAC International, Gaithersburg.MDATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc.,London.ISO standard 7932 (1993) General guidance for the enumeration of B. cereus. Colony count at 30ºC.

Nitrate Broth

Nutrient Media

Nutrient Agar (APHA)

Ref. 01-144

SpecificationSolid culture medium for the general purposes according ISO standard.

Formula (in g/L)Peptone ......................................................5,0Meat extract ................................................3,0Agar ..........................................................15,0Final pH 7,0 ± 0,2

DirectionsSuspend 23 g of powder in 1 L of distilled water and heat to boiling. Dispense into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

Nutrient Agar (Brit. Phar.)

Ref. 01-140

SpecificationSolid culture medium for general purposes and less fastidious organisms acc. EN 12780:2002

Formula (in g/L)Meat extract ................................................1,0Yeast extract ...............................................2,0Peptone ......................................................5,0Sodium chloride ..........................................5,0Agar ..........................................................15,0Final pH 7,4 ± 0,2

DirectionsSuspend 28 g of powder in 1 L of distilled water and bring to the boil to dissolve completely. Sterilize by auto-claving at 121°C for 15 minutes.

DescriptionThe Nutrient Agar is a simple medium in the range of meat infusions, complemented by a formulation which reinforces its nutrient qualities as well as its growth fac-tors by adding yeast extract. It is most suitable for gen-eral routine work and can support the growth of common

118

organisms, even those considered mildly fastidious with regard to nutrient elements. Besides this, by incorporat-ing sodium chloride it allows the addition of blood, even though it is not an optimal medium for it.

Nutrient Broth (APHA)

Ref. 02-144

SpecificationLiquid medium for the cultivation of non fastidious micro-organisms according ISO standard.

Formula (in g/L)Peptone ......................................................5,0Meat extract ................................................3,0Final pH 7,0 ± 0,2

DirectionsDissolve 8 g of powder in 1 L of distilled water heating up only if necessary. Dispense into suitable containers and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionNutrient Broth is a modern version of the classical general culture medium based on meat infusion. It is a simple medium that may be used in general purposes (i.e. maintenance of strains) as well as a base for other specialized media. However, in this way, there are other media with more nutrient capacity and better perform-ance.Nutrient broth is the liquid version of the Nutrient Agar, and it is a classical medium for normal tasks with non fastidious microorganisms. It is the ideal medium for the subculture of general bacteria, especially staphyloco-cci, to carry out later the coagulase and other biochemi-cal tests. It may also be used to determine the Phenol Coefficient by following the technique and microorgan-isms suggested by the AOAC.

Nutrient Broth (Brit. Phar.)

Ref. 02-140

SpecificationA general purpose liquid culture medium for the less fastidious microorganisms.

Formula (in g/L)Meat extract ................................................1,0Yeast extract ...............................................2,0Peptone ......................................................5,0Sodium chloride ..........................................5,0Final pH 7,4 ± 0,2

DirectionsDissolve 13 g of powder in 1 L of distilled water, heating if necessary to help dissolve the medium. Distribute into final containers and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionThe Nutrient Broth is the liquid version of the solid me-dium which bears the same name. It is a classical broth in the range of meat infusions. It is useful for the routine laboratory purposes since its yeast extract supplement allows the growth of most common organisms. It is also suitable for the preparation of inocula and for the ef-ficiency testing of bactericides, as well as for determina-tion of the Phenol Coefficient and others.

Nutrient Broth No. 2

Ref. 02-561

SpecificationLiquid medium for general purposes.

Formula (in g/L)

Meat Extract ..........................................10,00Peptone ..................................................10,00Sodium chloride ........................................5,00Final pH 7,5 ± 0,2

DirectionsDissolve 25 g of the powder in 1 L of distilled water, heating if necessary. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

DescriptionThis medium in the classical way of the meat infusions, presents a specially rich nutritional characteristics that facilities the growth of very low inocula, even with fastidi-ous microorganisms. Its formulation is according the BSI for the determination of Rideal-Walker Coefficient of disinfectants, where it is used at double concentration.

Nutrient DEV Agar

Ref. 01-451

SpecificationSolid culture medium for the total enumeration of micro-organisms in water, according to the German legislation and ISO standard.

Nutrient Media

119

Formula (in g/L)Meat peptone ...........................................10,0Meat extract ..............................................10,0Sodium chloride ..........................................5,0Agar ..........................................................18,0Final pH 7,3 ± 0,2

DirectionsSuspend 43 g of powder in 1 L of distilled water. Bring to the boil with constant stirring . Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionThis medium is formulated according to the German legislation but it differs from the other Anglo-Saxon media with the same name in the concentration of its compounds. This change intends to aid the recovery and growth of damaged microorganisms.

TechniqueGerman standards state a deep inocule of the water sample, following the mass seed technique, directly in the Petri plate. Incubation is performed at 20±2°C for 44±4 hours in most of the cases, but incubations at 37±1°C for the same period of time are also allowed. If the water is chlorinated, the incubation time must last up to 72 hours.

ReferencesAPHA (1948) Standard Methods for the Examination of Dairy Products. Washington.BRITISH PHARMACOPOEIA (1968), 357.BRITISH STANDARD 541 (1934). Determining the Ri-deal-Walker Coefficient of Disinfectants. BSI London 9.

DOWNES F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food.4th ed APHA. Washington.ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc. London.EUROPEAN STANDARD EN 12780:2002 Water Quality. Detection and enumeration of Pseudomonas aeruginosa by membrane filtrationBUNDESGESUNDHEITAMT: Amtliche Sammlung von Untersuchungsverfahren nach §35 LMBG. Beuth Verlag Berlin- Köln.VERORDNUNG von 12/12/1990 über Trinkwasser und über Wasser fur Lebensmittelbetriebe. Bundesgesetz-blatt: Teil I:2613-2629.DEUTSCHE EINHEITSVERFAHREN zur Wasser-, Abwasser- Und Schlammuntersuchung. VCH Verlags-gesellchaft, D-6940 Weinheim.ISO 8523 standard (1991) General guidance for the detection of enterobacteriaceae with pre-enrichment.ISO 6785 standard (2001) Milk and milk-products - De-tection of Salmonella spp.ISO 6340 standard (1995) Water Quality - Detection of Salmonella species.ISO 6579 standard (2002) Horizontal method for the detection of Salmonella spp.ISO 10273 standard (1994) General guidance for the detection of presumptive pathogenic Yersinia enteroco-litica.ISO 21567. Standard (2004) . Horizontal method for the detection of Shigella ssp.ISO 16266:2006 Standard. Water Quality.– Detection and enumeration of Pseudomonas aeruginosa. Method by membrane filtration

Nutrient Media

Nutrient Gelatin Media

Nutrient Gelatin

Ref. 03-088

SpecificationCulture medium for determination of gelatin liquefaction.

Formula (in g/L)Meat extract ................................................3,0Gelatin peptone ..........................................5,0Gelatin ....................................................120,0Final pH 6,7 ± 0,2

DirectionsSuspend 128 g of powder in 1 L of cold distilled water and heat gently in boiling bath up to 50°C. Keep at that temperature until total dissolution of gelatin. Dispense in tubes or flasks and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionGelatin Nutrient is used, essentially, to identify pure cultures that have no specific nutritional requirements. On the other hand, gelatin liquefaction is considered very important to differentiate enteric bacilli on the basis of their proteolysis. Gelatin was one of the first solidify-ing agents employed in bacteriology, and helped in the development of the Plate Count Technique, performed by Koch. Nonetheless, nowadays it is not used in that way, since it was replaced by Agar, which bears incuba-tions at higher temperatures and was not so attacked or degraded as gelatin.

However, the Plate Count Method is still used with gelatin. Standard Methods still recommend it for aerobic counting at 20-22°C.

120

TechniqueThe gelatin liquefaction test may be performed in plates or tubes, that are inoculated by stabbing,and incubated at 20-22°C or at the optimum temperature for the micro-organism to assay. Because gelatin is liquid above 20°C, it is necessary to put the plates or tubes in the refrigera-tor before reading.High temperature assays (35-37°C) eliminate wrong results from bacteriolysis, but they require more care in readings and also inclusion of a control (uninoculated) in order to verify effect of heat on the medium.

Gelatin tube readings must be carried out carefully, since often they have diagnostic value. In the case of using several plates, Stone’s reaction may be used to include different strains in the same dish, seeding them by paral-lel streak. After incubation period, cool in the refrigerator and then, over each streak, put a few drops of saturated solution of Ammonium sulfate or Sulfosalicilic acid 20% solution (freshly prepared).

Liquefaction (positive test) is shown by the presence of a clear halo or zone around the growth, 10 minutes after reagent addition. Incubation periods for gelatinase activity assay vary, ranging from a few days to weeks, at 20-22°C. Some Klebsiella and Enterobacter strains take up to 3 weeks before they show activity. Though incubation times are not standarized, it is recommended a maximum of 14 days with regular intermittent readings every 3 days.A modern version of this methodology is the usage of photographic film strips, not exposed, for the liquid me-dia, but this te chnique has a drawback that sometimes photosensitive particles included in the film are toxic to the microorganisms, and then they give false - negative results.

ReferencesAPHA/AWWA (1995) Standard Methods for the Exami-nation of Water and Wastewater. 19th. Ed. APHA Inc.New York. 596-597.ASM (1981) Manual of Methods for General Bacteriol-ogy, ASM, Washington, D.C.

Nutrient Gelatin DEV

Ref. 03-453

SpecificationCulture medium for the enumeration of total bacteria in not very polluted waters, according to the German legislation.

Formula (in g/L)Gelatin ..................................................120,00Meat extract ............................................10,00Meat peptone .........................................10,00Sodium chloride ........................................5,00Final pH 7,3 ± 0,2

DirectionsSuspend 145 g of powder in 1 L of cold distilled water and heat up, in boiling water bath, to 50-60°C. Maintain this temperature until total dissolution. Distribute into containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionThis medium has the same applications as the Gelatin Nutrient (Ref. 03-088) recommended by the APHA, AWWA and Standard Methods. The only difference is the concentration of nutrients and the inclusion of sodium chloride.

TechniqueGerman legislation states that water samples are inocu-lated by deep inoculum (in mass) in Petri plates. Incuba-tion is performed at 20±2°C for 44±4 hours. After the incubation, carry out the counting of total bacteria. If water is chlorinated, incubation must be 24 hours more in order to let damaged cells recover and form visible colonies.

ReferencesDEUTSCHE EINHEITSVERFAHREN zur Wasser-, Abwasser- Und Schlammuntersuchung. VCH Verlags-gesellschaft D-6940 Weinheim.

Nutrient Gelatin Media

121

Ref. 03-037

SpecificationFluid medium according to the Hugh and Leifson formu-lation, for determining the oxidative and/or fermentative metabolism of gram-negative bacilli.

Formula (in g/L)Casein peptone ........................................2,00Sodium chloride ........................................5,00Dipotassium phosphate ............................0,20Bromothymol Blue ....................................0,08Agar ..........................................................2,50Final pH 7,1 ± 0,2

DirectionsSuspend 9,8 g of powder in 1 L of distilled water and bring to the boil. Add sugar in the desired concentration and distribute in fermentation tubes. Add the vaseline seals or vaspar to half of them. Sterilize by autoclaving at 121°C for 15 min.

DescriptionHugh and Leifson obtained a clear differentiation of gram-negative bacteria with this medium. They classi-fied them into three categories: fermentative, oxidative and inactive. The strain to be studied is inoculated in two long narrow tubes (12x120 mm) by deep stab and one is covered with a vaseline layer to induce an anaerobic environment that forces the strain to carry out the fer-mentative metabolism.Fermentative organisms give a copious production of acid in both the tubes, and it is indicated by the yel-

low colouration of the bromothymol blue indicator. The bacteria following the oxidative metabolism carry out this reaction only in the tube without the vaseline but in the other one, which is closed, they change insignificantlly or simply do not grow. Inactive strains do not use sugars and therefore do not induce any change in both the tubes. However, some times there is a slight blue col-ouration in the open tube, probably due to alkalinization by peptone degradation.Some authors have proposed the usage of just one tube for this assay, but in that case the medium must be modified to be the solid (with 1,5% Agar) and with yeast and/or cystine extract. In these tubes the stab must be, at least, 8 cm.Hugh and Leifson recommend simultaneous assay with glucose, lactose and sucrose of 1% concentration, add-ing the sterilized sugars to the medium by filtration.

ReferencesHUGH, R. and E. LEIFSON, (1953) The taxonomic significance of fermentative vs. oxidative metbolism of cabohidrates by various gram negative bacteria J.Bact. 66:24ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological media. CRC Press, Inc.LondonFDA (1998) bacteriological Analitycal Manual. 8th ed. Rev. A. AOAC International. Gaithersburg. MDDOWNES, F.P. & K. ITO (2001) Compendium of meth-ods for the microbiological examination of food. 4th ed. APHA WashingtonISENBERG, H.D. (1992) Clinical Microbiology Proce-dures Handbook Vol I ASM Press. Washington

Oxidation-Fermentation Fluid Medium (O/F Medium)

Ref. 01-500

SpecificationSolid culture medium used as seed agar in the inhibitory substances test in milk.

Formula (in g/L)Meat extract ..............................................3,00Peptone ....................................................5,00Soy peptone .............................................0,30Dextrose ...................................................5,25Polysorbate 80 .........................................1,00Sodium chloride ........................................0,50Dipotassium phosphate ............................0,25Bromcresol purple ....................................0,06Agar ........................................................15,00Final pH 7,8 ± 0,2

P Medium Agar

DirectionsSuspend 30,36 g of powder in 1 litre of distilled water and bring to the boil. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

DescriptionThe present formulation is according the Food and Drug Administration Bacteriological Analytical Manual for the investigation of Inhibitory substances (Antibiotics and preservatives) in milk. This medium that in previous edi-tions of the BAM was called “PM Indicator Agar”, is used as seed agar with B. stearothermophilus spores in the qualitative method or disk assay.

ReferencesMATURIN, L.J. (1998) Inhibitory substances in milk. Qualitative Method II: B. stearothermophilus disk assay. In FDA Bacteriological Analytical Manual. 8th Ed. Revi-sion A. AOAC International Inc. Gaithersburg MD.

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Ref. 01-570

SpecificationSolid culture medium used for the enumeration of contaminants in dairy products, according the FIL-IDF standard

Formula (in g/L)Casein Peptone ........................................7,50Gelatine Peptone ......................................7,50Sodium chloride ........................................5,00Agar ........................................................15,00Final pH 7,5 ± 0,2

DirectionsSuspend 35 g of powder in 1 L of distilled water and bring to the boil. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

DescriptionPeptone Agar is produced according the formulation of the FIL-IDF for its use in the detection and enumera-tion of non-lactic contaminants in butter, fresh cheese and fermented milk. All the components of the medium are sugar-free and the pH is adjusted to 7,5 that’s is the used for cheese and butter. If the sample is fermented

Peptone Agar

milk, the medium will be adjusted to 8.00 with 0,1N NaOH sterile solution after the sterilization.

TechniqueThe inoculum or its dilution (in duplicate) is deposited over the surface of the medium in the plate in volumes of 0,1 mL. The inocula are spreaded quickly with a Drigalski rod (Ref. 5-010) and let stand 15 minutes to be absorbed in the medium. The plates are incubated at 30ºC for 72±2 hours. Select plates with less than 150 colonies to the count. In the counting the needle-bite colonies are not considered because they are probably lactic bacteria. The contaminant must be confirmed by its active catalase.For the sampling, processing and dilution of the products refer to the corresponding FIL-IDF standard.

ReferencesFIL-IDF (1991) Provisional Standard 13: Butter, fresh cheese and fermented milk. Enumeration of non-lactic contaminants. Plate Count at 30ºC technique.

Ref. 02-568

SpecificationLiquid non-selective pre-enrichment medium for entero-bacteria

Formula (in g/L)Meat Peptone .........................................10,00Sodium chloride ........................................5,00Disodium phosphate .................................3,50Monopotasium phosphate ....................... 1,50Final pH 7,2 ± 0,2


DescriptionThis medium is produced according the formulation of the Schwezerisches Lebensmittelbuch and is recom-mended as non-selective pre-enrichment for sub-lethally damaged cells of the enterobacteria group in food or in others samples.

Peptone Water Phosphate-Buffered

ReferencesBEKERS, H.J. (1987) Studies with salmonellae. J. Appl. Bact. 62:97-112SCHWEIZERISCHES LEBENSMITTELBUCH (1992) 5th ed. Chapter 56A

123

Ref. 02-032

SpecificationLiquid culture media,suitable for the sugar and other substrate fermentation studies according ISO 10273 standard.

Formula (in g/L)Casein peptone ....................................10,000Sodium chloride ......................................5,000Phenol red ..............................................0,018Final pH 6,8 ± 0,2

DirectionsDissolve 15 g of powder in 1 L of distilled water. Add sugar in the desired concentration and distribute into suitable containers with Durham’s tubes. Sterilize in the autoclave at 121°C for 10 minutes. Heat up the autoclave before putting the tubes into it to avoid sugar caramelization. Addition of some kinds of sugars may need a pH adjustment.

DescriptionPhenol Red Base Broth is a liquid version of the agar base for the fermentation studies, which is preferred by many authors to use with Durham’s tubes inclusion, to verify the gas production.

Sugar addition can be done in sterile solution after autoclav-ing the medium, or by adding impregnated discs to 10 mL of medium. Addition of some sugars may cause the acidifica-tion of the medium, in which case the original pH must be maintained by adding a few drops of 0,1 N NaOH.Should you be working with anaerobics, it is advisable to use a freshly prepared medium, or put the medium in boiling water bath for a few minutes, in order to eliminate dissolved oxygen. Many authors recommend the addition of 0,04% agar for these purposes to avoid convection streams and subsequent incorporation of the air.To study the sugar fermentations of enterobacteria, Brom-cresol Purple Base Broth (Ref. 02-031) is more suitable, as it is a better indicator of choice which is less toxic than the phenol red.

ReferencesISO 10273 Standard (1994) General guidance for the detec-tion of presumptive pathogenic Yersinia enterocolitica.ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press,Boca Raton,Fla.DOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food. 4th ed. APHA. Washington.FDA (1998) Bacteriological Analytical Manual 8th ed. Rev. A. AOAC International. Gaithersburg. MD

Phenol Red Broth Base

Ref. 01-083

SpecificationCulture medium for Enterobacteria, according Ewing et al. formulation.

Formula (in g/L)Yeast extract ...............................................3,0DL-Phenylalanine .......................................2,0Di-sodium phosphate .................................1,0Sodium chloride ..........................................5,0Agar ..........................................................15,0Final pH 7,3 ± 0,2

DirectionsSuspend 26 g of powder in 1 L of distilled water and heat to boiling. Dispense in tubes or flasks and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionThis formulation corresponds to the solid form, proposed by Ewing et al. which is a modification of the medium developed by Buttiaux et al. in order to achieve the green colour that confirms the positive reaction which lasts longer.Capacity to deaminate the phenylalanine oxidatively to convert it in phenylpiruvic acid is property of the Proteus type in enterobacteria. Phenylalanine is revealed by

the presence of a characteristic greenish colour in the medium when it reacts with iron. Nowadays, this test and the urease production test, have a lot of importance in the taxonomy of Proteus type .

TechniqueA recommended technique is the following:Inoculate the slant surface with plenty of inoculum, and incubate it for 12-16 hours. Add 0,2 mL of 10% ferric chloride solution so that the solution floods all over the growth.Phenylpiruvic acid presence (positive test) is shown by the presence of a characteristic green-blue colour on the surface, after approximately 1 minute.

ReferencesATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc.LondonBUTTIAUX,R., R. OSTEUX, R. FRESNOY & J. MO-RIAMEZ (1954) Les propietés biochimiques du genre Proteus.Ann.Inst. Pasteur 87:357-386EDWARDS and EWING (1973). Identification of Entero-bacteriaceae. Burges Pub.Cod. Minneapolis.ISENBERG, H.D. (1992) Clinical Microbiology Proce-dures Handbook. Vol I ASM Press Washington.

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Plate Count Agar (PCA)

Ref. 01-161

SpecificationMedium for the aerobic plate count by surface inocula-tion method (Standard Plate Count Agar) according ISO 4833 and 17410 standards.

Formula (in g/L)Casein peptone ..........................................5,0Yeast extract ...............................................2,5Dextrose .....................................................1,0Agar ..........................................................15,0Final pH 7,0 ± 0,2

DirectionsSuspend 23,5 g of powder in 1 L of distilled water. Dissolve by bringing to the boil with frequent stirring. Dis-tribute into final containers and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionThe Plate Count Agar follows the directions given by Buchbinder et al. in their study about media for the plate count of microorganisms.The original formulation of the standardized agar for dairy microbiology has been modified in order to avoid the addition of milk. This new composition allows the growth of most microorganisms without any further ad-ditions.This medium’s formulation is equivalent to that pre-scribed by the ‘Standard Methods for the Examination of Dairy products’, the USP’s ‘Tryptone Glucose Yeast Agar’, the ‘Deutsche Landswirtchaft’ and to the APHA and AOAC’s Plate Count Agar. Nowadays this is the medium selected for the plate count of any type of the sample.

Plate Count Modified Agar

Ref. 01-329

SpecificationModification of Plate Count Agar (Ref. 01-161), with a lesser amount of agar, especially recommended for the aerobic enumeration in plates, by the poured plates method.

Formula (in g/L)Casein peptone ..........................................5,0Yeast extract ...............................................2,5Dextrose .....................................................1,0Agar ............................................................9,0Final pH 7,0 ± 0,2

DirectionsDissolve 17,5 g of powder in 1 L of distilled water. Heat to the boiling by constant stirring. Distribute in the suit-able containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionPlate Count Modified Agar follows the same specifica-tions as Plate Count Agar, with the exception that of the agar concentration has been reduced. This modification provides a better growth of colonies if massive inocula-tion method is used, as the medium is softer and hence the colony expansion is improved.

Plate Count Skim Milk Agar

Ref. 01-412

SpecificationSolid medium for the plate counts of milk and dairy prod-ucts, according to DIN and FIL/IDF standards.

Formula (in g/L)Casein peptone ..........................................5,0Yeast extract ...............................................2,5Skimmed milk .............................................1,0Dextrose .....................................................1,0Agar ..........................................................10,5Final pH 7,0 ± 0,2

DirectionsSuspend 20 g of powder in 1 L of distilled water and let it soak . Bring to the boil with constant stirring. Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionThis medium, with the added milk, has a major nutri-ent richness than other standard media, however, the opalescence of the medium makes early observations sometimes difficult.Due to its lesser agar concentration, it may be used by the pouring plate method or by the surface inoculation method.

TechniquePrepare a decimal dilution bank of the sample and take 1 mL in duplicate from each dilution and put them in sterile Petri plates. Pour 20 mL approx. of sterile cooled medium (around 47°C) in each of the plates. Mix gently by moving the plate in eight (8) shape. Leave the plates undisturbed to solidify and incubate in inverted position. Time and temperature of incubation depend on the type of microorganism under study. For a general aerobic count, incubate for 3 days at 30°C, by observing also after 24 and 48 hours.The plate count method proposed by the APHA consists of a massive inoculum by pouring the molten agar at 50°C on plates containing the diluted samples. The final

Plate Count Media

125

count is carried out after 48 hours of incubation at 32-35°C. As for the microorganisms with other temperature requirements, the following incubations have been sug-gested: 2 days at 32-35°C, 2-3 days at 45°C, 2 days at 55°C, 3-5 days at 20°C, 7-10 days at 5-7°C.Sample dilutions are prepared with solutions of 1/4 of Ringer solution (Ref. 06-073), 1% of Peptone Water(Ref. 03-156) or 0,1M of phosphate buffer at pH 7,0; depend-ing on their nature.The poured plate count method is preferred to the surface inoculation method, since it gives higher results. Nevertheless, the latter gives a more appropriate isola-tion of the colonies.

ReferencesMARSHALL, R.T. (1992) Standard Methods for the Examination of Dairy Products, 16th Ed. APHA. Wash-ington.CLESCERI. L.S., A.E. GREENBERG, and A.D. EATON (1998) Standard Methods for the Examination of Water and Wastewater, 20th ed.. APHA, AWWA, WEF. Wash-ington.DOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food. 4th. Ed. APHA. Washington.HORWITZ, W. (2000) . Official Methods of Analysis. AOAC International. Gaithersburg

DIN 10192 Standard. Prüfungesbestimmungen für Milch und Milcherzeugnisse (Deutsche Landwirtsachft, Fach-bereit und Ernahrung). 1971.FIL/IDF Standards 3 (1958), 100 (1981), 101 (1981), 109 (1982) and 132 (2004).PASCUAL ANDERSON, MªRª (1992) Microbiología Alimentaria. Diaz de Santos, S.A.,Madrid,SPAIN.ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc.,Boca Raton,Fla.BUCHBINDER, L., Y. BARIS, L. GOLDSTEIN (1953) Further studies on new milk-free media for the stand-ard plate count of dairy products. Am. J. Public Health 43:869-872.ISO 4833 Standard (2003) Microbiology of food and ani-mal feeding stuffs. Horizontal method for the enumera-tion of microorganisms. Colony count technique at 30°C.ISO 17410 Standard (2001) Horizontal Method for the enumeration of psychrotrophic microorganisms.ISO 8552 Standard (2004) Milk - Estimation of psychro-trophic microorganisms - Colony-count technique at 21°C (Rapid method).

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Potato Dextrose Agar

Ref. 01-483

SpecificationSolid culture medium for the detection and enumeration of yeast and moulds in food, specially recommended in butter and other dairy products.

Formula (in g/L)Potato peptone .........................................4,00Glucose ..................................................20,00Agar ........................................................15,00Final pH 5,6 ± 0,2

DirectionsSuspend 39 g of powder in 1 L of distilled water and bring to the boil. Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes. Do not overheat.

DescriptionPotato Dextrose Agar is a weakly selective medium for fungi due to its high sugar content and acidic pH. The pigment production and aerial mycelium development is enhanced by the potato peptone, specially in the Fusarium, Aspergillus and Penicillium species.The selectivity can be increased by adding antibacterial antibiotics like chloramphenicol or tetracyclines, or by simply decreasing the pH to an acidic level. At pH 3,5 the bacterial growth is almost totally inhibited without significant effect on fungi. This acidification can be obtained by the aseptic addition of an adequate amount of organic acid to the medium after sterillization: 10-15 mL/L of a 10% sterile solution of tartaric or lactic acid is usually sufficient.After its acidification the medium should not be over-heated or reheated since it can hydrolyze the agar and hence there can be a loss in solidification property of the medium.

TechniqueDistribute the diluted samples into sterile petri plates. Pour the molten agar melted cooled to 45-50°C and gen-tly mix to homogenize the mixture. After the solidification, plates are incubated for 5-7 days at 20-25°C to permit the complete development of the fungal colonies.The weak consistency of the agar due to its original acidity makes this medium inadequate for streaking.

Potato Dextrose Broth

Ref. 02-483

Specification Liquid culture medium for the maintenance and multipli-cation of yeast.

Formula (in g/L)Potato peptone ...........................................4,0Glucose ....................................................20,0Final pH 5,6 ± 0,2

DirectionsDissolve 24 g of powder in 1 L of distilled water, heating up only if necessary. Distribute into suitable containers and sterilize by autoclaving at 121ºC for 15 minutes.

DescriptionPotato Dextrose Broth is the liquid version of the agar with the same name. This broth is mainly used to detect and enumerate yeast and moulds, since it does not contain any solidifying agent it may be acidified without altering its physical properties.At pH 3,5, the bacterial growth is totally inhibited without significant influence on fungi. This acidification may be achieved by the aseptic addition of an adequate amount of organic acid to the medium after sterilization:10-15 mL/L of a 10% sterile solution of tartaric or lactic acid. This addition may also be made before sterilization, but it must be considered that in acidic conditions Maillard reactions are strong and hence the medium may turn slightly brownish.

ReferencesATLAS, R.M. & PARKS,L.C. (1995) Handbook of Microbiological Media for the Examination of Food. CRC Press, London.RICHARDSON, G. H. (1985) Standard Methods for the examination of dairy products.15th Ed. APHA Washing-ton.DOWNES, F.P. & K. ITO (2001) Compendium of meth-ods for the microbiological examination of food. 4thEd. APHA WashingtonUS PHARMACOPOEIA (2002) 25th ed. <61> Microbial Limit Test. US Pharmacopoeial Convention Inc. Ltd. Rockville. MDFDA (1998) Bacteriological Analitycal Manual. 8th ed. Rev. A. AOAC International. Gaithersburg. MD.

Potato Dextrose Media

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Ref. 01-540

SpecificationSolid medium for the enumeration of heterotrophic micro organisms in treated waters

Formula (in g/L)Yeast Extract ..........................................0,500Proteose peptone ...................................0,500Casein hidrolysate ..................................0,500Glucose ..................................................0,500Starch .....................................................0,500Dipotassium hydrogen phosphate ......... 0,300Magnesium sulphate, anhydrous .......... 0,024Sodium pyruvate ...................................0,300Agar ......................................................15,000Final pH 7,2 ± 0,2

DirectionsSuspend 18,1 g of powder in 1 L of distilled water and bring to the boil with constant stirring. Distribute into suit-able containers and sterilize by autoclaving at 121ºC for 15 minutes.

Description The R2A Agar was proposed in 1979 by Reasoner and Geldenreich and few years later accepted by the APHA as an alternative medium for stressed cells in treated potable water. The use of nutrient rich media like PCA or TSA allows to the growth of normal microbiota, but do not permits the recuperation of the stressed or chlorine resistant biota. By the use of a medium like R2A of low nutrients in com-bination with a lower temperature and longer incubation time it is possible induce the resuscitation of this dam-aged cells. In the R2A Agar the source of nitrogen is the peptone and the Yeast Extract supplies the vitamins and growth factors. The source of carbon is the dextrose and mag-nesium sulphate and potassium phosphate maintains the osmotic pressure. The starch is a detoxifier and sodium piruvate increases the recuperations of stressed cells. The agar acts as gelling agent.

R2A Agar (Eur. Phar. Medium S)

TechniqueThe water sample must be processed as quickly as possible. If it is no possible within the first 6 hours, the sample must be refrigerate, but not for more than 30 hours: then the sample is rejected. R2A Agar is used with pour plates, streak plates or filtration but must be keep in mind that the pour plates method can affect the recovery capacity of the medium because the thermal shock. The incubation period at 35ºC is of 3-5 days but is more effective a incubation temperature of 20-28ºC an a time of 5-7 days. In any case the plates must be protected against an excessive drying.The fast-growing or non-stressed microorganisms in these conditions of incubation produce different and minute colonies than in the rich media.

ReferencesATLAS, R.M. (1995) Handbook of Media for Environ-mental Microbiology. CRC Press. Boca Raton USA.EATON, A.D., A.E. GREENBERG and L.S. CLESCERI (1995). Standard Methods for the Examination of Water and Wastewater. 19ª Ed. APHA Washington D.C. USA .EUROPEAN PHARMACOPOEIA 4th Ed. Suppl. 4.6 (2004) 2.6.13 Test for specified Microorganisms (pg 2621)GREENBERG, A.E., R.R TRUSSELL and L.S. CLESCERI (1985). Standard Methods for the Examina-tion of Water and Wastewater. 16ª Ed. APHA-AWWA-WPCF Washington D.C. USA REASONER, D.J. and E.E. GELDREICH (1979) A new medium for the enumeration and subculture of bacteria from potable water. Abstracts of Annual Meeting . ASM 79th Meeting. Paper #N7. Van SOETSBERGER, A.A. and C.H. LEE (1969) Pour plates or streak plates? Appl. Microbiol 18:1092-1094.

128

Rappaport Vassiliadis Broth

Ref. 02-379

SpecificationLiquid medium for the selective enrichment of Salmo-nella in foodstuffs and other materials.

Formula (in g/L)Soy peptone ...........................................4,500Sodium chloride ......................................7,200Monopotassium phosphate ....................1,260Dipotassium phosphate ..........................0,180Magnesium chloride ............................13,580Malachite green ......................................0,036Final pH 5,2 ± 0,2

DirectionsDissolve 26,8 g of powder in 1 L of distilled water, heat-ing if necessary to help dissolve the powder. Dispense into test tubes or flasks and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionThe Rappaport Vassiliadis medium complies with the recommendations of the APHA for the examination of food.This culture medium is the modificaction of the R10 me-dium (from Rappaport et cols) or RV broth (from Vas-siliadis et cols.)by van Schothort & Renaud. The modi-fications are an adjustement in the magnesium chloride concentration and a buffered reaction of the medium. It shows a higher selectivity towards Salmonella and produces better yields than other similar media, espe-cially after preliminary enrichment and at an incubation temperature of 43°C.Malachite green and magnesium chloride inhibit the growth of the microorganisms normally found in the in-testine but do not affect the proliferation of most Salmo-nellae. Malachite green inhibits the growth of Shigella. Soy peptone improve the growth of Salmonella. The low pH of the medium increases the selectivity.

TechniqueInoculate the culture medium with the sample or mate-rial from a pre-enriched culture in Buffered Peptone Water (Ref. 02-277) and incubate for up to 18-24 hours at 41±1°C. Streak the sample material from the resulting cultures onto selective culture media.

ReferencesATLAS, R.M., LC. PARKS (1993) Handbook of Microbio-logical Media. CRC Press Inc.,LondonVASSILIADIS,P (1983) The Rappaport-Vassiliadis(RV) enrichment medium for the isolation of salmonellas: An overview. J.Appl.Bact.54;54, 69-76.

VASSILIADIS, P, PATERAKI, EPAPAICONOMOU,N , PAPADAKIS, J.A. A. TRICHOPOULOS, D (1976) Nouveau procédé d’enrichissement de Salmonella. Ann. Microbiol. (inst. Pasteur) 127B (195-200)RAPPAPORT, F. N. KONFORTI & B. NAVON (1956) A new enrichment medium for certain salmonellae. J. Clin Pathol. 9:261-266VAN SCHOTHORST M. & A.M. RENAUD (1983) Dynamics of salmonellae isolation with modified Rappa-port’s medium (R10) J.Appl. Bact. 54: 209-215FIL-IDF Standard 93B:1995. Milk and Milk products. Detection of Salmonella. BrusselsFDA (1998) Bacteriological Analytical Manual 8th ed. Rev A. AOAC International. Gaithersburg. MD.HORWITZ, W. (2000) Official Methods of Analysis. AOAC International. Gaithersburg. MDDOWNES, F.P. & K. ITO (2001) Compendium of meth-ods for the microbiological examination of foods. 4th ed. APHA Washington.

Rappaport Vassiliadis Modified Semi-Solid Medium Base (MSRV)

Ref. 03-376

SpecificationSemi-solid medium for the isolation of mobile strains of Salmonella.

Formula (in g/L)Tryptose .................................................4,590Casein Peptone ......................................4,590Sodium chloride ......................................7,340Mono-Potassium phosphate ..................1,470Magnesium chloride .............................10,930Malachite green ......................................0,037Agar ........................................................2,700Final pH 5,2 ± 0,2

DirectionsSuspend 31,6 g of powder in 1 L of distilled water. Heath in a water bath until boil and complete dissolution. Cool to 50ºC an add 20 mg/L of Novobiocin. Without autoclav-ing nor reheating, homogenize and pour plates. Keep plates in a fresh place to settle the gel (1 hour minimum) and handle it with care because the medium is only semi-solid. It is recommended to keep MSRV plates in a cooler at 2-8ºC at the dark.

Description The Modified Semi-Solid Rappaport-Vassiliadis Medium Base is formulated according DeSmedt and cols. That shows its higher efficiency over the traditional enrich-ment methodology.

Rappaport Vassiliadis Media

129

The rapid migration of mobile strains of Salmonella in the semisolid medium allows to the early detection by the production of an halo of growth around the inocula-tion zone.The other competitive mobile organisms are inhibited by the novobiocin, the malachite green and the high concentration of magnesium chloride.The low concentration of agar produces a very soft and fragile gel but, at the temperature of incubation (42ºC), it is an special environment in which the mobile strains of Salmonella moves easy and quickly.

Technique 1. Three drops (~0,1 mL) of a pre-enrichment culture

are inoculated in a three different spots on the dry surface of the medium in a room-temperate plate.

2. Incubate the plates aerobically in an upright position for no longer than 24 hours at 42ºC.

3. The formation of a turbid or opaque halo around the initial inoculation zone shows the presence of mobile salmonellae.

4. To confirm the purity of the isolation and to follows with the identification tests, samples of the external border of the halo can be used.

Rappaport Vassiliadis Media

5. To prevent false negatives results due to the ab-sence of mobile strains of Salmonella is convenient to performs simultaneously a traditional enrichment in liquid medium.

ReferencesDe SMEDT, J.M., R. BOLDERDJIK, H. RAPPOLD and D. LAUTENSCHLAEGER (1986). Rapid Salmonella detection in foods by motility enrichment on a Modi-fied Semi-Solid Rappaport-Vassiliadis Medium. J. Food Protect. 49:510-514De SMEDT, J.M. and R. BOLDERJIK (1987) Dynamics of Salmonella isolation with Modified Semi-Solid Rappa-port-Vassiliadis Medium. J. Food Protect. 50:658-661HOLBROOCK, R., J.M. ANDERSON, A.C. BAIRD-PARKER, L.M. DODDS, D. SAWHNEY , S.H. STRUCH-BURY and D. SWAINE (1989) Rapid detection of Salmo-nella in food: A convenient two-day procedure. Lett. Appl. Microbiol. 8:139-142

Reinforced Clostridial Media

Reinforced Clostridial Agar

Ref. 01-289

SpecificationSolid medium for the cultivation and enumeration of clostridia and other anaerobic bacteria.

Formula (in g/L)Casein peptone ........................................10,0Yeast extract ...............................................3,0Meat extract ..............................................10,0Dextrose .....................................................5,0Sodium chloride ..........................................5,0Sodium acetate ..........................................3,0Soluble starch .............................................1,0L-Cysteine HCl ...........................................0,5Agar ..........................................................15,0Final pH 6,8 ± 0,2

DirectionsSuspend 52,5 g of powder in 1 L of distilled water and heat to boiling with constant stirring. Distribute into suit-able containers and sterilize in the autoclave at 121°C for 15 minutes.

Reinforced Clostridial Medium(RCM) (Eur. Phar. Medium P)

Ref. 03-289

SpecificationFluid medium for the cultivation and enumeration of clostridia by the MPN method.

Formula (in g/L)Casein peptone ........................................10,0Yeast extract ...............................................3,0Meat extract ..............................................10,0Dextrose .....................................................5,0Sodium chloride ..........................................5,0Sodium acetate ..........................................3,0Soluble starch .............................................1,0L-Cysteine HCl ...........................................0,5Agar ............................................................0,5Final pH 6,8 ± 0,2

DirectionsSuspend 38 g of powder in 1 L of distilled water and heat to boiling with constant stirring. Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

130

DescriptionReinforced Clostridial Agar was originally described by Hirsch and Grinstead to initiate the growth of small in-oculums and get a higher Clostridial count. Later, Barnes and Ingram used the medium to develop vegetative cells in assays of Clostridium perfringens. Barnes also used this medium to count clostridia in food, moreover other authors used this medium in enumeration assays of Cl. thermoscharolyticum in sugar, study of intestinal flora, and bacterial count in human or animal faeces, etc.For the enumeration by the MPN method, the liquid ver-sion is the preferred one.

TechniqueMaterial to be examined is ground in a Turmix or Stom-acher, and a decimal dilution bank is prepared. From each of the dilutions, take an aliquote to Petri plates or tubes, and pour the molten medium at 50°C over them. Let it solidify. Incubate at 30-55°C (depending on the microorganism that is anticipated to be found) for 1-10 days. An anaerobic environment can be achieved if tubes are used and they are covered with Sealing Anaer-obic Agar (Ref. 01-174) immediately after the Reinforced Clostridial Medium is solidified. If the plates are used, they have to be incubated in the anaerobic jars.Muñoa and Parés added a filter sterilized solution of Nalidixic acid 0,02 g/L, Polymyxin 0,025 g/L, Kanamy-

cin sulfate 0,05 g/L, Sodium iodoacetate 0,025 g/L and triphenyl-tetrazolium HCl 0,025 g/L to obtain a selective and differential medium for bifidobacteria in water and wastewater.

ReferencesATLAS, R.M., LC. PARKS (1993) Handbook of Microbio-logical Media. CRC Press, Inc.,Boca Raton,Fla.INGRAM, M. and BARNES, E.M. (1956) A Simple Modi-fication of the Deep Shake Tube for Counting Anaerobic Bacteria. Lab. Practise 5, 4:145.HIRSCH, A. and GRINSTEAD, E. (1954) Methods for the Growth and Enumeration of Anaerobic Sporeformers from Cheese, with Observations on the Effect of Nisin. J.Dairy Res. 21:101.MUÑOA, F.J., R. PARÉS (1988) Selective medium for isolation and enumeration of Bifidobacterium spp. Appl. Environm. Microgiol 54:1715-1718.EUROPEAN PHARMACOPOEIA,(2002) 4th ed. Suplle-ment 4.2 Chap. 2.6.13.Test for specified micro-organ-isms. Council of Europe. Strasbourg.

Reinforced Clostridial Media

Ref. 03-109

SpecificationMedium for rinsing the membrane filters according to the USP and European Pharmacopoeia specifications.

Formula (in g/L)Meat peptone .............................................5,0Meat extract ................................................3,0Final pH 6,9 ± 0,2

DirectionsDissolve 8 g of powder in 1 L of distilled water with 10 mL of Polysorbate 80 (Ref. 06-088). Distribute into suit-able containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionThis nutrient solution which is formulated according to the USP (Rinsing Fluid K) and European Pharmacopoe-ial specifications, removes all the fat and carobohydrate residues, due to the surfactant effect of Polysorbate, and at the same time, it avoids the osmotic shock to the microorganisms.

TechniqueAfter the filtration, wash the membrane by passing 3 volumes of 100 mL of solution through it.When the sample has a high concentration of fats or sugars it is recommended to double the concentration of polysorbate (2 mL/L).

ReferencesUS PHARMACOPOEIA (2002) 25th ed.<71> Sterility Tests. US Pharmacopoeial Convention Inc. Rockville. MDEuropean Pharmacopoeia. (2002) 4th Ed. V.2.18 Control of microbial contamination in no sterile products

Rinse Fluid K

131

Ref. 01-300

SpecificationSelective solid medium for the isolation and enumeration of lactobacilli.

Formula (in g/L)Glucose ................................................20,000Tryptone ...............................................10,000Sodium acetate ....................................10,000Monopotasium phosphate ......................6,000Yeast extract ...........................................5,000Ammonium citrate ..................................2,000Sorbitan monooleate ..............................1,000Magnesium sulphate ..............................0,575Manganese sulphate ..............................0,120Ferrous sulphate ....................................0,034Agar ......................................................15,000Final pH 5,5 ± 0,2

DirectionsSuspend 69,7 g of powder in 1 L of distilled water and add 1,32 mL of glacial acetic acid and the complemen-tary amount of sodium acetate to fit standard. Heat with gently stirring until boiling. Pour plates without autoclav-ing nor overheating.

DescriptionRogosa Agar was developed in 1951 for the isolation and enumeration of oral and faecal lactobacilli, but with changes in the acetate concentration it was used for several kinds of samples, including foods.

Rogosa Agar Base

The low pH and high acetate concentration, that can be adjusted to the sample confers to this medium a high selectivity for the lactobacilli. Nevertheless, it is no suit-able for the dairy samples, for which is recommended the addition of 20% sterile tomato serum (Ref. 06-092) or using the MRS Agar (Ref. 01-135) that offers a best performance with the dairy lactobacilli.Rogosa Agar, due to its high acidity, is not suitable for the maintenance of the microorganisms.

References ROGOSA, M., J.A. MITCHELL & R.F. WISEMAN (1951) A selective medium for the isolation and enumeration of oral and faecal lactobacilli. J. Bacteriol. 62:132.ROGOSA, M., J.A. MITCHELL & R.F. WISEMAN (1951) A selective medium for the isolation and enumeration of oral and faecal lactobacilli. J. Dental Res. 30:682DOWNES, F.P. & K. ITO (Eds) (1991) Compendium of methods for the microbial examination of foods. 4th ed. APHA. Washington D.C.MACFADDIN J.D. (1985) Media for isolation-cultivation-identification-maintenance of medical bacteria. William & Wilkins, Baltimore. MD.ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press. London.

132

Ref. 01-301

SpecificationSolid and selective medium for the isolation of yeast and moulds from the environment and food products.

Formula (in g/L)Peptone ....................................................5,00Dextrose .................................................10,00Potassium phosphate ...............................1,00Magnesium sulfate ...................................0,50Rose bengal .............................................0,05Chloramphenicol ......................................0,10Agar ........................................................15,00Final pH 7,2 ± 0,2

DirectionsSuspend 32 g of powder in 1 L of distilled water and heat to boiling with constant stirring. Distribute in suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionRose Bengal Agar is a selective medium to detect and enumerate moulds and yeast in food samples. Apart from the nutritional requirements for moulds and yeast,this medium also contains Rose Bengal, which apart from tainting the yeast with a pink colour, also fa-cilitates their count, avoiding massive growth of moulds such as Rhizopus and Neurospora, therefore it is easier to detect other moulds with slower growth.Chloramphenicol and also the Rose bengal, restrains bacterial growth, but does not interfere with fungi growth.

TechniqueAfter making a dilution bank, take 0,1 mL from each dilu-tion and inoculate with a Drigalsky Loop (Ref. 5-010) or glass spreader on Rose Bengal Agar plates. Should the massive seed method be preferred, take 1 mL from each dilution and put it in an empty plate. Pour the molten medium at 50°C and homogenize it by gently moving the plate in an eight (8) shape. Incubate at 22°C for 5 days and proceed to enumerate the fungi.

ReferencesATLAS, R.M., & PARKS, L.C. (1993) Handbook of Micro-biological Media. CRC Press, Inc.,Boca Raton,Fla.DOWNES F.P. & K. ITO (2001) Compendium of Methods for the Microbiological Examination of Food. 4th Ed. APHA. Washington.MARSHALL, R.T. (1993) Standard Methods for the ex-amination of dairy products. 16th ed. APHA Washington.APHA-AWWA-WEF (1998) Standard Methods for the examination of water and wastewater. 20th. ed. APHA Washington.

Rose Bengal Agar

R-45S-53-45

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Yeast and moulds colonies after 5 days at 22ºC

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Sabouraud Media

Sabouraud ChloramphenicolAgar

Ref. 01-166

SpecificationSolid culture medium for the isolation of fungi.

Formula (in g/L)Casein peptone ..........................................5,0Meat peptone .............................................5,0D(+) Glucose ............................................40,0Chloramphenicol ........................................0,5Agar ..........................................................15,0Final pH 5,6 ± 0,2

DirectionsSuspend 65,5 g of powder in 1 L of distilled water and bring to the boil. Distribute into final containers and sterilize by autoclaving at 121°C for 15 minutes. Do not overheat or reheat the medium since it will affect the solidification.

DescriptionThis culture medium differs from the classical Sabour-aud Agar only in the addition of Chloramphenicol. This thermostable antibiotic has a wide antibacterial spectrum which ensures the selective isolation of fungi from highly contaminated samples, such as eudates, faeces, nails and hair.

Sabouraud Dextrose Agar (Eur. Phar. Agar Medium C)

Ref. 01-165

SpecificationMedium for the enumeration and cultivation of fungi.

Formula (in g/L)D(+) Glucose ............................................40,0Casein peptone ..........................................5,0Meat peptone .............................................5,0Agar ..........................................................15,0Final pH 5,6 ± 0,2

DirectionsDissolve 65 g in 1 L of distilled water and bring to the boil with frequent stirring. Distribute into final containers and sterilize by autoclaving at 121°C for 15 minutes. Do not overheat the medium as its acidic pH may partially hydrolize the agar. Alternatively,if the European Pharma-copoeia formulation is desired, add before sterilization 50 mg/L of chloranphenicol (Ref. 06-118CASE)

DescriptionSabouraud Dextrose Agar is a modification of the clas-sical Sabouraud medium for the cultivation of fungi. This new formula helps to maintain the morphological aspects of fungi and thus permits a reliable cultivation and dif-ferentiation.Its selectivity is due to a low pH and a high glucose con-centration, which together with incubation at a relatively lower temperature (25-30°C) favours the growth of fungi while discouraging that of bacteria. Besides, the com-position of this peptone has been studied to provide the fungi with all their nitrogenated nutrient requirements.Since the Sabouraud medium’s strong acid reaction partially hydrolyzes the agar, only the required amount should be prepared and it should not be remelted. Any overheating will considerably diminish its gelling capac-ity.Should a higher selectivity be required, a variety of in-hibitors or selective agents may be added after steriliza-tion, while the medium is still in the molten form. It can even be made differential by adding the indicator agents. Some of the inhibitory and differential mixtures most commonly used are listed below:Penicillin: at 20,000 units/litre, encourages the selectivity

of the medium by inhibiting most of the bacteria.Penicillin and Streptomycin: at 20,000 u/L and 40,000

u/l each, favours the isolation of Histoplasma in dogs.

Penicillin and Neomycin: at 20,000 u/L and 40 mg/L each, is used for the isolation of yeast.

Streptomycin and Chloramphenicol: at 40 mg/L and 500 mg/L each, for the isolation of Trichophyton ver-rucosum .

Colistin, Novobiocin and Cycloheximide: at 8 mg/L, 0.1 mg/L and 30 mg/l each, for the isolation of Can-dida albicans .

Potassium Tellurite: at 150 mg/L, is used for the primary isolation of fungi from scales and scabs.

Cupric Sulfate, Crystal Violet and Brilliant Green: at 500 mg, 2 mg and 5 mg each, achieves considerable bacterial inhibition.

Triphenyltetrazolium chloride (TTC): at 100 mg/L, it is the basis of a Pagano-Levin medium for the isola-tion of Candida albicans, unpigmented, among other pathogenic yeast which form pink coloured colonies.

Sabouraud Broth

Ref. 02-165

SpecificationLiquid medium for the sterility control.

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134

Formula (in g/L)Casein Peptone ..........................................5,0Meat Peptone .............................................5,0D (+) Glucose ...........................................20,0Final pH 5,8 ± 0,2

DirectionsDissolve 30 g of powder in 1 L of distilled water, heating up only if necessary. Dispense into suitable containers and sterilize in preheated autoclave for 15 minutes at 121°C. Avoid overheating, since it may caramelize the glucose.

DescriptionThis medium is especially adapted to fungi and acido-philic bacteria culture.Sabouraud USP Broth is according to the formulations of US Pharmacopeia, US NF and 21 CFR guidelines. In the latest editions of these books it is also mentioned and allowed to use Tryptone and Soy Broth for the sterility checking in parenteral pharmaceutical products. This for-mulation is similar to the Antibiotic Medium 13 by Grove and Randall and 21 CFR.This medium is not a selective one, but the strong acidic pH notably inhibits the growth of non acidophilic microor-ganisms. Nonetheless, special measures must be taken while reconstituting and heating the medium due to this strong acid reaction and the high content of glucose. It is important to preheat the autoclave and thereby reach the sterilization temperature as soon as possible and in a regular way, since otherwise, glucose becomes caramel-ized turning the medium dark and effectiveless.

TechniqueIt has been recommended to use this medium in many tests and assays, but for a long time it has been the me-dium of choice for the verification of sterility of the sterile pharmaceutical products.Efficacy of the medium and absence of fungistatic prod-ucts is verified by checking if there is a growth from a loop of inoculum of Candida albicans, from a 1:1000 di-lution of a fresh 24 hours grown culture. Sterility assay or test is carried out in controlled and verified medium. To check the fungistatic activity of any product, prepare an inoculum as mentioned above and inoculate two series of tubes with the same medium as follows:a) Add to one batch the specified amount of product.

This is the test series.b) Add to the another batch only the inoculum and

simultaneously incubate with the test series. This is the control series.

Incubation of both the series must be carried out at 22°C for 10 days. After this period compare the results or observations of both the series. If the assay series has a lesser growth than the control one, product has the fun-gistatic activity. If the growth is equal or more, then it has not any fungistatic properties. For the quantitative assay of the fungistatic activity, perform the assay with several series of different concentrations (one lower than the previous) until reaching an equal growth in both control and test series.

Sabouraud Oxytetracycline Agar Base (OGYEA)

Ref. 01-275

SpecificationSolid culture medium for the total enumeration of moulds and yeasts.

Formula (in g/L)Glucose ....................................................20,0Yeast extract ...............................................5,0Agar ..........................................................20,0Final pH 7,0 ± 0,2

DirectionsSuspend 45 g of powder in 1 L of distilled water and let it soak for a few minutes. Distribute into suitable contain-ers and sterilize by autoclaving for 10 minutes at 115ºC. Cool to 50°C and then add oxytetracycline (Ref. 06-115CASE) to reach a 0,1 mg/mL concentration.

DescriptionThis formulation differs with others as it has no peptone and has a neutral reaction or pH. Unlike the others, it has a high oxytetracycline concentration that makes it almost impossible for the growth of bacteria.

TechniqueSome authors suggests an inoculum of 1 mL in each dilution, in duplicate and in mass. Perform an incubation at 22-25°C for 5 days with the intermittent observations or readings after 3 days of incubation.

ReferencesAJELLO, L.(1957) Cultural Methods for Human Patho-genic Fungi J. Chron. Dis. 5:545-551.PAGANO, J., LEVIN,J.D. and TREJO, W.(1957-58) Diagnostic Medium for Differentiation of Species of Can-dida. Antibiotics Annual, 137-143.SABOURAUD, R.(1910) Les Tignes. Masson, Paris. HANTSCHKE, D.(1968) Mykosen, 11:769-778.EUROPEAN PHARMACOPOEIA (2002), 2.6.13 Tests for specified micro-organisms Supplement 4.2, 4th Ed., EDQM. Council of Europe, Strasbourg.US PHARMACOPOEIA (2002) 25th Ed. <51>Antimicro-bial efectiveness Testing; <61> Microbial Limit Tests. US Pharmacopoeial Convention Inc. Rockville. MDISO 13681 Standard (1995) Enumeration of yeasts and moulds - Colony count technique.

Sabouraud Media

135

Ref. 01-171

SpecificationSolid and highly selective medium for the isolation of Salmonella and some Shigella species.

Formula (in g/L)Meat extract ........................................5,00000Peptone ..............................................5,00000Lactose .............................................10,00000Bile salts .............................................8,50000Sodium citrate ..................................10,00000Sodium thiosulfate ..............................8,50000Ferric citrate ........................................1,00000Brilliant green .....................................0,00033Neutral red ..........................................0,02500Agar ..................................................15,00000Final pH 7,0 ± 0,2

DirectionsSuspend 63,1 g of the dehydrated medium in 1 L of distilled water. Slowly bring to the boil, stirring until com-plete dissolution. Boil for 2 minutes. Do not autoclave. Cool to 50°C and pour into sterile Petri dishes.Do not overheat.

DescriptionThe SS Agar is a highly selective agar for the isolation of Salmonella and Shigella species from very contaminated samples.Selectivity is obtained by a high concentration of bile salts and brilliant green, which inhibits the growth of gram-positive bacteria. As for the other gram-negative flora, its growth is highly repressed by the presence of citrate and thiosulfate. Nevertheless, some coliforms may still grow on this medium. In such case, differentia-tion between pathogenic species and coliforms becomes evident by the colour change of the pH indicator neutral red. Lactose fermenters produce a pink or red colored medium and colonies, while non-fermenting species form colourless colonies and turn the medium yellow. Should any species eventually produce H

2S, it will be

easily detected by the ferrous sulfide’s black precipitate, which turns the colonies black.The peptone and the meat extract are usually capable of inducing the growth of most pathogenic species, nev-ertheless some Shigella are very fastidious and grow poorly.

TechniqueWhile using the samples suspected of being exposed to the treatments that might have damaged the viability of microorganisms (processed food, faeces from the patients under antibiotic treatment, etc.) it is advisable to proceed with a previous enrichment in Selenite Cystine Broth Base (Ref. 02-602) or Tetrathionate Base Broth (Ref. 02-033/Ref. 02-335). Afterwards, inoculate SS Agar plates heavily with the specimen and proceed in the same way with other specimens of a less selective

medium, such as Brilliant Green Agar (Ref. 01-203) or MacConkey Agar (Ref. 01-118). Incubate the inoculated plates at 37°C for 18-24 hours. The suspicious colonies should then be subcultured on differential media to be identified biochemically or serologically.Appearance of the colonies after 24 hours on SS Agar:Shigella: Colourless, transparent and flat.Salmonella (Non H

2S producers): Colourless, transpar-

ent and flat.Salmonella (H

2S producers): Black or black centered,

flat, with transparent borders.Proteus: Similar appearance as Salmonella colonies, but

smaller in size.Escherichia coli: If they grow, they are small, convex and

pink or red coloured.Coliforms (in general): Big, opaque, smooth and col-

oured in white or pink shade.

ReferencesLEIFSON, E.(1935) New culture media based on sodium deoxycholate for the isolation of intestinal pathogens and for the enumeration of colon bacilli in milk and water. J. Pathol. Bacteriol., 40.581.DOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food. 4th Ed. APHA. Washington DC.HORWITZ,W.(2000). Official Methods of Analysis 17th ed. AOAC International. Gaithersburg. MD.ATLAS, R.M.,and L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, LondonGRAY, L.D. (1995) Escherichia, Salmonella, Shigella and Yersinia. In Murray, Baron, Pfaller Tenover & Yolken (eds) Manual Clinical Microbiology. 6th ed. ASM Wash-ington DC.

Salmonella Shigella Agar (SS Agar)


136

Schaedler Agar

Ref. 01-231

SpecificationSolid medium with high reducing and nutrient capacity for the cultivation of fastidious anaerobic microorgan-isms.

Formula (in g/L)Casein peptone ........................................5,60Soy peptone .............................................1,00Meat peptone ...........................................5,00Yeast peptone ...........................................5,00Glucose ....................................................5,80Sodium chloride ........................................1,70Dipotassium phosphate ............................0,80Tris buffer .................................................3,00L-Cysteine HCl .........................................0,40Hemine .....................................................0,01Agar ........................................................15,00Final pH 7,6 ± 0,2

DirectionsSuspend 43,3 g of powder in 1 L of distilled water and heat to boiling. Dispense into suitable containers and sterilize in the autoclave at 121°C for 15 minutes. Pour into sterile plates just before the use.

Schaedler Broth

Ref. 02-231

SpecificationLiquid version of the agar with the same name, especial-ly suitable for fastidious anaerobic microorganisms.

Formula (in g/L)Casein peptone ........................................5,60Soy peptone .............................................1,00Meat peptone ...........................................5,00Yeast peptone ...........................................5,00Glucose ....................................................5,80Sodium chloride ........................................1,70Dipotassium phosphate ............................0,80Tris buffer .................................................3,00L-Cysteine HCl .........................................0,40Hemine .....................................................0,01Final pH 7,6 ± 0,2

DirectionsDissolve 28,3 g of powder in 1 L of distilled water, heat-ing up only if necessary. Distribute into suitable contain-ers and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionThese media viz. Schaedler Agar and Broth, were developed to create the selective conditions to allow the growth of fastidious anaerobic microrganisms from a mixed flora, like gastrointestinal tract, where there are many antagonistic activities between fast growing facultatives and the delicate fas-tidious anaerobic organisms. For this aspect, the media with thioglycolate are widely used, but this compound seems to inhibit some delicate anaerobic organisms. On the other hand, Schaedler media have L-Cystine as a reducing agent, thus some gramnegative do not grow. Effective separation or isolation in several biotypes is achieved with the addition of selective agents to the nu-trient base. For example, this medium can be rendered selective for lactic bacteria by adding 10 g/L of sodium chloride and 0,002 g/L of neomycin.For the selection of Clostridium and Bacteroides, it is more advisable to add 2 g/L of placenta powder and 0,002 g/L of neomycin. Should a selective medium for Flavobacterium be desired, add 7 mL of alcoholic solu-tion of tyrothricin 0,5% to 1 L of medium base. In any case, incubation must be carried out at 37°C and in an anaerobic atmosphere.

ReferencesSCHAEDLER, R.W., DUVOS, R. and COSTELLO, R. (1965) The development of the bacterial flora in the gas-trointestinal tract of mice. J. Exp. Med. 122:59.ATLAS, R.M., LC. PARKS (1993) Handbook of Microbio-logical Media. CRC Press, Inc.,LondonSTALONS, D.R., C.THORNSBERRY and V.R. DOWELL (1974) Effect of culture medium and CO

2 concentration

of growth of anaerobic bacteria commonly encountered in clinical specimens. Appl. Microbiol 27:1098-1104.ISENBERG H.D. (1992) Clinical Microbiology Proce-dures Handbook. ASM. Washington DC.MARSHALL, R.T. (1992) Standard Methods for the ex-amination of Dairy Products. APHA. WashingtonMacFADDIN, J.F. (1985) Media for Isolation-Cultiva-tion- Identification and Maintenance of Medical bacteria. William & Wilkins. Baltimore, MD, USA.WILKINS, T.D. and S. CHALGREN (1976) Medium for use in the susceptibility testing of anaerobic bacteria. Antimicrob. Agents. Chemother 10:926:928.

Schaedler Media

137

Ref. 01-174

SpecificationSolid substrate with high reducing capacity to cover and seal the cultures anaerobically.

Formula (in g/L)L-Cysteine HCl .........................................0,30Sodium Thioglycolate ..............................0,70Resazurine ...............................................0,01Agar ........................................................20,00Final pH 7,0 ± 0,2

DirectionsSuspend 21 g of powder in 1 L of distilled water and bring to the boil. Dispense into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionThis solid substrate, without the capacity to support any growth but with strong reducing power, is made to cover the tubes for anaerobic growth. Once liquified, let it cool to 50-55°C and pour into the tubes, taking care not to mix it with the medium.A minimum column of 1,5 cm over the medium is recom-mended to assure an anaerobic cover.Should cultures be stored for a long period of time, it is advisable to put a sterile vaseline layer over the cap to avoid desiccation.

ReferencesSANCHO, J. (1977) Personal communication.

Sealing Anaerobic Agar

Selenite Media

Selenite Brilliant Green Broth Base (SBG Broth Base)

Ref. 02-603

SpecificationSelective enrichment medium for Salmonella types.

Formula (in g/L)Gelatine peptone ....................................5,000Yeast extract ...........................................5,000D-Mannitol ..............................................5,000Sodium taurocholate ..............................1,000Potassium phosphate .............................3,500Sodium sulfapyridine ..............................0,500Brilliant green .........................................0,005Final pH 7,2 ± 0,2

DirectionsDissolve 20 g of powder. in 1 L of distilled water and add 4 g of sodium biselenite (Ref. 06-615). Homogenize and bring to the boil. Distribute in suitable containers. Termo-labile medium: Use immediately. Do not autoclave.

DescriptionSBG Broth is a modification to the classical Osborne and Stockes medium for enrichment of Salmonella from eggs and egg derivative products.The medium is maintained at a neutral pH, in spite of the acid products are liberated from the mannitol fermenta-tion, due to the strong phosphate buffer. On the other hand, inhibitor effect of sulfamide in gram-negative bacteria is helped by the classical selective agents for salmonellae like brilliant green, selenite and bile salts. Notwithstanding, presence of these substances, makes the medium thermolabile and thus autoclaving must be

avoided. Is not advisable to store the prepared medium for more than eight days, since it loses its selectivity notably.

Selenite Broth Base

Ref. 02-598

SpecificationLiquid medium for Salmonella and Shigella enrichment.

Formula (in g/L)Peptone ....................................................5,00Lactose .....................................................4,00Potassium phosphate .............................10,00Final pH 7,0 ± 0,2

DirectionsDissolve 19 g of powder. in 1 L of distilled water and add 4 g of sodium biselenite (Ref. 06-615). Homogenize and bring to the boil. Distribute in suitable containers. Termo-labile medium: Use immediately. Do not autoclave.

DescriptionSelenite Broth is formulated according to an original formulation by Leifson for selective enrichment of Salmo-nellae from very contaminated samples. Enrichment is especially effective during the first 12 hours of cultivation, since in this period it seems that only Salmonellae, some Proteus and some strains of Pseudomonas grow easily. For this reason, it is advis-able not to extend the enrichment phase and go quickly for the selective medium, either liquid or solid. According to Bänffer, the efficacy of the medium is improved nota-bly if enrichment is performed at 43°C. Presence of a red precipitate in the medium before inoculation, indicates

R-22-43S-24-37-46

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138

that there was a overheating in which case the selective properties of the medium are reduced.

Selenite Cystine Broth Base

Ref. 02-602

SpecificationLiquid enrichment medium for Salmonella sp. acc. USP and ISO 6785 and 6340 standards

Formula (in g/L)Peptone ....................................................5,00Lactose .....................................................4,00Potassium phosphate .............................10,00L-Cystine ..................................................0,01Final pH 7,0 ± 0,2

DirectionsDissolve 19,01 g of powder. in 1 L of distilled water and add 4 g of sodium biselenite (Ref. 06-615). Homog-enize and bring to the boil. Distribute in suitable con-tainers. Termolabile medium: Use immediately. Do not autoclave.

DescriptionSelenite Cystine Broth has been developed according to Leifson’s formulation with the addition of L-Cystine to comply with FDA specifications, since it was proved that the medium was better in reduced CO

2 atmosphere.

Essencially, it is an enrichment medium for Salmonella coming from food or pathological materials, such as fae-ces or urine, as a previous step to isolation in selective media plates such as SS Agar (Ref. 01-171) or Hektoen Agar (Ref. 01-216).

TechniqueFor normal assays or experiments, an incubation at 37°C for a period not exceeding 18 hours is recommend-ed, since within this period a good nutrition of coliforms and an enhancement of pathogens is achieved, but after 24 hours this effect seems to disappear and the growth of accompanying organisms may mask the growth of Salmonella.Appearance of red precipitate before inoculation is the indication of overheating the medium, in which case the selective properties are significantly reduced. Presence of abundant sample residues may also inactivate the selective property of the medium, if the sample is e.g. faeces and or egg powder. In those cases, it is better to make a dilution of 1:10 and let the bigger particles separate by settling down the dilution tube, and then inoculate Selenite Cystine Broth with an aliquot portion of it. Maintain a proportion of 1:10 between the sample and the medium.It has been demonstrated that when it is desired to iso-late Salmonella from faeces,the results are better if the enrichment medium is incubated at 43°C. However this procedure does not work with the isolation of Salmonella typhi.

When starting material is urine, the best procedure is to use Selenite Cystine Broth in double concentration, and to inoculate it with an equal volume of urine. Anyway, subculturing must always be done after 6 hours of incu-bation but before 24 hours. Most authors recommend the simultaneous use of another enrichment broth, such as Tetrathionate Base Broth (Ref. 02-033).

ReferencesUS PHARMACOPOEIA (2002) 25th ed Chapter <61> “Microbial Limit Tests” The U.S. Pharmacopoeial Con-vention. Rockville MD.DOWNES F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food. 4th ed. APHA. Washington.FDA (1998) Bacteriological Analytical Manual 8th ed. Rev. A. A.O.A.C.International.Gaitherburg VA.LEIFSON, E. (1936) “A new Selenite Selective Enrich-ment media for the Isolation of Typhoid and Paratyphoid (Salmonella) Bacilli” Am.J.Hyg. 24:423-432.US FDA (1962) “The determination of Salmonellae in Food”.BÄNFFER, J.R. (1971) Comparison of the isolation of Salmonellae from human faeces by enrichment at 37ºC and 43ºC Zbl. Bakt. I Orig. 217:(35-40)STOCKES, J.L. and OSBORNE, W.W. (1955) A Selenite Brilliant Green Medium for isolation of Salmonella. Appl. Microbiol 3-4:217-227.ATLAS, R.M., LC. PARKS (1993) Handbook of Microbio-logical Media. CRC Press, Inc LondonDIN - Standard 10160: Untersuchung von Fleisch u. Fleischerzneugissen. Nachweiss von Salmonella (Ref-erenzverfahren).ISO 6785 Standard (2002) Milk and Milk products - De-tection of Salmonella spp.ISO 6340 Standard (1995) Water Quality Detection of Salmonella spp.

Selenite Media

Left: Salmonella typhimu-rium ATCC 14028; right:

control.

Ref. 02-602Selenite Cystine

Broth Base

139

Ref. 01-175

SpecificationSolid differential medium for gram-negative non ferment-ing coccobacilli.

Formula (in g/L)Gelatin peptone ....................................20,000Sodium chloride ......................................2,000Sodium nitrate ........................................1,000Sodium nitrite .........................................0,350D-Mannitol ..............................................2,000L-Arginine ...............................................1,000Yeast extract ...........................................1,000Magnesium sulfate .................................1,500Dipotassium phosphate ..........................1,000Bromothymol Blue ..................................0,040Phenol red ..............................................0,008Agar ......................................................13,500Final pH 6,7 ± 0,2

DirectionsSuspend 43,5 g of powder in 1 L of distilled water and bring to the boil. Distribute into tubes and sterilize by autoclaving at 121ºC for 15 minutes. Let it solidify in slanted position with short slant and good butt.Just before the inoculation, allow 2 drops of sterile glu-cose 10% solution to slide down on the opposite side of the slant.

DescriptionSellers Agar is formulated to differentiate the gram nega-tive bacilli that do not produce characteristic acidification by the fermentation in the usual diagnostic media, such as TSI (Ref. 01-192) or Kligler Iron Agar (Ref. 01-103).Criteria for the differentiation among the microorganisms that can be obtained from this medium are as follows:- Fluorescence production enhanced by the magnesium

sulfate and mannitol.

- Glucose oxidation and pH changes that can be fol-lowed with the colour variation of the pH indicators included (phenol red and bromo thymol blue)

- Nitrogen gas release, indicated by the bubbles which may eventually break the agar.

TechniqueThe medium is inoculated with a pure culture by streak-ing on the surface and also by stabbing deeply. Incu-bation is performed at 35-37ºC for 24-48 hours. After the incubation period, observe the fluorescence under Wood’s light and also observe the change in pH and gas production. The yellow band on the surface of the medium due to glucose oxidation may disappear within 24 to 48 hours.On the slant, fluorescence may appear under UV light in some species of the fluorescent Pseudomonas group, especially Ps. aeruginosa. On the surface of the me-dium, most of Acinetobacter species produce a yellow band due to glucose oxidation. This band may disappear after 24 hours, and this phenomenon is very common with some strains of A. calcoaceticus. A blue colour at the bottom of the tube indicates ar-ginine-dehydrolase positive or any other reaction like anaerobic degradation of nitrate or the mannitol utilisa-tion by some strains of Alcaligens faecalis. Nitrogen gas released in the form of small bubbles or splitting of agar indicates denitrification reaction from nitrate or from nitrite. In the table below all these typical reactions are tabulated.

ReferencesATLAS, R.M. and R.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, LondonMacFADDIN, J. (1985) Media for isoltion-cultivation-iden-fication-maintenance of medical bacteria. Vol I. William & Wilkins. Baltimore,MD.SELLERS, W. (1964) J. Bact. 87:46

Sellers Agar

140

Ref. 02-072

SpecificationLiquid selective medium for faecal streptococci detection in several materials.

Formula (in g/L)Casein peptone ....................................20,000Dextrose .................................................5,000Di-potassium phosphate .........................4,000Sodium azide ..........................................0,500Sodium chloride ......................................5,000Brom cresol purple .................................0,032Monopotassium phosphate ....................1,500Final pH 6,9 ± 0,2

DirectionsDissolve 36 g of powder in 1 L of distilled water. Dis-pense in tubes or flasks with Durham´s tubes and steri-lize in the autoclave at 121°C for 15 minutes.

DescriptionThe efficacy of this diagnostic medium for detecting the faecal streptococci (enterococci) from several materials has been widely proved since its publication by Hajna and Perry, who utilized the advantage of the azide effect and combined it with the fermentative ability of strepto-

cocci to ferment glucose at 44,5°C. Inhibition of accom-panying bacteria is achieved by the high concentration of azide, which does not affect the growth of streptococci.Enterococci growth is indicated by the indicator brom cresol purple turning to yellow, when the tubes are incu-bated at 44,5°C.In this medium, the indicator turning yellow in pres-ence of enterococci is evident after 18-20 hours, but to proceed for the isolation, a supplementary incubation in petri plates is recommended.If the MPN method is used, do not excessively dilute the medium with the sample.

ReferencesHAJNA, A.A. and PERRY, C.A. (1943) Comparative study of presumptive and confirmativemedia for bacteria of the coliform group an fecal streptococci. Am.J.Pub.Hlth.,33:550.ATLAS, R.M. and L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Inc.,LondonAPHA-AWWA-WPCF (1998) Standard Methods for the examination of Water and Wastewater,20th Ed.,APHA, Washington.DOWNES, F.P. and K. ITO (2001)Compendium of Meth-ods for the Microbiological Examination of Food,4th Ed. American Public Health Association,Washington D.C.

SF Medium

Ref. 03-176

SpecificationDifferential fluid medium for detecting the motility, H

2S

production and indole formation.

Formula (in g/L)Yeast extract .............................................10,0Casein Peptone ........................................10,0Meat peptone .............................................6,0Ferric-ammonium sulfate ............................0,2Sodium thiosulfate ......................................0,2Agar ............................................................3,7Final pH 7,3 ± 0,2

DirectionsSuspend 30 g of powder in 1 L of distilled water and let it soak for a few minutes. Heat to boiling and dispense into suitable containers. Sterilize by autoclaving at 121°C for 15 minutes.

DescriptionThis classical medium was originally developed to dis-tinguish several types of enterobacteria, on the basis of motility test , detection of indole and H

2S production.

It is a semisolid or fluid medium, and so the motile micro-organisms can move freely. At the same time, richness of sulfur containing amino acids and presence of thiosul-

SIM Medium

fate allow those microorganisms that are able to produce sulfides, and then this reacts with iron and produces black precipitates which in turn make the medium darker. The amount of thiosulfate present in medium does not affect the motility mechanisms, instead it assures H

2S

production by those microorganisms that are not able to produce it from cystine or cysteine.Finally, the medium allows the production of indole from Tryptophan present in the peptone, which can be easily detected with the addition of Kovac’s Reagent (Ref. 06-018) (directly or with extraction) or with paper stripes impregnated with the reagent.These three characteristics are common for enterobac-teria, and on the basis of these properties, more differen-tial or even selective media can be used.

TechniqueRecommended technique is to inoculate by deep stab from a pure culture (or from an isolated colony). After an incubation period of 16-18 hours at 37°C, observe for the clarity in stab. Immotile microorganisms produce growth only in the stab, whereas motile ones may be easily detected by their displacement which is indicated by the turbidity in the medium.H

2S production is indicated by the general blackening of

the medium when there is a great amount of FeS pro-duced or by blackening of the stab when there is a little amount of FeS produced.

R-45-22-32-52/53S-53-45-7-61

T

141

Indole formation is the last test to be performed if the soaked or impregnated paper stripes are not used. Despite the fact that many authors suggest a previ-ous extraction of indole by stirring on the surface of the culture medium with chloroform. If Kovacs’ Reagent (Ref. 06-018) is employed then this is not necessary and the observations can be done by pouring a few drops of reagent on the surface of the medium. A positive test will produce the interphase in the medium turning to pur-ple red colour, whereas a negative test will produce no colour change. Many a times, chloroform extraction may give erroneous results, since the appearance of colour must be observed immediately after the reagent addi-tion. However if it is delayed by more than 30 seconds, the test must be considered negative.

ReferencesHARRIGAN, W.F. and McCANCE, M.E. (1966) Labora-tory Methods in Microbiology. Academic Press, .BLAZEVIC, D.J. (1968) Improved Motility Indole Me-dium. Appl. Microbiol. 16, (4),668.BULLMASH, J.M. and FULTON, M (1964). Discrepant Test for Hydrogen Sulfide. J.Bact. 88(6)1813.

SIM Medium

Ref. 01-177

SpecificationMedium for verifying the citrate utilization by enterobac-teria according ISO 10273 standard.

Formula (in g/L)Magnesium sulfate ...................................0,20Monoammonium phosphate .....................1,00Dipotassium phosphate ............................1,00Sodium citrate ..........................................2,00Sodium chloride ........................................5,00Brom thymol blue .....................................0,08Agar ........................................................15,00Final pH 6,8 ± 0,2

DirectionsDissolve 24 g of powder in 1 L of distilled water. Bring to the boil. Dispense in tubes and sterilize by autoclaving at 121°C for 15 minutes. Allow to solidify with long slant.

DescriptionSimmons Citrate Agar is the solid version of the classical Koser citrate medium, and it can be used in the plates as well as in slanted tubes. Slanted tubes can be inoculated by surface streaking or by deep stab.Although, originally, it was described as an isolation and identification medium for certain fungi, Edwards and Ewing had recommended it for the IMViC test since it has the advantage over the Koser’s medium that the readings can be made by the indicator colour change, instead of turbidity of the medium, which is sometimes difficult to detect.

TechniqueThe techique is simple and one has to take care in us-ing an inoculum as small as possible and the medium should be freshly prepared, because if it is very dry, false turning (colour change) may appear, even before the inoculation, especially at the bottom of the slant.The basis of this medium is in the capacity of microor-ganisms to use citrate as carbon source and ammonium compounds as the nitrogen source for their growth. Among enterobacteria, these properties are possessed by the following genera: Enterobacter, Klebsiella, Ser-ratia, Citrobacter and some species of Salmonella as S.schottumelleri, S.typhimurium, S.arizona etc. whereas Escherichia, Shigella, Salmonella typhi and S.paratyphi are unable to grow.Although, the test result must be read as the growth pro-duced, the presence of an indicator makes it easy, as the citrate degradation yields an alkaline reaction, which is indicated by the turning of the indicator to intense blue. This is evident even when the growth rate is high.

ReferencesSIMMONS J.S. (1926) A culture medium for differentiat-ing organisms of typhoid-colon aerogenes group and for isolatig certain fungi.J.Inf.Dis. 39:209FDA (1998) Bacteriological Analytical Manual. 8th Ed. Revision A. AOAC International. Gaithersburg.APHA-AWWA-WEC (1998) Standard Methods for the examination of water and wastewater. APHA. Washing-ton DC.HORWITZ, W. (2000) Official Methods of Analysis. 17th Ed. AOAC International. Gaithersburg. MD.ISO 10273 Standard (1994) General guidance for the detection of presumptive pathogenic Yersinia enteroco-litica.

Simmons Citrate Medium

142

Slanetz Bartley Agar

Ref. 01-178

SpecificationDifferential and selective medium for the enumeration of enterococci,

Formula (in g/L)Tryptose ....................................................20,0Yeast extract ...............................................5,0Dextrose .....................................................2,0Potassium phosphate .................................4,0Sodium azide ..............................................0,4 TTC .............................................................0,1Agar ..........................................................12,0Final pH 7,0 ± 0,2

DirectionsSuspend 43,5 g of powder in 1 L of distilled water and bring to the boil. Cool to 50°C and distribute into sterile petri plates immediately. Do not autoclave or overheat.

DescriptionThe Slanetz Bartley Agar is recommended for the determination of faecal streptococci by the membrane filtration technique. Although originally this medium was described by the authors only for the above technique, it gives such excellent results that it is now recommended for viable enumeration of enterococci from food samples.

Slanetz Bartley Agar Base

Ref. 01-579

SpecificationDifferential and selective medium for the detection and enumeration of enterococci acc. ISO Standard 7899-2:2000

Formula (in g/L)Tryptose ....................................................20,0Yeast extract ...............................................5,0Dextrose .....................................................2,0Potassium phosphate .................................4,0Sodium azide ..............................................0,4Agar ..........................................................12,0Final pH 7,0 ± 0,2

DirectionsSuspend 43,4 g of powder in 1 L of distilled water and bring to the boil. Sterilize by autoclaving at 121ºC for 15 minutes. Cool to 50°C and add 10 mL/L of 1% TTC sterile solution (Ref. 06-023). Mix well and distribute into sterile petri plates immediately.

DescriptionThis formulation, without TTC allows its sterilization by autoclaving without the development of the pink colour due to the formazan which is formed as a result of the partial-thermal reduction of TTC. This modification is more tedious in its preparation but provides a colour-less medium, making the results easier to read and the colonies are more sharply defined.

TechniqueFor the membrane filtration technique, take 100 mL of water sample which is previously homogenized, and pass it through a sterile membrane filter. Then wash it with 30 mL of sterile dilution water to wipe away the things left on the funnel of the filtering system. By using sterile forceps, transfer the membrane asepti-cally on to the culture medium contained in a Petri dish, making sure that the filter surface faces upwards. Close the lid and invert the plate. lncubate at 37°C for 48 hours. All the developed colonies which appear red or purple coloured must be considered as enterococci, since those bacteria reduce Triphenyltetrazolium-HCl to an insolu-ble formazan which is red in colour. The secondary or accompanying gram negative bacteria are inhibited by sodium azide.As regards, food samples from a decimal dilution bank of the product to be examined, incoculate 0,1 mL on the surface of the petri dishes using the Drigalski technique. lncubation and examination is carried out as same as in the membrane filtration technique.

ReferencesSLANETZ L.W. & BARTLEY, C.H. (1957). Numbers of Enterococci in Water, Sewage and Faeces Determined by The Membrane Filter technique with an lmproved Medium. J.Bact., 74:591-596.ATLAS, R.M., & LC. PARKS (1993) Handbook of Micro-biological Media,CRC Press Inc.,LondonISO Standard 7899-2:2000 Water Quality. Detection and enumeration of enterococci by membrane filtration method.LACHICA, L.V.F. & P.A., HARTMAN (1968) Two im-proved media for isolating and enumerating enterococci in certain frozen foodsJ. Appl. Bact. 31:151-156

Slanetz Bartley Media

R-22-32-52/53S-7-46-61

Xn

R-22-32-52/53S-7-46-61

Xn

Ref. 01-579 Slanetz-Bartley Agar Base + Ref. 06-023 TTC 1% Sterile Solution. Enterococcus faecalis ATCC 29212.

143

Ref. 02-523

Specification Liquid medium for the cultivation of recombinant strains of Escherichia coli.

Formula (in g/L)Tryptone ..............................................20,000Yeast Extract ..........................................5,000Sodium Chloride ....................................0,500Magnesium sulphate .............................2,400Potasium chloride ...................................0,186Final pH 7,0 ± 0,2

DirectionsDissolve 28 g of powder in 1 L of distilled water heating if it is necessary. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

DescriptionThis medium was developed by Hanahan in 1983 as a nutritionally rich base for growth preparation and trans-formation of competent cells that allows the introduction of foreign DNA onto cells..

The addition of 4 g/L of dextrose to this medium pro-duce the SOC Medium that is used in the final phase of transformation and in the recovery of electroporated E. coli cells.

ReferencesHANAHAN, D. (1983) Studies on transformation of Es-cherichia coli wth plàsmids. J. Mol. Biol. 166:557SAMBROOK, J., E.F. FRITSCH & T. MANIATIS (1989) Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

SOB Broth

Sporulating Agar (AK Agar) (USP Antibiotic Medium 32)

Ref. 01-069

SpecificationThis medium is according to the Arret and Kirshbaum formulation, adopted by US FDA, for the preparation of spore suspension for antibiotic assay. It is the same medium as the Antibiotic Medium 32.

Formula (in g/L)Gelatin peptone ..........................................6,0Casein peptone ..........................................4,0Yeast extract ...............................................3,0Meat extract ................................................1,5Dextrose .....................................................1,0Manganous sulfate .....................................0,3Agar ..........................................................15,0Final pH 6,6 ± 0,2

DirectionsDissolve 30,8 g of powder in 1 L of distilled water. Bring it to the boil with constant stirring . Dispense in suitable containers and sterilize at 121°C for 15 minutes.

DescriptionSporulating Agar is made according to the original for-mulation of Arret and Kirshbaum, and later adopted by FDA for the preparation of Bacillus subtilis ATCC 6633 spores suspension. To prepare this suspension, suspend

in 10 mL of Ringer Solution the growth from a slant. With this suspension, inoculate on the surface a Roux flask (bottle) containing 300 mL of solidified and controlled Sporulation Agar.The roux flask must be incubated at 35°C for 5 days. Growth is harvested with 50 mL of sterile Ringer So-lution, helping with a Drigalsky loop or sterile crystal balls (pearls) if necessary. The suspension obtained is carefully centrifuged at 5000 rpm for 15 minutes. Discard the supernatant liquid and suspend the pellet in a fresh volume of Ringer Solution. Put it in a boiling water bath at 70°C for 30 minutes. This suspension is active upto 6 months if stored refrigerated. Depending on the objective of the experiment,it can be standardized by turbidimetry.

ReferencesARRET and KIRSHBAUM (1959) J.Milk. Food Tech. 22:329SANCHO, GUINEA, PARES (1980) Microbiología Analítica Básica. Ed. JIM. Barcelona,US PHARMACOPOEIA (2002) 25th ed. <81> Antibiotic Microbial Assays. US Pharmacopoeial Convention Inc. Rockville. MD

144

Ref. 01-050

SpecificationSolid medium for the detection of Clostridium perfrin-gens in food.

Formula (in g/l)Sodium sulfite ...........................................0,50Polymyxin (B) sulfate ................................0,01Sodium sulfadiazine .................................0,12Casein peptone ......................................15,00Yeast extract ...........................................10,00Ferric citrate ..............................................0,50Sodium thioglycolate ................................0,10Polysorbate 80 .........................................0,05Agar ........................................................15,00Final pH 7,0 ± 0,2

DirectionsSuspend 41,3 g of powder in 1 L of distilled water and bring to the boil. Distribute into tubes or screw-cap containers and sterilize by autoclaving at 121°C for 15 minutes. Cool the sterilized medium quickly by placing in the refrigerator or in cold water.

DescriptionThe SPS Agar (Sulfite-Polymyxin-Sulfadiazine) is a modification of the original Wilson & Blair medium for the detection of clostridia. The present medium excels the formulation of Mossel and also the later modification of Angelotti et al. It achieves a higher selectivity for Cl. perf-ringens with the addition of sulfadiazine and polymyxin.On the other hand, the nutritional substrates constituted by the tryptone and the yeast extract are complemented by the Polysorbate, which also allows the recovery of the most delicate cells. The anaerobic conditions are im-proved by the thioglycolate, which permits the use of the medium on the plates without the Miller-Prichett tubes, used by Mossel and Wilson-Blair.

The differential system consists of sodium sulfite and ferric citrate. It allows the detection of sulfite-reducing organisms, which form black colonies due to the ferrous sulfide precipitate.

TechniqueThe usual technique for the use of this medium is as follows:The samples to be examined are ground or homog-enized with a vortex in a stomacher and then a decimal dilution bank is prepared. Take a sample aliquot from each one of these dilutions and place in the petri dishes. The medium, molten and cooled to 50°C, is now poured in the dishes and allowed to solidify. The dishes are incu-bated in an anaerobic system at 35°C for 24-36 hours.Usually, 90% of the black colonies which are formed can be attributed to the Clostridium perfringens. Neverthe-less, and since the medium is not extremely selective, it is advisable to verify the black colonies formed by gram-positive sporulated immotile organisms incapable of reducing the nitrates to nitrites. The Indole Nitrite Fluid Medium (Ref. 03-101) is suitable for such purposes, still a small quantity of agar has to be occasionally added in it.Most clostridia are sulfite reductors. Among them are Cl. perfringens and the Cl. botulinum which along with Cl. bifermentans are the species most frequently involved in food poisoning.

ReferencesDOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food,4th Ed. APHA,Washington.FDA (1998) Bacteriological Analytical Manual, 8th ed. Revision A. AOAC International. Gaithersburg. MD

SPS Agar

control

Clostridium perfringens ATCC 13124

145

Ref. 01-587

SpecificationSelective medium for the isolation and presumptive iden-tification of staphylococci.

Formula (in g/L)Casein Peptone ......................................10,00Yeast extract .............................................2,50Gelatin ....................................................30,00Sodium chloride ......................................75,00Di-potassium phosphate ...........................5,00Lactose .....................................................2,00D-Mannitol ..............................................10,00Agar ........................................................15,00Final pH 7,2 ± 0,2

DirectionsSuspend 149,5 g of powder in 1 L of distilled water and heat up to 55-60°C in boiling water bath until total dis-solution of gelatin. Bring to the boil. Dispense in suitable containers and sterilize by autoclaving at 121°C for 15 minutes. Before pouring in the petri dishes, gently shake to suspend possible precipitate. Should a blood addi-tion be desired, cool to 45°C and add sterile defibrinated blood and homogenize before pouring in the plates.

DescriptionThis medium combines the basic criteria of staphylococci identification i.e.: halotolerance, pigmentation, mannitol fermentation and gelatin liquefaction as were marked by Stone and Chapman. Smucker and Appleman suggests the addition of sodium azide (5mg/L) to improve the inhibition of Bacillus spp.It is widely known that pathogenic staphylococci are co-agulase positive, pigment former, highly halotolerant and able to liquefy gelatin and produce acids by fermenting mannitol. They are also strongly hemolytic.In the 110 medium, the following reactions can be observed: pigmentation, mannitol fermentation, gelatin liquefaction and saline tolerance. From this medium, hemolysis and coagulase production can also be proved afterwards.

TechniqueThe sample is inoculated in the petri dish by wire loop to obtain isolated colonies, and it is incubated for 48 hours at 30°C.At 37°C early reactions or results are obtained, but pigmentation is not good,as the pigmented colonies form a characteristic golden yellow colour, which in this case are white.Acid production from mannitol is verified by adding a few drops of Bromthymol Blue 0,04% solution on suspected colonies. Positive reaction produces a yellow coloura-tion.Gelatin hydrolysis (Stone’s reaction) is visualised by a clear halo surrounding the colony which forms 10 min-utes after a few drops of 20% aq. soln.of Sulfosalicylic acid or sat. aq. solution of Ammonium sulfate are added over them.These last two reactions are helped by placing Oxford cups around the discrete colonies, isolating them from affecting the other colonies. Identification is completed by verifying hemolysis tests, subculturing on Blood Agar plates, and verifying plasmacoagulase activity on rabbit serum.Although the subcultures can be done from isolated colonies in 110 medium, results are always better if a short (4-5 hours) incubation on Tryptic Soy Broth (Ref. 02-200) is carried out.

ReferencesSTONE, R.V. (1935) A cultural method for classifying staphylococcias of the “food poisoning” type. Proc.Soc. Exptl.Biol Med.33:185-187CHAPMAN, G.H. (1945) The significance of sodium chloride in studies of staphylococci. J. Bacteriol 50:201-205FDA (1998) Bacteriological Analytical Manual. 6th ed. Revision A. AOAC International. Gaithersburg

Staphylococcus 110 Agar

146

Starch Agar

Ref. 01-283

SpecificationSolid medium to detect the starch hydrolysis by microor-ganisms,

Formula (in g/L)Meat extract ................................................3,0Peptone ......................................................5,0Soluble starch .............................................2,0Agar ..........................................................15,0Final pH 7,2 ± 0,2

DirectionsSuspend 25 g of powder into 1 L of distilled water and let it soak. Heat to boiling and distribute into suitable containers. Sterilize in the autoclave at 121°C for 15 minutes.

DescriptionAlthough this medium was initially formulated to perform the test for the identification of Bacillus cereus, it can be applied to any kind of microorganism where starch hydrolysis activity is required to be analyzed.

TechniqueOver the medium plates with Starch Agar, inoculate in straight streaks the strains to be examined, (maximum four per plate). Incubate at 30-35°C for 48 hours if the strains are of Bacillus cereus and up to 5 days for dubi-ous cases.After the incubation, flood the plates with an alcoholic iodine solution 2%. Starch hydrolysis is seen by the ap-pearance of a clear halo surrounding the growth streak, whereas the rest of the medium in the plate acquire a dark blue colour. The bigger the clear zone, the starch activity is consid-ered higher of the strain under study.

ReferencesCOLLINS, C.H., LYNE, P.M. (1976) Microbiological Methods. 4th Ed. Butterworths,London.ISENBERG H.D. (1992) Clinical Microbiology Proce-dures Handbook. ASM Washington.ATLAS, R.M., & LC. PARKS (1993) Handbook of Micro-biological Media. CRC Press,Inc.,London

Starch Media

147

Ref. 03-454

SpecificationMedium for the conservation and transport of pathologi-cal specimens or fastidious microorganisms.

Formula (in g/L)Sodium glycerophosphate ....................10,000Sodium thioglycolate ..............................1,000Calcium chloride .....................................0,100Methylene blue .......................................0,002Agar ........................................................8,000Final pH 7,4 ± 0,2

DirectionsSuspend 19 g of powder in 1 L of distilled water and bring to the boil. Distribute in tubes or flasks close with air tight cap in such a way that the medium forms a vertical column of 7-10 cm. Sterilize in the autoclave at 121°C for 15 minutes and cool quickly in the vertical position.

DescriptionThe growth of microorganisms in this medium is restrict-ed by the total lack of nitrogen, but they remain alive and inactive for a long period. Thanks to the buffering and protective effect of glycerophosphate. Thioglycolate pro-

vides a reducing environment which is aided and main-tained by to the low concentration of agar, that avoids convection streams and restricts oxygen difussion. Progressive oxidation of the medium can be seen by the change of the methylene blue, which acts as an Eh (redox) indicator.

TechniqueSample is placed directly inside the tube, taking care that it is beneath the blue band. If the sample is taken with a swab, it is advisable to impregnate it with a suspension of active carbon (activated charcoal) before puting it into the transport medium. The sample must always be in the centre of the medium and beneath the blue band that indicates oxidation. If the depth of the blue band is bigger than the half of the medium, do not use the tube.

ReferencesSTUART, R.D. (1959) Transport medium for specimens in public health bacteriology. Publ. Hlth. Rep. 74:431-438.RINGERTZ, O. (1960) A modified Stuart medium for the transport of gonococcal specimens. Acta Path. Microbiol. Scand. 48:105-112

Stuart-Ringertz Transport Medium

R-22-43S-24-37-46

Xn

148

TCBS Media

TCBS Agar

Ref. 01-190

SpecificationTCBS Agar is specially recommended for the selective isolation of Vibrio that causes cholera or dysenteric diar-rhoea, and for the examination of food that might have Vibrio.

Formula (in g/L)Casein peptone ........................................5,00Meat peptone ...........................................5,00Yeast peptone ...........................................5,00Sodium citrate ........................................10,00Sodium thiosulfate ..................................10,00Ox bile ......................................................5,00Sodium Cholate ........................................3,00Sucrose ..................................................20,00Sodium chloride ......................................10,00Ferric citrate ..............................................1,00Thymol blue ..............................................0,04Bromo thymol blue ...................................0,04Agar .......................................................14,00Final pH 8,6 ± 0,2

DirectionsSuspend 88 g of powder in 1 L of distilled water and bring to the boil. Boil it for 1 minute. Cool to 45-50°C and pour it into sterile petri plates. Do not autoclave it.

TCBS Modified Agar

Ref. 01-567

Specification TCBS Agar is specially recommended for the selective isolation of Vibrio parahaemolyticus according ISO 8914 standard.

Formula (in g/L)Proteosa Peptone ...................................10,00Yeast Extract ............................................5,00Sucrose ..................................................20,00Sodium citrate ........................................10,00Sodium chloride ......................................10,00Ox bile ......................................................8,00Sodium thiosulphate ...............................10,00Iron citrate ................................................1,00Thymol Blue .............................................0,04Bromthymol blue ......................................0,04Agar ........................................................14,00Final pH 8,6 (± 0,2)

DirectionsSuspend 88 g of powder in 1 L of distilled water and bring to the boil. Boil it for 1 minute. Cool to 45-50°C and pour it into sterile petri plates. Do not autoclave it.

DescriptionNowadays TCBS Agar is universally accepted as the medium of choice for differential isolation of enteropatho-genic Vibrio, and it achieves a great inhibition of all the accompanying organisms. This formulation allows a high growth of Vibro cholerae and V.parahaemolyticus. V.alginoliticus and NAG vibrios also grow well.Enterobacteria are strongly inhibited by high concentra-tions of citrate, thiosulfate, bile and sodium chloride. Although some enteric bacteria may also grow in this medium, their colony morphology is quite different than Vibrios.The organisms that may be confused with vibrios are some biotypes of Proteus and Pseudomonas. There are some resistent enterococci which may form exception-ally small and yellow colonies on this medium. Usually, in this medium, colonies are selected or chosen and then identified with primary tests (oxidase reactions in Kliger Iron Agar (Ref. 01-103), MRVP Broth (Ref. 02-207), and antibiogram) before performing serological identification and phage typing.Due to its high selectivity, the medium allows massive inoculation of pathological material. Once solidified and cooled, the medium is turbid, but the observations are not affected. This medium is very thermolabile and so it must not be autoclaved, overheated or remelted.

Colonial appearance on TCBS Agar after 24 hours at 37°C:Vibrio alginolyticus: Big, yellowVibrio cholerae and sucrose fermentative strains of Vi-

brio parahaemolyticus: Average size, dirty yellow with yellow halo in the center

Non sucrose fermentative strains of Vibrio parahaemolyticus: Small, yellow, without halo

and with a green core.Streptococcus faecalis: Very small and convex, yellow

with yellow haloEnterobacteria: Small and transparentPseudomonas, Aeromonas, Proteus: Average size and

blue.

ReferencesKOBAYASHI, T., ENOMOTO, S. SAKAZARI, R. and KUWAHARA, S. (1963) A new selective medium for pathogenic vibrios TCBS, (modified Nakanishi Agar)Jap.J.Bact.18:387.FDA (1998) Bacteriological Analytical Manual 8th ed. Revision A. AOAC INTERNATIONAL. GaithersburgISO 8914 Standard (1990) General guidance for the detection of Vibrio parahemolyticus.

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DOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food,4th Ed. American Public Health Association,Washington D.C.PASCUAL ANDERSON, MªRª (1992) Microbiología Alimentaria. Diaz de Santos, S.A.,Madrid,.BHATTACHARYA, M.K., S.K. BATTACHARYA, S. GARG, P.K. SAHA, D.DUTTA, G.B. NAIR, B.C. DEB &

TCBS Media

Ref. 02-474

SpecificationLiquid medium for the cultivation of recombinant strains of Escherichia coli.

Formula (in g/L)Casein peptone ......................................12,00Yeast extract ...........................................24,00Dipotassium phosphate ............................9,40Monopotassium phosphate ......................2,20Final pH 7,3 ± 0,2

DirectionsDissolve 47,6 g of powder in 1 L of distilled water, heat-ing up only if necessary. Distribute in suitable containers and sterilize by autoclaving at 121ºC for 15 minutes.

Description Terrific Broth was developed by Tartoff and Hobbs to improve yield in plasmid bearing E. coli. This medium supports a high cellular density and mass and maintains the growth in the logarithmic phase for a long time. Due to this fact, it provides greater yields of recombinant proteins and plasmid DNA. On many occasions it sub-stitutes the classical LB Broth (Ref. 02-385, 2-384 and 2-406) therefore usually increases yields of plasmid DNA and recombinant proteins.

ReferencesSAMBROC, J., E.F. FRITSCH, T. MANIATIS (1989) Mo-lecular Cloning: A laboratory Manual. 2ª Ed.,Cold Spring Harbor Press. Cold Spring Harbor,USA.TARTOFF, K.D. & C.A. HOBBS (1987) Improved media for growing plasmids and cosmid clones. Bethesda Re-search Laboratoires Focus 9:12

Terrific Broth

KP DAS (1993). Outbreak of Vibrio cholerae non-01 in India and Bangladesh. Lancet, 341:1346-1347HORWITZ, W. (2000) Official Methods of Analysis of AOAC International 17 ed. Gaithersburg. MD

control

Proteus mirabilis ATCC 14273

Vibrio alginolyticus

Ref. 01-567TCBS Modified Agar

150

Tetrathionate Media

Tetrathionate Broth Base

Ref. 02-033

SpecificationMedium for the selective enrichment of Salmonellae (AOAC 17th, ICMSF 1968, USP 25th)

Formula (in g/L)Meat peptone .............................................2,5Casein peptone ..........................................2,5Bile salts .....................................................1,0Calcium Carbonate ...................................10,0Sodium Thiosulfate ...................................30,0

DirectionsSuspend 46 g of powder to 1 L of distilled water, heat to boiling and cool to 40-45°C. Add 20 mL of iodine-iodide solution and 2 vials of the Selective Supplement of Bril-liant Green-Novobiocin ref. 06-017CASE and distribute in sterile tubes.Do not heat after adding the iodine solution. Medium must be used immediately. Without the iodine solution, medium can be stored in refrigeration for some days. The appearance of white medium precipitate is normal, and it comes from calcium carbonate.

DescriptionThis is the version originally used, which has been modi-fied or improved the Muller-Kauffmann formulation, since the latter one has more efficacy.

Muller-Kauffmann Medium Base

Ref. 02-335

SpecificationMedium for selective enrichment of Salmonellae, acc. ISO standard.

Formula (in g/L)Bile salts #3 ..............................................4,78Meat extract ..............................................4,30Casein peptone ........................................8,60Sodium chloride ........................................2,60Calcium carbonate .................................38,70Sodium thiosulfate ..................................47,80

DirectionsAdd 107 g of powder to 1 L of distilled water. Heat to boiling and let it cool to 40-45°C. Add 20 mL of iodine-iodide solution and 2 vials of the Selective Supplement of Brilliant Green-Novobiocin Ref. 06-017CASE and distribute into sterile tubes.Do not heat after adding the iodine solution. Complete medium must be used immediately; the base, without iodine, may be stored in the refrigerator for some days.

White precipitate is due to calcium carbonate and it must be considered as normal.

DescriptionTetrathionate Broth is a classic medium for the enrich-ment of enteric or intestinal pathogens, and for all the members of Salmonella type, from very polluted sam-ples, like faeces, urine, waste water and others.During the preparation, when iodine is added, tetrathion-ate is produced from the sulfate, and this salt together with the bile salts in the medium, provoke a strong inhibi-tion to most of the normal intestinal bacteria, except for those which are capable of reducing tetrathionate, e.g. Salmonellae. Reduction reaction liberate sulfuric acid, which is neutralized by the carbonate, avoiding a de-crease of the pH, which is harmful even for Salmonellae.However, many Proteus species resist the bile salts concentration and moreover, they may even reduce tetrathionate. So, many authors recommend the addition of other inhibitors simultaneously, such as 0,1% Brilliant Green Solution (10 mL/L) which at the same time inhibits gram-positive flora, or Novobiocin in a concentration between 4 and 40 mg/L. Basal medium can be kept indefinitely in the refrigerator, but after the addition of inhibitors, efficacy of the medium decreases with time.Sorting the inhibitors depending on their stability if they are kept in the refrigerator produce the following list: bril-liant green, novibiocine, which is effective for 2 months in the refrigerator but only 48 hours at 37°C, and finally iodine solution, which is only effective for 40 hours once the inhibitor is added to the medium.

TechniqueIt is recommended to prepare the Base Broth, distribute it into tubes, sterilize and cool it. Add the Brilliant Green

151

solution and store it in the refrigerator. If you are going to use the medium before 60 days, you may also add novobiocin. Iodine-iodide solution has to be added just before its use. Do not reheat the medium after one of these additions.Usual technique consists of adding the sample to the medium (1:10) and then homogenizing it well. Incubate at 37°C for a period not longer than 48 hours, since after this time the medium loses its selectivity and the suppressed flora may also grow. Some authors suggest to incubate at 43°C and perform observations after 18, 24 and 48 hours, but one can get better results if the sample from the surface of the broth is taken after 30-36 hours.Take aliquotes with a loop and inoculate on the surface of the selective media like SS Agar (Ref. 01-171) or Hëktoen Enteric Agar (Ref. 01-216), etc...

ReferencesDIN Standard 10160 Untersuchung von Fleisch und Fleischerzeugnissen: Nachweis von Salmonellen. Ref-erenzverfahren.DIN Standard 10181 Mikrobiologische Milchuntersuc-hung: Nachweis von Salmonellen. Referenzverfahren.DOWNES, F.P. & K.ITO (2001) Compendium of meth-ods fort he microbiological examination of foods. 4th ed. APHA. Washington DC. USAFDA (1998) Bacteriological Analytical Manual. 8th ed. Revision A. AOAC International. Gaithersburg. Md. USA.

Tetrathionate Media

Thioglycollate Broth (USP Alternative Thioglycollate Medium)

Ref. 02-186

SpecificationA medium for sterility test and the cultivation of microaer-ophilic and anaerobic organisms. It is specially used for viscous or turbid samples.

Formula (in g/L)Peptone from casein ................................15,0Yeast extract ...............................................5,0Dextrose .....................................................5,5Sodium chloride ..........................................2,5Sodium thioglycollate .................................0,5 L-Cystine ....................................................0,5 Final pH 7,1 ± 0,2

Thioglycollate Media

DirectionsDissolve 29 g of powder in 1 L of distilled water, heating if necessary to help dissolution. Distribute into suitable containers and sterilize by autoclaving at 121°C for 15 minutes. This culture medium should always be freshly prepared or heated at 100°C for 10 minutes before use.

DescriptionThe Thioglycolate broth is a standard medium, named also Alternative Thioglycollate Medium, formulated and recommended by USP, NF, NIH and FDA.It is used for sterility testing of biological products or samples of turbid appearance where Fluid Thioglycol-late Medium (Ref.3-187) is not suitable because of its viscosity.The formula of Thioglycollate broth is the same as Thioglycollate USP Fluid Medium without resazurin and agar.Media must be freshly prepared, boiled, sterilised, cooled and used within 4 hours for its inoculation.

FIL-IDF Standard 93. (2001) Milk and milk products: Research of Salmonella.HORWITZ, W. (2000) Official Methods of Analysis. 17th ed. AOAC International. Gaithersburg. Md.USAISENBERG, H.D. (1992) Clinical Microbiology Proce-dures Handbook. Vol. 1. APHA. Washington DC. USAISO Standard 6579 (2002) Microbiology of food and ani-mal feeding stuffs – Horizontal method fort he detection of Salmonella spp.ISO Standard 6785 (2001) Milk and Milk Products – De-tection of Salmonella spp.ISO (1975) Standard 3565. Meat Products: Reference Method for detection of Salmonellae..U.S. PHARMACOPEIA. (2002) 25th ed. <61> Microbial Limits Test. US Pharmacopoeial Convention Inc. Rock-ville. Md. USA.KAUFFMAN, F. (1931) Ein Kombiniertes Anreicherungus verfahren für Typhus und Paratyphus Bazillen.Zblt. Bakt Microbiol. Hyg Abt. I. Orig. 119:148MARSHALL, R.T. (1993) Standard methods for the ex-amination of dairy products. 16th ed. APHA Washington DC. USA.MULLER, L. (1923) Un nouveau milieu d’enrichiessement pour la recherche du bacille typhique est des partyphyques. Comp. Rend. Soc. Biol. 89:434-437.

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152

DescriptionThe Thioglycollate Fluid Medium is a standard medium formulated and recommended by European Pharmaco-poeia, USP, NIH and FDA.The reducing agents thioglycollate and L-cystine ensure an anaerobiosis which is adequate even for fastidious anaerobes. The sulfhydryl groups of these substances also inactivate arsenic, mercury and other heavy metal compounds. The Thioglycollate media are thus suitable for the examination of materials which contain heavy metals or heavy metal preservatives. The higher viscosity of the fluid thioglycollate medium prevents rapid uptake of oxygen. Any increase in the oxygen content is indicated by the redox indicator so-dium resazurine which changes its colour to pink.

TechniqueInoculate the culture medium with the sample material taking care that the sample reaches the bottom of the tubes. Incubate for at least 14 days at the optimal temperature.Anaerobes grow in the lower part of the culture medium container.

ReferencesATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-biological Media, CRC Press Inc., LondonBREWER J.H (1940) Clear liquid medium for the “aerobic” cultivation of anaerobes. J. Amer. Med. Assoc. 113:598-600DOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food. 3rd ed., A.P.H.A., Washington D.C.EUROPEAN PHARMACOPOEIA, (2005) § 2.6.1 Steril-ity. 5th Ed., EDQM Council of Europe. Strasbourg.FDA (1998).Bacteriological Analytical Manual, 8th ed. Revision A., AOAC International. Gaithersburg. MDHORWITZ, W., (2000) Official Methods of Analysis, 17th ed., A.O.A.C. International. Gaithersburg. MDISENBERG, H.D. (Ed), (1998) Essential Procedures for Clinical Microbiology. ASM., Washington. USAMacFADDIN, J.F. (1985) Media for Isolation-cultivation-identification-maintenance of medical bacteria. Vol. I., Williams & Wilkins. Baltimore. MD. USAUS PHARMACOPOEIA (2005) 25th Ed. § <71> Steril-ity Test., US Pharmacopoeial Convention Inc., Rockville MD.

ReferencesATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc. LondonDOWNES, F.P. & K. ITO. (2001) Compendium of Meth-ods for the Microbiological Examination of Foods. 4th Ed. APHA. Washington DC. USAHORWITZ, W. (2000) Official Methods of Analysis. 17th ed. AOAC International. Gaithersburg. Md. USAUS PHARMACOPOEIA (2002) <71> Sterility Test. 25th ed. US Phamacopoeial Convention Inc. Rockville. Md. USA.

Thioglycollate Fluid Medium

Ref. 03-187

SpecificationFluid medium for sterility testing acc. to Eur. Phar., USP, FDA, and ISO 7937:2004 for the cultivation of microaer-ophilic and anaerobic organisms. It is specially used for viscous or turbid samples.

Formula (in g/L)Peptone from casein ............................15,000Yeast extract ...........................................5,000Dextrose .................................................5,500Sodium chloride ......................................2,500Sodium thioglycollate .............................0,500 L-Cystine ................................................0,500 Resazurine .............................................0,001Agar ........................................................0,750Final pH 7,1 ± 0,2

DirectionsDissolve 30 g of powder in 1 L of distilled water, slowly bring to the boil, stirring until complete dissolution. Dis-tribute into final containers and sterilize by autoclaving at 121°C for 15 minutes. Store in dark at room tempera-ture for not more than one month. If after the storage the medium is pink coloured (sign of oxidation) for more than 1/3 of its depth, recreate anaerobic conditions by heating at 100°C for 10 minutes.

Thioglycollate Media

Left: anaerobical growth; center: control; right: fermentative growth

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Ref. 03-187 ThioglycollateFluid Medium

153

Triple Sugar Iron Agar (TSI Agar) (Eur. Phar. Agar Medium M)

Ref. 01-192

SpecificationDifferential medium for identification of enterobacteria, according ISO standard.

Formula (in g/L)Peptone ................................................20,000Meat extract ............................................3,000Yeast extract ...........................................3,000Lactose .................................................10,000Sucrose ................................................10,000Dextrose .................................................1,000Sodium chloride ......................................5,000Ferric ammonium citrate .........................0,500Sodium thiosulfate ..................................0,300Phenol red ..............................................0,025Agar .....................................................12,000Final pH 7,4 ± 0,2

DirectionsDissolve 64,8 of powder in 1 L of distilled water and bring to boiling. Dispense into tubes and sterilize at 121°C for 15 minutes. Leave to solidify with short slant and good butts.

DescriptionTSI Agar is a modification of the classical Kliger’s agar. 1% of sucrose has been added to this medium to dif-ferentiate Proteus and Hafnia (sucrose positive) from Salmonella and Shigella (sucrose negative).Sugar degradation with acid formation is detected by the turning of an indicator (phenol red) to yellow, whereas if there is alkalinization, it turns to purple. When there is only glucose degradation, the acid production is weak and is evaporated on the surface, so indicator may be reoxidised producing an alkaline surface (red) and an acid butt (yellow). If lactose or sucrose are degradated, acid production is intense and then all of the medium (surface and depth) turns yellow. Gas production is detected by the formation of bubbles and occasionally cracks in the agar.Hydrogen sulfide production, from thiosulfate or sulfured aminoacids of peptones, is detected by the formation of black FeS precipitate when medium reacts with iron salts.Use the medium in slanted tubes with good depth and short slant. Inoculate by streaking on surface and stabbing deeply. It is advisable to use tubes with cotton plugs, in order to allow a reoxidation of the indicator. If screw caps are used, they must be loose.Following will find the table of reading (observations) and interpretation of results in TSI Agar.

ReferencesDOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the microbiological examination of Foods. 4th ed. APHA. Washington DC. USA

EDWARD, S.P. and EWING, W.H. (1962). Identification of Enterobacteriaceae. Burgess. Pub. Co. Minneapolis.EUROPEAN PHARMACOPOEIA.(2005) Supp. 5.8 § 2.6.13 Test for specified micro-organisms. EDQM. Stras-boug E.U.FIL-IDF (1991) International Standard 93A. Milk and Milk Products. Detection of Salmonella species.HAJNA, A.A. (1945) Triple Sugar-Iron medium for the identification of the intestinal group of bacteria. J.Bact. 49:516-517HORWITZ, W. (2000) Official Methods of Analysis. 17th ed. AOAC International. Gaithersburg. Md. USA.

154

ISO 3560 Standard. (1975) Reference Method for the Detection of Salmonella in meat and meat products.ISO 6579 Standard. (2002) Microbiology of foods and animal feeding stuffs - Horizontal method for the detec-tion of Salmonella spp.ISO 6785 Stanbdard (2001) Milk and milk Products – Detection of Salmonella spp.ISO 10272 Standard (1995) Microbiology of foods and animal feeding stuffs - Horizontal method for the detec-tion of thermotolerant Campylobacter.ISO 21567. Standard (2004) . Horizontal method for the detection of Shigella ssp.KRUMWIEDE, C. & L. KOHN (1917) A triple sugar modi-fication of the Russell Double Suigar Medium. J. Med. Res. 37:225-229US PHARMACOPOEIA (2002)<61> Microbial Limit Tests. 25th ed. US Phamacopeial Convention Inc. Rock-ville. Md. USA

Triple Sugar Iron Agar (TSI AGAR) (Eur.Phar. Agar Medium M)

Ref. 02-191

SpecificationLiquid culture medium for the propagation of beta-hemo-lytic streptococci and for studies about serologic typing.

Formula (in g/L)Meat extract ..............................................10,0Casein peptone ........................................20,0Glucose ......................................................2,0Sodium bicarbonate ...................................2,0Sodium chloride ..........................................2,0Disodium phosphate ...................................0,4Final pH 7,8 ± 0,2

DirectionsDissolve 36,4 g of powder in 1 L of distilled water and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionThis classical medium formulation has been modified to achieve optimal results in the growth and production of hemolysins, which are not inhibited due to the high buffer composition of the medium. Many official organi-sations, such as APHA, have recommended this medium

as an alternative type in epidemiologic studies of group A streptotocci as well as pathogenic microorganisms.With the addition of 15g/L of agar, medium can be solidi-fied and then it is an excellent substrate for the produc-tion of capsules in streptococci.

ReferencesTODD, E.W. and HEWITT, L.F. (1932) A new culture medium for the production of antigenic streptococcol haemolysin.J. Path. Bact. 35:973-974MacFADDIN, J. (1985) Media for isolation-cultivation-identification-maintenance of medical bacteria. Vol. 1. Williams & Wilkins. Baltimore. USA.ATLAS, R.M. and L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press Inc.LondonISENBERG H.D. (1992) Clinical Microbiology Proce-dures Handbook. ASM. Washington DC.

Todd Hewitt Broth

155

Tryptic Soy Agar (TSA)(Eur. Phar. Agar Medium B)(Casein Soybean Digest Agar)

Ref. 01-200

SpecificationGeneral purpose solid medium containing animal and plant peptone according ISO 9308-1 standard.

Formula (in g/L)Casein peptone ........................................15,0Soy peptone ...............................................5,0Sodium chloride ..........................................5,0Agar ......................................................... 15,0Final pH 7,3 ± 0,2

DirectionsMix 40 g of powder in 1 L of distilled water. Let it soak and bring to the boil to dissolve the agar. Sterilize by autoclaving at 121°C for 15 minutes.

DescriptionTSA is a widely used medium containing two peptones which support the growth of a wide variety of organisms, even that of very fastidious ones such as Neisseria, Listeria, Brucella, etc. It is frequently used for routine diagnostic purposes due to its reliability and its easily reproducible results.The following list includes some of its most common applications:

1.- Sensitivity testing either by the Kirbky-Bauer system or by following the WHO guidelines. Both the sys-tems recommend the use of the Mueller Hinton Agar (Ref. 01-136) for verification purposes.

2.- The medium provides, with added blood,perfectly de-fined hemolysis zones, while preventing the lysis of the erythrocytes due to its sodium chloride content.

3.- It can be used for the preparation of an exceptionally nutrient ‘chocolate´ agar, thanks to the richness of its peptones.

4.- In a reducing environment or with a CO2 enriched

atmosphere, its plates provides an excellent medium for the isolation of Brucella and Neisseria . It may be made selective by using certain additives.

5.- Most streptococci grow in this medium though clear differences can be observed from one species to another.

6.- The Tryptic Soy Agar is the selective medium for the count of urine samples although the differentiation must be done on selective differential media.

7.- Several tests for the differentiation and identification of staphylococci can be obtained in this medium, provided with suitable additives.

8.- Yeast, particularly Candida species, can grow in this medium forming very characteristic colonies.

9.- Chromogenic pseudomonads frequently produce pigmentation on the TSA and are therefore easily recognized.

10.- It is widely used for testing contaminated samples. A vast bibliography documents its applications in the food industry.

11.- It has been frequently used in the Health industry to produce antigens, toxins,etc...

12.- Its simple and inhibitors-free composition makes it suitable for the detection of antimicrobial agents in food and other products.

13.- A balanced and highly nutrient value together with a lack of fermentable carbohydrates make this medium one of the most recommended for the strain mainte-nance.

Tryptic Soy Broth (TSB)(Eur. Phar. Broth Medium A)(Casein Soybean Digest Broth)

Ref. 02-200

SpecificationHighly nutrient liquid medium for the general purposes, formulated according to USP, FDA and Eur. Phar. regula-tions.

Formula (in g/L)Casein peptone ........................................17,0Soya peptone .............................................3,0 Sodium chloride ..........................................5,0Dipotassium phosphate ..............................2,5Dextrose .....................................................2,5Final pH 7,3 ± 0,2

DirectionsDissolve 30 g of powder in 1 L of distilled water and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionThe Tryptic Soy Broth was initially developed for the cultivation of very fastidious microorganisms without the addition of serum, blood or any other enrichment agent.As a general purpose culture medium it supports the growth of most organisms, both aerobes and faculta-tives, even if their requirements are high. Due to its high vitamin content the development of Brucella, Pasteurel-la and Streptococcus is perfectly viable, moreover a CO

2 enriched atmosphere can further favour it.

In anaerobic conditions this broth will easily bear the growth of Bacteroides and Clostridium species. For this purpose, the best results can be obtained by adding 0.3% agar and 0.05% sodium azide for Clostridium. The Tryptic Soy Broth’s superior growth-promoting prop-erties makes it particularly suitable for the tube dilution method for antibiotic sensitivity testing. It also achieves good results in the detection of gram-positive cocci. The broth can be used for bile solubility testing in pneumo-

Tryptic Soy Media

156

cocci, and also used for catalase and coagulase assays and for the preparation of hypersaline broths. It is a most suitable medium for the preparation of anti-gens and toxins in bacteria, moulds and yeasts.TSB is used as a primary enrichment medium for food examination. In the dairy industry it is employed for test-ing resazurine reduction.The medium is not suitable for maintenance purposes since carbohydrate fermentation liberates many acids which may threaten the organisms’ viability. Therefore, though it allows the growth of streptococci and Neisse-ria, these species tend to die if repeatedly subcultured in this medium. Such fastidious organisms are best main-tained on Cystine Tryptone Fluid Medium (CTA) (Ref. 03-045) or even TSA (Ref. 01-200) if it is not suitable or convenient to the use of solid media.

ReferencesUS PHARMACOPOEIA (2002) 25th ed. <61> Microbial Limit Tests. US Phsarmacopoeial Convention. Rockville. MD. EUROPEAN PHARMACOPOEIA (2002) 2.6.13 Tests for Specified Microorganisms. 4th Ed. Suppl.4.2 EDQM Council of Europe Strasbourg.ATLAS, R.M., & LC. PARKS (1993) Handbook of Micro-biological Media. CRC Press, Inc.LondonDOWNES, F.P. & K. ITO (2001). Compendium of Meth-ods for the Microbiological Examination of Food, 4th ed,ASM,Washington,D.C.PASCUAL ANDERSON, MªRª (1992) Microbiologia Alimentaria. Diaz de Santos, S.A.Madrid,.FDA (1998) Bacteriological Analytical Manual. 8th ed. Revision A. AOAC International. Gaithersburg. MDISO 9308-1 Standard (2000) Water Quality - Detection and enumeration of E.coli and coliform bacteria. Mem-brane filtration method.

Tryptic Soy Broth w/o Dextrose(TSB w/o Dextrose)

Ref. 02-227

SpecificationLiquid culture medium for the massive production of spores of B. stearothermophilus for the inhibitory sus-tances in food acc. FDA-BAM

Formula (in g/L)Tryptone .................................................17,00Soy peptone .............................................3,00Sodium chloride ........................................5,00Dipotasium phosphate ..............................2,50Final pH 7,3 ± 0,2

DirectionsDisolve 27,5 g of powder in 1 L of distilled water, heat-ing if necessary. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

DescriptionTSB w/o Dextrose is produced according the formulation from Bacteriological Analitycal Manual of Food and Drug Administration for the massive production of spores of Bacillus stearothermophilus used to determine the pres-ence of inhibitory sustances in milk and dairy products.This medium is not recommended for sugars fermenta-tion studies because the great amount of fermentable carbohidrates in the soy peptone.

ReferencesMATURIN, L.J. (1998) Inhibitory substances in milk. Qualitative Method II: B. stearothermophilus disk assay. en FDA Bacteriological Analitical Manual. 8th Ed. Revi-sion A. AOAC International Inc. VA.

Tryptic Soy Media

157

Ref. 01-526

SpecificationSelective solid medium for the rapid enumeration of Escherichia coli according ISO 9308-1 standard.

Formula (in g/L)Tryptone .................................................20,00Bile salts #3 ..............................................1,50Agar ........................................................15,00Final pH 7,2 ± 0,2

DirectionsSuspend 36,5 g of powder in 1 L distilled water and bring to the boil. Distribute into suitable containers and steri-lize at 121ºC for 15 minutes.

DescriptionThis medium is formulated according the Anderson & Baird-Parker Direct Plating Method (DPM) for the rapid enumeration of Escherichia coli in raw meats. The method is based on the characteristically production of indol from tryptophane when the bacteria growth at 44ºC over a cellulose acetate membrane in the surface of the Tryptone bile agar.The International Commission on Microbiological Stand-ards for Food noticed that he DPM was lesser variable and offer a better recovery and rapidity than the MPN method for frozen samples of meat. The ISO Standard 6391:1988 also uses this medium for the E. coli enu-meration.

The indol producer microorganisms other than E. coli are inhibited by the bile salts and the incubation tempera-ture, but in sugar-rich samples the indol production can be inhibited due to the sugar concentration interferes the tryptophanase synthesis.

Technique A cellulose acetate membrane of 0,45 ìm pore is extend-ed on the surface of the medium. A 0,5 mL of the sample dilution (with Tryptone Water Ref. 03-156) inoculum is spreaded on the membrane and the plates are incubate in upright position at 44±1ºC for 18-24 hours.After incubation the membrane is immersed in indole reactive using the petri dish cover as container and exposed the direct sunlight for 5 minutes or to the Wood lamp for 10 minutes. The indole positive colonies turns to the reddish colours (pink to deep red). The results are expressed as number of Escherichia coli per g or mL of sample.

ReferencesANDERSON, J.M. & A.C. BAIRD-PARKER (1975) Appl. Bact. 39:111-117International Commission on Microbiological Specifica-tions for Foods (1979) Can. J. Microbiol. 25:1321-1327ISO 9308-1 Standard (2000) Water Quality - Detection and enumeration of E.coli and coliform bacteria. Mem-brane filtration method.

Tryptone Bile Agar

158

Tryptone Glucose Extract Agar(TGE Agar)

Ref. 01-082

SpecificationPlate count medium for the milk and dairy products, ac-cording to Standard Methods for Dairy Products.

Formula (in g/L)Meat extract ................................................3,0Casein peptone ..........................................5,0D (+) Glucose .............................................1,0Agar ..........................................................15,0Final pH 7,0 ± 0,2

DirectionsAdd 24 g of powder in 1 L of distilled water. Heat to the boil by constantly stirring. Dispense in suitable contain-ers and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionSolid medium Tryptone Glucose Extract was adopted long ago as an alternative to Nutrient Agar acc. APHA (Ref. 01-144) and Nutrient Agar acc. British Pharmaco-poeia (Ref. 01-140) for milk bacteria enumeration, being a complement to Plate Count Agar (Ref. 01-161).

TechniqueFor the enumeration purposes, it is suggested to use the poured plate method, and incubation at 30-32°C for 48 hours. If the dilutions in the plate is more than 10% it is advisable to add milk to the medium. To do this, prepare the supension of skimmed milk (Ref. 06-019) separately, and sterilize it for 10 minutes at 118°C. Autoclaving must be as short as possible. Homogenize with the culture medium which is sterilized and cooled to 50°C. The use of natural milk is not recommended due the wide varia-tion .Medium must be quickly poured into Petri dishes be-cause if it remains hot for too long, floccules and abnor-mal precipitates may appear. If the sample under study is not diluted or the volume in the plate is more than 2 mL, it is not necessary to add the skimmed milk (Ref. 06-019) because it is assumed that the sample provides the required growth factors.

ReferencesFDA.(1998) Bacteriological Analytical Manual 8th ed Revision A. AOAC International,Gaitherburg MDDOWNES, F.P. & K. ITO (2001) Compendium of meth-ods for the Microbiological Examination of Foods, 4th Ed. APHA, Washington.MARSHAL, R.T.. (Ed.) (1992) Standard Methods for the Examination of Dairy Products. 16th Ed. APHA. Washing-ton.

APHA-AWWA-WEF (1998) Standard Metods for the Examinationof Water and Wastewater. 20th ed. APHA. WashingtonHORWITZ, W. (2000) Official Methods of Analysis. AOAC International. Gaitherburg. MD

Tryptone Glucose Extract Brothfor Membrane Filtration (m-TGE Broth)

Ref. 02-082

SpecificationNon-selective liquid medium for enumerating microor-ganisms by membrane filtration method.

Formula (in g/L)Tryptone ................................................10,00Meat extract ..............................................6,00Dextrose ...................................................2,00Final pH 7,0 ± 0,2

DirectionsDissolve 18 of powder in 1 L of distilled water, heating if it is necessary. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

DescriptionThis medium is a liquid nutritive substrate that can be used in the colony count by the membrane filtration method for the absorbent pad impregnation The broth has the same formulation as Tryptone Glucose Extract Agar , except that the broth contains no agar and the ingredients are at twice the concentration.

Technique The sample is filtered through a membrane. In a plate the absorbent pad is impregnated with the medium avoiding any excess. The membrane is transferred on the pad and the complete system is incubated at 35±2ºC for 18-24 hours. After this time total colonies are counted and results recorded.

ReferencesDOWNES, FP. & K. ITO (2001) Compendium of methods for the microbiological examination of foods 4th ed APHA WashingtonAPHA-AWWA-WEF (1998) Standard Methods for the examination of water and wastewater.

Tryptone Glucose Extract Media

159

Ref. 02-277

SpecificationDilution and nonselective pre-enrichment liquid medium acc. to ISO 6579, 8523, 8261 and 6785 standards.

Formula (in g/L)Peptone ....................................................10,0Sodium chloride ..........................................5,0Disodium phosphate ...................................9,0Potassium phosphate .................................1,5Final pH 7,0 ± 0,2

DirectionsDissolve 25,5 g of powder in 1 L of distilled water. Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionThis formulation of Tryptone Phosphate Water has the advantages of the two classical diluents for food sam-ples: it has the property of revitalization of the peptoned

water and the pH change absorbing capacity of the phosphate buffer.The composition of this diluent is made according the specification of the ISO Standard 6579 for the detection of Salmonella in foods.

ReferencesATLAS, R.M.,& L.C. PARKS (1993) Handbook of Micro-biological Media,CRC Press, Inc.,LondonPASCUAL ANDERSON, MªRª (1992) Microbiologia Alimentaria. Diaz de Santos, S.A.,Madrid,.ISO 6579 Standard (2002). Microbiology of food and animal feeding stuffs. Horizontal method for the detec-tion of Salmonella spp.ISO 8523 Standard (1991) General guidance for the detection of Enterobacteriaceae with pre-enrichment.ISO 8261 Standard (2001) Milk and milk products - General guidance for the preparation of test samples for microbiological examination.ISO 6785 Standard (2001) Milk and milk products - De-tection of Salmonella spp.

Tryptone Phosphate Water (Buffered Peptone Water)

Ref. 01-195

SpecificationSolid selective medium for Clostridium perfringens isola-tion.

Formula (in g/L)Casein peptone ......................................15,00Sodium sulfite ...........................................1,00Neomycin sulfate ......................................0,05Polymyxin B ..............................................0,02Yeast extract ...........................................10,00Ferric citrate ..............................................0,50Agar ........................................................13,50Final pH 7,2 ± 0,2

DirectionsSuspend 40 g of powder in 1 L of distilled water and bring to the boil. Dispense in suitable containers and sterilize by autoclaving at 121°C for 15 minutes.To obtain better results, add 20 mL/L of a solution con-taining 1 g/L dipotassium phosphate, 0,5 g/L sodium car-bonate and 1 g/L sodium thioglycollate just before use.

DescriptionThis culture medium was formulated by taking the advantage of the tolerance of Cl. perfringens to the high concentration of sulfite, which apart from being an inhibitor agent, provides a strong reducing environment. Selection of Cl.perfringens is almost complete when it is incubated at 46°C, since neomycin and polymyxin

included in the medium restrain the development of Cl.bifermentans and all the accompanying gram nega-tive bacteria.The medium is especially suitable for the investigation of food products, and it may be used in tubes as well as in plates. If the incubation is not performed in an anaerobic jar, thioglycolate buffered solution must be added or the inoculated surface must be covered with a sterile layer of medium.Colonies of Cl.perfringens form very characteristic black colonies that, if exposed to air, become decolourised by oxidation. TSN have a very short storage period once prepared, so it is advisable to rehydrate or reconstitute it in small amounts and use it on the day of its preparation.

ReferencesMARSHALL, R.S., STEENBERGEN, J.F., MacCLUNG, L.S. (1955) Rapid Technique for the enumeration of Clostridium perfringens. Appl. Microbiol. 13:559-563.MOSSEL, D.A.A (1959) Enumeration of sulfite reducing clostridia occurring in nfoods. J. sci. Food Agr. 10:662-669MacFADDIN, J.F. (1985) Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria,Williams & Wilkins. Baltimore,USA.ATLAS, R.M., & L.C. PARK (1993) Handbook of Micro-biological Media,CRC Press Inc.,London

Tryptone Sulfite Neomycin Agar (TSN Agar)

160

Ref. 03-156

SpecificationSubstrate with low nutrient capacity, for the research of indole production in coliform micro-organisms according ISO 7251 standard.

Formula (in g/L)Casein peptone ........................................10,0Sodium chloride ..........................................5,0Final pH 7,2 ± 0,2

DirectionsDissolve 15 g of powder in 1 L of distilled water and dis-pense into suitable containers. Sterilize by autoclaving at 121°C for 15 minutes.

DescriptionThe standard protocol requires to reinoculate one loop from each suspected tube in 10 mL of Tryptone Water. Incubate for 48 hours at 44°C before investigating the indole production with the Kovacs’ Reagent for indole (Ref. 06-018).

As an alternative method, the Ehrlich’s Reagent (Ref. 06-024) could also show indole production. After 48 hours of incubation at 37°C, take 0,5 mL of growth and mix it with 0,5 mL of Ehrlich’s Reagent. Let them settle a few minutes. If it takes on a pink colour,then the test is positive. Colour appearance is accelerated if a few drops of saturated solution of potassium persulfate is added.Other authors prefer extraction and concentration of in-dole with 1 mL of ether, and then observe on the extract with any of the reagents mentioned above.

ReferencesATLAS, R.M.& L.C. PARKS (1993) Handbook of Micro-biological Media,CRC Press Inc.,LondonDOWNES, F.P. & K. ITO (2001). Compendium of Meth-ods for the Microbiological Examination of Food. 4th Ed. APHA,Washington APHA-AWWA-WEP (1998) Standard Methods for the examination of water and wastewater. 20th ed. APHA. Washington, DC.ISO 7251 Standard (1993) General guidance for the enumeration of E.coli by the MPN technique.

Tryptone Water (Peptone Water)

Ref. 01-590

SpecificationSolid medium for the enumeration of water microorgan-isms acc. ISO Standard 6222.

Formula (in g/L)Tryptone .....................................................6,0Yeast extract ...............................................3,0Agar ..........................................................15,0Final pH 7,2 ± 0,2

DirectionsSuspend 24 g of powder in 1 L of distilled water and bring to the boil. Distribute into containers and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionThis medium, formulated according to ISO Standard 6222, is for the enumeration of heterotrophic microor-ganisms from water.

TechniqueFrom the water sample, obtained according the ISO Standard 5667-2 and 5667-3, make a decimal dilution bank (see ISO Standard 6887) with Ringer Solution (Ref. 6-073) and take aliquots to 2 parallel series of plates. Pour the Tryptone Yeast Extract Agar melted and cooled to 45°C, and homogenize with sample (see ISO Stand-ard 8199). Once solidified, incubate one of the series at

Tryptone Yeast Extract Agar

36±2°C for 44±2 hours and the other one at 22°C for 3 days (68±4 hours).In order to achieve a good count, select plates with 30-300 colonies. Express the results as number of colony forming units per milliliter (cfu/mL) of the sample for each temperature of incubation. If there are no colo-nies with the undiluted sample express the results as “no detected in one mL”. If there are more than 300 colonies in the highest dilution express the results as “>300/mL”

ReferencesISO Standard 6222 Watewr Quality – Enumeration of culturable microorganisms. Colony count by inoculation in a nutrient agar culture.ISO Standard 5667-2 (1991) Water Quality-Sampling – Guidance on sampling techniquesISO Standard 5667-3 (1996) Water Quality – Sampling. –Guidance on the presevation and handling of samplesISO Standard 6887 (1999) Microbiology- General – Guidance for the preparation of dilutions for microbio-logical examination.ISO Standard 8199 (1988) Water Quality – General guide to the enumeration of microorganisms by culture.

161

Ref. 02-418

SpecificationLiquid medium for the indole production according to ISO standard.

Formula (in g/L)Meat peptone ...........................................10,0L-Tryptophan ..............................................1,0Sodium chloride ..........................................5,0Final pH 7,2 ± 0,2

DirectionsDissolve 16 g of powder in 1 L of distilled water. Distrib-ute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionThis broth allows the indole production from the tryp-tophan, and therefore it is suitable for the differentiation and identification of coliforms from water and food. Its formulation is according to the German standards for waters and foods.

TechniqueMedium is inoculated with the previously isolated culture, and then incubated at 30-32°C for 24-48 hours.Indole production is observed adding a few drops of Kovacs’ Reagent (Ref. 06-018) over the broth (with or without previous extraction) and shaking gently. Forma-tion of a red ring indicates indole presence.

ReferencesVERORDNUNG über Trinkwasser und über Wasser für Lebensmittelbetriebe vom 12-12-1990. Bundesgesatbl. I. 2613-2619.BUNDESGESMELHEITSAMT: Amtliche Samnulung von Untersuchungs verfahren nach #35LMBG. Beuth Verlag.Berlin-Köln.ISO 9308-1 Standard (2000) Water Quality. Detection and enumeration of Escherichia coli and coliform bacte-ria. Part 1: Membrane filtration method.ISO 6785 Standard (2001) Milk and milk products. De-tection of Salmonella spp.ISO 21567. Standard (2004) . Horizontal method for the detection of Shigella ssp.

Tryptophan Broth

Tryptose Agar

Ref. 01-197

SpecificationSolid medium for isolation, cultivation and differentiation of Brucella, streptococci and fastidious pathogens.

Formula (in g/L)Tryptose ................................................20,000Dextrose .................................................1,000Sodium chloride ......................................5,000Thiamine HCl ..........................................0,005Agar ......................................................15,000Final pH 7,2 ± 0,2

DirectionsSuspend 41g of powder in 1 L of distilled water and heat to boiling. Dispense in tubes or flasks and sterilize it in the autoclave at 121°C for 15 minutes.To obtain better results, add to the molten medium, 20 mL/L of a solution composed by dipotassium phosphate 1 g/L, sodium carbonate 0,5 g/L and sodium thioglyco-late 1 g/L just before using the medium.

Tryptose Broth

Ref. 02-197

SpecificationLiquid culture medium for massive culturing of fastidious microorganisms.

Formula (in g/L)Tryptose ................................................20,000Dextrose .................................................1,000Sodium chloride ......................................5,000Thiamine HCl ..........................................0,005Final pH 7,3 ± 0,2

DirectionsDissolve 26 g of powder in 1 L of distilled water, heating up if necessary. Dispense into suitable containers and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionTryptose culture media are suitable, essencially for the growth of fastidious strains, such as Listeria, Pasteurella, Brucella, etc. despite the fact that nowadays it seems there is a trend towards the use of more defined media such as Brucella Agar (Ref. 01-042) and Broth (Ref.

Tryptose Media

162

02-042), which provide better results. Tryptose Agar and Broth have been recommended by several National and International Organisations for Brucelosis Control and for the maintenance, propagation and cultivation of stand-ardized strains. They may be successfully used to dif-ferentiate the several types of Brucella with the addition of suitable indicators. In this case, its use is analogous to the Brucella Broth (Ref. 01-042).With the adequate additives (crystal violet, sodium azide etc.) the media may become very selective and efficient, and their nutrient conditions may be improved if citrate and blood are added, although glucose is not very suit-able for observing hemolysis.

ReferencesCASTAÑEDA, M.R. (1947) A practical method for rutine blood cultures in brucellosis. Proc. Soc. Exp.Biol. Med. 64:114-115HAUSSLER, W.J. (1976) Standard Methods for the Examination of Dairy Products,9th Ed. APHA.MARSHALL, R.T.. (1992) Standard Methods for the Ex-amination of Dairy Products. 16th Ed. APHA. Washington.RENNER, E.D., K.J. McMAHUN (1981) Brucellosis in Di-agnostic Procedures for Bacterial Mycotic and Parasitic Infections. 6th Ed.,APHA, Washington.DCATLAS, R.M. & LC. PARKS (1993) Handbook of Micro-biological Media. CRC Press Inc.,London.DOWNES,F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food. 4rdEd. APHA.WashingtonFDA (1998) Bacteriological Analytical Manual. 6th ed. Revision A. AOAC International. Gaithersburg. MDMURRAY,P.R., E.J. BARON, M.A PFALLER, F.C. TENO-VER, & R.H. YOLKEN (1995) Manual of Clinical Microbi-ology. 6th ed. APHA. Washington DC .

Tryptose Phosphate Broth

Ref. 02-199

SpecificationLiquid culture medium, with glucose and buffer, for cultivation of fastidious microorganisms and for tissues culture media.

Formula (in g/L)Tryptose ....................................................20,0Dextrose .....................................................2,0Sodium chloride ..........................................5,0Disodium phosphate ...................................2,5Final pH 7,3 ± 0,2

DirectionsDissolve 29,5 g of powder into 1 L of distilled water. Dis-pense into suitable containers and sterilize by autoclav-ing at 121°C for 15 minutes.

DescriptionTryptose Phosphate Broth is a recommended medium for the cultivation and propagation of microorganisms that have strong needs, such as streptococci, menin-gococci, and Brucella. It has also been also used to determinate antibiotic sensitivity testing by the dilution in tube method.This medium has been used as primary diluent and emulsifier in dairy products for determination of Brucella, but it is really effective for the cultivation of many strepto-cocci and to test the bile solubility of these organisms.When it is used to isolate streptococci, it is suggested to add 0,1% Agar to render it into a fluid medium. Should a very selective medium be desired, add 2,5% of sodium azide. To get a solid medium, add 1,5% of Agar.

ReferencesGINSBERG, H.S. (1955) Tryptose Phosphate Broth as Supplementary Factor for Maintenance of Hella Cell Tis-sue Cultures. Proc. Soc. Exper. Biol. Med. 89(1):66-71.WAISBREN, B.A. (1951). The Tube Dilution Method of Determining Bacterial Sensitivity to Antibiotics. Am. J. Clin. Path 21:884.BALOWS, A., W.J. HAUSSLER (1981) Diagnostic Pro-cedures for Bacterial Mycotic and Parasitic Infections. 6thEd,APHA. Washington.ATLAS, R.M., & J.W. SNYDER (1995) Handbook of Me-dia for Clinical Microbiology. CRC Press. LondonFDA (1998) Bacteriological Analytical Manual. 8th ed. Revision A. AOAC International. Gaithersburg. MD.

Tryptose Media

163

Tryptose Lauryl Sulfate Media

Tryptose Lauryl Sulfate Broth

Ref. 02-108

SpecificationLiquid medium for the detection and enumeration of coliform bacteria according IDF-FIL 73B and ISO 4831 and 7251 standards.

Formula (in g/L)Tryptose ..................................................20,00Sodium Lauryl sulfate ...............................0,10Lactose .....................................................5,00Dipotassium phosphate ............................2,75Monopotassium phosphate ......................2,75Sodium chloride ........................................5,00Final pH 6,8 ± 0,2

DirectionsDissolve 35,6 g of powder in 1 L of distilled water. Dis-tribute into tubes or containers fitted with the inverted Durham tubes (for gas). Sterilize at 121°C for 15 min-utes. As for the double concentration medium, dissolve 71,2 g/L and proceed as indicated above.Preferably store the broth at room temperature, and use screw-capped bottles to prevent evaporation of water. Refrigerated broth generally becomes cloudy or forms precipitates but clears at incubation at room tempera-ture. However, clarity is not important as only the gas production is significant criterion.

DescriptionLaurylsulfate broth is used for MPN Presumptive Test of coliforms in water and sewage, confirmatory test of lactose fermentation with gas production for milk and detection of coliforms in food.The high nutrient quality and the presence of phosphate buffer in this medium en-sures rapid growth and increased gas production, even by slow lactose-fermenting coliforms.This medium can be used as Presumptive broth for E.coli (by fluorescent reaction) if before sterilization MUG (Ref. 06-102CASE) is added.

TechniqueIf the volume of sample to inoculate is substantial, then reconstitute the medium at such a concentration which would remain normal, once the sample has been added to it.Incubate at 37°C for 24-48 hours. Lactose fermenta-tion within 48 hours, shown by the appearance of gas in the Durham tubes , indicates the presence of coliform bacteria.Verification can be done by the isolation and identifica-tion of coliforms on an appropiate medium.

ReferencesF.D.A. (1998) Bacteriological Analytical Manual. 8th ed. Revision A. AOAC International Gaitherburg, MD.FIL-IDF Standard 73B (1998) Milk and milk products. Enumeration of coliforms. IDF. Brussels.DOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food. 4th. ed. APHA. Washington.MARSHALL R.T. (1992) Standard Methods for the ex-amination of dairy products. 16th ed. APHA. WashingtonAPHA-AWWA-WPCF (1995) Standard Methods for the examination of water and wastewater. APHA. Washing-tonHORWITZ, W. (2000) Official methods of Analysis.17th ed. AOAC International. Gaithersburg. MD. ISO 4831 Standard (1991) General guidance for the enumeration of coliforms - MPN technique.ISO 7251 Standard (1993) General guidance for enu-meration of E.coli by MPN technique.

Tryptose Lauryl Sulfate Mannitol Tryptophan Broth

Ref. 02-460

SpecificationLiquid medium for the production of indole and gas in a single tube, according to the ISO 9308-1 and 9308-2 standards.

Formula (in g/L)Tryptose ..................................................20,00Mannitol ....................................................5,00Sodium chloride ........................................5,00Monopotassium phosphate ......................2,75Dipotassium phosphate ............................2,75Sodium Lauryl sulfate ...............................0,10L-Tryptophan ............................................0,20Final pH 6,8 ± 0,2

DirectionsDissolve 35,8 g of powder in 1 L of distilled water, heating up if necessary. Distribute in tubes containing Durham´s tubes and sterilize by autoclaving at 121ºC for 15 minutes. Do not overheat.

DescriptionThis broth is proposed in the ISO 9308-1 standard as an alternative medium for the production of indole and gas in a single tube and to confirm the presence of thermo-tolerant coliforms and the presumptive presence of E.coli in the water sample.

164

Tryptose Lauryl Sulfate Media

TechniqueTubes with medium are inoculated from suspicious colonies on the already incubated membrane and then are incubated at 44ºC for 24 hours. Gas production, that appears in the Durham tubes, confirms the presence of thermotolerant coliforms. If after the addition of 0,2-0,3 mL of Kovacs Reagent (Ref. 06-018) a cherry red colour appears on the top surface of the medium (Indole +), a presumptive pres-ence of E.coli is considered and it should be confirmed with other tests.

ReferencesISO Standard 9308-1 (1990) Water Quality Detection and Enumeration of coliform organisms, thermotolerant coliform organisms and presumptive E.coli. Part 1. Mem-brane filtration method. Part 2. MPN method.

165

Ref. 01-278

SpecificationSolid selective and differential medium for isolation and presumptive identification of Clostridium perfringens ac-cording to ISO 7937 Standard.

Formula (in g/L)Tryptose ..................................................15,00Soya peptone ...........................................5,00Yeast extract .............................................5,00Sodium metabisulfite ................................1,00Ferric ammonium citrate ...........................1,00Agar .......................................................20,00Final pH 7,6 ± 0,2

DirectionsSuspend 47 g of powder in 1 L of distilled water and let it soak . Heat to boiling and distribute into suitable contain-ers, but not more than 250 mL in each one. Sterilize in the autoclave at 121°C for 15 minutes. Let it cool to 60°C and add 1 flask of CycloserineSelective Supplement (Ref. 06-116CASE) to every 250 mL of medium. Mix well and pour it into plates. If it is desired to include egg yolk, then add Egg Yolk Sterile Emulsion (Ref. 06-016) in a concentration of 80 mL/L, simultaneously to the antibi-otic,

DescriptionThe medium is a modification of the classical TSN Agar (Ref. 01-195) in which the traditional antibiotics, polymix-in and neomycin have been replaced by cycloserine. Cycloserine has been found more selective for Clostrid-ium perfringens, and it seems also to reduce the trend to produce diffuse blackening. On the other hand, Clostrid-ium perfringens is more resistant to cycloserine than to sulfadiazine, polymixin and neomycin, which permits a better dosage.The medium has sodium metabisulfite and ferric ammonium citrate to manifest the reducing capac-ity of sulfite, and in this way, three differential characteristics of this anaerobic species may be verified with just one assay. These characteristics are

sufite reduction, growth at 46°C and cycloserine resist-ance.Nevertheless, it has to be noted that cycloserine does not tolerate temperatures above 100°C and its stability in a solution is very restrained, even then it is used in alkaline media. Therefore, it is advisable to prepare the exact number of plates that are going to be used.Anyway, if it is desired, an active solution of cycloserine in phosphate buffer at pH 8,0 may be prepared (Dipotas-sium phosphate 16,73 g/L and monopotassium phophate 0,52 g/L) and if it is maintained refrigerated, it can be used for approx. 5 days.

TechniqueThe standard procedure recommends surface inocula-tion of the samples or their dilutions, and once absorbed, to pour a second layer as cover and seal for anaerobio-sis. After an incubation at 46°C for 18-20 hours, proceed to enumerate the black colonies that appear in the plate.

ReferencesSMITH, L.D. (1981) Clostridial Anaerobic Infections, in Diagnostic Procedures for Bacterial,Mycotic and Para-sitic Infections. 6thEd. APHA,Washington.ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press Inc.,LondonDOWNES, F:P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food. 4rd ed. APHA Washington ISO 7937 Standard (2004) Microbiology of food and animal feeding stuffs- Horizontal method for enumeration of Clostridium perfringens - Colony-count technique.DIN Standard 10165. Referenz Verfahren fur Bes-timmung von Clostridium perfringens. Fleisch und Fleischerzeugnissen.FDA (1998) Bacteriological Analytical Manual 8th ed. Revision A. AOAC International. Gaithersburg. MD.ISO 6461-2:1986 Standard Water Quality - Detection and enumeration of the spores of sulfite - reducing anaerobes (Clostridia) - Part 2: Method by MF.

Tryptose Sulfite Cycloserine Agar Base (TSC Agar Base)

Left and center: Clostridium perfringens ATCC 13124; right: control.

166

Urea Agar Base acc. to Christensen

Ref. 01-261

SpecificationSolid medium for detection of urea lysis, according to ISO 6579, 6340 and 6785 standards and DIN 10160 standard.

Formula (in g/L)Gelatin peptone ......................................1,000Dextrose .................................................1,000Sodium chloride ......................................5,000Monopotassium phosphate ....................2,000Phenol red ..............................................0,012Agar ......................................................15,000Final pH 7,0 ± 0,2

DirectionsSuspend 24 g of powder in 950 mL of distilled water and bring to the boil. Sterilize in the autoclave at 121°C for 15 minutes. Let it cool to 50-55°C. Add 50 mL of Urea Sterile Solution 40% (Ref. 06-083) and mix well. Distrib-ute aseptically in tubes and let them solidify in slanted position.

DescriptionUrea Agar complies with Christensen’s specifications, and it is recommended for the detection of urolytic or urea degrading microorganisms, especially Enterobacte-riaceae, although it can be used also with gram positive bacteria.

TechniquePure culture is inoculated by surface streaking, and then incubated at 37°C. Generally, organisms with strong urease activity can be read after 3-5 hours.Reaction is evident as the medium changes its colour. It turns form orange to pink-fuchsia, due to a strong alkali-nization produced by ammonia release.

ReferencesCHRISTENSEN, W.B. (1946) Urea decomposition as means of differentiating Proteus and Paracolon cultures from each other and from Salmonella and Shigella types. J.Bact. 52:461.EDWARDS and EWING (1962) Identification of Entero-bacteriaceae. Burgess Pub. Co.ATLAS, R.M., & L.C. PARK (1993) Handbook of Micro-biological Media. CRC Press Inc.LondonDOWNES, F.P. & K. ITO (2001) Compendium of meth-odscfor the Microbiological Examination of Foods 4th ed. APHA. WashingtonMARSHALL, R.T. (1992) Standard Methods for the ex-amination of Dairy Products. 16th ed APHA. Washington DCISO 6785 Standard (2001) Milk and milk products - De-tection of Salmonella spp.

ISO 6340 Standard (1995) Water Quality - Detection of Salmonella spp.ISO 6579 Standard (2002) Microbiology of food and ani-mal feeding stuffs. Horizontal method for the detection of Salmonella spp.DIN Standard 10160. Untersuchung von Fleisch und Fleischerzeugnissen. Nachweiss von Salmonellen. Ref-erenzverfahren.FIL-IDF 93 Standard (2001) Detection of Salmonella.

Urea Broth Base

Ref. 02-202

SpecificationLiquid diagnostic medium according to Rustigian and Stuart formulation.

Formula (in g/L)Monopotassium phosphate ......................9,10Disodium phosphate .................................9,50Yeast extract .............................................0,10Phenol red ................................................0,01Final pH 6,8 ± 0,2

DirectionsDissolve 19 g of powder into 950 mL of distilled water and sterilize by autoclaving at 121°C for 15 minutes. Let it cool to 50-55°C and then add 50 mL of Urea Sterile Solution 40% (Ref. 06-083). Mix well and dispense in hemolysis tubes (3,0 mL/tube).

DescriptionAccording to Rustigian and Stuart, this Urea Broth is excellent for diagnosing enterobacteria, since within this family, only Proteus may alkalinize the medium over pH 8,1. Despite the fact that some authors prefer a buffer of potency 10 or 100 times lower to obtain faster results for saving the time (about 2 hours) does not compensate for the instability of the medium.Urease production is shown by the indicator turning to dark pink, produced by strong alkalinization by ammo-nium. With plenty of inoculum (2-3 loops in 3-5 mL of medium), Proteus produces the colour change after 6-8 hours, meanwhile other positive enterobacteria need up to 24-48 hours.

ReferencesRUSTIGIAN, R., STUART, C.A. (1941) Decomposition of urea by Proteus. Proc. Soc. Exp. Biol. Med. 47:108DOWNES, FP & K ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food, 4rd ed. APHA,Washington.FDA (1998) Bacteriological Analytical Manual. 8th ed. Revision A. AOAC International. Gaithersburg. MDPASCUAL ANDERSON, MªR. (1992) Microbiología Alimentaria. Diaz de Santos, S.A.,Madrid.

Urea Media

167

Violet Red Bile Agar (VRB Agar)

Ref. 01-164

SpecificationSelective and differential agar medium for the detection and enumeration of coliforms in milk and other dairy products, according to APHA, ICMSF, FIL-IDF and ISO standard.

Formula (in g/L)Yeast extract ...........................................3,000Gelatin peptone ......................................7,000Bile salts #3 ............................................1,500Lactose .................................................10,000Sodium chloride ......................................5,000Neutral red ..............................................0,030Crystal violet ...........................................0,002Agar ......................................................13,000Final pH 7,4 ± 0,2

DirectionsSuspend 39,5 g in 1 L of distilled water. Bring to the boil and distribute into final containers. Sterilization at 121°C for 15 minutes is optional, but If the medium is to be used on the same day of preparation it need not be sterilized. No sterilization improves the recovery of stressed microorganisms.

DescriptionThe Violet Red Bile Agar corresponds to the classic formulation of standardized media for the screening of coliforms in milk and other dairy products.This medium has been adopted for the enumeration of coliforms as well as for differentiating between lactose fermenting and non-lactose fermenting organisms, due to its contents of crystal violet and bile salts, whose inhibiting or selecting properties have been widely confirmed.This medium can be used as Presumptive medium for E.coli (by fluorescent reaction) if before sterilization MUG (Ref. 06-102CASE) is added.

TechniqueThe recommended procedure is the massive inocula-tion directly on Petri dishes, with the molten agar cooled to 47°C. Observations can be read after 24 hours of incubation at 37°C.The size of the colonies ranges from 2 to 5 mm, depend-ing on the amount per plate. The enterococci that might eventually develop will appear small in size and pink coloured. Lactose fermenting enterobacteria acquire a dark red colour with a clearing zone around them, while lactose non-fermenting ones form colourless colonies.

ReferencesDOWNES, F.P. & K. ITO (2001). Compendium of Meth-ods for the Microbiological Examination of Food. 4rd ed. APHA, Washington. DCMARSHALL, R.T. (1992) Standard Methods for the Ex-amination of Dairy Products,16thEd. APHA, Washington. DCICMSF (1978). Microorganisms in Food, University of Toronto Press.ISO (1986) Standard 5541-1 Milk and Milk Products. enumeration of coliforms. Colony count technique at 30ºCFIL-IDF. (1998) Standard 73B. Enumeration of coliform bacteria.PASCUAL ANDERSON, MªR. (1992) Microbiología Alimentaria. Diaz de Santos, S.A.,Madrid,.

Violet Red Bile Dextrose Agar(VRBD Agar)

Ref. 01-295

SpecificationSolid medium for the enumeration of enterobacteria ac-cording ISO 8523 standard.

Formula (in g/L)Yeast extract ...........................................3,000Gelatin peptone ......................................7,000Bile salts #3 ............................................1,500D (+) Glucose .......................................10,000Sodium chloride ......................................5,000Neutral red ..............................................0,030Crystal violet ...........................................0,002Agar ......................................................13,000Final pH 7,4 ± 0,2

DirectionsSuspend 39,5 g in 1 L of distilled water and let it soak. Bring to the boil and sterilize by autoclaving at 121°C for 15 minutes. If the medium is to be used on the same day of preparation it need not be sterilized. Prolonged heat-ing in thermostatic bath could cause slight precipitates.

DescriptionThis medium is a modification of the Violet Red Bile Agar (Ref.1-164) and the MacConkey Agar (Ref.1-118) as described by Mossel et al. These authors proved that the addition of glucose to the Violet Red Bile Agar favoured both the growth of the most fastidious enterobacteria and the recovery of those having suffered from adverse conditions. Later on, Mossel himself realized that by removing the lactose and keeping the glucose, the medium’s efficiency remained stable. Furthermore, an economic improvement occurred since the same amount of product allows the reconstitution of more litres of the medium.

Violet Red Bile Media

168

This medium can be used as presumptive medium for E.coli (by fluorescent reaction) if before sterilization MUG (Ref. 06-102CASE) is added.

TechniqueThe Violet Red Bile Dextrose Agar is widely used in the analysis of food, medicines and cosmetics. It is particu-larly indicated for the recovery of bacteria which have been damaged during preparation. In such cases, a progressive enrichment is recommended in TSB (Ref. 02-200) first and in EE Broth (Ref. 02-064) next. Once the enriched culture is ready it can be inoculated by profound inoculation in tubes or by isolation in Violet Red Bile Dextrose Agar plates.For the count of enterobacteria, the technique to use will be the massive inoculum described for the Violet Red Bile Agar.Observations can be read after 24 hours of incubation at 31°C. Enterobacterial colonies form an intense purple colouring surrounded by a clearer zone . If enterococci colonies eventually develop, then they will be small and pink coloured.

Violet Red Bile Lactose Dextrose Agar (VRBLD Agar)(Eur. Phar. Medium F)

Ref. 01-220

SpecificationSolid selective medium for the detection of Enterobacte-riaceae according the European Pharmacopoeia.

Fórmula (in g/L)

Yeast extract ...........................................3,000Peptone ..................................................7,000Sodium chloride ......................................5,000Bile salts # 3 ...........................................1,500Lactose monohydrate ...........................10,000Glucose monohydrate ..........................10,000Neutral red ..............................................0,030Crystal violet ...........................................0,002Agar ......................................................15,000Final pH 7,4 ± 0,2

DirectionsSuspend 51.5 g of powder in 1 L of distilled water and heat to the boil. Pour into Petri dishes inmediately. Do not sterilize in autoclave nor overheat.

DescriptionThis medium developed in 1962 by Mossel et al. as more effective than MacConkey Agar for the detection of Enterobacteriaceae in foods, has been officially adopted by the European Pharmacopoeia for the microbiological examination of non-sterile products. The medium is spe-

cially used in the recovery of process-stressed bacteria using a progressive enrichment technique.This medium can be used as presumptive medium for E.coli (by fluorescent reaction) if before sterilization MUG (Ref. 06-102CASE) is added.

TechniqueSample is diluted 1:10 in Lactose Broth (Ref. 02-105) and incubate 2-5 hours at 35-37 ºC. Then a volume of this pre-enrichment is ten fold dilute in EE Broth (Ref. 02-064) and incubate at 35-37ºC for 18-24 hours. From this enrichment the surface of several plates of VRBDL Agar are inoculated. The product passes the test if after 18-24 hours of incubation at 35-37ºC there is no growth of gram negative bacteria in any plate.In the surface of the VRBDL Agar the Enterobacteriace-ae colonies are deep purple in colour surrounded by a clearing zone. Sometimes are present little colonies from Pseudmonas or Aeromonas that can be easy differenti-ated by the oxidase test.

References MOSSEL, D.A.A., MENGERINK and SCHOLTS H.H. (1962) Use of a modified MacConkey Agar medium for the selective growth and enumeration of all Enterobacte-riaceae. J. Bact. 84:381.MOSSEL, D.A.A., VISER, M. and CORNELISSEN, A.M.R. (1963) The examination of food for Enterobacte-riaceae using a test of the type generally adopted for the detection of Salmonellae. J. Appl. Bact.(26) 444-452.MOSSEL, D.A.A. (1985) Media for Enterobacteriaceae. Int. J. Food Microbiol. 2:27-35ISO 5552 Standard (1997) Meat and Meat Products. Detection and enumeration of Enterobacteriaceae without resuscitation. MNP technique and colony-count technique.PASCUAL ANDERSON, MªR. (1992) Microbiología Alimentaria. Diaz de Santos, S.A.,Madrid.MOSSEL, D.A.A. and M.A. RATTO (1970) Rapid detec-tion of sub-lethally impaired cells of Enterobacteriaceae in dried foods Appl. Microbio¡ 20: 273-275.EUROPEAN PHARMACOPOEIA 3ª Edición (Suppl. 1999) Cap. 2.6.13 Microbiological examination of non sterile products. Tests for specified organisms. Council of Europe. StrasbourgISO 8523 Standard (1991) General guidance for the detection of Enterobacteriaceae with pre-enrichment.

Violet Red Bile Media

169

Ref. 01-206

SpecificationSolid and very selective medium for isolation and identifi-cation of staphylococci according ISO 22718 standard.

Formula (in g/L)Casein Peptone ....................................10,000Yeast Extract ..........................................5,000Mannitol ................................................10,000Dipotassium phosphate ..........................5,000Litium chloride ........................................5,000Glycine ................................................10,000Phenol Red .............................................0,025Agar ......................................................15,000Final pH 7,2 ± 0,2

DirectionsSuspend 60 g of powder in 1 L of distilled water and bring to the boil. Dispense in suitable containers and sterilize at 121°C for 15 minutes. Cool it to 50°C approx. and add aseptically 20 mL of Potassium Tellurite Solu-tion 1% (Ref. 06-089) or 6,0 mL of Potassium Tellurite Solution 3.5% (Ref. 06-011). Do not reheat after tellurite addition.

DescriptionVJ Agar is a selective medium for detection and enu-meration of pathogenic staphylococci.The medium’s strong selective action is due to lithium chloride, glycine and potassium tellurite presence. They inhibit almost all the accompanying organisms, mean-while staphylococci are not affected. Although staphy-

lococci may reduce tellurite to tellurium, lithium may per-form some action that is compensated by glycine.Moreover a high correlation between tellurite reduction and mannitol fermentation has been proved, and this is shown in the medium by the indicator turning to yellow due to the amount of acid produced.The medium’s selectivity avoids, in the first 24 hours, the development of any other bacteria, so massive inocu-lation is permited. Nonetheless, after this period, it is possible that other bacteria may appear like micrococci, which produce tiny colonies, and staphylococci that ferment mannitol and coagulase negative, therefore it is recommended to verify this last test separately.Due to reduced tellurite, staphylococci generally appear as black colonies over red medium (if they do not fer-ment mannitol) or yellow medium (if they do, and these are presumptive pathogen). Saprophytic staphylococci (S.epidermidis, S.saprophiticus and S.intermedius) have a grey-black colour and are mannitol negative. Complete medium may be stored up to 1 week in the refrigerator. Do not remelt it after tellurite is added.

ReferencesVOGEL and JOHNSON (1960) A modification of the tellurite-glycine medium for the use in the identification of Staphylococcus aureus. Pub. Health. Lab. 18:131-133.US PARMACOPOEIA (2002) 25th ed. <61> Microbial Limit Tests. Pharmacopoeial Convention. Rockville. MDATLAS, R.M.,& L.C. PARK (1993) Handbook of Microbio-logical Media. CRC Press Inc.,LondonFDA (1998) Bacteriological Analytical Manual. 8th ed. Revision A. AOAC International. Gaithersburg. MDISO 22718:2006 Standard. Cosmetics – Detection of Staphylococcus aureus.

Vogel Johnson Agar (VJ Agar)

Ref. 01-164 Violet Red Bile Agar

control

Escherichia coli ATCC 25922Salmonella typhimurium

ATCC 14028

170

Ref. 03-408

SpecificationFluid medium for the cultivation and enumeration of anaerobic bacteria by the impedometry technique.

Formula (in g/L)Tryptone .............................................10,0000Gelatin peptone ..................................10,0000Gelatin ..................................................8,0000Dextrose .............................................41,0000Yeast extract .........................................5,0000Sodium chloride ....................................5,0000Ammonium sulfate ................................5,0000L-Arginine .............................................1,0000Sodium pyruvate ..................................1,0000Sodium bicarbonate .............................1,0000Ferrous sulfate .....................................0,1000Calcium chloride ...................................0,1000Hemine .................................................0,0050Menadione ............................................0,0005Agar ......................................................0,3000Final pH 7,1 ± 0,2

DirectionsSuspend 87,5 g of powder in 1 L of distilled water. Bring to the boil. Sterilize in the autoclave at 121°C for 10 minutes. Prepared medium takes on a dark colour due to the high concentration of sugar. Do not reheat.

DescriptionThis medium is a modification of the Wilkins-Chalgren Anaerobic Medium for impedometric methods.

ReferencesWILKINS, T.D. and S. CHALGREN (1976) Medium for use in antibiotic susceptibility testing of anaerobic bacte-ria. Antimicrob. Agents Chemorther 10:6:926.

Wilkins-Chalgren CN Modified Fluid Medium (WCCN Modified Fluid Medium)

WL Nutrient Media

WL Nutrient Agar

Ref. 01-210

SpecificationSolid medium for the cultivation and enumeration of yeast and bacteria for microbiological control in brewing and other fermentation industries.

Formula (in g/L)Yeast extract .........................................4,0000Tryptone ...............................................5,0000Dextrose .............................................50,0000Monopotassium phosphate ..................0,5500Magnesium sulfate ...............................0,1250Calcium chloride ...................................0,1250Potassium chloride ...............................0,4250Iron (III) chloride ...................................0,0025Manganese sulfate ...............................0,0025Bromcresol green .................................0,0220Agar ....................................................20,0000Final pH 5,5 ± 0,2

Directions Suspend 80 g of the powder in 1 L of distilled water. Mix thoroughly. Heat with frequent agitation and boil for one minute. If a final pH of 6,5 is desired, the pH may be adjusted with one percent aqueous sodium carbonate, using about 30 mL per litre of medium.Dispense and sterilize the medium by autoclaving at 121°C for 15 minutes.The WL Differential Agar has the same formula as the WL Nutrient Agar with the addition of 2 flasks/L of Cy-cloheximide Selective Supplement Ref. 06-022CASE.

DescriptionWL Nutrient Agar was formulated by Green and Gray in the Wallerstein Laboratory for use in the control of indus-trial fermentations, particularly the processing of beer. It is recommended for examination of worts, beers, liquids containing yeast and other materials.WL Nutrient Agar has a pH of 5,5 which is optimal for the enumeration of brewers´yeast . If bakers or distiller´s yeast is to be examined, the pH should be adjusted to 6,5 (better yields). When cultivating the microorganisms from an alcoholic mash, tomato juice should be added to the medium.

171

WL Differential Agar contains cycloheximide to suppress yeast and any other moulds which may be present; this medium allows reliable counting of all bacteria which may be encountered in the tests performed in brewery laboratories.

TechniqueDilute the sample material and spread 0,1 mL onto WL Nutrient Agar or WL Differential Agar.The WL Nutrient Agar and WL Differential agar are used together, one plate with nutrient agar and two with the differential agar. The WL Nutrient Agar plate is incubated aerobically to obtain a total count, mainly of yeast colonies. One WL Differential Agar plate is incubated aerobically for growth of acetic acid bacteria, Flavobacterium, Proteus, and other organisms; the second plate is incubated anaerobi-cally for detection of such organisms as lactic acid bacilli and Pediococcus species.Plates prepared with both the media are generally incu-bated at 25°C, if brewing materials are being studied, and at 30°C for baker´s yeast and alcohol mash sam-ples. Incubation may be continued for a week, or even for ten days to two weeks, depending upon the flora present. Counts are made at the intervals during the incubation period.

WL Nutrient Broth

Ref. 02-210

SpecificationLiquid medium for the microbial control of industrial fer-mentations and massive cultivation of yeast.

Formula (in g/L)Yeast extract .........................................4,0000Tryptone ...............................................5,0000Dextrose .............................................50,0000Monopotassium phosphate ..................0,5500Magnesium sulfate ...............................0,1250Calcium chloride ...................................0,1250Potassium chloride ...............................0,4250Ferric chloride .......................................0,0025Manganous sulfate ...............................0,0025Bromcresol green .................................0,0220Final pH 5,5 ± 0,2

DirectionsDissolve 60 g of powder in 1 L of distilled water and dis-pense into suitable containers. Sterilize by autoclaving at 121°C for 15 minutes. Should a pH 6,5 be desired, ad-just it by adding 30 mL of Sodium carbonate solution 1%.To obtain 1 L of WL Differential Broth just add, asepti-cally, 2 flasks of Cycloheximide Selective Supplement (Ref. 06-022CASE) to 1 L of WL Nutrient Broth, after sterilization.

DescriptionWL Medium was developed in the Wallerstein Labora-tories for industrial uses, since it allows to differentiate between beer yeast and wild yeasts contaminants.Adjusting pH to 6,5 is very advisable to enumerate bread and alcohol yeast. The medium also allows bacterial growth and it is possible then to count the contaminant bacteria of fermented liquors, but it is recommended to use WL Differential Broth for this assay, since it inhibits yeast growth.The WL Nutrient Broth is useful to enumerate cells by the MPN technique. Alternatively, it can be used as an enrichment broth previous to the colony plate count.

TechniqueUsual the technique is to inoculate one plate of WL Nutri-ent Broth and two plates of WL Differential. Incubate all the plates at 25°C for 5-15 days. It is advisable to incu-bate one of the plates with differential agar anaerobically to enhance development of contaminants that produce lactic acid. Green and Gray stated that to perform the observations of viable yeast in bread, WL Nutrient broth at pH 5,5 may be used, but to do it in distilleries, the pH has to be adjusted to 6,5.Analogously, time and temperature of incubation vary depending on material to be analysed. Beer samples are incubated at 35°C, but bread, and alcohol fermenta-tion ones are incubated at 30°C. Incubation time varies between 2 and 7 days, and in some cases, depend-ing on the flora found, it may be up to 14 days. When Differential type (with cycloheximide) is employed for a bacterial count, it should be incubated anaerobically to detect cocci in beer and lactic bacilli, and it should be incubated aerobically to detect acetic acid bacteria and thermobacteria.

ReferencesGREEN, S.R. & GRAY, P.P. (1950) A differential proce-dure applicable to bacteriological investigation in brew-ing. Wallerstein Lab. Comm. 13:357GREEN, S.R. & GRAY, P.P. (1950) Paper read at Am.Soc. of Brewing Chemists Meeting; Wallerstein Lab. Comm. 12:43GREEN, S.R. & GRAY, P.P. (1951) A differential proce-dure for bacteriological studies useful in the fermentation industries. Wallerstein Lab. Comm. 14:289GRAY, P.P. (1951) Some advances in microbiological control for beer quality. Wallerstein Lab. Comm. 14:169 ATLAS, R.M., & L.C. PARK (1993) Handbook of Micro-biological Media for the examination of Food. CRC Press Inc. Boca Ratón,Fla.MASTERS BREWERS ASSOCIATION OF THE AMERI-CAS (2002) The Practical Brewer 3rd ed. St. Paul. Min-nesota

WL Nutrient Media

172

Wort Agar

Ref. 01-132

SpecificationSolid medium for the cultivation, isolation and enumera-tion or enrichment of fungi, especially of yeast.

Formula (in g/L)Malt extract ...............................................15,0Casein Peptone ..........................................1,0Maltose .....................................................12,5Dextrine ......................................................2,5Dipotassium hydrogen phosphate .............. 1,0 Ammonium chloride ....................................1,0Agar ..........................................................17,0Final pH 4,8 ± 0,2

DirectionsSuspend 50 g of powder in 1 L of distilled water and add 2-3 mL of glycerol and bring to the boil to dissolve completely. Distribute into final containers and sterilize by autoclaving at 121°C for 15 minutes. Do not over-heat. Prolonged heating will diminish the gelling strength of the medium.

DescriptionWort Agar is used for the cultivation, isolation and enu-meration of yeast and moulds.It is particulary well adapted for counting osmophilic yeast in butter, sugar and syrups, in lemonade and more generally in sweet or soft drinks.For a more selective utilization it is possible to adjust the pH to 4,5 or 3,5. Never heat the medium after adding acid, in order to prevent the loss of solidifying properties of the agar. The acid pH inhibits the growth of bacteria and favours that of yeast.

Wort Broth

Ref. 02-132

SpecificationThis medium is the liquid version of the classical Wort Agar (Ref. 01-132).

Formula (in g/L)Malt extract ...............................................15,0Casein Peptone ..........................................1,0Maltose .....................................................12,5Dextrine ......................................................2,5Dipotassium hydrogen phosphate .............. 1,0 Ammonium chloride ....................................1,0Final pH 4,8 ± 0,2

DirectionsSuspend 37 g of powder in 1 L of distilled water and add 2-3 mL of glycerol and bring to the boil to dissolve completely. Distribute into final containers and sterilize by autoclaving at 121°C for 15 minutes.

DescriptionIt is especially designed to propagate the multiplication of yeast, and often it has been employed as a semise-lective or enrichment medium, due to its high acidity, which makes it inhibitory for bacteria. This effect may be more enhanced by adding, before sterilization, 10 mL/L of a 10% solution of lactic or tartaric acid. To avoid precipitate it is recommended to sterilize by filtration.

ReferencesSCARR, M.P. (1959) Selective media used in the micro-biological examination of sugar products. J. Sci. Food Agric. 10:678-681RAPP, M (1974) Indikator-zusätze zur Keimdifferen-zierung auf Würze und Malzextrakt-Agar. Milchwiss 29:341-344ATLAS, R.M.,& L.C. PARKS (1993) Handbook of Micro-biological Mediafor the examination of Food. CRC Press Inc.LondonMASTERS BREWERS ASSOCIATION OF THE AMERI-CAS (2002) The Practical Brewer. 3rd ed. St. Paul. Min-nesota

Wort Media

173

Xylose Lysine DeoxycholateAgar (XLD Agar) (Eur. Phar. Agar Medium K)

Ref. 01-211

SpecificationSolid medium for the isolation of enteropathogenic species, especially Salmonella according to ISO 6340 standard.

Formula (in g/L)Xylose .......................................................3,50L-Lysine ....................................................5,00Lactose .....................................................7,50Sucrose ....................................................7,50Sodium chloride ........................................5,00Yeast extract .............................................3,00Phenol red ................................................0,08Sodium Deoxycholate ..............................2,50Sodium thiosulfate ....................................6,80Ammonium ferric citrate............................0,80Agar ........................................................15,00Final pH 7,4 ± 0,2

DirectionsSuspend 56,68 g of powder in 1 L of distilled water. Heat up constantly with stirring until boiling. Pour it immediate-ly into plates. Do not autoclave and avoid remelting.

DescriptionXylose Lysine Deoxycholate Agar is a differential me-dium, slightly selective, very suitable for the detection of pathogenic enterobacteria, especially Shigella. Gram negative flora is inhibited by the low amount of deoxy-cholate, but Shigella grows easier in this medium than in any other selective media. Xylose, lactose or sucrose fermentation produce the acidification of the medium, and this is seen by an indicator turning to yellow, surrounding the colonies. This colour disappears after 24 hours, so observations must be carried out between 18 and 20 hours.Hydrogen sulfide production from thiosulfate is easily de-tected because colonies become darker, due to the ferric sulfure precipitate. Lysine decarboxylation to cadaverine may also be observed in the medium, since it produces alkalinization and consequently the indicator turns to red.All these reactions allow a good differentiation of Shig-ella, which besides Edwardsiella and Proteus inconstans are the single enterobacteria that do not ferment xylose and therefore show negative fermentation reaction. Salmonella type members do ferment xylose, but it is consumed quickly and then alkalinization of the medium, due to lysine decarboxylation, may mask the reaction. The difference between Shigella and Salmonella is that with the latter colonies become darker due to ferrous sulfure precipitates, and this is a common property with Edwardsiella. The other types of enterobacteria do not suffer this phenomenon, since acid acumulation due to lactose and sucrose fermentation is so high that it avoids

pH reversion by decarboxylation and even ferrous sul-fure precipitate in the first 24 hours.In the table below, typical colonial appearances on XLD medium after 24-36 hours of incubation at 37°C are described.

ReferencesTAYLOR, W.J. (1965) Isolation of Shigella. I. Xylose Lysine Agars: New media for isolation of enteric patho-gens. Am. J.Clin. Path 44:471-475DOWNES, F.P. & K. ITO (2001) Compendium of Meth-ods for the Microbiological Examination of Food,4th ed. APHA,Washington.ICMSF (1978) Microorganisms in Food 1. Univ. Toronto Press.FDA (1998) Bacteriological Analytical Manual. 8th ed. Revision A. AOAC International Gaithersburg. MD.ATLAS, R.M.,& L.C. PARK (1993) Handbook of Micro-biological Mediafor the examination of Food. CRC Press Inc. LondonPASCUAL ANDERSON, MªR. (1992) Microbiología Alimentaria. Diaz de Santos, S.A. Madrid, .EUROPEAN PHARMACOPOEIA, (2002) 2.6.13 Test for specified micro-organisms 4th Ed.,Suppl. 4.2 EDQM Council of Europe, Strasbourg,US PHARMACOPOEIA (2002) <61> Microbial Limit Tests. 25th Ed. US Pharmacopoeial Convention. Rocville. MD.ISO 6340 Standard (1995) Water Quality - Detection of Salmonella spp.

Xylose Lysine Deoxycholate Modified Agar (XLD Modified Agar)

Ref. 01-552

SpecificationMedium for isolation of enteropathogenic species, espe-cially Salmonella in food and animal feeding stuffs, acc. ISO Standard 6579:2002

Formula (in g/L)Xylose ......................................................3,75L-Lysine ...................................................5,00Lactose ....................................................7,50Sucrose ...................................................7,50Sodium chloride .......................................5,00Yeast extract ............................................3,00Phenol red ...............................................0,08Sodium Deoxycholate ..............................1,00Sodium thiosulfate ...................................6,80Ammonium iron(III) citrate .......................0,80Agar .......................................................15,00Final pH 7,4 ± 0,2

Xylose Lysine Deoxycholate Media

174

DirectionsSuspend 55,43 g of powder in 1 L of distilled water. Heat up constantly stirring until boiling. Pour it immediately into plates. Do not sterilize and avoid remelting.

DescriptionXylose Lysine Deoxycholate Agar is a differential me-dium, slightly selective, very suitable for the detection of pathogen enterobacteria in food, especially Shigella, with a modification in the original formulation of Tay-lor to perform the specifications of the ISO standard 6579:2002Gram negative flora is inhibited by the low amount of deoxycholate, but Shigella grows easier than in other selective media.Xylose, lactose or sucrose fermentation produce medium acidification, and this is shown by an indicator turning to yellow, surrounding the colonies. This colour disappears after 24 hours, so readings must be carried out between 18 and 20 hours.Sulfhydric production from thiosulfate is easily detected because colonies become darker, due to the ferric sulfide precipitate. Lysine decarboxylation to cadaverine may also be observed in the medium, since it produces alkalinization and consequently the indicator turns to red.All these reactions allow a good differentiation of Shig-ella, which besides Edwarsiella and Proteus inconstans are the single enterobacteria that do not ferment xylose and therefore show negative fermentation reaction. Salmonella-type members do ferment xylose, but it is consumed quickly and the medium alkalinization, due to lysine decarboxylation, may hide the reaction. The difference between Shigella and Salmonella is that with the latter colonies become darker due to ferrous sulfide

precipitates, and this is a common character with Edwar-siella. The other types of enterobacteria do not suffer this phenomenon, since acid accumulation due to lactose and sucrose fermentation is so big that it avoids pH reversion by decarboxylation and even ferrous sulfide precipitate in the first 24 hours.

In the table below, typical colonial appearances on XLD Agar after 24-36 hours of incubation at 37°C are de-scribe.

ReferencesTAYLOR, W.J. (1965) Isolation of Shigella. I. Xylose Lysine Agars: New media for isolation of enteric patho-gens. Am. J. Clin. Path 44:471-475VANDERZANT & SPLITTSTOESSER (1992). Compen-dium of Methods for the Microbiological Examination of Food. 3rd. Ed. APHA. Washington.ICMSF, ( 1978) Microorganisms in Food 1. University of Toronto Press.FDA (1990) Bacteriological Analytical Manual AOAC International Arlington. VA. USA.ATLAS, R.M., L.C. PARK (1993) Handbook of Microbio-logical Media for the examination of Food. CRC Press Inc. Boca Ratón.PASCUAL ANDERSON, MOR. (1992) Microbiología Alimentaria. Diaz de Santos, S.A. Madrid.ISO Standard 6579 (2002) Microbiology of foods and animal feeding stuffs. Horizontal method for the detec-tion of Salmonella spp.

Xylose Lysine Deoxycholate Media

control Salmonella typhimurium ATCC 14028

Colony appearance Microorganism

Red colonies, transparent Shigella sp., Proteus incontans, Salmonella paratyphi A., sometimes S.cholerasuis and S. Pullorum

Red colonies, transparent with black core. Edwardsiella and most of biotypes of SalmonellaOrange and slightly opaque colonies Salmonella typhiRed and translucent colonies, without halo. Pseudomonas, Proteus rettgeri.Yellow and opaque Escherichia when it grows, Enterobacter, Aeromonas, Citro-

bacter.Yellow, opaque, mucose and with black core. Klebsiella, Citrobacter intermedius when it growsYellow, transparent and with black core. Most of Proteus mirabilis, P.vulgaris.Yellow, opaque and without halo Serratia, Hafnia.

Ref. 01-552XLD Modified Agar

175

Yeast Extract Agar

Ref. 01-465

SpecificationSolid medium for the enumeration of microorganisms from water.

Formula (in g/L)Tryptone ....................................................5,0Yeast extract ...............................................3,0Agar ..........................................................15,0Final pH 7,2 ± 0,2

DirectionsSuspend 23 g of powder in 1 L of distilled water and bring to the boil. Distribute into suitable containers and sterilize by autoclaving at 121ºC for 15 minutes.

DescriptionThis medium, formulated according to Windle Taylor, is the most used in the UK for the enumeration of hetero-trophic microorganisms from water. Distinction between bacteria, yeast and filamentous fungi must be carried out by morphology after differential incubations at 35º and 20ºC.

TechniqueFrom the water sample, make a decimal dilution bank with Ringer Solution (Ref. 06-073) and take aliquotes to 2 parallel series of plates. Pour the Yeast Extract Agar, molten and cooled to 45ºC, and homogenize with sam-ple. Once solidified, incubate one of the series at 35ºC for 24 hours and the other one at 20ºC for 3 days. In order to achieve a good count, select the plates with 30-300 colonies.

ReferencesWINDLE TAYLOR, E. (1958) The examination of water and water supplies. 7th Ed. Churchill Ltd. London.ATLAS, R.M., & L.C. PARKS (1993) Handbook of micro-biological media, CRC Press, London

Yeast Extract Peptone DextroseAgar (YPD Agar)

Ref. 01-473

SpecificationSolid medium for the cultivation of yeast in molecular biology procedures.

Formula (in g/L)Peptone ....................................................20,0Yeast extract .............................................10,0Dextrose ...................................................20,0Agar ..........................................................20,0Final pH 6,8 ± 0,2

DirectionsSuspend 70 g of powder in 1 L of distilled water and bring to the boil. Distribute into suitable containers and sterilize by autoclaving at 121ºC for 15 minutes.

Yeast Extract Peptone DextroseBroth (YPD Broth)

Ref. 02-473

SpecificationLiquid medium for the cultivation of yeast in molecular biology procedures.

Formula (in g/L)Peptone ....................................................20,0Yeast extract .............................................10,0Dextrose ...................................................20,0Final pH 6,8 ± 0,2

DirectionsDissolve 50 g of powder in 1 L of distilled water, heating up if necessary. Distribute into suitable containers and sterilize by autoclaving at 121ºC for 15 minutes.

DescriptionThese media support the growth of most heterotrophic microorganisms, but due to their simple composition they have been adopted as the basal media for the rou-tine cultivation of yeasts for molecular biology studies.

ReferencesSHERMAN, F. (1991) Studies on the phenotype switch-ing with Candida albicans. Meth. Enzimol 194:3-17.MARTINEZ, J.P., M.L. GIL, M. CASANOVA, J.L. LOPEZ-RIBOT, J. GARCIA DE LOMAS, R. SENTANDREU (1990) Wall mannoproteins in the cells from colonial phenotypic variants. J. Gen. Microbiol. 136:2421-2432.ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro-biological Media. CRC Press. London AUSUBEL, F.M., R.BRENT,R.E. KINGSTON, D.D. MOORE, J.G. SEIDMAN, J.A. SMITH & K. STRUHL (1994) Current Protocols in Molecular Biology. Current Protocols. Brooklyn. N.Y.

Yeast Extract Media

176

Yeast Malt Agar (YM Agar)

Ref. 01-219

SpecificationSolid medium for the cultivation of fungi and actinomyc-ete.

Formula (in g/L)Dextrose ...................................................10,0Peptone ......................................................5,0Malt extract .................................................3,0Yeast extract ...............................................3,0Agar ..........................................................20,0Final pH 6,2 ± 0,2

DirectionsSuspend 41 g of powder in 1 L of distilled water and let it soak. Bring to the boil and distribute into suitable containers. Sterilize in the autoclave at 121°C for 15 minutes.

Yeast Malt Broth (YM Broth)

Ref. 02-219

SpecificationLiquid medium for the cultivation of fungi and actinomyc-etes.

Formula (in g/L)Dextrose ...................................................10,0Peptone ......................................................5,0Malt extract .................................................3,0Yeast extract ...............................................3,0Final pH 6,2 ± 0,2


DescriptionThis is a classical culture medium for the cultivation of moulds, yeasts and acidophilic actinomycetes. Medium may become selective to one or other group of micro-organisms by adding antibiotics when the medium is at 50°C.

ReferencesATLAS, R.M.,& L.C. PARK (1993) Handbook of Micro-biological Mediafor the examination of Food,CRC Press Inc.London.SAMSOM, R.A., E.S. HOEKSTRA, J.C. FRISVAD, O. FILTENBORG (2002) Introduction to food- and airborne fungi. 6th. Ed. CBS. Utrech.

Yeast Malt Media

Culture Media Ingredients

179

DefinitionsEXTRACTS are concentrated preparations of liquid, solid or intermediate consistency, usually obtained from dried vegetable or animal matter. For some preparations, the matter to be extracted may undergo a preliminary treatment, for example, inactivation of enzymes, grind-ing or defatting. Extracts are prepared by maceration, percolation or other suitable, validated methods, using water, ethanol or another suitable solvent. After extrac-tion unwanted matter is removed if necessary.

PEPTONES are complex water-soluble mixes of free amino acids, peptides, sugars, mineral salts and other components obtained by acid, alkaline or enzymatic hydrolysis of protein substrates. Their very variable char-acteristics depend on:- The nature of the substrate(s).- The nature of the hydrolysis: enzyme(s), alkali(s),

acid(s), term of hydrolysis.- The technique of purification (filtration, ultrafiltration,

…).- The other operating conditions used in the production

process.

The term peptone is more commonly applied to the hydrolisates obtained by enzymatic digestion. The en-zymes more frequently used are:

Papain that acts on the links adjacent to arginine, lysine, phenylalaline and glycine. Bromelain and Ficin are also used because are similar to papain but from other plants and with pH-range and temperature-range different.

Pepsin, that acts on the links adjacent to phenylalanine or leucine.

Pancreatin (a variable mixture of trypsin and chimo-trypsin) that acts on the links adjacent to arginine, lysine, tyrosine, tryptophane, phenylalanine and leucine.

Microbial Proteases are obtained from microbial cultures as extracellular enzymes and used for the peptones production. Acid and Neutral microbial pro-teases works in similar way as papain and alkaline microbial proteases as pancreatin.

The papain is obtained from plant material and pepsin and pancreatin, that are animal enzymes, are of swine origin.

Production Process The general production process is eschematized in the following flow-chart.


180

Warranty of health and originAll the animal tissue raw materials used in the elabora-tion of the Scharlau Microbiology Peptones come from approved slaughterhouses and are covered by certifi-cates obtained from the veterinary authorities. The country of origin of bovine animal tissues and casein used in the manufacture of each batch of peptone is specified in the health certificate.These documents certify that animals from which tissues have been taken were in good health and fit for human consumption. They are used by Scharlau Microbiology Quality Control Department to edit a health certificate for each batch of product manufactured: a copy of this certif-icate is submitted to our customers upon each delivery.

Analytical data and control methodsAll data figuring in the following documentation result from the analysis of a significant number of batches of every product. These data may be:Typical data, which are in fact average values (com-

parative tables and technical data sheets). Norms of acceptability (Technical data sheets).

Bacteriological controls as well as most general physico-chemical controls are carried out each batch system-atically. The other characters are verified according to routine. For the control methodology, a norm is defined spe-cifically for each character. When a pharmacopoeial monograph (Eur. Phar. 4th ed. 2002; USP 25th ed 2002) is available, it is adopted as routine method. If it is not possible an other documented methodology is accepted. The typical data are show in the following Tables 1 to 6 and in the specific description of any product. The information contained in this publication is based in own research and development work and is the best of our knowledge true and accurate. Users should however conduct their own tests to determine the suitability of our products for their own purpouses. Statements contained herein should not be considered as a warranty of any kind, expressed or implied, and no liability is accepted for the infringement of any patents.

ReferencesATLAS, R.M. & L.C. PARKS ( 1993) Handbook of Micro-biological Media CRC Press London.BRYDSON, E.Y. (1978) Natural and Synthetic Culture Media for Bacteria. In M. Rechcigl Jr. (ed) Handbook


Series in Nutrition and Food. Section G. Vol III. CRC Press. Cleveland SYKES, J. (1956) Constituents of Bacteriologic Culture Media. Cambridge University Press. Cambridge.

181


182


183


184


185


186


187

Agar Bacteriological

Ref. 07-004

Agar is the dried, hydrophilic, colloidal substance ex-tracted from the algae known as Agarophyites (several species and genera of the Class Rodophyceae). It con-sists of two polysaccharides, agarose and agaropectine, in a variable proportion depending on the geographical origin zone.

The Agar Bacteriological is a solidifying agent selected and prepared by mixing different agar from several zones of origin and is especially recommended for gelling the microbiological culture media where a great transparency and brightness is required.

The most important characteristics are shown in the fol-lowing tables. Data are average values, which may vary from batch to batch.

Agar-Agar

Ref. 07-490

Agar is the dried, hydrophilic, colloidal substance ex-tracted from the algae known as Agarophytes (several species and genera of the Class Rodophyceae). It con-sists of two polysaccharides, agarose and agaropectine, in a variable proportion depending on the geographical origin zone.

The Agar-Agar is a solidifying agent with the same gel-ling strength as the Agar Technical but with an inferior grade of purification, and shows a greater opacity and a higher salts content. Its use among the culture media is recommended only when the brightness and clarity is not a critical requirement.


188

Agar Technical

Ref. 07-521

Agar is the dried, hydrophilic, colloidal substance ex-tracted from the algae known as agarophyites (several species and genera of the Class Rodophyceae). It con-sists of two polysaccharides, agarose and agaropectine, in a variable proportion depending on the geographical origin zone.

The Agar Technical is a solidifying agent with a gel strengh higher than Agar Bacteriological, especially sug-gested when the culture medium does not require a total brightness, since it shows a slight turbidity.


Beef Extract

Ref. 07-515

For a long time beef extract has been the basic com-ponent of culture media, and initially it substituted meat infusions due to its easy usage. Now, there is a trend to substitute it by peptones and different mixtures with a more defined composition, because they allow a greater reproducible result.

Scharlau Microbiology Beef Extract is obtained from free tendons and fat beef muscle, enzymatically predigested. Its production also includes the elimination of fermenta-ble sugars.

Among the raw materials and auxiliaries used in its preparation, the bovine constituents belong to the cat-egory 4 of the WHO classification. The bovine tissues are sourced from New Zealand, and come from herds free from Bovine Spongiform Encephalopathy virus and foot-and-mouth disease after examination by the Veteri-nary Authorities. The enzymatic preparation is of porcine origin.

The product does not contain and is not derived from specified risk material as defined in the European Com-mission Decision 97/534/EC.The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instantaneous heating at 200ºC on spray drying.

The totally desiccated version is easier to use than the paste form, and require less quantity in order to obtain the same effects. Beef extract solutions are clear, slightly coloured and with pH near to neutral. In culture media they are used in concentrations varying from 0,3-0,5%.

The most important characteristics are shown in the fol-lowing figures and tables and in Tables 1 to 6 at the beg-gining of this chapter. Data are average values, which may vary from batch to batch.

189

Beef Extract

Bile

Ref. 07-039

Ox bile powder is obtained by spray drying the fresh bile at high temperature, assuring the maintenance of the most important characteristics or properties of fresh bile. 1g of ox bile powder is corresponds to approx. 10 g of fresh bile.

The bile used in its preparation is naturally of bovine origin. It comes from animals which are raised and slaughtered in Holland, and are in good health and suit-able for human comsumption on ante- and post- mortem examination by the Veterinary Authorities. These animals come from herds free from Bovine Spongiform Encepha-lopathy and Foot-and-Mouth disease.

The manufacturing process includes the rapid heating at 200ºC on spray drying.

In the culture media it is employed at concentrations varying from 1-2%. Ox bile act as a non enteric micro-bial flora inhibitor, and it is used as selective agent in enterobacterial isolation media. Ox bile solutions at low concentrations are clear and have a slight colour, but at higher concentrations they are opalescent and have darker colours.

Dry powder has a colour varying form yellow-beige to yellow-green, and it allows transparent 5% solutions in water, that have a pH between 6 and 7,5. In alcoholic solutions (ethanol 84%) it provides less than 0,1% of insoluble substances.

The most important characteristics are shown in the fol-lowing tables and in Tables 1 to 6 at the beggining of this chapter. Data are average values, which may vary from batch to batch.

07-515 Beef ExtractMolecular Weigth Distribution

07-515 Beef ExtractAmino Acids (Free/Total) x 100

190

Bile Salts #3

Ref. 07-525

The bile salts for microbiological applications are ob-tained from fresh animal (sheep, pig) bile by precipitation with chlorhidric acid, in a process that removes pigments and other toxic substances and concentrate the bile salts. Nevertheless the standardization of the results is very difficult because the composition of the final mixture depends not only on the process but on the raw material that is very variable in origin.In the normal preparation of bile salts several compo-nents like gluconate, taurocholate, cholate and deoxy-cholate, and others, can be identified, and the inhibitory character of the mixture depends on the relative rate between all these substances. Usually, in the micro-biological culture media, the bile salts are dosed in a concentration of 0,5% (w/v) to inhibit the growth of gram-positive bacteria.

There are other type of preparations of mixture o bile salts, with a higher level of purification and concentration of actives substances. These ones are used, in micro-biological applications, in lower concentration, that is, a third o the concentration of the normal preparations. Because its efficacy, they are called Bile Salts #3 and its usual concentration in culture media is 0,15%.

The Scharlau Bile Salts #3 is standardized batch by batch intending to supply a product as homogeneous as possible.

Dry powder has a flowable blurred white aspect, with a bitter in odour and taste, but provides clean, transparent and pale yellow 2% solutions in water, which present an alkaline reaction (pH 8,0) and therefore its addition to culture media may require pH adjustment. When this product is used as culture media component it is advis-able do nod exceed never the 0,3 % (w/v) concentration.


Brain Extract

Ref. 07-076

For a long time animal brain infusions have been one of the basic components of some culture media for fastidi-ous microorganisms, and nowadays, in most cases they are still necessary.

Scharlau Microbiology Brain Extract produces clear, clean and stable solutions at 121ºC, and provide the cul-ture medium with very complex nutrients. It is obtained from brains of healthy pigs with ante- and post-mortem sanitary certification.

Among the raw materials and auxiliaries used in its prep-aration, the bovine constituents belong to the category 4 of the WHO classification. The product does not contain and is not derived from specified risk material as defined in the European Commission Decision 97/534/EC. All the constituents are of swine origin.

The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instantaneous heating at 200ºC on spray drying.


191

Casein Acid Hydrolysate

Ref. 07-151

Casein Acid hydrolysate is a protein hydrolysate ob-tained by acid digestion, where all the casein com-pounds reduced to their aminoacids, except tryp-tophane which almost disappears. Vitamines also almost disappear due to the acid digestion process.

Among the raw materials and auxiliaries used in its prep-aration the bovine constituents belong to the category 4 of the WHO classification. The lactic casein from cow milk is sourced from New-Zealand, and come from herds free from Bovine Spongiform Encephalopathy and foot-and-mouth disease after examination by the Veterinary Authorities.The product does not contain and is not derived from specified risk material as defined in the European Com-mission Decision 97/534/EC. The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instantaneous heating at 200ºC on spray drying.


07-151 Casein Acid HydrolisateMolecular Weigth Distribution

07-151 Casein Acid HydrolisateAmino Acids (Free/Total) x 100

192

Casein Pancreatic Peptone

Ref. 07-154

Casein Pancreatic Peptone is a protein hydrolysate, obtained by digestion with pancreatic extracts. It differs from Tryptone (Ref. 07-119) only in the way it is ob-tained, which produces a different aminoacid composi-tion and a lesser molecular size. It is the most used type of peptone in industrial fermentation operations.

Among the raw materials and auxiliaries used in its preparation the bovine constituents belong to the cat-egory 4 of the WHO classification. The Milk casein from cow milk is sourced from New-Zealand, and come from herds free from Bovine Spongiform Encephalopathy and foot-and-mouth disease after examination by the Veteri-nary Authorities.

The enzymatic preparation is of porcine origin. The product does not contain and is not derived from specified risk material as defined in the European Com-mission Decision 97/534/EC.



07-154 Casein Pancreatic PeptoneMolecular Weigth Distribution

07-154 Casein Pancreatic PeptoneAmino Acids (Free/Total) x 100

193

Ref. 07-119

The Casein Trypsic Peptone or Tryptone, is a protein hydrolysate obtained by digestion of casein with an especially tripsin-enriched pancreatic enzymatic prepa-ration. Both, its nitrogen content and balanced amino-acid com-position makes it more suitable for the manufacturing of culture media, producing exceptionally clear solutions.

Among the raw materials and auxiliaries used in its preparation the bovine constituents belong to the cat-egory 4 of the WHO classification. The lactic casein from cow milk is sourced from New-Zealand, and come from herds free from Bovine Spongiform Encephalopathy and foot-and-mouth disease after examination by the Veterinary Authorities. The enzymatic preparation is of porcine origin.

The product does not contain and is not derived from specified risk material as defined in the European Com-mission Decision 97/534/EC.

Casein Trypsic Peptone (Tryptone)



07-119 Casein Trypsic PeptoneMolecular Weigth Distribution

07-119 Casein Trypsic PeptoneAmino Acids (Free/Total) x 100

194

Ref. 07-153

Gelatin peptone is a cream coloured powder, with characteristic odour, obtained by pancreatic digestion of gelatin.

The gelatin is of porcine origin. None of the raw materi-als and auxiliaries used in its preparation are of bovine origin. The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instantaneous heating at 200ºC on spray drying.

It has a low content of Tryptophan, and has no fermenta-ble sugars. Their solutions, even at high concentrations (10%), take a light colours, without precipitate, due to their elaborated manufacturing process. It produces a slight acid reaction (after sterilization) and has no indole. Its nutritional capacity is low, but it may be used for non fastidious microorganisms, and complies with the pep-tone specifications for fermentation studies.

The most important characteristics are shown in the fol-lowing figures and tables and in Tables 1 to 6 at the beg-

Gelatin Pancreatic Peptone

gining of this chapter. Data are average values, which may vary from batch to batch.

07-153 Gelatin Pancreatic PeptoneMolecular Weigth Distribution

07-153 Gelatin Pancreatic PeptoneAmino Acids (Free/Total) x 100

195

Heart Extract

Ref. 07-077

Bovine heart extract has been widely used as an alterna-tive to meat extract where very special nutrient require-ments are needed. Scharlau Microbiology Heart Extract is obtained from the bovine cardiac muscle of healthy animals with explicit sanitary certification.

Among the raw materials and auxiliaries used in its preparation, the bovine constituents belong to the cat-egory 4 of the WHO classification. The bovine tissues are sourced from New Zealand, and come from herds free from Bovine Spongiform Encephalopathy and foot-and-mouth disease after examination by the Veterinary Authorities.

The product does not contain and is not derived from specified risk material as defined in the European Com-mission Decision 97/534/EC. The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instantaneous heating at 200ºC on spray drying.



07-077 Heart ExtractMolecular Weigth Distribution

07-077 Heart ExtractAmino Acids (Free/Total) x 100

196

Lecithin

Ref. 07-342

Phosphatidylcholine, a mixture of diglycerides of the stearic, palmitic and oleic acids, linked to the cholic ester of the phosphoric acid.

The SCHARLAU Lecithin is a clear brown powder, obtained from soy beans by extraction. It is especially treated to be included in culture media as an emulsifier or as a nutrient factor for fastidious microorganisms.


Liver Peptone

Ref. 07-614

Liver peptone is a proteic hydrolized by enzymatic diges-tion of fresh swine liver, followed by a careful desiccation process to maintain its fundamental characteristics.

Liver peptone is very employed in culture media for Tri-chomonas and other fastidious protozoa, and for some pathogen and saprofitic fungi, micoplasms and anaero-bic bacteria.

Liver peptone provides clear solutions of a dark colour, and is perfectly compatible with other medium com-ponents. It accepts sterilization and does not loose its characteristics. Generally it uses to replace peptones, at the same weight. In culture media for protozoa, its con-centration uses to be high (25-30 g/L), but for bacteria it uses to vary from 0,5 to 1 %, except for anaerobics enrichment media, where concentration may be higher.



197

Malt Extract

Ref. 07-080

Malt extract is used in the culture media for fungi, as much as enrichment as a true nutritive base, because very often it substitutes the peptone. It is obtained by extraction of soluble fraction of malted barley, followed by a drying proccess at low tempertature so that there is only minimal alteration in its nitrogenated composition and high sugar content, especially maltose. All the raw materials and auxiliaries are of plant origin. It has no diastatic activity. Very hygroscopic product.

Malt extract solutions are usually opalascent or turbid. Should clear solutions are required, filtration is neces-sary.


Liver Peptone

07-614 Liver PeptoneMolecular Weigth Distribution

07-614 Liver PeptoneAmino Acids (Free/Total) x 100

198

Meat Extract

Ref. 07-075

Meat extract has been the basic component of culture media for a long time, and initially it substituted meat infusions due to its easy usage. Now, there is a trend to substitute it by peptones and different mixtures with a more defined composition, because they allow a greater reproductivity result.

Meat extract is obtained from free tendons and fat tissues of the animals (sheep and pork), which are enzymatically predigested. Its production involves the elimination of fermentable sugars.

Among the raw materials and auxiliaries used in its preparation the bovine constituents belong to the cate-gory 4 of the WHO classification. The bovine tissues are sourced from New-Zealand, and come from the herds free from Bovine Spongiform Encephalopathy and foot-and-mouth disease after examination by the Veterinary Authorities.The product does not contain and is not derived from specified risk material as defined in the European Com-mission Decision 97/534/EC. The other constituents are of porcine origin. The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instan-taneous heating at 200ºC on spray drying.

The totally desiccated (dried) version is easier to use and requires less quantity in order to obtain the same effects. Meat extract solutions are clear, slightly coloured and with pH near to neutrality. In the culture media they are used in concentrations varying from 0,3-0,5%.


07-075 Meat ExtractMolecular Weigth Distribution

07-075 Meat ExtractAmino Acids (Free/Total) x 100

199

Meat Peptone

Ref. 07-152

Meat peptone is an hydrolysate obtained by a partial digestion of meat by pepsine. It complies with the USP/NF25 and Eur. Phar. 4th. Ed. specifications for the peptic digestion of animal tissues.

It is a fine powder, cream or brown coloured, that gives very clear and light solutions and is specially prepared for using in the culture media.

Among the raw materials and auxiliaries used in its preparation the bovine constituents belong to the cat-egory 4 of the WHO classification. The bovine tissues are sourced from New-Zealand, and come from the herds free from Bovine Spongiform Encephalopathy and Foot-and-Mouth Disease after the examination by the Veterinary Authorities.The product does not contain and is not derived from specified risk material as defined in the European Com-mission Decision 97/534/EC. The other constituents are of porcine origin.



07-152 Meat PeptoneMolecular Weigth Distribution

07-152 Meat PeptoneAmino Acids (Free/Total) x 100

200

Ref. 07-489

The Peptone from casein is a protein hydrolysate ob-tained from trypsin-digested casein according to the USP specifications. Both its nitrogen rate and its balanced amino-acid composition provide an adequate support for the production of culture media, producing exceptionally clear solutions.

Among the raw materials and auxiliaries used in its preparation the bovine constituents belong to the cat-egory 4 of the WHO classification. The bovine tissues are sourced from New-Zealand, and come from the herds free from Bovine Spongiform Encephalopathy and Foot-and-Mouth Disease after the examination by the Veterinary Authorities.The product does not contain and is not derived from specified risk material as defined in the European Com-mission Decision 97/534/EC.

Peptone from Casein

The digestion process by an exceptionally trysin-rich enzymatic preparation produces a high content of tryp-tophane and assures the absence of fermentable sugars and enzymatic activity.


07-489 Peptone from CaseinMolecular Weigth Distribution

07-489 Peptone from CaseinAmino Acids (Free/Total) x 100

201

Proteose Peptone

Ref. 07-213

This peptone is obtained after a partial enzymatic (pep-tic) digestion process of animal tissues. It is obtained in such a way that there is a high proportion of peptides of low molecular weight, with free amino acids and other growth factors. Although all these things make its definition very difficult, it has a high nutritive capacity that makes it suitable for obtaining toxins and as a basic growth support for very fastidious microorganisms.

Among the raw materials and auxiliaries used in its pro-duction, the bovine constituents belong to the category 4 of the WHO classification. The bovine tissues are sourced from New-Zealand, and come from herds of cat-tle which are free from Bovine Spongiform Encephalopa-thy and foot-and-mouth disease after examination by the Veterinary Authorities.The product does not contain and is not derived from specified risk material as defined in the European Com-mission Decision 97/534/EC. The other constituents are of porcine origin.



07-213 Proteose PeptoneMolecular Weigth Distribution

07-213 Proteose PeptoneAmino Acids (Free/Total) x 100

202

Soy Peptone

Ref. 07-155

Soy peptone is a proteic hydrolysate obtained by papaic digestion of soy flour. It complies with the USP/NF25 and Eur. Phar. 4th. Ed. specifications for these type of prod-ucts, and it is a useful compound in laboratory culture media. However, due to its high content of sugar it is not recommendable for fermentation assays.


07-155 Soy PeptoneMolecular Weigth Distribution

07-155 Soy PeptoneAmino Acids (Free/Total) x 100

203

Tryptose

Ref. 07-197

Tryptose is a mixed peptone with high nutrient properties that make it appropiate for the use in culture media for very fastidious microorganisms.

Among the raw materials and auxiliaries used in its preparation the bovine constituents belong to the cat-egory 4 of the WHO classification. The bovine tissues are sourced from New-Zealand, and come from herds free from Bovine Spongiform Encephalopathy and foot-and-mouth disease after ex-amination by the Veterinary Authorities.

The product does not contain and is not derived from specified risk material as defined in the European Com-mission Decision 97/534/EC. The other constituents are of porcine origin.



07-197 TryptoseMolecular Weigth Distribution

07-197 TryptoseAmino Acids (Free/Total) x 100

204

Yeast Extract

Ref. 07-079

A water soluble extract of fresh autolyzed yeast cells. Prepared and standardized for use in microbiological culture media.

It is commonly added to culture media in concentrations between 0.2% and 1%.


07-079 Yeast ExtractMolecular Weigth Distribution

07-079 Yeast ExtractAmino Acids (Free/Total) x 100

Additives

207

Dextrose Powder (D(+)- Glucose Powder)

Ref. 06-048

Presentation500 g Flask

DescriptionPurified and standardized carbohydrate for the use in microbiological cultrure media as energy source for bacteria. Carbohydrate are adapted to the suitable basic media.Scharlau carbohydrates are pure and without mixtures, and this important characterístic is assured in order to get always right results.

Physical DataBulk density............................................... ~ 630kg/m3

Solubility in water (20ºC) .......................... ~ 470g/LMelting point ............................................. ~ 146ºCIgnition temperature ................................. ~ 500ºCpH (100 g/L H

2O, 20ºC) ................................ 6-7

SpecificationsSpecific rotation ([α]20ºC/D, c=10, H

2O) ......+52,6 - +53,2 º

Molecular weight ..............................180,16 g/molAcidity/alkalinity ................................passes testInsoluble in water ................................. max. 0,01 %Chlorides (Cl)........................................ max. 0,01 %Sulfates (SO4) ...................................... max. 0,02 %Sulfite (as SO2) .................................... max. 0,001 %Arsenic (As) .......................................... max. 0,0001 %Calcium (Ca) ......................................... max. 0,02 %Heavy metals (as Pb) ........................... max. 0,0005 %Iron (Fe) ................................................ max. 0,0005 %Lead (Pb) .............................................. max. 0,00005%Sulfated ash ......................................... max. 0,1 %Water (K.F.) .......................................... max. 1 %Foreign sugars,starchs,dextrines .....passes testResidual solvents (Eur. Phar./ICH) ......excluded

Egg’s Yolk Sterile Emulsion

Ref. 06-016

Presentation100 mL Flask

DescriptionSterile egg’s yolk emulsion stabilized for use in bacte-riology, especially with Bacillus cereus Agar Base (Ref. 01-262), Bacillus cereus Selective Agar Base (Ref. 01-487) and Tryptose Sulfite Cycloserine Agar Base (Ref. 01-278).

It is also used for the detection of lecithinase in spe-cies such as Bacillus, Clostridium and Staphylococcus, and in all processes related with this enzyme which are present in dairy microorganisms and most psychrotroph-ics.

Lecithinase AssayThe medium is prepared by aseptically adding 0,5-1,0 mL of yolk emulsion to 10 mL of sterile melted solid medium, cooled to 55-60°C. Tryptic Soy Agar (Ref. 01-200), Nutrient Agar (APHA) (Ref. 01-144) and Nutrient Agar (B. Ph.) (Ref. 01-140) are very adequate for these purposes. The solid medium is inoculated with the assay strain and incubated at 35-37° C for 5 days. If there is lecithinasic activity the broths will turn opalescent and solid media will present an opaque zone of clearing around the colonies.Bacillus cereus, with a strong lecithinase, produces vis-ible results in just a few hours.

The Egg’s Yolk Sterile Emulsion has been widely used as bactericide neutralizer on sampling liquids. Such an effect can be considerably intensified by adding a bit of Polysorbate with the yolk in the formulation. 1% (v/v) is usually enough.

Egg’s Yolk Tellurite SterileEmulsion

Ref. 06-026


DescriptionThis emulsion has been especially formulated for its ad-dition to the Baird Parker Agar Base (Ref. 01-030)

Aseptically add 50 mL of Egg’s Yolk Tellurite Sterile Emulsion to 1 litre of sterile Baird Parker Agar Base (Ref. 01-030), melted and cooled down to approximately 55-60°C. Mix uniformly avoiding bubbles and foam, and pour into Petri dishes.The presumptive Staphylococcus aureus colonies show the lecithinase activity by the halo digestion around the colonies and simultaneously a black center due to the tellurite reduction.

Egg’s Yolk Sterile Emulsions

R-22S-46

Xn

208

Ref. 06-052



Lactose Powder

Ref. 06-051



Physical DataBulk density.............................................~ 500 kg/m3

Solubility in water (20ºC) ............freely solubleMelting point ...........................................~ 223 ºCpH (50 g/L H

2O, 20ºC) ................................4-6


2O) ......+54,4 - +55,9 º

Molecular weight ..............................360,32 g/molAcidity/alkalinity ................................passes testAppaerance of solution (10% water) .......passes testProteins ............................................passes testArsenic (As)...........................................max. 0,00005 %Copper (Cu) .......................................... max. 0,0025 %Heavy metals (as Pb) ........................... max. 0,0005 %Lead (Pb)...............................................max. 0,00005 %Zinc (Zn) ............................................... max. 0,0025 %Sulfated ash ......................................... max. 0,1 %Water (K.F.)............................................4,5 - 5,5 %Residual solvents (Eur. Phar./ICH)...........excluded

Maltose Powder

Physical DataBulk density.............................................~ 320 kg/m3

Solubility in water (20ºC) ............freely solubleMelting point ..................................~ 160 - 165 ºCpH (50 g/L H

2O, 20ºC) ..........................4,5-6,0


2O) ......+137 - +139 º

Molecular weight ..............................342,31 g/molAcidity/alkalinity ................................passes testAppaerance of solution (10% water) .......passes testProteins ............................................passes testArsenic (As).......................................max. 0,00005 %Copper (Cu).......................................max. 0,0005 %Heavy metals (as Pb)........................max. 0,002 %Barium (Ba).......................................max. 0,0005 %Calcium (Ca)......................................max. 0,005 %Sulfated ash.......................................max. 0,1 %Water (K.F.)........................................4,5 - 5,5 %Residual solvents (Eur. Phar./ICH) ......excluded

209

Mannitol Powder

Ref. 06-050



Physical DataBulk density...................................~ 400 - 500 kg/m3

Spec. density.................................................1,49 g/m3

Solubility in water (25ºC)...........................213 g/LMelting point ...................................~ 164 -169 ºCBoiling point (4 hPa) ......................~ 290 - 295 ºCpH (100 g/L H

2O, 20ºC) ..............................5-7

SpecificationsAssay (iodometric) ............................min. 98 %Acidity/alkalinity ................................passes testAppaerance of solution (20% water) .......passes testConductivity (20ºC, 20% water) ........passes testSpecific rotation ([α]20ºC/D, c=8, Na

2B

4O

7) ...+23 - +25 º

Molecular weight ............................. 182,17 g/molChlorides (Cl)........................................ max. 0,005 %Sulfates (SO

4) ....................................... max. 0,01 %

Arsenic (As) .......................................... max. 0,0001 %Copper (Cu) .......................................... max. 0,001 %Heavy metals (as Pb) ........................... max. 0,0005 %Lead (Pb)...............................................max. 0,00005 %Nickel (Ni) ............................................. max. 0,0001 %Zinc (Zn) ............................................... max. 0,0025 %Related substances (sorbitol) ............... max. 2 %Red. impurities (as glucose) ................. max. 0,05 %Sulfated ash ......................................... max. 0,1 %Loss on drying (105ºC, 4h) ................... max. 0,3 %Residual solvents (Eur. Phar./ICH)...........excluded

Polysorbate 80 (Polyoxyethylene sorbitan monooleate)

Ref. 06-088

Presentation100 mL Flask1 L Flask

DescriptionUnder the name of Polysorbate 80 or polyoxyethylene sorbitanmonooleate are included a serie of derivates of polyose-1,2-ethanodiol sorbitan-mono-9-octodecenoate.

The product supplied by SCHARLAU is verified to be a nutrient in some cases and an emulsionant in others, but it is always compatible with the rest of components of the culture medium.It is a very thick liquid, amber colour, and density 1,08 approx. It is very soluble in water, it has an average solu-bility in organic diluents but it is not soluble in mineral lipids.

The incorporation of Polysorbate 80 to culture me-dia may slightly affect the final pH, if the SCHARLAU medium is not originally formulated to be composed by Polysorbate.

Although it may bear sterilization in the autoclave when it is more than 1% in the medium, it is usual to homog-enize the medium after the sterilization, since with the autoclaving sometimes the polysorbate is separated from the medium.

Polysorbate is a tensioactive agent that makes decrease the superficial tension of the cell, modifying at the same time the cellular exchange speed. The response use to be a quicker growht or the increase of some bacterial activities.

210

Potassium Tellurite Solutions

Potassium Tellurite Solution 1%

Ref. 06-089


Potassium Tellurite Solution 3,5%

Ref. 06-011


DescriptionAquouse solutions of potassium tellurite at 1% or 3,5%, sterilized by filtration and suitable to be used as an inhibitor additive in culture media.

The Potassium Tellurite Solution is added to culture me-dia as an inhibitor. Its purpose is to prevent the growth of most gram-negatives and of those gram-positives unable of reducing it.

It is used in media such as Giolitti Cantoni Broth (Ref. 02-230), Vogel-Johnson Agar (Ref. 01-206) and other selective media for staphylococci. This solution is also contained in selective media for corynebacteria, strepto-cocci and vibrios.

There is high relation between the ability to reduce po-tassium tellurite to tellure and the staphylococci’s patho-genity. Therefore, the presence of potassium tellurite in a medium helps to determine staphylococci of clinical interest, together with other tests.

The Potassium Tellurite Solution should be stored at room temperature, since low temperatures will cause the crystallization and later precipitation of the product. Should this occur, intense agitation will help redissolve the precipitate. Due it its thermolabile qualities, the po-tassium tellurite is supplied sterile filtered.

Ref. 06-073

Presentation500 g Flask100 g Flask

SpecificationIsotonic solution for the cellular suspensions.

Formula (in g/L)Sodium chloride ......................................2,250Potassium chloride .................................0,105Calcium chloride .....................................0,120Sodium bicarbonate ...............................0,050

DirectionsTo obtain an isotonic solution for eukaryotic cells, dis-solve 10 g of powder in 1 L of distilled water. To obtain an isotonic solution for prokaryotic cells, dissolve 2,5 g of powder in 1 L of distilled water. Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

DescriptionRinger saline solution is an isotonic medium which is more balanced than the simple sodium chloride saline solution, and its formulation permits the autoclaving without producing any precipitation.

For the routine work with bacteria the solution is diluted one fourth (to the fourth part) (Ringer 1/4), and is em-ployed to get cell suspensions or to prepare the dilution banks.Nonetheless, to dilute the food samples or substances that have undergone thermal treatment, it is more advis-able to use Peptone Water (Ref. 3-156) for the dilutions, since the Peptone Water acts as a revitalizer also.

ReferencesDAVIS, J.G. (1956) Laboratory Control of Dairy Plant. Dairy Industries Ltd. London.ANONYMOUS (1937) Bacterial Tests for Graded Milk. Memo 139-Food. Dept. of Health and Social Security,London.

Ringer Powder

R-22S-46

Xn

R-22S-46

Xn

211

Ref. 06-019


DescriptionPowder of skimmed milk for bacteriology is obtained after a depurated atomization process that keep it free from thermophil organismsm that use to interfere with its use.

100 g of powder produce 1 L of skimmed milk. Water addition must be gradual, until getting an homogene-ous paste. Then fullfil with water to the desired volume. Sterilization may be under fluent vapor for 30 minutes and three consecutive days or in the autoclave at 121°C for 15 minutes or at 114°C for 15-20 minutes (this last way is the better). Anycase, do not overheat prepared milk since natural sugars may become caramelizated and produce toxic compounds.

Skimmed milk may be use alone or as an additive to other culture media. It is a very suitable medium for the culture of acid lactic bacteria and for identification in general, in base to its capacity to coagulate o peptonize milk. With the additon of suitable indicators as brom-cresol purple at 0,004% the pH variations in the transfor-mation may be screened. It also accepts oxid-reduction indicators like methylen blue, resazurine or TTC (Ref. 6-023) to verify microorganisms development.

Skimmed milk may be added to media as Tryptone Soy Agar (Ref. 01-200) or Nutrient Agar (Ref. 01-144 and 01-140) to detect caseolytic activity.

Fat ............................................................. 0,5 %Protein ..................................................... 33,0 %Ash ............................................................ 8,0 %Moisture ..................................................... 5,0 %Lactic acid ..................................................1,5 %Antibiotic test .....................................Negative

Skimmed Milk

Sodium Biselenite

Ref. 06-615


DescriptionChemical compound to be added to selenite based culture media.

The toxic and potetially theratogenicity of this product recommend its exclusion from the dehydrated powder mixture of culture media, to minimize the hazard of acci-dental inhalation or contact. The supply of this product in a separate form from the base medium, with all the risk considerations, enhances its safe and responsible use.

The intended use of this product is to complete the fol-lowing culture media by adding the specified amounts:

02-602 Selenite Cystine Broth Base .....................4 g/L02-603 Selenite Brilliant Green Broth Base ...........4 g/L02-598 Selenite Broth Base ..................................4 g/L

The use of this product is restricted to technically quali-fied personnal. Keep attention to the job limitations in inexpert personnal.

R-23/25-33-50/53S-20/21-28-45-60-61

NT

212

Sucrose Powder (D(+) Saccharose Powder)

Ref. 06-049



Physical DataBulk density....................................~ 800 - 950 kg/m3

Solubility in water (25ºC) ............freely solubleMelting point ...................................~ 169 -170 ºCpH (100 g/L H

2O, 20ºC) ..............................~ 7


2O) ......+66,3 - +67,0 º

Molecular weight ..............................342,30 g/molAcidity/alkalinity ................................passes testAppaerance of solution (50% water) .......passes testConductivity .....................................max. 35 μS/cmChlorides (Cl)........................................ max. 0,0035 %Sulfites (as SO

3) ................................... max. 0,0015 %

Sulfates (SO4) ....................................... max. 0,005 %

Dextrines ..........................................passes testGlucose and invert sugar .................passes testCalcium (Ca) ......................................... max. 0,001 %Heavy metals (as Pb) ........................... max. 0,0005 %Lead (Pb) .............................................. max. 0,00005 %Sulfated ash ......................................... max. 0,01 %Organic volatile impurities (NF) .........passes testResidual solvents (Eur. Phar./NF) .......max. 0,5 %

213

Ref. 06-023


DescriptionSterile solution at 1% of 2-3-5-triphenyl-2H-tetrazolium chloride. It is used as an additive for culture media to show biological activity, since the colourless form gets hydrogenizated or reduced to a red insoluble pigment: triphenylformazan, which may be easily observed.

Despite of TTC decomposes at 243°C, it is not advis-able to incorporate it to culture media before steriliza-tion, because it lose efficacy. Very good results may be achieved when the addition is carried out asseptically with cold medium at 60°C maximum. TTC is photolabile and becomes yellow by the effect of light, therefore keep it in the refrigerator and avoid direct light.

Concentration of use vary depending on the medium, but generally it goes between 0,3 and 1% (v/v).

TTC Sterile Solution 1%

The general structure is the following:

This product is especially produced to be added to the following media: Chapman TTC Agar Base (Ref. 01-053), KF Media (Ref. 01-294 and Ref. 02-294) and Slanetz Bartley Agar Base (Ref. 01-579).

Urea Sterile Solution 40%

Ref. 06-083


DescriptionAquose urea solution 40%, sterilized by filtration and suitable to be used as an additive in culture media.

It is supplied to be used with the dehydrated media Urea Agar acc. to Christensen (Ref. 01-261) and Urea Broth (Ref. 02-202). It must be added to these media after the sterilization and with the media cooled below 55°C.

Once it is added, do not reheat the media.

Vaseline Sterile

Ref. 06-077


DescriptionLiquid media may be kept in anaerobic conditions if sterile compounds, as vaseline, are used to grant an hermetic lock.To achieve an hermetic lock in tubes with liquid medium, heat them up in boiling bath for 10 minutes to remove the oxygen and add after the vaseline to avoid the

incorporation of air to the medium. To seed the tubes, use a capilar pipette or inoculate them previously to the vaseline addition.

Vaspar is another method used to achieve an hermetic lock. It is prepared melting vaseline and solid parafine toghether in equal parts.Vaseline sterile is especially suggested for the addition to O/F Medium (Ref. 03-037). For solid media is more suitable to use the Sealing Agar (Ref. 01-174).

Supplements

216

REFERENCE DESCRIPTION06-012CASE SC Selective Supplement for Clostridium VIAL CONTENTS: 120 mg of Sodium azide, 90 mg of Neomycin sulphate and solvent. Each vial is sufficient to supplement 500 ml of Blood Columbia Agar Base (Ref. 01-034) or Blood Agar Base (Ref. 01-352) in order to prepare Clostridium spp. Selective Agar. 06-013CASE CP Gram-positive cocci in Blood Agar Selective Supplement VIAL CONTENTS: 5 mg of Colistin sulphate, 7,5 mg of Nalidixic acid and solvent. Each vial is sufficient to supplement 500 ml of Blood Columbia Agar Base (Ref. 01-034) or Blood Agar Base (Ref. 01-352) and obtain Staphylococcus and Streptococcus Selective Agar. 06-017CASE Brilliant Green + Novobiocin Selective Supplement VIAL CONTENTS: 5 mg of Brilliant green, 20 mg of Novobiocin and solvent. Each vial is sufficient to supplement 500 ml of Tetrationate Base Broth (Ref. 02-033) and Muller-Kauffmann Medium (Ref. 02-335). 06-021CASE Polymyxin B Sulphate Selective Supplement VIAL CONTENTS: 50 mg Polymyxin B sulphate and solvent. Each vial is sufficient to supplement 500 ml of Bacillus cereus Agar Base (Ref. 01-262). 06-022CASE Cycloheximide Selective Supplement VIAL CONTENTS: 2 mg of Cycloheximide and solvent. Each vial is sufficient to supplement 500 ml of WL Nutrient Agar (Ref. 01-210) or WL Nutrient Broth (Ref. 02-210) and converted in WL Differential Agar or Broth. 06-025CASE Brucella Selective Supplement VIAL CONTENTS: 50 mg of Cycloheximide, 3000 u.i. of Polymyxin B Sulphate, 12500 u.i. of Bacitracin sulphate and solvent. Each vial is sufficient to supplement 500 ml of Brucella Selective Agar (Ref. 01-042) and Brucella Selective Broth (Ref. 02-042). 06-085CASE Rosolic Acid Selective Supplement VIAL CONTENTS: 50 mg of Rosolic acid and solvent. Each vial is sufficient to supplement 500 ml of Faecal Coliforms Agar (m-FC Agar) (Ref. 01-287) or Faecal Coliforms Broth (m-FC Broth) (Ref. 02-287). 06-091CASE CPB Selective Supplement for Campylobacter VIAL CONTENTS: 2,5 mg of Trimethoprim, 5 mg of Vancomycin, 7,5 mg of Cephalotin, 1250 u.i. of Polymyxin B sulphate, 1 mg of Amphotericin B and solvent. Each vial is sufficient to supplement 500 ml of Blood Columbia Agar Base (Ref. 01-034) and obtain 500 ml of Campylobacter Selective Agar acc. Blaser-Wang. 06-102CASE MUG Supplement. Fluorescent Agent for Escherichia coli VIAL CONTENTS: 50 mg of MUG (4-methilumbeliferil-ß-D glucuronide) and solvent. Each vial is sufficient to supplement 500 ml of Coliforms Agar or Broth. 06-106CASE Listeria Selective Supplement for Primary Enrichment (UVM I) VIAL CONTENTS: 10 mg of Nalidixic acid, 6 mg of Acriflavine and solvent. Each vial is sufficient to supplement 500 ml of Listeria Enrichment Broth Base (UVM) (Ref. 02-472) in order to prepare 500 ml of Listeria Primary Enrichment Medium (UVM I formulation). 06-107CASE Listeria Selective Supplement for Enrichment acc. FDA and IDF/FIL VIAL CONTENTS: 20 mg of Nalidixic acid, 25 mg of Cycloheximide, 7,5 mg of Acriflavine and solvent. Each vial is sufficient to supplement 500 ml of Listeria Enrichment Broth Base acc. Lovett (Ref. 02-498). 06-109CASE Oxford Agar Selective Supplement for Listeria VIAL CONTENTS: 5 mg of Phosphomycin, 1 mg of Sodium Cephotaxim, 10 mg of Colistin, 200 mg of Cycloheximide, 2,5 mg of Acriflavine and solvent. Each vial is sufficient to supplement 500 ml of Oxford Agar Base (Ref. 01-471) in order to prepare Listeria Selective Agar (Oxford formulation). 06-110CASE Palcam Agar Selective Supplement for Listeria VIAL CONTENTS: 5 mg of Polymyxin B sulphate, 10 mg of Sodium ceftazidime, 2,5 mg of Acriflavine and solvent. Each vial is sufficient to supplement 500 ml of Palcam Agar Base (Ref. 01-470) in order to prepare Listeria Selective Agar (Palcam formulation).

REFERENCE DESCRIPTION06-111CASE Listeria Selective Supplement for Secondary Enrichment (UVM II/Fraser) VIAL CONTENTS: 10 mg of Nalidixic acid, 12,5 mg of Acriflavine and solvent. Each vial is sufficient to supplement 500 ml of Listeria Enrichment Broth Base (UVM) (Ref. 02-472) in order to prepare 500 ml of Listeria Secondary Enrichment Medium (UVM II formulation); or to supplement 500 ml of Listeria Enrichment Broth acc. Fraser (Ref. 02-496) in order to prepare 500 ml of Fraser Broth. 06-112CASE Ferric Ammonium Citrate for Bacteriology VIAL CONTENTS: 250 mg of Ferric ammonium citrate and solvent. Each vial is sufficient to supplement 500 ml of Listeria Enrichment Broth Base acc. Fraser (Ref. 02-496) 06-113CASE Ferric Ammonium Citrate for bacteriology VIAL CONTENTS: 312 mg of Ferric ammonium citrate and solvent. Each vial is sufficient to supplement 500 ml of Lactose Sulphite Broth Base (Ref. 02-519). 06-114CASE Disodium disulphite Selective Supplement for bacteriology VIAL CONTENTS: 375 mg of Disodium disulphite and solvent. Each vial is sufficient to supplement 500 ml of Lactose Sulphite Broth Base (Ref. 02-519). 06-115CASE Oxytetracycline Selective Supplement VIAL CONTENTS: 50 mg of Oxytetracycline and solvent. Each vial is sufficient to supplement 500 ml of Sabouraud with Oxytetracycline Agar Base (OGYEA) (Ref. 01-275). 06-116CASE Cycloserine Selective Supplement VIAL CONTENTS: 100 mg of Cycloserine and solvent. Each vial is sufficient to supplement 250 ml of Tryptose Sulphite Cycloserine (TSC) agar Base for Clostridium perfringens Ref. 1-278). 06-118CASE Chloramphenicol Selective supplement VIAL CONTENTS: 25 mg of Chloramphenicol and solvent. Each vial is sufficient to supplement 500 ml of Sabouraud Dextrose Agar (Eur. Phar. Agar Medium C), (Ref. 01-165). 06-124CASE Nalidixic Acid Selective Supplement VIAL CONTENTS: 7,5 mg of Nalidixic acid and solvent. Each vial is sufficient to supplement 500 ml of CN Selective Agar Base for Pseudomonas (Ref. 01-609). 06-125CASE m-CP Selective Supplement VIAL CONTENTS: 200 mg of D-Cycloserine, 12,5 mg of Polymyxin B sulphate, 30 mg of 3-indoxyl-ß-D-glucopyranoside, 50 mg of Phenolphthalein diphosphate, 45 mg of Iron III Chloride, and solvent. Each vial is sufficient to supplement 500 ml of m-CP Agar Base (Clostridium perfringens Agar) (Ref. 01-513).

Storage conditions: Should be stored at 2-8ºC in the dark.Shelf life:18-24 months depending the reference. Larger vial-larger variety of supple-ments.

PRESENTATION: 10 VIALS INSIDE A RESISTANT CASE

MORE INFORMATION AVAILABLE IN OUR WEB

AND CD-ROM CATALOGUE

Improved NEW presentation

217

Selective Supplements

Culture media supplements in a practical presentation: an extemporaneous solution.Main advantages:

• fast• simple• easy• safe• ready to add• easy storage• longer shelf life• less risk of contamination

With this 10 vial-case format you no longer have to worry about things like sterile solvents, sterile syringes, sterilis-ing the supplements that must be added to the medium by filtration ...With one simple pressure on the lid you obtain the sterile supplement solution, ready to add to the medium base.Selective supplements should be stored at 2-8°C in the dark. When stored as directed the reagents remain stable until the expiry date shown on the label.

Method of use: 4 simple steps

1 Press on the cap brea-king the container that holds the solid.

2The solid then falls into the solvent. Shake vigorously for total dissolution.

3 The supplement is now ready. Open the vial aseptically, close to a fla-me or in a safety cabinet. Pour over the medium base, which has been cooled down to 45-50°C.

4 Homogenize and distribute into the suitable container: flasks, tubes or plates.

218

Ref. 06-617CASE

ContentsThe box contains 10 vials. Each vial is sufficient to sup-plement 250 mL of Endo LES (Ref. 01-604).

Vial contentsNecessary amount for 250 mL of medium.Basic Fuchsin ............................................200 mgEthanol ..........................................................5 mL

DirectionsMix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 250 mL of Endo LES Agar Base.Note: Don’t heat the media once the supplement has been added.

Precautions• This product should be for laboratory use only.• Do not use beyond stated expiry date.

Applicable mediaRef. 01-604 Endo LES Agar Base

Basic Fuchsin 200 Selective Supplement

Ref. 06-607CASE

ContentsThe box contains 10 vials. Each vial is sufficient to sup-plement 500 mL of Endo Agar Base (Ref. 01-589), Endo DEV Agar Base (Ref. 01-606) and only 250 mL of Endo Base Broth (Ref. 02-605).

Vial contentsNecessary amount for 500 mL of solid medium or 250 mL of liquid broth.Basic Fuchsin ............................................250 mgEthanol ..........................................................5 mL

DirectionsMix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to the boiled medium. Homogenize and use as per each monography medium. Do not remelt the solid media.


Applicable mediaRef. 01-589 Endo Agar BaseRef. 01-606 Endo DEV Agar Base

Basic Fuchsin 250 Selective Supplement

R-11S-7-16

F

R-11S-7-16

F

219

Brilliant Green + Novobiocin Selective Supplement

Ref. 06-017CASE

ContentsThe box contains 10 vials. Each vial contains inhibitors sufficient to add to 500 mL of Tetrathionate Broth Base Ref. 02-033 or 02-335 Muller Kauffmann Medium.

Vial contentsNecessary amount for 500 mL of medium.Brilliant Green ................................................5 mgNovobiocin, sodium salt ..............................20 mgDistilled water ................................................5 mL

DirectionsMix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the vial contents to 500 mL of boiled broth base cooled to 50°C. Homogenize and use as per each monography medium.Note: Don’t heat the media once the supplements have been added.


Applicable mediaRef. 02-033 Tetrathionate Base BrothRef. 02-335 Muller-Kauffmann Medium

220

Chloramphenicol Selective Supplement

Ref. 06-118CASE

ContentsThe box contains 10 vials. Each vial is sufficient to supplement 500 mL of Sabouraud Dextrose Agar (Ref. 01-165).

Vial contentsNecessary amount for 500 mL of medium.Chloramphenicol .........................................25 mgDistilled water ................................................5 mL

DirectionsMix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500 mL of sterile agar base cooled to 50°C. Ho-mogenize and distribute the complete medium into the containers. Note: Don’t heat the media once the supplement has

been added.


Applicable mediaRef. 01-165 Sabouraud Dextrose Agar (Eur. Phar. Agar

Medium C)

m-CP Selective Supplement

Ref. 06-125CASE

ContentsThe box contains 10 vials. Each vial is sufficient to supplement 500 mL of m-CP Agar Base, Ref. 01-513, in order to prepare 500 mL of m-CP (Clostridium perfrin-gens) Agar.

Vial ContentsNecessary amount for 500 mL of complete medium.D-Cycloserine .......................................200,0 mgPolymixin B sulfate .................................12,5 mg3-Indoxyl-ß-D-Glucopyranoside .............30,0 mgPehnolphthalein di-phosphate ................50,0 mgIron III Cloride .........................................45,0 mgDistilled water ...........................................5,0 mL

DirectionsMix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500 mL of sterile agar base cooled to 50ºC. Note: Don’t heat the media once the supplement has been added.


Applicable mediaRef. 01-513 m-CP Agar Base

R-45S-53-45

T

221

CP Gram-positive cocci in Blood Agar Selective Supplement

Ref. 06-013CASE

ContentsThe box contains 10 vials. Each vial is sufficient to sup-plement 500 mL of Blood Columbia Agar Base Ref. 01-034 or Blood Agar Base Ref. 01-352 in order to prepare 500 mL of Staphylococcus and Streptococcus selective blood agar.

Vial contentsNecessary amount for 500 mL of complete medium.Colistin sulfate ........................................ 5,00 mgNalidixic Acid, sodium salt ...................... 7,50 mgDistilled Water ........................................ 5,00 mL

DirectionsMix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500mL of blood agar. Homogenize and use as per each monography medium.Note: Don’t heat the media once the supplements have been added.


Applicable mediaRef. 01-034 Blood Agar Base (Columbia)Ref. 01-352 Blood Agar Base

222

Cycloheximide Selective Supplement

Ref. 06-022CASE

ContentsThe box contains 10 vials. Each vial is sufficient to supplement 500 mL of WL Nutrient Agar or Broth Ref. 01-210 or 2-210 in order to prepare 500 mL of WL Dif-ferential Agar or Broth.

Vial contentsNecessary amount for 500 mL of complete medium.Cycloheximide ............................................. 2 mgSodium chloride ........................................... 8 mgDistilled water .............................................. 5 mL

DirectionsMix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500 mL of sterile agar base cooled to 50°C.Note: Don’t heat the media once the supplement has been added.


Applicable mediaRef. 01-210 WL Nutrient AgarRef. 02-210 WL Nutrient Broth

Cycloserine Selective Supplement

Ref. 06-116CASE

ContentsThe box contains 10 vials. Each vial is sufficient to sup-plement 250 mL of Tryptose Sulfite Cycloserine Agar Base (TSC Agar) Ref. 01-278.

Vial contentsNecessary amount for 250 mL of medium.D-Cycloserine ..........................................100 mgDistilled water ..............................................5 mL

DirectionsMix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solu-tion to 250 mL of sterile agar base cooled to 50°C. If it is desired, add 20 mL of Egg Yolk Sterile Emulsion (Ref. 06-016). Homogenize and distribute the complete medium into the plates. Note: Don’t heat the media once the supplements has been added.


Applicable mediaRef. 01-278 Tryptose Sulfite Cycloserine Agar (TSC

Agar)

223

Listeria Selective Supplement for Enrichment acc. FDA/IDF

Ref. 06-107CASE

ContentsThe box contains 10 vials. Each vial is sufficient to sup-plement 500 mL of Listeria Enrichment Broth Base acc. Lovett Ref. 02-498 in order to prepare 500 mL of Listeria enrichment broth according FDA and IDF/FIL.

Vial contentsNecessary amount for 500 mL of complete medium.Nalidixic acid, sodium salt ...................... 20,0 mgCycloheximide ........................................ 25,0 mgAcriflavine ................................................. 7,5 mgDistilled water ........................................... 5,0 mL

DirectionsMix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500 mL of sterile broth base cooled to 50°C.Note: Don’t heat the media once the supplement has been added.


Applicable mediaRef. 02-498 Listeria Enrichment Broth Base acc. Lovett.

Ferric Ammonium Citrate for Bacteriology

Ref. 06-112CASE

ContentsThe box contains 10 vials. Each vial is sufficient to sup-plement 500 mL of Listeria Enrichment Broth acc. Fraser Ref. 02-496 in order to prepare 500 mL of Fraser broth.

Vial contentsNecessary amount for 500 mL of complete medium.Ferric ammonium citrate ......................... 250 mgDistilled water ............................................. 5 mL



Applicable mediaRef. 02-496 Listeria Enrichment Broth Base acc. to Fraser

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Ref. 06-113CASE

ContentsThe box contains 10 vials. Each vial is sufficient to supple-ment 500 ml of Lactose Sulfite Broth Base Ref. 02-519 in order to prepare 500 ml of Lactose Sulfite Broth.

Vial contentsNecessary amount for 500 ml of complete medium.Ferric ammonium citrate ......................... 312 mgDistilled water ............................................. 5 mL

DirectionsMix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500 ml of sterile broth base cooled to 50°C.Note: Don’t heat the media once the supplement has been added.


Applicable mediaRef. 02-519 Lactose Sulfite Broth Base

224

Listeria Selective Supplement for Primary Enrichment (UVM I)

Ref. 06-106CASE

ContentsThe box contains 10 vials. Each vial is sufficient to supplement 500 mL of Listeria Enrichment Broth Base (UVM) Ref. 02-472 in order to prepare 500 mL of Liste-ria primary enrichment medium.

Vial contentsNecessary amount for 500 mL of complete medium.Nalidixic acid, sodium salt ......................... 10 mgAcriflavine .................................................... 6 mgDistilled water .............................................. 5 mL



Applicable mediaRef. 02-472 Listeria Enrichment Broth Base (UVM)

Listeria Selective Supplement for Secondary Enrichment(UVM II / Fraser)

Ref. 06-111CASE

ContentsThe box contains 10 vials. Each vial is sufficient to supplement 500 mL of Listeria Enrichment Broth Base (UVM) Ref. 02-472 in order to prepare 500 mL of Liste-ria secondary enrichment medium (UVM II formulation); or to supplement 500 mL of Listeria Enrichment Broth acc. Fraser Ref. 02-496 in order to prepare 500 mL of Fraser Broth.

Vial contentsNecessary amount for 500 mL of complete medium.Nalidixic acid, sodium salt .....................10,0 mgAcriflavine ..............................................12,5 mgDistilled water ..........................................5,0 mL



Applicable mediaRef. 02-472 Listeria Enrichment Broth Base (UVM)Ref. 02-496 Listeria Enrichment Broth Base acc. to

Fraser

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225

Ref. 06-124CASE

ContentsThe box contains 10 vials. Each vial is sufficient to sup-plement 500 mL of CN Selective Agar for Pseudomonas (Ref. 01-609).

Vial ContentsNecessary amount for 500 mL of complete medium:Nalidixic acid sodium salt ........................ 7,5 mgDistilled water .......................................... 5,0 mL

MUG Supplement

Ref. 06-102CASE

ContentsThe box contains 10 vials. Each vial is sufficient to sup-plement 500 mL of Coliforms Agar or Broth.

Vial contentsNecessary amount for 500 mL of complete medium.MUG (4-methilumbeliferil-ß-D-glucuronide) .....50 mgDistilled water ..............................................5 mL

DirectionsMix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500 mL of agar or broth cooled to 50°C.MUG supplement may be added to almost all the media that allow the growth of Escherichia coli for its identifica-tion. However, results will be more reliable in all those media that are selective for coliforms. Attached is a list of the most currently used. In our Culture Media Handbook you will find a table indicating the aspect of Escherichia coli in each medium.Note: Don’t heat the media once the supplement has been added.


Applicable mediaRef. 01-047 CLED Agar (Brolacin Agar)Ref. 01-118 MacConkey Agar (Eur. Phar. Medium H)Ref. 01-164 Violet Red Bile Agar (VRB Agar)Ref. 01-220 Violet Red Bile Dextrose Lactose Agar (VRBDL Agar)Ref. 01-484 E.coli Direct Agar (ECD Agar)Ref. 02-041 Brilliant Green Bile 2% BrothRef. 02-060 EC BrothRef. 02-105 Lactose Broth (Eur. Phar. Broth Medium D)Ref. 02-108 Tryptose Lauryl Sulfate BrothRef. 02-118 MacConkey BrothRef. 02-120 MacConkey Modified BrothRef. 02-611 MacConkey G Broth (Eur. Phar. Medium G)

Nalidixic Acid Selective Supplement

DirectionsMix the liquid with the powder by pressing downd on the cap. Shake to dissolve and asseptically add the solution to 500 mL of sterile agar base cooled to 50ºC.Note: Don’t heat the media once the supplement has been added.


Applicable mediaRef. 01-609 CN Selective Agar Base for Pseudomonas.

226

Oxytetracycline Selective Supplement

Ref. 06-115CASE

ContentsThe box contains 10 vials. Each vial is sufficient to sup-plement 500 mL of Sabouraud with Oxytetracycline Agar Base Ref. 01-275.

Vial contentsNecessary amount for 500 mL of medium.Oxytetracycline HCl .................................. 50 mgDistilled water ............................................. 5 mL

DirectionsMix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500 mL of sterile agar base cooled to 50°C. Homoge-nize and distribute the complete medium into the plates. Note: Don’t heat the media once the supplement has been added.


Applicable mediaRef. 01-275 Saboraud with Oxytetracycline Agar (OG-

YEA)

Oxford Agar Selective Supplement

Ref. 06-109CASE

ContentsThe box contains 10 vials. Each vial is sufficient to sup-plement 500 mL of Oxford Agar Base Ref. 01-471 in order to prepare 500 mL of Listeria selective agar (Oxford formulation).

Vial contentsNecessary amount for 500 mL of complete medium.Acriflavine ...................................................2,5 mgFosfomicyn .................................................5,0 mgSodium cefotaxim .......................................1,0 mgColystin .....................................................10,0 mgCycloheximide ........................................200,0 mgDistilled water .............................................5,0 mL


Precautions• This product should be for laboratory use only.•Do not use beyond stated expiry date.

Applicable mediaRef. 01-471 Oxford Agar Base

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227

Polymixin B Sulfate Selective Supplement

Ref. 06-021CASE

ContentsThe box contains 10 vials. Each vial is sufficient to supplement 500 mL of Bacillus cereus Agar Base Ref. 01-262.

Vial contentsNecessary amount for 500 mL of medium.Polymixin B sulphate ................................ 50 mgDistilled water ............................................. 5 mL

DirectionsMix the liquid with the powder by pressing down on the cap Shake to dissolve and aseptically add the solution to 450 mL of sterile agar base cooled to 50°C.Add also 50 mL of sterile Egg Yolk Emulsion. Homoge-nize and distribute the complete medium into the plates. Note: Don’t heat the media once the supplements have been added.


Applicable mediaRef. 01-262 Bacillus cereus AgarRef. 01-487 Bacillus cereus Selective Agar

Palcam Agar Selective Supplement

Ref. 06-110CASE

ContentsThe box contains 10 vials. Each vial is sufficient to supplement 500 mL of Palcam Agar Base Ref. 01-470 in order to prepare 500 mL of Listeria Selective Agar (Palcam formulation).

Vial contentsNecessary amount for 500 mL of complete medium.Acriflavine ............................................... 2,5 mgPolymixin B sulphate .............................. 5,0 mgSodium ceftazidime ............................. 10,0 mgDistilled water ......................................... 5,0 mL



Applicable mediaRef. 01-470 Palcam Agar Base

228

Rosolic Acid Selective Supplement

Ref. 06-085CASE

ContentsThe box contains 10 vials. Each vial is sufficient to sup-plement 500 mL of Fecal Coliforms Agar or Broth (m-FC) Ref.1-287 or 2-287 in order to prepare 500 mL of m-FC complete medium.

Vial contentsNecessary amount for 500 mL of complete medium.Rosolic Acid ............................................. 50 mgEthanol ...................................................... 5 mL

DirectionsMix the liquid with the powder by pressing down on the cap. Shake till total dissolution and aseptically add the solution to 500 mL of agar or broth cooled to 50°C.Use medium newly made.Note: Don’t heat the media once the supplement has been added.


Applicable mediaRef. 01-287 Fecal Coliforms Agar (FC Agar)Ref. 02-287 Fecal Coliforms Broth (FC Broth)

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229

SC Selective Supplement

Ref. 06-012CASE

ContentsThe box contains 10 vials. Each vial is sufficient to sup-plement 500 mL of Blood Columbia Agar Base Ref. 01-034 or Blood Agar Base Ref. 01-352 in order to prepare 500 mL of Clostridium ssp. selective agar.

Vial contentsNecessary amount for 500 mL of complete medium.Sodium Azide .......................................120,0 mgNeomycine sulfate ..................................90,0 mgDistilled water ...........................................5,0 mL

DirectionsMix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 475 mL of sterile agar base cooled to 50°C. Add 25 mL of defibrinated blood . Homogenize and distribute into the plates.Note: Don’t heat the media once the supplements have been added.


Applicable MediaRef. 01-034 Blood Columbia Agar BaseRef. 01-352 Blood Agar Base

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Disodium disulfite (meta-bisulfite) for Bacteriology

Ref. 06-114CASE

ContentsThe box contains 10 vials. Each vial is sufficient to supple-ment 500 ml of Lactose Sulfite Broth Base Ref. 02-519 in order to prepare 500 ml of Lactose Sulfite Broth.

Vial contentsNecessary amount for 500 mL of complete medium.Disodium sulfite ....................................375,0 mgDistilled water ...........................................5,0 mL

DirectionsMix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500 ml of sterile broth base cooled to 50°C.Note: Don’t heat the media once the supplement has been added.

Precautions• Reagent for laboratory use only.• Do not use beyond stated expiry date.

Applicable MediaRef. 02-519 Supplement for Lactose Sulfite Broth

Reagents

233

Barrit’s Reagent

Ref. 06-027

Presentation100 mL dropper flask

SpecificationReagent for the Voges-Proskauer test in enterobacte-riaceae.

DescriptionAll the enterobacteria ferment dextrose, but some spe-cies like Klebsiella, Enterobacter, etc..., do it following the 2-3-butanediol path and other species like E.coli, Salmonella, etc..., do it by the mix acid path.Voges Proskauer test shows the production of 2-3-butanediol and acetoine, that are only produced in big amounts in the 2-3-butanediol path. The basis of the test is that these compounds, in alkaline medium and with air, bear an oxidation and become diacethyl, which at the same time reacts with guanidine producing very visible coloured compounds.O’Meara, in 1931, observed that adding creatine to the alkaline solution (O’Meara’s Reagent, Ref. 06-006) aids diacethyl reaction with guanidine, and then it was easier to detect the red coloured compounds.Later, in 1936, Barrit demonstrated that the addition of an alcoholic solution of alpha-naphtol 5% (Barrit’s Reagent, Ref. 06-027) increased very much sensibility, and it was possible to obtain positive reaction even when the final concentration of diacethyl was very low. It is important to add the Barrit’s Reagent before the alkaline solution.

TechniqueMicroorganism to be assayed is inoculated in MRVP Broth (Ref. 02-207) and is incubated at 30°C for a period between 3 and 5 days maximum.Just before read, add Barrit’s Reagent (Ref. 06-027) until all the medium gets a milky look. Following, add O’Meara’s Reagent (Ref. 06-006) until the milky look disappears and then shake chiefly. Relative volumes of each reagent depend on the initial volume of inoculated medium.When test is positive a violet pinked colour appears be-fore 5 minutes, starting from top. When test is negative there is no change ol colour.There is a quicker way to perform the Voges-Proskauer test, with very little volumes of medium and massive inocules. This way allows very short incubations (18-20 hours) and the read may be accelerated by heating up the culture almost to boiling after adding the reagents. However, this method increases the possibility of getting wrong results.

ReferencesBARRY, A.L., K.L. FEENEY (1967) Two Quick Methods for the Voges Proskauer Test. Appl. Microbiol. 15:1138-1141.BARRIT, M.M. (1936) The intensification of The Voges Proskauer Reaction by the Addition of alpha-Naphtol. J. Pathol. Bacteriol. 42:441-453.BLAZEVIC, D.J. and EDERER, G.M. (1975) Principles of Biochemical Tests in Diagnostics Microbiology. John Wiley Sons. N.Y.O’MEARA, R.A.Q. (1931) A simple, Delicated and Rapid Method of Detecting the Formation of Acetylmethyl-car-binol by Bacteria Fermenting Carbohydrate. J. Pathol. Bacteriol. 34:401-406.McFADDIN, J.E. (2000) Biochemical tests for identifica-tion of medical bacteria. 3rd. Ed. Cippincott William & Wilkins. Philadelphia.

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234

Crystal Violet Dye Solution for Gram Staining

Ref. 06-029

Presentation100 mL dropper flask1 L dropper flask

DescriptionThis solution has been prepared according to the speci-fications by Hucker for the Gram staining, and it is very stable though, when it is too old, it may require filtration immediately before the use. Elderliness does not affect the staining properties but may force to make decoulor-ing times longer.

TechniqueFix the smear following the habitual method and let it cool or dry.Cover the extension with Crystal Violet Dye Solution (Ref. 06-029) and let it act for 1 minuteWash the exceed of colouriser. The best way is to put the preparation in a precipitate glass with fluent water. Do not wash excessively. This step may be critical for the rest of the test.

Cover the preparation with Lugol Solution (Ref. 06-030) and drain immediately. Cover again with new solution and let it act for 1 minute.Wash softly again with water. To put the preparation in water fluent for 5 seconds will be enough.

Decolourate, pouring the Gram Decoluriser (Ref. 06-031), drop to drop, over the slanted microscopical slide until total decolourising. Anycase, this step may not be longer than 60 seconds.Wash with water to stop the decolouring action.

Cover the preparation with Safranine Dye Solution (Ref. 06-032) and let it act for 1 minute.Wash gently to remove the excess of colouriser, putting the preparation in fluent water for 1-2 seconds.

Dry and observe under microscope in homogeneous inmersion.

Microorganisms that get coloured by the first colouriser, Crystal Violet, become dark blue coloured and it is said that they take the gram, and they are call grampositive (G+). Those microorganisms that just get coloured by the contrast colouriser become red and they are called gramnegative (G-).

Most of eukariote cells, except yeasts, are coloured as gramnegative and thus the staining is not very significa-tive. In spite of, it is one of the first levels in the system-atic identification of prokariote: between the bacteria, all the coci, except Neisseria and Veillonella, are gramposi-tive, and all the sporogen bacilli and some part of the other bacilli are grampositive too. Spiriles, vibria, rikett-sia, clamidia and most bacilli are gramnegative.

Should actinomycete presence is suspected or microor-ganisms are not well coloured as grampositives, it is ad-visable to use Crystal Violet with Anilin for Actinomycete.

ReferencesBARTHOLOMEW, J.W. (1962) Variables Influencing Re-sults, and the Precise Definition of Steps in Gram Stain-ing as a Means of Standardizing the Results Obtained. Stain Technol. 37:139-155.PAIK, G. (1980) Reagents, Stain and Miscellaneous Procedures, in Manual of Clinical Microbiolgy by Lenette, Balows, Hausler and Truant (eds.). ASM, Washington.CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed. William & Wilkins. Baltimore.

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235

Decolouriser for Gram Staining

Ref. 06-031


DescriptionGram’s decolouriser is a mixture of alcohol and acetone especially adapted to act softly and quickly over base colourings. It use to be enough 15 or 20 drops to achive an total decolourising of a correctly coloured smear.

TechniqueFix the smear following the habitual method and let it cool.Cover the extension with Crystal Violet Dye Solution (Ref. 06-029) and let it act for 1 minuteWash the exceed of colouriser. The best way is to put the preparation in a precipitate glass with fluent water. Do not wash excessively. This step may be critical for the rest of the test.








ReferencesBARTHOLOMEW, J.W. (1962) Variables Influencing Re-sults, and the Precise Definition of Steps in Gram Stain-ing as a Means of Standardizing the Results Obtained. Stain Technol. 37:139-155.PAIK, G. (1980) Reagents, Stain and Miscellaneous Procedures, in Manual of Clinical Microbiolgy by Lenette, Balows, Hausler and Truant (eds.). ASM, Washington.CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed. William & Wilkins. Baltimore.

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236

Ref. 06-018

Presentation100 mL flask1L in flask

DescriptionMany microorganisms can produce indole (=benzopyr-role) from Tryptophane thanks to a Tryptophanase, in a process favored by oxygen and inhibited by glucose. We therefore recommend that media used in this test contain no glucose, present a high Tryptophane content and are incubated aerobically.The indole production property constitutes a classical test for the differentiation of Escherichia and Entero-bacter, integrated in the IMViC. It is also widely used for the differentiation of other non enteric microorganisms.The indole test can be conducted by various means, still the biochemical basis of the reaction remain the same. When a Pyrrol is mixed with a heated alcoholic p-dimeth-ylaminobenzaldehyde solution, a peculiar cherry-red coloring develops (Rosindole). If the reagent’s solution is prepared with concentrated hydrochloric acid it will not be necessary for it to progress in hot, as is the case with our reagents.The reagent which was initially used was that of Ehrlich-Böhme (Ref. 06-024) with a previous extraction and con-centration in Xylene, Ether, Chloroform or Toluol. Later on Kovacs modified the original reagent by replacing the ethanol with amyl-alcohol, so that a previous extrac-tion was no longer necessary. In 1956 Gadenbusch and Gabriel proved that the Kovacs’s reagent was much stable if instead of Amyl alcohol, Butilic or Isoamyl were used. Nevertheless, the indole test reaction with paradiami-nobenzaldehyde is not very specific since at least 17 compounds close to Indole are known to react similarly. Although other reagents such as Oxalic acid and Hy-droxilamine HCI have been proposed, their use has not been widespread.

Kovac’s Reagent

Since indole is one of the most volatile compounds of the group, some authors choose to conduct the test using strips impregnated in reagent. Others suggest placing the reagent directly on the cap’s swabs, avoiding all contact with the culture medium so that the reaction will occur with just the indole’s steam. Isenberg and Suddenheim demonstrated that if a previous extraction with Toluene was performed, only indole and Alpha-Methylindole (=indoleacetic acid) were detected. Such is the most common practice nowadays when using the Ehrlich-Böhme reagent.

TechniqueWhen conducting the indole production test on vari-ous groups of bacteria, an appropriate reagent for each group must be considered. Kovacs’s reagent (Ref. 06-018) is recommended for enterobacteria while the Ehrlich reagent is for non-fermentating and anaerobes in general.The directions to follow during the assay are:Inoculate the pure culture to be verified in a high Tryp-tophane content medium, as for example the Indole-Ni-trite Fluid Medium (Ref. 03-101), the SIM Medium (Ref. 03-176) or a non-glucose tryptone broth. Incubate at 35°C for 48 hours. Incubation time can be reduced to 4 hours if a massive inoculum in solid medium is done fol-lowed by seeding of a small volume (0,5 mL) of culture medium.

In both cases, examination after incubation in the follow-ing way:a) Kovacs’s Reagent (Ref. 06-018, for enterobacteria)Add 0,5 mL of reagent to the broth’s surface, shaking lightly to help extraction. If a cherry-red color develops in less than a minute it will be considered a POSITIVE RE-ACTION. No change in the original coloring constitutes a negative reaction.

b) Ehrlich-Böhme TestAdd 1 mL of Xylene or Toluene to the broth and shake energically to help extraction. Allow to stand for 2 min-utes until both layers separate. Then slide 0.5 mL of the reagent carefully down the sides of the tube, making sure there is no agitation. Should a dark red colored ring appear in the interface, it will be considered a POSITIVE REACTION.

StorageReagents must be stored refrigerated and avoiding direct light.

ReferencesBÖHME, A. (1905) Die Anwendung der Ehrlichschen In-dolreaktion für bakteriologische zwecke. Zentralbl. Bakt. Parasit. Abt 1, Jena 40:129-133

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237

Lactophenol Blue

Ref. 06-037

Presentation100 mL flask1 L flask

SpecificationReagent for staining of fungi in fresh and fixed prepara-tions.

DescriptionLactophenol Blue is an excellent colouriser for fresh preparation of fungi, since it has, in a single solution, the propierties of a mordant, a fixer and a colouriser.In fungi preparations for microscopical examination is not usual to use water neither aquose colouriser solu-tions, since most of the moulds expel water and remain trapped in air microbubbles. This reason makes Lac-tophenol Blue an idoneous medium for the examination, becase it moistens the structures at the same time that it acts like a fixer and soft mordant. In other hand, its nature makes the preparations useful longer because evaporation is smaller. This effect can be enhanced if the preparations are sealed with vaspar or nails varnish.The Cotton Blue, China Blue or Soluble Anilin Blue is probably, an impurified and complex colouriser but, its

efficacy in the staining of moulds and plants material has been demonstrated.

TechniquePut a bit of mould to be assayed in a clean microscopical slide and pour one or two drops of Lactophenol Blue.Disperse the material with two needles, mixing it with the colouriser.Add a couple of drops of water and homogenize all before putting the overglass.Heat slightly the preparation over a flave until it will be almost boiling. In that precise moment, press to remove all the excess of liquid and seal the borders with vaspar or nails varnish.Preparation is ready for the microscopical examination.

ReferencesHARRIGAN, W.F. and McCANCE, M.E. (1976) Labora-tory Methods in Food and Dairy Microbiology. Academic Press. London.CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed. William and Wilkins. BaltimoreLARONE, D.H. (2002) Medically important fungi. ASM Press. Washington, DC.

KOVACS, N. (1928) Eine vereinfachte Methode zum Nachweis der Indolbihdung duch bakterien. Z. Immuni-tats. Forsch. Exp. Ther. 55:311-315GADEBUSCH, H.H. and GABRIELS, S. (1956) Modified Stable Kovacs’s Reagent for the detection of Indol. Am. J. Clin. Pathol. 26:1373-1375ISENGERG, H.D. and SUNDHEIM, L.H. (1958) «Indole Reactions in Bacteria» J. Bact. 75:682-690CENTER FOR DISEASE CONTROL (1968)»Identifica-tion of Unusual Pathogenic Bacteria» Atlanta G.VIRGINIA POLYTECHNICAL INSTITUTE (1972) Anaer-obe Laboratory Manual Blaksburg,Va.EDWARDS, P.R. and EWING, W.H. (1972) «Identifica-tion of Enterobacteriaceae» 3rd Ed. Burgess Pub. Co. MinneapolisMcFADDIN, J.F. (2000) Biochemical tests for identifica-tion of medical bacteria. 3rd. Ed. Lippincott William & Wilkins. Philadelphia.ISO 9308-2 Standard (1990) Water Quality - Detection of coliforms thermotolerant coliforms and presumptive E.coli - MPN method.

Kovac’s Reagent

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Left: control; center: Salmonella typhimurium ATCC 14028 (negative reaction); right: Escherichia coli ATCC

25922 (positive reaction).

Kovac’s reaction on: Ref. 02-277 Tryptone Phosphate Water, Ref. 02-460 Tryptose Lauryl Sulfate Mannitol

Tryptophan Broth, Ref. 02-494 Buffered Peptone Water Eur. Phar., Ref. 03-156 Tryptone Water.

238

Lugol Solution for Gram Staining

Ref. 06-030


DescriptionIodine solution has been prepared according to the specifications by Burke, therefore it is more stable than the classical Lugol formulation, and it does not affect the colouring. The solution may be stored for months at room temperature, but if a characteristic amber colour is observed, it must be discarded.









ReferencesBARTHOLOMEW, J.W. (1962) Variables Influencing Re-sults, and the Precise Definition of Steps in Gram Stain-ing as a Means of Standardizing the Results Obtained. Stain Technol. 37:139-155.PAIK, G. (1980) Reagents, Stain and Miscellaneous Procedures, in Manual of Clinical Microbiolgy by Lenette, Balows, Hausler and Truant (eds.). ASM, Washington.CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed. William and Wilkins. Baltimore.

239

Malachite Green

Ref. 06-038


DescriptionMalachite Green for Spores has been prepared accord-ing to the formulation by Barholomew and Mittwer, in 1950, which was a modification over the procedure by Schaeffer and Fulton, in 1933, that was based in the original one by Wirtz.

Essencially, the modification is the supression of the step where the preparation was heated up. To achieve this without affecting the test, they prepared a more concen-trated colouriser and let it act longer, and afterwards they did the contrast with a softer colouriser, Safranine Dye Solution (Ref. 06-032).

This stain is a saturated and stabilized aquose solution of malachite green. To use it with the Schaeffer tech-nique it has to be diluted at 50% to avoid the formation of precipitates.

TechniquePrepare a smear of microorganism, in the habitual way, and let it dry. Fix strongly by passing the microscopical slide over a flame about 20 times.Before doing the staining, let cool the microscopical slide. Cover all the smear with Malachite Green for Spores Stain and let it act for 10 minutes.Wash with water to remove the excess of colouriser.Contrast by covering the smear with Safranine Dye Solu-tion (Ref. 06-032) and letting it act for 15-30 seconds. Wash again, dry and perform the microscopical exami-nation in homogeneous inmersion.Cellular bodies appear red or pinked coloured, whereas spores are green. Should the Schaeffer’s technique is wanted to be used diluting the colouriser, once the smear is covered with Malachite Green, do not let it act for 10 minutes and instead of this, bring it to the boiling 3 consecutive times. Then follow the described technique.

ReferencesBARTHOLOMEW, J.W., MITTWER, T. (1950) A Simpli-fied Bacterial Spore Stain. Stain Technol. 24:153-156.SCHAEFFER, A.B., M. FULTON (1933) A Simplified Method of Staining Endospores. Science, 77, 194.CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed. William and Wilkins. Baltimore.

240

Methyl Red

Ref. 06-007


SpecificationIndicator solution for the fermentation test in enterobac-teria.

DescriptionClark and Lubs, in 1915, described the Methyl Red test to distinguish between the E. coli group and Entero-bacter.All the enteric bacteria ferment dextrose, some do it following the 2-3-butanediol path, like Klebsiella, En-terobacter, etc..., and other follow the mix acid path, like E.coli, Salmonella, etc...In this last case, there is an accumulation of acid products produced by the sugar unfoldment. This causes a decrease of pH, reaching a value up to 4 after the incubation. Adding the methyl red alcoholic solution the change of the indicator may be de-tected: it remains yellow over pH 5,1 and red below pH 4,4. If the change is positive it means that the dextrose fermentation followed the mix acid path, since in the 2-3-butanediol path, after 3 days of incubation, predomi-nate the neutral or alkaline products.Too early reads may produce false positive results.

TechniqueInoculate a tube of MRVP Broth (Ref. 02-207) and incubate at 30°C for 3-5 days. Take 5 mL of culture and transfer them to a clean tube and then add 5 or 6 drops of indicator.Positive reaction is shown by the presence of a red colour, whereas the negative reaction produce a yellow or orange colour.Most cases, a 48 hours incubation at 37°C is enough, but if the results are doubtous, the assay must be re-peated incubating at 30°C for 5 days.There is a quicker way to perform the test: suspend a loop of bacterial growth from a solid medium in 0,5 mL of Azide Dextrose Broth acc. to Rothe (Ref. 02-027) and incubate at 37°C for 18 hours. Add a couple of drops of indicator and read the results as in the last case.

ReferencesBARRY, A.L., K.L. BERNSOHN, A.P. ADAMS, L.D. THRUPP (1970) Improved 18-hour methyl red test. Appl. Microbiol. 20:886-870.CLARK, W.M. and LUBBS, H.A. (1915) The Differentia-tion of Bacteria of the Colon-Aerogenes Family by the use of indicators. J. Infect. Dis. 17:161-173.EDWARDS, P.R., W.H. EWING (1972) Identification of Enterobacteriaceae. 3rd. Ed. Burgess Pub Co. Minne-apolis.BLAZEVIC, D.J. and EDERER, G.M. (1975) Principles of Biochemical Tests in Diagnostics Microbiology. John Wiley Sons. N.Y.McFADDIN, J.F. (2000) Biochemical tests for identifica-tion of medical bacteria. 3rd. Ed. Lippincott William and Wilkins. Philadelphia.

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241

Nitrates Reduction Reagents

Nitrates A Solution

Ref. 06-003


Nitrates B Solution

Ref. 06-004


SpecificationGriess-Ilosvay’s Reagents for the verification of the nitrates reduction through nitrites detection.

DescriptionTo use them, mix equal parts of the solutions A and B. Once they are mixed, the reagents are stable just for a few hours. Alone they may be stored for several months at room temperature. Nitrates B solution may produce a slight cristalization that does not affect its efficacy. This process is accelerated with refrigeration, therefore it is recommended not to store them in the refrigerator.

Nitrates reduction in bacteria is performed through sev-eral ways and it obeys different procedures.

Nitrates Assimilation involves a reduction to ammonia in several steps where nitrite may be detected. The am-monia that is produced is finally incorporated to the cel-lular material. However, in the Deassimilation process, nitrite is used as the final receiver of electrons, and thus, more than an assimilation process it is an energetic re-action of respiration without oxygen, and this fact allows the facultative growth of many aerobic in anaerobiosis. In this case, it is usual the presence of nitrite acumula-tions, which may be toxical for the microorganism. In other cases, nitrate may reduce itself to gas states and it is expeled as free nitrogen bubles. This process is called Denitrification, since it makes the active ion (NO

3) an

inert gas (N2).

The scheme for the global process is the following:

Generally, the Griess-Ilosvay’s reagents detect the presence of nitrites with bacterial origin in a medium that initially has no nitrites (Indole Nitrite Fluid Medium, Ref. 03-101 and Nitrate Broth, Ref. 02-138). The scheme for the complete reaction is the following:

ReferencesWALLACE, G.I., S.L. NEAVE (1927) The nitrite test as applied to bacterial cultures. J.Bact. 14:377-384.BLAZEVIC, D.J., G.M. EDERER (1975) Principles of Biochemical Tests in Diagnostics Microbiology. John Wiley Sons. NY.FORBES, B.A., D.F. SAMM, A.S. WEISSFELD (1998) Bailey & Scott’s Diagnostics Microbiology. 10th. Ed. Mosby. St. Louis.GRIESS, P. (1879) Liebereinige Azoverbindungen. Ber. Deutsch. Chem. Geselkch. 12:426-427.McFADDIN, J.F. (2000) Biochemical Tests for identifica-tion of medical bacteria. 3rd. Ed. Lippincott William and Wilkins. Philadelphia.

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242

O’Meara’s Reagent

Ref. 06-006


SpecificationReagent for the Voges-Proskauer test in enterobacte-riaceae.

DescriptionAll the enterobacteria ferment dextrose, but some spe-cies like Klebsiella, Enterobacter, etc..., do it following the 2-3-butanediol path and other species like E.coli, Salmonella..., do it by the mix acid path.Voges Proskauer test shows the production of 2-3-butanediol and acetoine, that are only produced in big amounts in the 2-3-butanediol path. The basis of the test is that these compounds, in alkaline medium and with air, bear an oxidation and become diacethyl, which at the same time reacts with guanidine producing very visible coloured compounds.O’Meara, in 1931, observed that adding creatine to the alkaline solution (O’Meara’s Reagent, Ref. 06-006) aids diacethyl reaction with guanidine, and then it was easier to detect the red coloured compounds.Later, in 1936, Barrit demonstrated that the addition of an alcoholic solution of alpha-naphtol 5% (Barrit’s Reagent, Ref. 06-027) increased very much sensibility, and it was possible to obtain positive reaction even when the final concentration of diacethyl was very low. It is important to add the Barrit’s Reagent before the alkaline solution.

TechniqueMicroorganism to be assayed is inoculated in MRVP Broth (Ref. 02-207) and is incubated at 30°C for a period between 3 and 5 days maximum.Just before read, add Barrit’s Reagent (Ref. 06-027) until all the medium gets a milky look. Following, add O’Meara’s Reagent (Ref. 06-006) until the milky look disappears and then shake chiefly. Relative volumes of each reagent depend on the initial volume of inoculated medium.When test is positive a violet pinked colour appears be-fore 5 minutes, starting from top. When test is negative there is no change ol colour.There is a quicker way to perform the Voges-Proskauer test, with very little volumes of medium and massive inocules. This way allows very short incubations (18-20 hours) and the read may be accelerated by heating up the culture almost to boiling after adding the reagents. However, this method increases the possibility of getting wrong results.

ReferencesBARRY, A.L., K.L. FEENEY (1967) Two Quick Methods for the Voges Proskauer Test. Appl. Microbiol. 15:1138-1141.BARRIT, M.M. (1936) The intensification of The Voges Proskauer Reaction by the Addition of alpha-Naphtol. J. Path. Bact. 42:441-453.BLAZEVIC, D.J. and EDERER, G.M. (1975) Principles of Biochemical Tests in Diagnostics Microbiology. John Wiley Sons. N.Y.O’MEARA, R.A.Q. (1931) A simple, Delicated and Rapid Method of Detecting the Formation of Acetylmethyl-car-binol by Bacteria Fermenting Carbohydrate. J. Pathol. Bact. 34:401-406.McFADDIN, J.F. (2000) Biochemical tests for identifica-tion of medical bacteria. 3rd. Ed. Lippincott William and Wilkins. Philadelphia.

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243

Oxidase Reagent

Ref. 06-057

Presentation5 g flask

SpecificationReagent for the detection and verification of bacterial citochrome-oxidase.

DescriptionThe chemical formulation for the Oxidase Reagent is N,N-dimethyl-P-phenyldiamine-2-HCl, and it is also designated as 4-amino-N,N-dimethylaniline-2-HCl. It is advisable to store it in powder at 4°C since it has a very short life time when is dissolved.

DirectionsPrepare an aquose solution of Oxidase Reagent 1% im-mediately before the use. It is recommended to prepare just the amount that is going to be used, since once diluted it will work just for a week, even if it is kept at 4°C and avoiding direct light. Althought self oxidation is restrained with the addition of ascorbic acid 0,01%, if the liquid is dark it must not be used. The normal colour for the solution is transparent or slightly pinked.

TechniqueThere are several techniques to determinate citrocrome-oxidase in the different genus. The more standardized are the following:a) Soak a filtration paper disc with the reagent and put it

over a clean Petri plate. Take a colony and spread it over the paper. This step must be performed with a Platinum-Iridium loop (Ref. 5-006) or a Pasteur pi-pette. The use of metallic objects (nicrom loop, etc...) may produce wrong positive results.

b) Flood the colony with reagent directly in the plate. Fol-lowing this way, colonies are not able to subculture, but the test does not interfere with the Gram staining and the colony may be observed at the microscope.

Positive reaction is shown by the presence of a pink colouring, that becomes dark red and finally black after 10 minutes.

The basis of the test is the following:Citochromes are hemoproteins that act as oxidant enz-imes in the transportant chains of oxidative phosphorila-tion electrons. Generally, citochromes are found just in the aerobic bacteria, whereas the strict anaerobic does not present them.Citochrome-oxidase found even in the enterobacte-riaceae is composed by a

3 citochrome. However, the

substance that the Oxidase Reagent is able to reduce is c citochrome, thus the test just determinates the pres-ence of c citochrome and therefore test is positive only for the bacteria that have c citochrome in their respira-tory chains.

Reagent is very unstable to oxygen, and light acts as a catalyst in the self oxidation and so the reagent may be stored in amber flask. When the reaction in the colony is very slow, it has to be considered negative or very weak, since the lately appaerance of colour is more attributable to the spontaneous oxidation than to the truly presence of c citochrome.Generally, except a few cases, oxidase production is linked to flagelation in the following way: Polar flagela-tion is related with oxidase positive; peritrichous flagela-tion is related with oxidase negative.The oxidation of c citochrome for the positive reaction of oxidase is the following and it may be observed that molecullar oxygen is absolutely necessary:

ReferencesGABY, W.L., C. MARTLEY (1957) Practical laboratory test for the identification of Pseudomonas aeruginosa. J. Bact. 74:356-358.BLAZEVIC, D.J., G.M. EDERER (1975) Principles of biochemical test in diagnostic Microbiology. John Wiley Sons. NY.FORBES, B.A., D.F. SAHM, A.S. WEISSFELD (1998) Bailey & Scott’s Diagnostic Microbiology. 10th. Ed. Mosby. St. Louis.ISO 9308-2 Standard (1990) Water Quality - Detection of coliforms, thermotolerant coliforms and presumptive E.coli - MPN method.

244

Safranin Dye Solution for Gram Staining

Ref. 06-032


DescriptionThe contrast colouriser is composed by the classic Safranine solution 0,25%. It is demonstrated that this solution is more effective than the fuchsine. This con-trast colouriser is employed also in many other staining methods.









ReferencesBARTHOLOMEW, J.W. (1962) Variables Influencing Re-sults, and the Precise Definition of Steps in Gram Stain-ing as a Means of Standardizing the Results Obtained. Stain Technol. 37:139-155.PAIK, G. (1980) Reagents, Stain and Miscellaneous Procedures, in Manual of Clinical Microbiolgy by Lenette, Balows, Hausler and Truant (eds.). ASM, Washington.CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed. William and Wilkins. Baltimore.

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