Download Training Manual for Food Testing, Microbiology and Physical Chemistry Course UPDATED 33

7/27/2019 Download Training Manual for Food Testing, Microbiology and Physical Chemistry Course UPDATED 33

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Dr.Ali Mohamed Ali Iye ( Ali kanu)

Food Safety Consultants

P. O. Box 7698-00100

Nairobi KENYA

Tel, 0722809835, 0710398280

E. mail: [email protected]

TRAINING MANUAL OF COURSE ON FOOD MICROBIOLOGY AND PHYSICAL

CHEMISTRY TECHNIQUES HELD ON JANUARY 2013

VENUE: IGAAD SHEIKH TECHNICAL VETERINARY SCHOOL

Prepared and conducted by:

Mr. Joseph Kimari and Mr. Duncan Ndegwa of food safety consultants

Mr. Joseph Kimari and Mr. Duncan Ndegwa1


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Table of Contents

1.0 GOOD LABORATORY PRACTICE.........................................................................4

2.0 LABORATORY SAFETY......................................................................................4

3.0 PERSONAL HYGIENE........................................................................................44.0 LABORATORY DOS AND DONTS..................................................................5

5.0 BACTERIOLOGY..............................................................................................6

5.1 GENERALIDENTIFICATIONOFBACTERIA.........................................................65.2 IDENTIFICATIONOFBACTERIA....................................................................7

5.2.1 Gram Staining .............................................................................75.3 MEDIAUSEDINBACTERIOLOGY...................................................................8

5.4 MEDIAPREPARATIONPROCEDURE ...............................................................9

5.5 SAMPLEPREPARATIONANDDILUTIONS.........................................................105.5.1 Procedure for sample preparation and dilution.................................11

5.6 TOTALVIABLECOUNTS (TVC)..................................................................13SEEPROCEDUREBELOW...............................................................................14

PROCEDUREFOR PERFORMING TOTAL VIABLE COUNTSINMILK, DAIRYPRODUCTS, FOODSANDSWABSBYTHE POUR PLATE METHOD.........................................................14

5.7 COLIFORMCOUNTS ( PLATEMETHOD )......................................................17

PROCEDUREFORTHE ENUMERATIONOF COLIFORM ORGANISMSBYTHE POUR PLATEMETHOD.................................................................................................18

5.8 YEASTAND MOULDSCOUNTS...................................................................225. 9 MOST PROBABLE NUMBERTESTING ( MPN) INWATER....................................25

5-10. DETECTIONOF SALMONELLASP..............................................................28

PROCEDUREFORTHE DETECTIONOF SALMONELLASP. IN ALL FOODS, MILK, AND DAIRYPRODUCTS & ENVIRONMENTALSWABS..............................................................29

..........................................................................................................295.11 ENUMERATIONOF STAPHYLOCOCCUSAUREUS...............................................33

PROCEDUREFORTHEENUMERATIONOF STAPHYLOCOCCUSAUREUS............................34

5.12 ACIDITY TEST - TITRATABLE ACIDITYOF MILK.............................................385.13 BUTTERFAT FATCONTENT GERBER METHOD ...............................................40

5.14 RESAZURIN TEST................................................................................425.15 ALCOHOL TEST .................................................................................45

5.16 MOISTURE ANALYSISIN FOODS...............................................................46

Procedure for the Determination of Moisture content by Ohaus MB45 Thermo gravimetricMethod .............................................................................................................47

5.17 PHOSPHATASE TESTFOR PASTEURIZED MILK...............................................48



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5.18 ANTIBIOTIC RESIDUEINMILKAND MEAT/LIVER...........................................50

5.18.CHLORINE CONTENTIN WATER...............................................................53

5.19 0BRIX.............................................................................................545.20 PROCEDUREFOR DETERMINATIONOF HYDROGEN PEROXIDEINMILK...................55

ANNEX 2 MAKINGSTERILEBLOODAGARPLATES.................................................60ANNEX 3 MAKING MCCONKEYAGAR...............................................................62

ANNEX 4 PERFORMINGTHE CATALASETEST......................................................63

ANNEX 5 DETECTING INDOLEPRODUCTION.......................................................64ANNEX 6 IDENTIFYING OXIDASEPOSITIVEORGANISMS.........................................65

ANNEX 7 MAKINGSTERILE UREAAGAR............................................................66ANNEX 9 PROCEDUREFORMAKING TRIPPLE SUGAR IRON......................................69

ANNEX 10 PROCEDUREFORMAKING STERILEBAIRD PARKERMEDIUM....................71



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1.0 GOOD LABORATORY PRACTICE

Good Laboratory Practices are generally accepted methods to perform activities or

operations in the laboratory. These practices are known or believed to be safe and

protects the workers and have a positive influence on the quality of the result. GoodLaboratory Practice (GLP) embodies a set of principles that provides a framework

within which laboratory work is planned, performed, monitored, recorded, reported

and archived.

The approach is meant to cover good laboratory practice as far as safety, personal

hygiene and good working practices are concerned.

2.0 LABORATORY SAFETY

The most important factor in the prevention of laboratory acquired

infections/accidents/incidences and maintenance of laboratory standards is good

laboratory practice. Safety in the laboratory is of paramount importance and all

employees have a duty to take reasonable care for the health and safety of

themselves and all other persons who may be affected by their acts or omissions at

work. The most efficient means of achieving this is to spend some time identifying all

safety hazards in a particular laboratory, assess them and the means of avoidance or

control determined. This together with good laboratory practice (GLP) will ensure a

safe working environment and improve standards of activities in the laboratory.

3.0 PERSONAL HYGIENE

Apart from good laboratory practice individual workers can contribute to the

prevention of self-infection and the infection of others by personnel hygiene. To

prevent infection in the laboratory, personnel have to identify the routes of infection,

most hazardous organisms that requires use of biosafety cabinet, different routes of

infections, which techniques are dangerous and how a worker can protect himself.

Below, is a list of laboratory dos and donts;

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4.0 LABORATORY DOS AND DONTS

1. Always wash your hands before starting work, after working and always wear a

laboratory coat while working in the laboratory.

2. Always wear gloves for procedures necessitating direct contact with infectious

materials.

3. Mouth pipetting is prohibited.

4. Always swab the workbench with 70% alcohol or any other suitable disinfectant

after each working session.

5. Always disinfect and wash your hands after handling infectious materials, before

eating or drinking during breaks in the day, when using the telephone and when

you leave the laboratory.

6. All spills, accidents and potential exposures to infectious materials must be

reported to a senior member of staff and entered in the ACCIDENT BOOK. Always

keep the laboratory neat, clean and free from materials not related to your work.

7. Eating, drinking, smoking, storing food, chewing pencils, biting nails or applying

cosmetics are not permitted in the laboratory working area.

8. All visitors must be signed in and wear laboratory coats when they enter the

laboratories.

9. Persons who are at increased risk of acquiring infection, e.g. children are not

allowed to the laboratory.



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10. Cuts, scratches, sores and other lesions on the hands and exposed parts of the

body should be covered with adhesive plasters.

5.0 BACTERIOLOGY

5.1 General identification of bacteria

The most common bacteria in human, veterinary and food microbiology can be classified i

three distinctive shapes;

a) Cocci ( spherical )

b) Bacilli ( rod shaped )

c) Vibrio ( comma shaped )

The cocci can be further divided into;

a) Diplococci

b) Streptococci ( chains )

c) Staphylococci ( clusters )



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5.2 Identification of bacteria

The first step in the identification of bacteria is the performance of the Gram stain.

5.2.1 Gram Staining

The purpose of this procedure is to differentiate bacteria into Gram negative and Gram

positive organisms. The Grams reaction is due to differences in their cell wall structure.

The reporting should include the following information: The Gram reaction of the

bacteria, whether Gram positive or Gram negative. Morphology of the bacteria, whether

cocci, diplococci, rods, coccobacilli or yeast cells.

Note:Cocci are round or oval bacteria measuring about 0.5 to 1 m in diameter.

Rods are stick like bacteria with rounded tapered, square or swollen ends. They

measure 1 to 10m in length by 0.3 to 1 m in width. The short rods with

rounded ends are often called coccobacillus. Rods and cocci are sometimes found

in chains, and this should be mentioned when describing the bacterial

morphology.

Discussion to include the principle of the test, interpretations and presentation of results.

Procedure:

3.1 Make a smear on the slide by emulsifying a sample of the colony in normal saline,

purulent specimens spread the material thinly using a wire loop and for a swab roll

swab on the slide.

3.2 Allow the smear to air dry.

3.3 Heat fix.

3.4 Cover with crystal violet for one minute and wash in tap water.

3.5 Cover with Lugols iodine for one minute and wash.

3.6 Decolourize rapidly with acetone and wash immediately.

3.7 Counter stain with Safranin for two minutes and wash.

3.8 Wipe the back of the slide, and place in a draining rack to air dry.



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3.9 Examine using oil immersion.

Observations:

Gram positive bacteria..... Dark purple

Yeast cells....... Dark purple

Gram negative bacteria......Pale to Dark red

After establishing the gram reaction of an organism further characterization is done

using biochemical tests. The biochemical tests mostly applied in a Food Microbiology

laboratory are as follows;

Catalase

Coagulase

Indole production

Oxidase

Dnase Testing for urea

Tripple sugar iron

API profiles etc

5.3 Media used in bacteriology

The main types of culture media are:

a) Basic e.g. Nutrient agar

b) Enriched or enrichment eg Blood agar

c) Selective e.g. XLD

d) Differential e.g.MaConkey agar

e) Transport e.g. Stuarts Transport media

The different types of media and their application will be discussed in detail.

A wide variety of media are available for cultivation of pathogenic bacteria and fungi. M

are available commercially in the dehydrated form. The formulations and directions for

preparation of the commercially available dehydrated media are normally given as

manufacturers instructions



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5.4 Media preparation procedure

The purpose of this procedure is to ensure that media production is standardized,

organized and recorded to ensure that media components are available when

required and that the media used in analyses is suitable for use, traceable back toproduction, components, methods used and that production operators are identifiable

if necessary.

Production of sterile media is important for proper testing of various types of

samples. Failure to produce media as per the laid down work instructions would lead

to production of media which may not sustain the growth of the target organisms.

Great care is therefore necessary in this area.

The water used in the preparation of culture media should be distilled or de-ionized.

The water should have a conductivity of < 15 micro-Siemens and a pH of not lessthan 5.5 and not more than 7.7. If the de-ionization process is inadequate, residue

acid may cause the final pH of the medium to drop.

Media should be tempered in water bath @ 45 oC. The temperature of the water bath

should be continuously monitored with a thermometer.

Note:

A. If pathogens are to be isolated successfully, culture media must be prepared carefu

Each of the following steps must be performed correctly;o Weighing and dissolving

o Addition of heat sensitive ingredients

o Dispensing

o Sterilization and sterility testing

o pH testing

o Quality control

o Storage

B: Record the day of opening a new bottle of media



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Dehydrated media is hygroscopic, i.e., it absorbs water. Dehydrated media should

weighed rapidly and tops replaced immediately and tightly

Use completely clean glassware

Use distilled water or deionized water

If possible check the Electrical conductivity of the distilled water When heating is required to dissolve the medium, stir while heating and control the h

to prevent boiling and foaming which may damage the medium. Overheating a med

can alter its nutritional and gelling properties, and its pH.

Practicals

Preparation of severalbatches of standard plate count agar; Buffered Peptone Water,

Violet Red Bile Agar, MacConkey Agar, XLD and BGA weighing, autoclaving, tempering,

pouring and storage.

Several batches of media were prepared and training on the all above aspects applied.

addition the following was covered;

a) Autoclaving

b) Media tempering

c) Media pouring

d) Quality control; to assess the performance of the media

The following procedures in microbiology and physical chemistry testing in foods were

discussed and practicals done;

5.5 Sample preparation and dilutions

The procedure describes how to prepare a 10-1 homogenate of food samples in a suitable

diluent for enumeration purposes and preparation of further dilutions for enumeration in

samples likely to contain high numbers of organisms.

For homogenous samples including powders and free flowing liquids and concentrate mix

well before removing a portion for testing. Do not shake powders immediately before

testing as the environment may become contaminated by dust particles. For

heterogeneous samples such as sandwiches it is usually appropriate to remove a

representative portion of the whole product so that all components are taken.



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Using sterile instruments and aseptic technique, weigh a representative 25g sample of

each food into either a sterile bottle. Aseptically add 225 ml of sterile buffered water or

the appropriate diluent according to the method.

See procedure below.

5.5.1 Procedure for sample preparation and dilution

1. Purpose

The procedure describes how to prepare a 10-1 homogenate of sample in a

suitable diluent for enumeration purposes and preparation of further dilutions for

enumeration in samples likely to contain high numbers of organisms

2. Scope

This procedure is applicable to the microbiological examination of food samples

3. ResponsibilityThe technician in-charge of the Bacteriology laboratory is responsible for the

implementation of the procedure.

4. Requirements:

Weighing balance 0.01

Sterile spoons

Mechanical blender

Bunsen burner

Vortex Mixer

Appropriate diluents

1000 l automatic pipetteSterile 1 ml pipette tips

5. Reference Documents:

Preparation of samples and dilutions 03/05/2005 Reference no: F 2 Health

Protection Agency, Standard Units, UK

6. Procedure

6.1 Sample preparation

For homogenous samples including powders and free flowing liquids

and concentrate mix well before removing a portion for testing. Do

not shake powders immediately before testing as the environment



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may become contaminated by dust particles. For heterogeneous

samples such as sandwiches/samosas it is usually appropriate to

remove a representative portion of the whole product so that all

components are taken.

6.2 Preparation of homogenate

Using sterile instruments and aseptic technique, weigh a

representative 25g sample of each food into either a sterile bottle.

Record the weight. For samples such as meat aseptically blend

samples using a Mechanical blender. Blend at 10,000 12, 000 rpm

for 2 minutes.

6.4 Add exactly nine times the weight or volume of Buffered Peptone

Water at ambient temperature to give a 1 in 10 (10 -1) suspension.

Record the weight or volume used. If the amount of food available is

less than 25g maintain the sample: diluent volume ratio at 1:9 (1 in

10dilution).

6.5 Using a mechanical blender to homogenize the suspension (See 6.2

above)

6.6 The time lapse between preparation of the homogenate and

inoculation of the counting media should not exceed 45 minutes.

6.7 Buffered peptone water (BPW) is used for the preparation of the

homogenate when a single 10-1 homogenate is made for both

detection ofSalmonella and enumeration of other organisms. In this

instance prepare the homogenate by weighing at least 27g of

sample, add an appropriate volume of BPW, remove 20mL for

enumeration and use the remainder of the homogenate for detection

ofSalmonella.

6.8 Preparation of dilutions. Use Buffered peptone water (BPW) at

ambient temperature for all dilutions.



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6.9 To prepare decimal dilutions transfer 1.0ml of the 10-1 homogenate

to 9.0mL of BPW avoiding contact between the pipette/pipette tips,

the diluent and the inside wall of the container. Mix carefully using a

vortex mixer for 5-10 seconds. This constitutes the 10-2 dilution.

6.11 Using a fresh pipette/pipette tip for each dilution repeat this

procedure to produce further decimal dilutions.

5.6 Total viable counts (TVC)

This procedure ensures that the number of colony forming units (cfu) per

millilitre or per gram of an original sample is determined. A defined test

portion or series of decimal dilutions of the sample are mixed with culture

media in Petri dishes and incubated. The number of cfu per millilitre or per

gram of the original sample is calculated from the number of colonies counted

on selected dishes. The calculation is carried out using a formula described inthe procedure for TVC. The test is carried out on raw milk, milk products, food

(eg meat) and animal feeds.

The TVC gives you the levels of contamination of the food and hence the

quality of the food. The media for performing this test must be cooled down to

45oC. Higher temperatures of the media will kill the bacteria. TVCs of products

are useful for indicating the overall microbiological quality of products and

potential spoilage in perishable products. National bodies have specifications

on various products and to establish whether a product passes TVC have to be

carried out.

In products where the bacterial load is expected to be high, decimalserialdilutions needs to done to determine the breakpoint. Plate with counts

between 10 and 300 will be used for calculation. Plates with >300 colonies will

considered as uncountable.

After counting the colonies on the Petri dishes, the calculation of the final

count is as follows;

N = C/ [V (n1 + 0.1n2) d]

Where = sum of all colonies on all Petri dishes counted

n1 = number of dishes in the first dilution counted



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n2 = number of dishes in the second dilution counted

d =dilution for which the first counts were obtained

V = volume applied to each dish

Example

Volume applied 1 ml

Dilution 1/100 (10-2) 278 and 290 colonies


N=(278+ 290 + 33 + 28)/1 x (2+[0.1 x 2]) x0.01]

= 629/0.022

= 28590 or 2.9 x 104 cfu/ml


Procedure for Performing Total Viable Counts in milk, dairy products, foods and swabs

by the Pour Plate Method.

1.Purpose

This procedure ensures that the number of colony forming units (cfu) per

milliliter or per gram of an original sample is determined. A defined test

portion or series of decimal dilutions of the sample are mixed with culture

media in Petri dishes and incubated. The number of cfu per milliliter or per

gram of the original sample is calculated from the number of colonies counted

on selected dishes.

2 .Scope

This covers the use of the test for milk, dairy products and swabs in the

Bacteriology Laboratories.

3. Responsibility

The Laboratory Head is responsible for the correct implementation of the

procedure



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4. Requirements

Weighing balance (0.01g)

Spatula

Autoclave

Autoclave tape

Bunsen burner

Vortex Mixer

Incubator @ 30oC 1oC

Water bath @ 45oC 1oC

Conical flask (250 mls, 500mls or 1000mls)

1000 l automatic pipette

Sterile 1ml pipette tips

9ml sterile quarter strength Ringers solution or Buffered Peptone Water in

Universal bottle. Autoclaved @ 121oC for 15 minutes. This is used as the

diluent.

Sterile Petri dishes

Sterile Standard Plate Count Agar in a sterile conical flask (CM0 463).

5. Reference Documents:

Plate count test at 30oC 03/05/2005 Reference no: D2 Health Protection

Agency, Standard Units, UK

6. Procedure:

6.1 Prepare the sample as described in the Procedure for preparation of

samples and dilution. The interval between mixing and pipetting should notexceed 3 minutes.

6.2 Transfer 1ml of the sample aseptically into 9ml of sterile diluent in a

Universal bottle and mix thoroughly.

This is the Primary Dilution.

6.3 Transfer 1ml from the Primary dilution (6.2 above) aseptically using a fresh

sterile pipette tip to a further 9ml of diluent and mix thoroughly. Further

dilutions are prepared by transferring 1ml of each successive dilution into a

further 9 ml. of diluent using a fresh sterile pipette tip in each case.

6.4 Transfer 1ml of each chosen dilution using a sterile pipette tip into labeled

sterile Petri dishes starting with the most dilute of the dilutions prepared.6.5 Add 15 18 ml of the tempered melted medium aseptically to each

inoculated Petri dish.



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6.6 Mix the contents of the Petri dish immediately after pouring by 5 to and fro

movements of the dish followed by 5 circular clockwise movements

followed by 5 to and fro movements at right angles to the first set, followed

by 5 circular anti-clockwise movements.

6.7 Allow the Petri dishes to stand on a clean horizontal surface until the

medium sets, invert and transfer to the incubator.

6.8 Incubate the Petri dishes at 30oC 1oC for 72 3 hours

For each batch of agar, pour one Control Plate per every1/2 litre of medium

(a sterile Petri dish) and incubate with the sample plates. If there is

bacterial growth and colonies are observed the test results should be

considered with caution and entered into the Day Book .If more than 10

colonies are observed the test results are void. The demonstration of

growth in the control plate(s) must be reported to the Head of Bacteriology

and action taken that he recommends.

7.0 Expression of Results

Calculate the number of cfu, N, per millilitre of sample as follows:-

N = C/ V [(n1 + 0.1n2) d]

Where =sum of all colonies on all Petri dishes counted.



d = dilution for which the first counts were obtained


Example

Volume applied 1 ml

Dilution 1/100 (10

-2

) 278 and 290 coloniesDilution 1/1000 (10-3) 33 and 28 colonies



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N = (278+ 290 + 33 + 28)/[1 x (2+ {0.1 x 2}) x0.01]

= 629/0.022

= 28590 or 2.9 x 104 cfu/ml

Enter the results in the Laboratory Day Book showing the calculation

Reporting of result

Record the count expressed as two significant figures and expressed as a

power of 10. When the third figure is less than five, do not change the

preceding figure, when the third figure is 5 or more increase the preceding

figure by one unit.

e.g28,500 is expressed as 2.9 x 104

If the plate prepared from the 10-1 .dilution contain no colonies, report the

number of organisms as less than 1.0 x 101per ml or g (derived from 1 x

1/d, where d the dilution is 10-1 ). If there are only plates containing more

than 300 colonies report the count as greater than 3.0 x 102 per ml or per

g multiplied by the dilution factor. For example, if -3=u/c -4=u/c -5=u/c ,

the count will be > 300x105 = 3x108 CFU/ml. If all plates have

uncountable colonies report as being an Estimated count based on the

highest dilution measured. If a sample is plated undiluted, and no growth

appears on plate, report as Nil CFU/ml .

8.0 Records Bacteriology Laboratory Day Book.

5.7 Coliform counts ( plate method )

This procedure ensures the identification and enumeration of coliform

organisms (E. coli, Citrobacter, Enterobacter or Klebsiellaspp.). Coliforms are

indicators of external contamination. Coliform organisms are able to ferment

lactose within 48 hrs at 35-37C with the production of both acid and gas.

They form characteristic purplish red colonies in crystal violet neutral red Bile

Lactose Agar (VRBL-Oxoid CM107). These colonies have a diameter of at least0.5mm surrounded by a reddish zone of precipitation .This Procedure covers

the use of the test for various food products.



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Some coliforms do not exhibit the characteristics described above and this

have be too confirmed by sub culturing into brilliant green broth.

Confirmation for the presence ofE.colican be demonstrated by the indole test

and the ability of this organism to grow at 44oC.

After counting the colonies on the Petri dishes, the calculation of the final

count is as follows;

N = C/[ V (n1 + 0.1n2) d]






Example

Volume applied 1 ml



N= (278+ 290 + 33 + 28)/ [1 x (2+ {0.1 x 2}) x0.01]

= 629/0.022

= 28590 or 2.9 x 10

4

cfu/ml


Procedure for the Enumeration of Coliform Organisms by the Pour Plate Method

1.Purpose

This procedure describes enumeration of coliform organisms (E. coli,Citrobacter, Enterobacter or Klebsiellaspp.). Coliforms are indicators of

external contamination. Coliform organisms are able to ferment lactose within



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24-48 hrs at 30C -37C with the production of both acid and gas. They form

characteristic purplish red colonies in violet red Bile Agar (VRBL-Oxoid CM107).

These colonies have a diameter of at least 0.5mm surrounded by a reddish

zone of precipitation

2.Scope

This Procedure covers the use of the Test for enumeration of coliform

organisms.

3.Responsibility

The Senior Bacteriological Technician is responsible for the implementation of

the procedure.

4.Requirements

1.1 Sterile Violet Red Bile Agar (Oxoid CM 107)4.2 Water bath @ 44 o C oC used for tempering the medium

after sterilization

4.3 9 ml of quarter strength Ringers Solution or Buffered Peptone Water in

Universal Bottles autoclaved at 1210C for 15 minutes. This is the diluent.

4.4 Sterile Petri dishes

4.5 One, 1,000l automatic pipette

4.6 Sterile blue 1ml pipette tips

4.7 Incubator at 30o C 1oC

4.8 Tryptone water

4.9 Water bath @ 444.10 Vortex mixer

4.11 Wire loop

4.12 Bunsen burner

4.13 Brilliant Green Bile Broth.(BGBB)

5.Reference Documents:

Enumeration of coliforms colony count at 30oC ( 03.05.05) D 4,

Standards Unit, Health Protection Agency, UK.

6. Procedure:



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6.1 Samples are prepared as in described in the procedure for sample

preparation and dilution.

6.2 Mix the sample thoroughly by shaking the sample container 25 times in 10

seconds over 30 cm arcs. The sample may be shaken mechanically. The

interval between mixing and pipetting should not be more than 3 minutes.

6.3 Transfer 1ml of milk into 9 ml of diluent and mix thoroughly, either

mechanically or by a Vortex mixer. This is the Primary dilution.

6.4 Transfer 1 ml from the Primary dilution aseptically using a fresh pipette tip

to a further 9 ml of diluent and mix. This is the 10 -2 dilution. Further

dilutions are made by transferring 1 ml of each successive dilution to a

further 9 mls of diluent using a fresh sterile pipette tip for each transfer.

6.5 Pipette 1 ml from each dilution using a sterile pipette tip onto a sterile Petri

dish starting with the highest dilution prepared.

6.6 15 ml of VRBL agar at a maximum temperature of 45Care poured into

each Petri dish and the agar and sample are mixed as below

6.8 Mix the plate thoroughly by moving the plate horizontally 5 times followed

by using a circular motion in a clockwise direction 5 times. Then repeat

using vertical motion 5 times followed by a circular anticlockwise rotation 5

times.

6.9 When the initial medium has set, a further thin layer (approx 4 ml) of

sterile VRBA is poured on the surface of the medium

6.10 When the medium has solidified, the Petri dishes are inverted and placed in

an incubator at 30oC for 24 2 hrs

6.11 Count the characteristic colonies which are dark red with a diameter of at

least 0.5mm. Colonies of coliform bacteria are counted, if necessary, by a

counter or by manually marking the underside of the plate with a marker

pen.

7.0 Confirmatory test

If uncharacteristic colonies are present , inoculate 5 such colonies ( or all

colonies if less than 5 present ) into (BGBB), including a representative of

each different type of colony. Incubate BGBB tubes at 37oC for 24 2

hrs. Consider colonies which produce gas in the Durham tube as confirmed

coliforms.



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For confirmation ofE.coliinoculate 5 colonies into tryptone water, incubate

at 44 oC for 24 2 hrs and test for indole production. Consider colonies

that are Indole Positive as confirmed E.coli.

Expression of Results

Calculate the number of cfu, N, per millilitre of sample as follows:-

N = C/[ V (n1 + 0.1n2) d]

Where C = sum of all colonies on all Petri dishes counted





Example

Volume applied 1 ml



N = (278+ 290 + 33 + 28)/[1 x (2+ {0.1 x 2}) x0.01]

= 629/0.022

= 28590 or 2.9 x 10

4

cfu/ml

6.12 Record Result in the Laboratory Day Book



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5.8 Yeast and Moulds counts

This is a procedure used to determine the level of contamination of food samples, Milk &

Milk Products with fungi,ie, yeast and moulds. The procedure is used in the Food & Dairy

Hygiene Laboratories.

When certain environmental conditions prevail, moisture, temperature etc fungal

contaminants in food grow and release toxic secondary metabolite. These substances

are called mycotoxins. Aspergillusflavusand Aspergillusparasiticusgrowing in cereals

produces aflatoxin which affects man and animals. Fungi also cause food spoilage.

National bodies have specifications on various products and to establish whether a

product passes or not, yeast and mould tests have to be carried out. Potato dextrose

agar, sabouraud dextrose agar and yeast extract agar are examples of media that can

be used for the enumeration of fungi. This media should have acid added onto them to

depress the growth of bacteria.

Procedure for enumeration of yeast and moulds

2. Purpose

This procedure describes the enumeration of yeasts and mouldsin milk and

dairy products.

3. Scope

This document covers the use of the procedure for milk & milk products in theBacteriology and Food & Dairy Hygiene Laboratories.

4. Responsibility

The Bacteriology technician is responsible for the implementation of the

procedure.

5. Requirements

4.1 Sterile Potato Dextrose Agar tempered at 450C 10C in a conical flask

4.2 Sterile 10% Lactic acid

4.2 Sterile Petri Dishes4.3 Incubator set at 30C 10C



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4.4 Water bath set at 45C 10C

4.4 Automatic 1,000l pipette

4.5 Sterile 1ml pipette tips

4.6 9 ml quarter strength Ringers Solution in Universal bottles.

6. Reference Documents :

KEBS.KS 05-11: Parts 1-4 1976 ( Confirmed, 1999)

7. Procedure :

6.1 Prepare serial ten-fold dilutions of the sample homogenate in 9ml BPW up

to the desired dilution using a sterile pipette tip for each dilution.

6.2 Pipette 1ml from each dilution onto sterile Petri dishes starting at the

highest dilution with a fresh sterile 1ml pipette tip.

6.3 Remove tempered medium from water bath checking that it is not abo

450C

6.4 Add 1ml of sterile 10% lactic acid for each 100 ml of medium mix by swirli

just before pouring onto Petri dishes

6.5 Add 15ml of Potato Dextrose Agar to each plate

6.6 Mix immediately after pouring by 5 to and fro movements followed

by 5 circular clockwise movements followed by 5 to and fro movements at

right angles to the first set, followed by 5 circular anti-clockwise movements.

6.7 Allow to solidify. Invert and incubate plates in the incubator at 30C for 5

days.

6.8 Count plates containing 10-150 colonies. If mainly yeasts are present,

plates with 150 colonies are usually countable.

6.9 Report results in colony forming units/g or colony forming units /ml

depending on the type of sample.

Expression of Results

Calculate the number of cfu,N, per milliliter or gramme of sample as

follows:-

N = C/ [V (n1 + 0.1 n2) d]



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Example

Volume applied 1 ml



N = (278 + 290 + 33 + 28)/ [1 x (2 + [0.1 x 2]) x 0.01

= 629/0.022

= 28590 or 2.9 x 104 cfu/ml

Enter the results in the Laboratory Day Book showing the calculation

Reporting of result

Record the count expressed as two significant figures and expressed

as a power of 10. When the third figure is less than five, do notchange the preceding figure, when the third figure is 5 or more,

increase the preceding figure by one unit.

e.g 28,500 is expressed as 2.9 x 104

If the plate prepared from the 10-1 .dilution contains less than 10

colonies, report the number of organisms as less than 1.0 x 10 2 per

ml or g (derived from 10 x 1/d, where d the dilution is 10-1 ). If there

are only plates containing more than 150 colonies report the count

as greater than 1.5 x 103per ml or per g. If all plates have

uncountable colonies report as being an Estimated count .

If sample is done undiluted, and no growth appears on plate, reportas Nil CFU/ml.



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7.0 Records

Bacteriology Day Book

5. 9 Most Probable Number testing ( MPN) in water

Procedure for Most Probable Number Method (MPN)is used for detection andenumeration of coliforms organisms, thermotolerant coliforms organisms and

presumptive Escherichia coliin water

The purpose of this procedure is to enumerate coliform organisms in water using the M

method. Coliforms organisms are used as indicator organisms. A water sample which

coliforms is most likely to have pathogenic organisms of faecal origin like Salmonella

Shigella sp. Coliform organisms ferment lactose in the testing media producing acid and gas

The gas production is indicated by the inverted Durham tubes. Fermentation is indicated

colour change like in MacConkey broth purple which changes from purple to yellow.

E.coliis a coliform organism which is able to grow at 44 oC and breakdown trytophan with

production of indole.

Procedure for testing faecal coliforms and E.coli in water using the Most probable Num

Technique.

1. Purpose

This procedure ensures the detection and the enumeration of coliform

organisms in water, thermotolerant coliform organisms and presumptive

Escherichia coliby culture in a liquid medium in multiple tubes and calculation

of their most probable numbers in the sample. It is applicable to all types ofwater. Coliform organisms are capable of aerobic growth at either 35oC

0.5oC or 37oC 0.5oC in a liquid lactose culture medium with the production of

acid and gas.Thermotolerant coliform organisms have the same fermentative

properties as coliforms within 24 hr at either 44oC 0.25oC or at 44.5oC.

0.25oC. E.coliis a thermotolerant coliform organism which also produces indole

from tryptophan within 24 hr, at either 44o 0.25oC or 44.5oC. 0.25oC.

2. Scope

This procedure is used in the Food Testing Laboratory.

3. Responsibility

The Technicians is responsible for the correct implementation of the procedure.



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4. Requirements

Weighing balance (0. 01g)

Spatula

Autoclave

Autoclave tape

Bunsen burner

Vortex Mixer

Incubator @ 37oC 0.5oC

Water bath @ 44oC 0.5oC

Conical flask


Sterile 1 ml pipette tips

Sterile 125ml bottles (one)

Sterile 20mls Universal bottles (five)

Water samples of at least 200mls

Kovacs reagent for indole

MacConkey Broth purple

Durham tubes (big and small)

Sterile tubes

pH meter

Tryptone water

5. Reference Documents: Water quality Detection and enumeration of coliform organisms,

thermotolerant coliform organisms and presumptive Escherichia coli

Part 2: Multiple tube (most probable number ) method ISO 9308-2

6. Procedure:

Test portions of the water sample are inoculated into a series of bottles and

tubes as follows:

Label the bottles and tubes (below) with the laboratory sample number

Mix the sample of water thoroughly by inverting the bottle at least 10

times.Inoculate the bottles of the sterile MacConkey Broth purple

as follows:



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Add 50 ml of water sample to the bottle containing 50ml (double strength)

of MacConkey broth

Add 10 ml of water sample to each of five Universal bottles containing 10ml

(double strength) of broth.

Add 1 ml of water sample to each of five tubes containing 5 ml of (single

strength) broth.

Note Each bottle or tube must contain an inverted Durham tube for the

collection of gas. Small Durham tubes are used for the tubes and medium

sized Durham tubes are used for medical flat bottles.

Mix the contents of each bottle or tube.

Incubate the inoculated broths in a water bath at 44 oC for 24-48 hours with

stoppers and caps loose.

6.2 Examination of the bottles/tubes

Examine the bottles/tubes cultures after incubation for 18-24 hrs and

regard as positive reactions those which show turbidity due to bacterial

growth and gas formation in the Durham tubes, together with acid

production (indicated by change of broth colour from purple to yellow).Re-

incubate those tubes which do not show any or all of these changes and

examine them again for positive reactions after 48hr.

6.3 Confirmatory Test

To confirm the presence of Presumptive E.coli, incubate a tube oftryptonewater,and test for indole formation after incubation at 44oC for 24

hr by add 0.2-0.3 ml of Kovacs reagent to the tryptone water tube, the

development of a red colour after gentle agitation denotes the presence of

indole .

7.0 Expression of results

From the number of tubes of isolation medium and confirmatory tests

giving positive reactions, calculate by reference to the statistical tables

below, the most probable numbers of coliform organisms, thermo tolerant

coliform organisms and presumptive E.coli in 100ml of the sample. Forexample if a sample gives the following results; 50ml bottle positive(i.e gas

and fermentation ), 3 bottles of 10 ml positive and 3 bottles of 5 ml positive



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the profile will be 1 3 3. Using the table below this interprets as 18 faecal

coliforms/ 100ml water.

MPN values per 100 ml of sample and 95 % confidence limits

(When one 50 ml, five 10 ml and five 1 ml portions are used)

Number of tubes giving positive

reaction

MPN

(per 100

ml)

95% confidence

limits

1 of 50 ml 5 of 10 ml 5 of 1 ml Lower Upper

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

3

3

3

3

4

4

4

4

4

4

5

5

5

5

5

5

1

2

3

4

0

1

2

3

4

5

0

1

2

3

4

5

11

14

18

21

13

17

22

28

35

43

24

35

54

92

161

>180

3

4

5

6

4

5

7

9

12

15

8

12

18

27

3

_

26

34

53

66

31

47

69

85

101

117

75

101

138

217

450

_

5-10. Detection of Salmonella sp

Members of the genus Salmonella are infectious pathogens capable of causing food

poisoning and clinical symptoms in humans. They reach food directly or indirectly from



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animal excreta at time of slaughter, from human excreta, water polluted by sewage and

in the kitchen by transfer from raw to cooked food by hands or utensils.

The genus is made up of a large group, which causes enteric fever; the main Salmonella

sp that cause fever are S. typhi and S. paratyphi which causes typhoid and

paratyhoidrespectively. Both are endemic in many developing countries.

Most Salmonellae are found in the intestines of animals of pig, cows, goats, sheep,

rodents and poultry. However S. typhi, and S.paratyphi are usually found only in

humans. The two are excreted in the urine and faeces of infected individuals.

Food poisoning with Salmonella sp and other bacteria is characterized by fever,

headache, and diarrhoea and vomiting.

Salmonella is usually present in much lower numbers in food. The organisms in food

have been subjected to processing and the surviving organisms are often injured. So the

method of Salmonella detection involves several stages to give the organism every

chance to grow. The stages are;

2. Pre-enrichment in Buffered Peptone Water.

3. Selective enrichment in two broths.

4. Subculturing the broths onto two selective agar plates.

5. Identification of the bacteria using serological and biochemical tests, followed by

API 20E.

The different stages would be discussed as described in the procedure. See procedure

below.

Procedure for the Detection of Salmonella sp. in All Foods, Milk, and Dairy Products &

Environmental swabs.

1.Purpose

The purpose of this procedure is to determine whether Salmonella sp. is presen

foods, environmental swabs, milk and dairy products.

2.Scope

This procedure is used in Bacteriology for detection ofSalmonellasp

in all food types including milk and dairy products. .



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3.Responsibility

The Laboratory Head is responsible for the correct implementation of the procedu

4.Requirements

Weighing balance 0.01g

Spatula

Autoclave

Autoclave tape

Bunsen burner

Vortex Mixer

Incubator @ 37oC 1oC


Water bath @ 41.5oC 1oC

Hot plate

Conical flask



Buffered Peptone Water (BPW),

Selenite CystineBroth ,

Rappaport-Vassiliadis Soya Peptone Broth,

Brilliant Green Agar (BGA)

Xylose Lysine Deoxycholate Agar (XLD)

Triple sugar iron agar slopeUrea agar slope

MacConkey agar

Nutrient agar

Salmonella polyvalent 'O' and 'H' antisera

Wire loop


Detection of Salmonella Species (16/09/2005) Reference no: F 13i2 Health

Protection Agency, Standard Units, UK



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Procedure

6.1:

Following the procedure described in the Procedure for preparation of samples

and dilutions prepare 10-1 homogenate of the sample in buffered peptone

Water (BPW).

6.2 Pre-enrichment Place the homogenate or swab suspension in an

incubator at 37C 1oC for 18hr 2 h. For dehydrated foods the incubation

period should be extended to 24hr 2 h

6.3 Selective enrichment:-

Transfer 1ml of the pre-enrichment buffered peptone water to 10ml of MKTTn

broth incubates at 37C 1oC for 24 - 48hrs 3 h.

Transfer 0.1ml of the pre-enrichment culture to 10ml of Rappaport-Vassiliadis

Soya Peptone Broth (RVS) and incubate at 41.5C 1C for 24 3 hours.

Subculture two loopfuls from each of the broths by streaking onto plates of

brilliant green agar and xylose lysine deoxycholate agar. Place in an incubator

at 37o 1C for 24hrs 3h. It is advisable to retain the incubated BPW under

refrigeration until investigations are complete.

6.4 Recognition of colonies.

On XLD Salmonella sp. colonies appears as red or red colonies with black

centers. Isolated colonies may appear yellow with black centers. Lactose

fermenting organisms may also appear as yellow with or without black centers.On BGA Salmonella sp. are red colonies surrounded by a bright red medium.

7.0 Confirmatory Tests

7.1 Typical (red or red colonies with black centers on XLD or red colonies

surrounded by a bright red medium on BGA) or suspect colonies of Salmonella

from each plate must be subjected to serological and biochemical confirmation.

7.2 Select at least five suspect Salmonella colonies including one from each

plate of the selective agar and inoculate purity plates by sub culturing onto

MacConkey agar. Incubate at 37

0

C for 21hrs 3hScreen discrete colonies from the MacConkey agar biochemically using TSI

(Triple sugar iron) agar slopes and urea agar. TSI needs streaking on the



32/77


surface of the slope and stabbing of the butt. Inoculation for the urea agar is

the same as for the TSI agar. Incubate all media at 370C for 21 3h.

Salmonella typically produce an acid (Yellow) butt with gas bubbles and an

alkaline (deep pink) slope, with blackening due to hydrogen sulphide

production. This blackening may mask the acid production in the butt, but is

occasionally absent. Strains of Salmonella do not produce urease (rare

exceptions) so no change in color is seen in urea agar.

If biochemical results exclude the presence of Salmonella and the strain is

pure no further action is required.

Identify at least one isolate giving biochemical and/or serological reactions

consistent with Salmonella with API 20E. Make sure that the API reagents

are up to date and stored as per the manufacturers instructions.

8.0 Serological confirmation

Subculture non-lactose fermenting colonies from the MacConkey agar to

nutrient agar (NA) slope. Ensure that some water of condensation is present

at the base of the NA slope, if none is present then add a few drops of sterile

water. Inoculate the colony into the water of condensation and streak up the

slope. Incubate at 370C overnight.

Using the growth from the NA slope prepare three saline suspension on a slide

using a loopful of saline and growth from the slope for 'O' antigens, the water

of condensation at the bottom of the slope for 'H' antigens and a mixture from

slope and condensate for auto agglutination. If auto agglutination occurs

proceed to biochemical confirmation.

Add a loop full of polyvalent 'O' and polyvalent 'H' antisera to two separate

saline suspensions and rock the slide gently for 30 seconds. If agglutination

occurs with the polyvalent antisera but not with the saline the reactions are

considered to be positive.

Final confirmation is done with the API 20E system.

REPORTING OF RESULTS

If Salmonella species are not isolated report as follows:Salmonella species not detected in 25g, 25ml or swab as appropriate.



33/77


If the isolate is confirmed as Salmonella species report as follows:

Salmonella species detected in 25g, 25ml, or swab.

The actual weight or volume of sample examined must be reported as, for

example, 10g or ml, 25g or ml,

100g or ml.

7.0Records

Bacteriology Laboratory Day Book

5.11 Enumeration of Staphylococcus aureus.

The purpose of this procedure is to isolate and enumerate Staphylococcus aureus from

foods, milk and dairy products.

Staphylococci are Gram-positive spherical bacteria that occur in microscopic clustersresembling grapes. Staphylococci are facultative anaerobes that grow by anaerobic

respiration or by fermentation that yields principally lactic acid. Staphylococcus aureus

forms a fairly large yellow colony on rich medium. The bacteria are catalase positive

and oxidase- negative. Staphylococcus aureus grows at a temperature range 15oC to

45oC degrees and at sodium chloride concentrations as high as 15 per cent

Characteristics ofStaphylococcusaureus

Gram positive, cluster- forming coccus

Non motile, non-spore forming facultative anaerobe Ferments glucose producing lactic acid

Catalase positive

Coagulase positive

Dnase- positive

Golden yellow colony on agar, usually haemolytic on blood agar, it produces the

enzymes coagulase, DNase and catalase which are used to identify it.

Normal flora of humans found on nasal passages, skin and mucous membranes.

Pathogen of humans, causes a wide range of suppurative infections, as well as food

poisoning and toxic shock syndrome.



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Staphylococcus aureus causes food poisoning by releasing enterotoxins into food, and

toxic shock syndrome by release of pyrogenic exotoxins into the blood stream.

In processed foods Staphylococcus aureus is readily destroyed by heating, drying, and

other processing conditions to which the food is subjected. Thus the presence of

Staphylococcus aureus indicates contamination from the skin, mouth, or nose of food

handlers. Contamination of processed foods may also occur when contaminated food

collects on processing surfaces to which food products are exposed. Large number of

Staphylococcus aureus cells in processed foods indicates that sanitiation, temperature

control or both were inadequate. This finding, however, is not sufficient evidence to

incriminate a food as the cause of food poisoning. The isolated Staphylococcus aureus

organisms must be shown to produce enterotoxins.

In the Staphylococcus group of organisms, only Staphylococcus aureusand

Staphylococcus epidermidis are significant in their interactions with humans.

Screening for Staphylococcus aureus in foods is done using a selective, Baird Parker

Medium where it grows as shiny black colonies. Further confirmation is done using the

enzymatic tests mentioned above; catalase,coagulaseand Dnase. See procedure below.

Procedure for the enumeration ofStaphylococcus aureus

1.Purpose

The purpose of this procedure is to isolate and enumerate Staphylococcus aur

from foods, milk and dairy products.

2.Scope

This procedure applies to Bacteriology Laboratories.

3.Responsibility

The Senior Bacteriology Technician is responsible for the implementation o

procedure.

4.Requirements

SpatulaBunsen burner

Vortex Mixer



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9ml sterile quarter strength Ringers solution in Universal bottles autoclaved @

121oC for 15 minutes

Sterile Petri dishes

Mechanical blender

Automatic 1ml pipette


Balance capable of weighing to 0.01g

Incubator at 37oC 1oC

Buffered Peptone Water (BPW)

Baird Parker (BP) medium

Sterile spoons

Sterile 1 ml and 10 ml glass pipettes graduated in 0.1ml volumes

Sterile spreading rods

DNase medium

Staphylase Test Reagent Kit (Oxoid,UK)


Enumeration of Staphylococcus aureus, Reference no F 12, Health

Protection Agency, Standard Units, UK, 03.05.05

6.Procedure:

6.1 Following the procedure described in Procedure for preparation of samplesand dilutions, prepare 10-1 homogenate in buffered peptone water (BPW) and

further decimal dilutions as required.

6.2 Starting with highest dilution to be plated, aseptically transfer 0.5ml of a

sample of each dilution suspension onto its own Baird Parker plate. Spread

inoculum over surface of the agar plate using a sterile bent glass streaking

rods and let the plates dry.

6.3 Invert the plates and incubate for 48 2 hours at 37oC 1oC.

6.4 Countand record colonies.

Examine the plates for typical colonies of Staphylococcus aureuson plates

containing up to 150 colonies. Typical colonies appear as black, shiny, convexcolonies up to 3mm in diameter, with a narrow zone of opacity surrounded by

a zone of clearing. Count and record the number of typical colonies. For foods



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of bovine origin, including dairy products, atypical colonies of Staph.aureus

may occur but do not show opacity or clearing. For foods of this type also

count and record atypical colonies.

6.5 Confirmatory tests

Sub culture five colonies of each type for (or all colonies if less than five)

for confirmatory testing using Dnase and coagulase production.

6.6 Inoculate each colony onto a DNase agar plate and plate out onto a segment

of a blood agar plate. Set up blood agar plates and DNase agar plates with

a positive control strain of Staphylococcus aureus and a negative control

strain of Staphylococcus epidermidis to verify performance and incubate

alongside the plates of the test organism. Transfer the plates to an

incubator at 37oC 1oC for 18-24 hours.

6.7 Examine the blood agar plates for purity and colonial morphology

consistent with S. aureus cream or golden coloured colonies up to 3mm in

diameter.

6.7 DNase production

Flood the DNase plates with normal hydrochloric acid (HCL). After about 30

seconds, discard the excess reagent (HCL) into a chemical waste container.

Positive reaction occurs when colonies show a defined zone of clearing.

6.8 Coagulase production

Using the growth on blood agars (BA), perform a slide agglutination test onthe strains giving a positive DNase test. Compare the results with the

growth from the Blood agar plates of the control Staphylococcus aureus and

Staphylococcus epidermidis.

7.0Control cultures

Positive and negative controls must be used for confirmatory tests. A

positive reaction shows clumping within ten seconds and a negative

reaction shows no clumping within ten seconds and thus no coagulase

produced.

Positive control S. aureus (Oxford strain) NCTC 6571



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Negative control S. epidermidisNCTC 11047

Record the results of the control strains in the day book.

8.0 Calculations

Counts should be calculated where possible using dilutions giving 15 or

more colonies on the plate. Calculate the count of Staph. aureus as

follows:-

Counts ofStaph.aureus per gram=

Number of typical colonies confirmed x Number of colonies counted

Number of colonies tested Volume tested x dilution

Added to;-

Number of atypical colonies confirmed x Number of colonies counted

Number of atypical colonies tested Volume tested x dilution

9.0 Reporting of results

If no colonies of the test organism are present on the 10 -1 dilution, report

as

Less than 20 cfu /g or ml. This indicates a LOD less than 20cfu.

If the test organism is detected with counts between 20 and 99 per gram

report in the form of:

acfu/g or ml ( Where a is a number between 20 and 99)

If the test organisms are detected at counts of 100 or higher per gram,

report with one figure before and one figure after the decimal point

expressed to the power of 10 in the form of :

ax 10bcfu/g or ml



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(Where a is never less than 1.0 or greater than 9.9 and b represents the

appropriate power of ten. Round counts up if the last figure is 5 or more

and down if the last figure is 4 or less: e.g 1920 cfu/g or ml is reported

as 1.9 x 103 cfu/g or ml

235,000 cfu/g or ml is reported as 2.4 x 105cfu/g or ml

Plates with more than 300 colonies.

When number of CFU per plate exceeds 300, for all dilutions, record the counts

as too numerous to count or uncountable (TNTC or UC) for all plates. Mark

calculated count as estimated to denote that it was estimated from counts

greater than 300 per plate.

If all plates have uncountable colonies report as being an Estimated count.

Based on the highest dilution measured.

7.10 Records

Laboratory Day Book.

5.12 Acidity Test - Titratable Acidity of Milk

Introduction:

The test is performed to determine whether the raw milk is stable to heat-treatment.

This is meant to measure the level of total acidity in milk and reported as lactic acid

(LA). The apparent acidity of fresh milk is a property and components of milk which falls

between 0.12 - 0.16% LA. The development of acidity is due to activities of micro-

organisms present in the milk. Acid development in milk affects the pH and its stability

to heating. Milk clots on boiling when its acidity is about 0.20% and clots at room

temperature when the acidity is about 0.5% lactic acid.

This test is carried out by titration and is based upon the chemical principle that equal

volumes of acids and alkaline of the same strength will exactly neutralize each other.



39/77


The point of neutralization is determined by means of an indicator which gives one

definite colour in an alkaline medium and another colour in an acid medium.

Procedure for titratable Acidity of milk.

1. References.

KS 05-30 (2001) Specifications for Pasteurised milk

KS 05-34 Specifications for yoghurt

2. Requirements

Standardised N/9 NaOH

10ml Pipettes and Pipette filler

2 Beakers (50 or 100ml)

Burette 10ml (with identity number and validated)

Phenolphthalein Indicator (2.5%) freshly and not exceeding 6 months since

preparation

Thermometer range (-100C to +1100C)

3. Procedure.

3.1 Mix each sample well and adjust the temperature of the milk if necessary to 200C

1.00C.

3.2 Fill the burette with standardised N/9 NaOH.

3.3 Remove any excess NaOH from the tip of the burette with a tissue and adjust

the volume to a convenient starting reading

3.4 Pipette 10 ml of the sample into a beaker.

3.5 Add 2-3 drops of phenolphthalein indicator to the milk in the beaker and agitate

by rotating the contents.

3.6 Note the initial volume of N/9 NaOH in the burette when starting to titrate

3.7 Titrate the sample quickly and continuously by adding the N/9 NaOH from the

burette into the beaker until the first permanent faint pink colour appears and



40/77


persists in the whole volume of the milk for at least 10 seconds, stop adding NaOH

and note the volume N/9 NaOH in the burette at the end (final).

3.8 Calculate the volume of NaOH used by subtracting the initial from final volume

shown on the burette used. The volume of NaOH used divided by 10 gives the

percentage acidity of the sample and this is expressed as % lactic acid (LA).

Example; Volume of NaOH solution used = 1.5ml,

Hence milk acidity shall be 1.5/10 = 0.15%LA

The natural acidity in milk is due to the presence of phosphates, calcium and carbon

dioxide.

Developed Acidity is due to microbial growth.

7. Results Standards.

Fresh milk acidity range is (0.13 0.15%) and Cream (0.08 0.11%) while the

acidity of fermented product depends on stage of fermentation.

9. RecordsLaboratory day book

5.13 Butterfat Fat content Gerber Method

Rapid volumetric methods are often used for routine purposes for determing fat in milk,

Gerber method is commonly used. This test method gives value for fat content in grams

of fat per 100g of milk.

Principle separation of the fat of milk in a butyrometer by centrifuging after dissolving

the protein with sulphuric acid, the separation being aided by the addition of a smallquantity of amyl alcohol. The butyrometer is graduated to give a direct of fat content.

The butter fat test of the same milk was compared with results generated by lacto scan

milk analyser,

Procedure for the Determination of Butter Fat in milk using the Gerber Method

1. Purpose

This procedure ensures that the butter fat analysis of raw and pasteurized milk is

properly conducted.



41/77


2. Scope

This test is only used in milk, raw or pasteurized regardless from which animal.

3 Responsibility

The Head of the Food and Milk Hygiene Laboratory is responsible for the proper

implementation of this test.

4. Reference Documents

KS 05-12: Parts 1-2: (1976) Confirmed (1999) Determination of Fat Content in

Milk.

5. Requirements:

Sulphuric acid (H2SO4) GP or Analar Grade with a specific gravity of 1.815

0.002g/ml at 200C or 90% Concentration of H2SO4

Amyl alcohol, Analar Grade with a specific gravity of 0.809 - 0.813 at 200C

10.94 ml Gerber pipette (with ID and validated)

1ml Dispenser or pipette (numbered and validated)

10ml aciddispenser (numbered and validated)

Milk butyrometers range (0- 8%) (with a numbered butyrometer)

Double ended stoppers for butyrometers

Water baths at 400C 1.00C and 650C 1.00C.

Gerber Centrifuge able to attain 1,100rpm

Thermometer with range -100C to + 110 0C that is internally calibrated and

engraved for identification

Timer with IDPipette filler

Rack for shaking butyrometers

Goggles for eye protection

6. Procedure:

6.1 Warm the milk sample in a water bath to 400C 1.00C, mix it well and then

cool to 20 0C 1.00C

6.2 Dispense 10 ml of Sulphuric Acid into each butyrometer.



42/77


6.3 Confirm the temperature, mix the milk and transfer 10.94 ml of milk using Ger

pipette into each butyrometer allowing it to flow slowly and gently down the s

of the butyrometer to avoid burning of the milk by the sulphuric acid.

6.4 Slowly add 1.0ml of amyl alcohol from a tilt bottle.

6.5 Cork the butyrometers with stopper without disturbing the contents until

half of the cork is into the butyrometer. Shake well in a shaker or by hand

until a chocolate brown colour is attained.

6.6 Centrifuge for 5 minutes at 1,100 rpm.

6.7 Place the butyrometer in the water bath at 65 0C 1.00C for 3 minutes.

6.8 Adjust the meniscus (by moving the stopper) formed by the junction of the m

and acid to the bottom of the butyrometer scale. Read off the upper level a

lower level of the butter fat using the bottom of the meniscus from

butyrometer scale.

6.9 The difference between the two readings gives the percentage butter fat in

milk by mass.

6.10 IQC regime, test to be carried out in duplicate.

Acceptable repeatability range for this test is 0.1, if the error exceed this

range a repeat of test will be carried out and mean calculated from results

of four tests.

5.14 Resazurin Test

This test is used to determine in a general way the bacteriological quality of raw milk

and determine its keeping quality. Resazurin, which is added to milk in liquid form, is aredox indicator. When the oxygen potential of the milk is normal, the colour will be blue

but if the potential is lowered because of metabolic activity by micro-organisms the

colour will change to pink or even white.

Resazurin reduction occurs in two stages, the first an irreversible change from the blue

resazurin to the pink resorufin and the second a reversible change from the pink

resorufin to the colourlessdihydroresorufin. This is a dye reduction test which is based on

the ability of micro-organisms to alter the oxidation-reduction potential (the redox

potential) if a medium which is reflected through a colour changes of the dye.

False reduction can be assumed to be brought about by the leucocytes if the colour inthe downgraded milk sample (for example, milk from animals suffering from mastitis)

remains unchanged for a longer time than observed normally.



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The solution of resazurin is prepared by adding one tablet to 50mls cold sterile distilled

water. When the tablet is completely dissolved a 0.005% standard resazurin solution is

obtained. When not in use the solution should be kept in a cool dark place preferably a

refrigerator and be discarded when more than 8 hours old.

Disc reading Colour Keeping Quality

6 and 5 Deep & light blue Satisfactory

4 Deep pink Acceptable

3 & 2 pink & pale pink Reject

1& 0 slight pink & white Reject

Procedure for the Resazurin Test on Raw or Pasteurized Milk


KS 10: 2006 Specification for Raw Whole Milk

2.Requirements

Water Bath at 370C 1.00C

Sterile Resazurin Test Tubes and sterile rubber stoppers

Resazurin Solution freshly made

10ml graduated pipettes, numbered and validated

1ml pipetteFreshly made Resazurin solution

Lovibond Comparator with standard Resazurin disc, having seven standa

numbered 0-6 (disc 4/9)

Thermometer with range -100C to + 110 0C that is Analabs internally calibrated

and coded for identification

Timer

Test tube rack for holding test-tubes.

50ml measuring cylinder

50 l of the milk sample to be tested

3.ReagentSterile standard resazurin solution



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4.Procedure:

1. Wash and clean the test tubes and rubber stoppers and rinse with

distilled water. Sterilize in the autoclave at 1210C for 15 minutes. Two

tubes are required, one for the sample and the other for the test control.

2. Heat a portion of the test milk to boiling point and cool to room

temperature. This is for the test control.

3. Mix the test sample and the control sample, separately, by inverting

each at least 25 times.

4. Aseptically pipette 10 mls of the test sample into one test tube and add

1 ml of resazurin solution. Prepare the control tube in the same manner

using boiled milk, stopper the tubes and invert three times.

5. Mix by inverting the tubes twice, place the tubes in the water bath at

37.50C 0.50C and note time. For Raw milk read and report after 10

minutes, while pasteurized milk read after 30min, 1, 2, and 3 hours.

Report the results of third hour.

6. Place the tube containing the prepared sample in the right hand

compartment (when viewed from the front of the instrument) and the

control tube in the left hand side.

7. Use indirect light, match the sample with one of the comparator

numbers. When the colour falls between two disc numbers, record the

sample as the lower number value and add a half.

8. IQC regimes set the test in duplicate.

5.0 Interpretation of Results

Colour of Sample Comparator No Milk Grade

White or complete reduction of pink colour 0 Reject

(stop test)

Pale Pink, pink & white mottling or

pink band at the top with paler pink below 1 or 2 Reject

Deep Pink 3 or 4 Reject

Light Blue 5 or 5.5 2nd Grade

Deep Blue 6 1st Grade

Control Sample 6

6.0 Records



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Laboratory Day Book

5.15 Alcohol Test

The alcohol test is used for rapid assessment of stability of milk to processing. The testis useful as an indication of the mineral balance of milk and not so much as an index of

developed acidity. The test aids in detecting abnormal milk such as colostrums, milk

from animals in late lactation, milk from animals suffering from mastitis and milk in

which mineral balance has been disturbed.

The test is based on diffusion of water from milk to alcohol, this migration results in

precipitation of milk. This only occurs when milk quality is questionable or its failing.

The concentration of alcohol used is 70% or 75%

Procedure for the alcohol Test in Raw Milk.

1. Purpose

The purpose of this test is to determine the stability of milk proteins when equal

parts of milk/alcohol are mixed. The test evaluates suitability of unprocessed milk for

further processing or shelf-life of processed milk. Chemical instability in milk is due to

colostrum, mastitis or high acidity.

2 Scope

This procedure covers raw and pasteurized milk regardless of its source.

3 Responsibility.

The senior Technician in Food & Dairy Hygiene is responsible for the proper

implementation of this procedure.

4 References.

KS 05 -10:1992 Specification for Unprocessed Whole Milk

5 Requirements



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Alcohol Gun

70 % Ethanol in distilled water

6 Procedure.

Fill alcohol gun with 70 % alcohol (Ethanol).

Hold the alcohol gun almost at perpendicular to milk sample with the receiver cup up.

Dip the tip of the gun into test milk, the open tip will sample approx. 1ml of milk

Close the opening of receiver cup with the thumb

Invert the gun, to allow milk and alcohol flow into the receiver cup.

Shake the mixture for 30 sec and observe for any coagulation or precipitates.

7 Report

Coagulation or precipitate Alcohol positive

No coagulation Alcohol negative

Interpretation

A negative test indicates low acidity and good heat stability of milk sample.

Note any flakes or clots. The presence of a flake or a clot denotes a positive. Milk

showing positive is not considered suitable for the processing, as it is unstable on

heating.

5.16 Moisture Analysis in Foods

Principle

Free water in foods or any other material evaporate on heating, hence change in weight.

The principle is weight to weight basis

The loss in weight is calculated as percentage of the original weight of sample. Moisture

content is of great value in food science because it determines the rheological

characteristic of food, moisture levels above set limits encourages growth of micro-

organisms especially moulds in dry foodsMoisture content can be carried out by heating in oven at 105 0C for 4 hrs, cooled in

desiccators and weighed.



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Moisture analysis can also be done using gravimetric method; the machine combines

heating and weighing simultaneously. Heating is by halogen lamp.

For the accurate results the test requires sample weight of at least 5g,

After preparing and setting the machine, start the test and allow time until the test

ends.

Allow the machine to cool to near room temperature before weighing second and third

repeat tests.

The cooling lowers rate of evaporation during weighing and spreading of the sample

which results to a lower moisture content of the material at the end of the test.

When the test is completed the machine beeps ones and the LCD display test over,

results of the test M/ content as percentage and time taken.

Procedure for the Determination of Moisture content by Ohaus MB45 Thermo gravimetric

Method

1. Purpose

This procedure ensures that moisture in dairy products, artemisia and other

food samples is correctly determined using the Ohaus Moisture Analyzer MB45

model.

2. Scope

This instruction covers all types of food and feed samples, dairy products

(butters, cheese, ghee, milk powder) and all dried food samples.

3. Responsibility

The Head of the laboratory is responsible for the correct implementation of this

procedure.

4. Reference Documents :

Instruction Manual MB45 Moisture Analyzer.Certified ISO 9001 QMS.Ohaus

Corp. New Jersey. USA

5. Requirements

MB45 Moisture Analyzer machine

Sample pan, Spatula or spoon



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Sample

Standard weight 5g

OTR Form

6. Procedure

6.1 Switch on the Moisture Analyzer

6.2 Allow the machine to warm-up for about 30 minutes

6.3 Open the cover on the Moisture Analyzer, place the empty pan in the

panhandle and place in the sample chamber and tare the weight

6.4 Place a 5 gram standard weight on the pan and confirm and record the

weight.

6.5 Program the machine to

Identify the sample either by - Name or Lab No.

Drying temperature - 1050C

Temperature program - Standard

Switch of criteria

6.7 Tare the pan

6.8 Weigh approximately 5g of sample onto the pan.

6.9 Close the cover of the Moisture Analyzer

6.10 Press start button to start the process.

6.11 Monitor the progress of the machine until Test Over is displayed on LCD

6.12 Record the result as percentage moisture

5.17 Phosphatase Test for Pasteurized Milk

Pasteurization

Heat treatment process applied to liquid milk the objective of eliminating possible health

hazards arising from pathogenic micro-organisms associated with milk.

This can be achieved through

a) Batch method

Milk is heated to 650C and maintained at this temperature for at least 30 minutes

and immediately and rapidly cooled to 100C or less.



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b) High temperature short time method (HTST)

Milk is heated to 730C and maintained at this temperature for at least 16 seconds


c) Flash pasteurization

Milk is heated to 800C and maintained at this temperature for at least 10 seconds


The principle on which the test is based is that although Phosphatase enzymes are

invariably present in raw milk. They are inactivated during pasteurization. It has been

shown that this enzyme is more difficult to destroy that the most heat-resistant

pathogenic organisms which are likely to be present in milk.

The test involves incubation of the milk with disodium p-nitrophenyl phosphate under

alkaline co

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