Differential stain techniques Gram Staining

1. Thedifferential stain techniquedistinguishes two kinds of organisms. An example is theGram stain technique.2. This differential technique separates bacteria into two groups, Gram-positive bacteria and Gram-negative bacteria. 3. Procedure : Crystal violet is first applied, followed by the mordant iodine, which fixes the stain . Then the slide is washed with alcohol, and the Gram-positive bacteria retain the crystal-violet iodine stain; however, the Gram-negative bacteria lose the stain. The Gram-negative bacteria subsequently stain with the safranin dye, the counterstain, used next. These bacteria appear red under the oil-immersion lens, while Gram-positive bacteria appear blue or purple, reflecting the crystal violet retained during the washing step. The process canbe diagrammatically represented as follows :

Figure 1The Gram stain procedure used for differentiating bacteria into two groups.

Acid Fast Staining

1. Another differential stain technique is theacid-fast technique.This technique differentiates species ofMycobacteriumfrom other bacteria. 2. Heat or a lipid solvent is used to carry the first stain, carbolfuchsin, into the cells. Then the cells are washed with a dilute acid-alcohol solution.Mycobacteriumspecies resist the effect of the acid-alcohol and retain the carbolfuchsin stain (bright red). 3. Other bacteria lose the stain and take on the subsequent methylene blue stain (blue). Thus, the acid-fast bacteria appear bright red, while the nonacid-fast bacteria appear blue when observed under oil-immersion microscopy.

Microorganisms Used in Alcohol Production

There is a limited number of microorganisms which ferment carbohydrates (pentose or hexose sugars) into alcohols and yield some by-products. Microorganisms utilize various pathways.

Following are some of alcohol producing micro-organisms:

Bacteria:Clostridium acetobutylicum, Klebsiella pneumoniae, Leuconostoc mesenteroides, Sarcina ventriculi, Zymomonas mobilis,etc.

Fungi:Aspergillus oryzae, Endomyces lactis, Kloeckerasp.,Kluyreromyees fragilis, Mucorsp.,Neurospora crassa, Rhizopussp.,Saccharomyces beticus, S. cerevisiae, S. elltpsoideus, S. oviformis, S. saki,Torulasp.,Trichosporium cutaneum,etc. A summary of alcohol production through different routes of microorganisms is given in Fig. 15.4.

Fermentable Substrates

i. Ethyl alcohol is produced from such organic material that contains sugar or its precursor as fundamental units. The cost of substrates (raw materials) in fermentation is of major consideration, because it directly affects the cost of products. The fermentable substrates used for alcohol production are given under 'fermentable substrates' .

ii. Before using in fermentation processes, the cellulosic, lignocellulosic and starchy materials are hydrolyzed by enzymes or acids just to render the complex substances into a simple forms (monosaccharides). Enzymes for hydrolysis are obtained from barley malt or moulds by heat treatment of acidified materials.iii. Klebsiella pneumoniais capable of utilizing the wood hemicellulose hydrolysate which consists of pentose and hexose sugars, low molecular weight oligomers and uronic acids. The products of fermentation are butanol, ethanol acetone, etc. These products can be utilized as solvent.iv. Clostridium acetobutylicumanaerobically ferments the starchy substrates of grains and potatoes to produce acetone, ethanol and butanol. Recently, it has been demonstrated thatSchwaniomyces castelliidirectly converts the soluble starch into ethanol.v. Sugarcane molassess, a by- product of sugarcane mill, contains high percentage of sucrose and fructose sugars. The fruit juice, for example grapes(Vitis vinifera)contains high amount of sugars (8-13% glucose and 7-12% fructose) when grapes are ripen. Moreover, percentage of sugar contents depends on ripeness of grapes. Juices are obtained from the apples, pear, palmyra and palm-flower stalk to prepare alcoholic beverages sold in market by different names in different countries. For instance, beverages named as wine, cider, perry and toddy are prepared from grapes, apple, pear and palmyra, respectively. Similarly, names of beverages prepared from different substrates are 'Kvass' (U.S.S.R.) and 'Bear' (India) from barley, 'Sonti'(India) and Sake (Japan) from rice, 'Merissa' (Sudan) and 'Kaffir bear' (Malawi), from sorghum, 'Thumba' (India) and 'Busa' (USSR) from millets.

Alcoholic Beverages and the microbes used in their preparations :Percentage of alcohol differs, in different alcoholic beverages . Some of them are briefly discussed:

i. WineIt is mainly an European drink produced from juice of fresh grapes(Vitis vinifera, V. rotundifolia).In ripen grapes, concentration of sugar (glucose and fructose) increases. Grape juice (27% sugar) is fermented by various strains ofS. ellipsoideusinto alcohol and also renders the chemical constituents which alters the flavor.ii. BeerBeer is produced after the fermentation of mixture of barley malt and starchy solutions by S.cerevisiae(a top fermenter yeast that do not settle at bottom) orS. carlsbergensis(the bottom fermentation yeast).Malt is prepared from barley. Grains are allowed to germinate. After 4-6 days amylase and protease are formed. The sprouted grains are gradually heated to about 80C. The dried rootlets are knocked off and the remaining grain is coarsely ground. Malt is mixed with coarsely ground starchy cereals (rice, maize, wheat) to produce grist. Mash is prepared by adding hot water to the grist holding for a period to allow enzymatic conversion and draining off the resultant sweet wart. Wart is filtered and then boiled. Finally it is fermented with yeast .After fermentation sugar is converted into alcohol and also brings about minor chemical changes, for example protein.iii. RumRum is the distilled product of culture fluid.S. cerevisiaeor other yeast is used as the fermenting microorganism. Culture medium is prepared from black strap molasses containing 12-14% fermentable sugar. Ammonium sulphate and some times phosphates are added as nutrients. When fermentation is over, culture fluid is distilled to remove the alcohol and used as rum.iv. WhiskeyIt is prepared through the fermentation of grain mash (cooked and saccharified with peated malt) by a top yeast(S. cerevisiae)when fermentation is over, the culture fluid contains alcohols, traces of acids and esters

v. SakeSake, the rice wine, is manufactured from the strach. It is a complicated process which implies the mastering of different fermentation techniques in semi-solid and sub merged conditions, and regulation of successive microbial populations. First of all a mould(Aspergillus oryzae)then bacteria(LactobacillusandLeuconostoc)and finally yeast (5.cerevisiae)are mixed with the fermentable medium. The culture fluid contains about 20% alcohol; therefore, before marketing the concentration of alcohol is adjusted to 16% (Sasson, 1984).

Uses of Alcohols(a)Ethanol is used as solvent, extractant and antifreeze. It is also used as a substrate for the synthesis of many other solvents of dyes, Pharmaceuticals, lubricants, detergents, pesticides, plasticizers, explosives and resins, and for the manufacture of synthetic fibers .

(b)N-butanol is used in the manufacture of plasticizers, brake fluids, urea-formaldehyde resins, extractants and pertrol additives.

(c)Glycerol is used in medicals, biosynthesis of D-fructose via mannitol, and in food industry(because of its sweetness and high solubility). Mannitol is used in industry and research.

(d)Butanol plus acetone acid 2, 3-butanediol are used as industrial solvent. Butanol plus acetone is used in the production of explosive materials(e.g.cordite) and 2, 3-butanediol in the synthesis of rubber.

(e)Ethanol is used as alcoholic beverages 1. Antibiotics are chemicals that kill or inhibit the growth of bacteria and are used to treat bacterial infections. They are produced in nature by soil bacteria and fungi. This gives the microbe an advantage when competing for food and water and other limited resources in a particular habitat, as the antibiotic kills off their competition.2. Antibiotics take advantage of the difference between the structure of the bacterial cell and the hosts cell.3. They eitherprevent the bacterial cells from multiplying so that the bacterial population remains the same, allowing the hosts defence mechanism to fight the infectionorkill the bacteria, for example stopping the mechanism responsible for building their cell walls.4. An antibiotic can also be classified according to the range of pathogens against which it is effective. Penicillin G will destroy only a few species of bacteria and is known as a narrow spectrum antibiotic. Tetracycline is effective against a wide range of organisms and is known as a broad spectrum antibiotic.

5. Penicillin :

a. The antibacterial effect ofpenicillinwas discovered by Alexander Fleming in 1929. He noted that a fungal colony had grown as a contaminant on an agar plate streaked with the bacteriumStaphylococcus aureus, and that the bacterial colonies around the fungus were transparent, because their cells were lysing.b. He then recognised the importance of a fungal metabolite that might be used to control bacteria. The substance was named penicillin, because the fungal contaminant was identified asPenicilliumnotatum. Fleming found that it was effective against many Gram positive bacteria and he even used locally applied, crude preparations of this substance, from culture filtrates, to control eye infections. Later on the product was purified by two other British scientists, Florey and Chain, working in the USA, who managed to produce the antibiotic on an industrial scale for widespread use. All three scientists shared the Nobel Prize for this work, and rightly so - penicillin rapidly became the "wonder drug" which saved millions of lives. It is still a "front line" antibiotic, in common use for some bacterial infections although the development of penicillin-resistance in several pathogenic bacteria now limits its effectiveness.c. The action of penicillin :

This shows an 'overlay plate', in which a central colony of the fungusPenicillium notatumwas allowed to grow on agar for 5-6 days, then the plate was overlaid with a thin film of molten agar containing cells of the yellow bacterium,Micrococcus luteus. The production of penicillin by the fungus has created a zone of growth inhibition of the bacterium. This demonstration parallels what Alexander Fleming would have observed originally, although he saw inhibition and cellular lysis of the bacteriumStaphylococcus aureus.

d. An expanded role for the penicillins came from the discovery that natural penicillins can be modified chemically by removing the acyl group to leave6-aminopenicillanic acid(see diagram above) and then adding acyl groups that confer new properties.

e. These modernsemi-synthetic penicillinssuch as Ampicillin, Carbenicillin and Oxacillin have various specific properties such as:

resistance to stomach acids so that they can be taken orally,

a degree of resistance to penicillinase (a penicillin-destroying enzyme produced by some bacteria)

extended range of activity against some Gram-negative bacteria.

Some examples of clinically important antibiotics produced by microbes are given as follows : Some clinically important antibiotics

AntibioticProducer organismActivitySite or mode of action

Penicillin

Penicillium chrysogenumGram-positive bacteria

Wall synthesis

Cephalosporin

Cephalosporium acremoniumBroad spectrum

Wall synthesis

Griseofulvin

Penicillium griseofulvumDermatophytic fungi

Microtubules

Bacitracin

Bacillus subtilisGram-positive bacteria

Wall synthesis

Polymyxin B

Bacillus polymyxaGram-negative bacteria

Cell membrane

Amphotericin B

Streptomyces nodosusFungi

Cell membrane

Erythromycin

Streptomyces erythreusGram-positive bacteria

Protein synthesis

Neomycin

Streptomyces fradiaeBroad spectrum

Protein synthesis

Streptomycin

Streptomyces griseusGram-negative bacteria

Protein synthesis

Tetracycline

Streptomyces rimosusBroad spectrum

Protein synthesis

Vancomycin

Streptomyces orientalisGram-positive bacteria

Protein synthesis

Gentamicin

Micromonospora purpureaBroad spectrum

Protein synthesis

Rifamycin

Streptomyces mediterraneiTuberculosis

Protein synthesis

1. Organic acids are valuable commodity chemicals or may be further processed into higher value chemicals, solvents, fuels, & bio-products.

2. Bio-based acids are produced through the fermentation of biomass sugars by a number of different microorganisms under different conditions, metabolic pathways, and efficiencies.

3. A variety of organisms in the production of organic acids and solvents through the fermentation of lactose, xylose, arabinose, & glucose.

4. The process involves developing and improving bacterial microorganism strains, fermentation systems, bio-reactor designs, product recovery & separation systems for the production of organic acids and bio-solvents in non-sterile industrial environments.

5. Iimprovements of the process mainly focus on: increased conversion rates & efficiencies, higher product concentrations, more efficient product recovery, and increasing reactor infection resistance.

6. The production of different organic acids (acetic acid, lactic acid, glucconic acid, succinic acid, etc.) through fermentation of biomass sugars can be a primary or secondary co-product in a bio-refining system. Organic acids can be produced and sold as commodity chemicals or further processed into higher value chemicals, biofuels, or bio-products.Advantages:

Conversion of 5-Carbon Sugars:Xylose / Ara4binose,

Higher Metabolic Conversion Efficiency & Rates,

Increased Product Diversity, Revenues, & Profitability,

Higher Biomass to Product Yield & Product Yield Rates,

Increased Product Concentrations in Fermentation,

Decreased Energy Usage in Separations & Product Recovery,

Decreased Fermentation Infection & Upsets,7. Fermentative production of a number of organic acids;lactic, acetic, gluconic, citric, succinic, propionic, & butyric acids, from biomass derived sugars through the development of improved processes, bio-reactor systems, and organisms for the processing and recovery of various of organic acids, bio-solvents, and chemical precursors. What are Biopesticides?Give some examples of microbial pesticides.Biopesticides are certain types of pesticides derived from such natural materials as animals, plants, bacteria, and certain minerals. For example, canola oil and baking soda have pesticidal applications and are considered biopesticides.Biopesticides fall into three major classes:1. Microbial pesticides : They consist of a microorganism (e.g., a bacterium, fungus, virus or protozoan) as the active ingredient. Microbial pesticides can control many different kinds of pests, although each separate active ingredient is relatively specific for its target pest[s]. For example, there are fungi that control certain weeds, and other fungi that kill specific insects.The most widely used microbial pesticides are subspecies and strains of Bacillus thuringiensis, or Bt.

2. Plant-Incorporated-Protectants (PIPs): They are pesticidal substances that plants produce from genetic material that has been added to the plant. For example, scientists can take the gene for the Bt pesticidal protein, and introduce the gene into the plant's own genetic material. Then the plant, instead of the Bt bacterium, manufactures the substance that destroys the pest. The protein and its genetic material, but not the plant itself, are regulated by EPA.

3. Biochemical pesticides: They are naturally occurring substances that control pests by non-toxic mechanisms. Conventional pesticides, by contrast, are generally synthetic materials that directly kill or inactivate the pest. Biochemical pesticides include substances, such as insect sex pheromones, that interfere with mating, as well as various scented plant extracts that attract insect pests to traps. Because it is sometimes difficult to determine whether a substance meets the criteria for classification as a biochemical pesticide, EPA has established a special committee to make such decisions.Some examples of biopesticides are as follows :

a. Viruses have been developed against insect pests such as Lepidoptera (butterflies and moths), Hymenoptera (bees, wasps, and ants), and Dipterans (flies).Gypsymoths and tent caterpillars, for example, periodically suffer fromepidemic virus infestations, which could be exploited and encouraged.b. Many commensal microorganisms (microorganisms that live on or in other organisms causing no direct benefit or harm) that occur on plant roots and leaves can passively protect plants against microbial pests by competitive exclusion (that is, simply crowding them out). Bacillus cereus has been used as an inoculumon soybeanseedsto prevent infection by fungal pathogens in the genus Cercospora.c. Some microorganisms used as biopesticides produce antibiotics, but the major mechanism in most cases seems to be competitive exclusion. For example, Agrobacterium radiobacter antagonizes Agrobacterium tumefaciens, which causes the disease crown gall.d. Species of two bacterial generaBacillus and Streptomyceswhen added as biopesticides tosoilhelp control the damping-off disease of cucumbers, peas, and lettuce caused by Rhizoctonia solani. Bacillus subtilis added to plant tissue also controls stem rot and wilt rot caused by species of the fungus Fusarium.e. Mycobacteria species produce cellulose degrading enzymes, and their addition to young seedlings helps control fungal infection by species of Pythium,Rhizoctonia, and Fusarium. Species of Bacillus and Pseudomonas produce enzymes that dissolve fungal cell walls.What are the advantages of using biopesticides?

Biopesticides are usually inherently less toxic than conventional pesticides. Biopesticides generally affect only the target pest and closely related organisms, in contrast to broad spectrum, conventional pesticides that may affect organisms as different as birds, insects, and mammals. Biopesticides often are effective in very small quantities and often decompose quickly, thereby resulting in lower exposures and largely avoiding the pollution problems caused by conventional pesticides. When used as a component of Integrated Pest Management (IPM) programs, biopesticides can greatly decrease the use of conventional pesticides, while crop yields remain high. Many plants and animals are protected from pests by passive means. For example, plant rotation is a traditional method of insect anddiseaseprotection that is achieved by removing the host plant long enough to reduce a regions pathogen and pest populations. Biopesticides have several significant advantages over commercialpesticides. They appear to be ecologically safer than commercial pesticides because they do not accumulate in the food chain. Some biopesticides provide persistent control, as more than asinglemutation is required to adapt to them and because they can become an integral part of a pests life cycle. In addition, biopesticides have slight effects on ecological balances because they do not affect nontargetspecies. Finally, biopesticides are compatible with other control agents.Bacillus thuringiensis as biopesticide : Explain.The most widely used microbial pesticides are subspecies and strains of Bacillus thuringiensis, or Bt. Each strain of this bacterium produces a different mix of proteins, and specifically kills one or a few related species of insect larvae. While some Bt's control moth larvae found on plants, other Bt's are specific for larvae of flies and mosquitoes. The target insect species are determined by whether the particular Bt produces a protein that can bind to a larval gut receptor, thereby causing the insect larvae to starve.

1. The bestexamplesof microbial insecticides are Bacillus thuringiensis (B.t.) toxins, which were first used in 1901. They have had widespread commercial production and use since the 1960s and have been successfully tested on 140 insects, including mosquitoes.

2. Insecticidal endotoxins are produced by B.t. during sporulation, and exotoxins are contained in crystalline parasporalproteinbodies. These protein crystals are insoluble in water but readily dissolve in an insects gut.

3. Once dissolved, the proteolytic enzymes paralyze the gut. Spores that have been consumed germinate and kill the insect. Bacillus popilliae is a related bacterium that produces an insecticidal spore that has been used to control Japanese beetles, a corn pest.4. The gene for the B.t. toxin has also been inserted into the genomes of cotton and corn, producing genetically modified, or GM, plants that produce their own B.t. toxin. GM cotton and B.t. corn both express the gene in theirroots, which provides them with protection from root worms.5. Ecologists and environmentalists have expressed concern that constantly exposing pests to B.t. will cause insects to develop resistance to the toxin. In such a scenario, the effectiveness of traditionally applied B.t.would decrease.

there are several characteristics that all culture media have in common: Media must be prepared in such a way that it is sterile prior to being inoculated with a bacterial sample, so that when a particular type of bacteria is cultured (cultivated) on that medium, it is the only type of bacteria present. Growth media must also provide everything the bacterial culture needs to live and grow, including water, nutrients, and the properpH. Media can be either liquid (nutrient broth) or solid (agar).Defined Media versus Complex MediaSome media formulations are very specific recipes in which certain ingredients must be present in specific amounts. These defined media (also known as synthetic media) are used to grow bacteria that have very particular needs.Most clinical cultures do not have such exacting requirements, and can be grown in what is referred to as complex media. Complex media are composed of partially digested yeast, beef, soy and additional proteins, in which the exact concentration and composition is unknown. In comparison with defined media, which are good for growing bacteria with very particular needs, complex media can be thought of as a crowd-pleaser, suitable for growing many different types of less fastidious microbes.In addition to growth media formulations being classified as either defined or complex, there are also media that are designed to do more than just grow bacteria, selective and differential media provide information about the bacteria growing.Selective Bacterial Growth MediaSelective media contain ingredients that inhibit the growth of certain types of bacteria and/or encourage the growth of others. This type of media is useful in helping to identify unknown bacteria and in encouraging the growth of only the types of bacteria that the microbiologist is interested in cultivating.Differential Bacterial Growth MediaDifferential culture media are formulated to display a color change when the bacteria growing metabolize a certain ingredient. For exampleMacConkeys Agar, in addition to being selective, contains the sugarlactoseand a pH sensitive dye. When bacteria growing on MAC ferment lactose (metabolize it for food), they generate acidic waste products that trigger the pH sensitive dye to turn the bacteria pink. So, when grown on MAC, colonies of Gram-negative, lactose fermenting bacteria are pink, the intensity of the pink color corresponds to how good the bacteria are at eating lactose. Colonies of Gram-negative non-lactose fermenting bacteria grow in colorless colonies.Mannitol Salt Agar also contain food (mannitol, a sugar alcohol) and a pH sensitive dye. When the bacteria growing on MSA ferment mannitol, the medium changes from its original pink color to a bright highlighter yellow.Bacteria that grow on MSA are all halophiles. If halophilic normal flora bacteria are growing on MSA (such asStaphylococcus epidermidis) the medium remains pink. If halophilic pathogens are present (such asStaphylococcus aureus), the medium changes to bright yellow.Another specialized medium,Blood Agar(BAP) contains sheeps blood,if bacteria growing on the medium produce exotoxins that hemolyze (cut up) the red blood cells, the medium changes color. BAP medium that has changed from red to transparent 9completely clear) indicates pathogenStaphylococcus pyogenes. BAP bruised or unaffected by bacterial growth present indicate normal flora, non-pathogenic bacteria.Want to see more Specialized Media Visuals?For exampleMacConkeys Agar(MAC) is used to cultivate Gram-negative bacteria, by discouraging the growth of Gram positive bacteria through the use of crystal violet dyes and bile salts. Another selective medium,Mannitol Salt Agar(MSA), has a high concentration of sodium chloride, which selects for halophiles (salt-loving bacteria) such as members of the genusStaphylococcus.Classification based on consistency:Culture media are liquid, semi-solid or solid. Liquid media are sometimes referred as broths (e.g nutrient broth).

Liquid mediaare available for use in test-tubes, bottles or flasks. In liquid medium, bacteria grow uniformly producing general turbidity. Certain aerobic bacteria and those containing fimbriae (Vibrio & Bacillus) are known to grow as a thin film called surface pellicle on the surface of undisturbed broth. Bacillus anthracis is known to produce stalactite growth on ghee containing broth. Sometimes the initial turbidity may be followed by clearing due to autolysis, which is seen in penumococci. Long chains of Streptococci when grown in liquid media tend to entangle and settle to the bottom forming granular deposits but with a clear medium. Culturing bacteria in liquid media has some drawbacks. Properties of bacteria are not visible in liquid media and presence of more than one type of bacteria can not be detected. Liquid media tend to be used when a large number of bacteria have to be grown. Culture media are suitable to grow bacteria when the numbers in the inoculum is suspected to be low. Inoculating in the liquid medium also helps to dilute any inhibitors of bacterial growth. This is the practical approach in blood cultures. Culturing in liquid medium can be used to obtain viable count (dilution methods). Solid media:Any liquid medium can be rendered by the addition of certain solidifying agents. Agar agar (simply called agar) is the most commonly used solidifying agent. The word "agar" comes from the Malay word agar agar (meaning jelly). It is also known as kanten, China grass, or Japanese isinglass. Agar is chiefly used as an ingredient in desserts throughout Japan. It is an unbranched polysaccharide obtained from the cell membranes of some species of red algae such as the genera Gelidium and Gracilaria, or seaweed (Sphaerococcus euchema). Commercially it is derived primarily from Gelidium amansii. Agar is composed of two long-chain polysaccharides (70% agarose and 30% agarapectin). It melts at 95oC (sol) and solidifies at 42oC (gel), doesnt contribute any nutritive property, it is not hydrolysed by most bacteria and is usually free from growth promoting or growth retarding substances. However, it may be a source of calcium & organic ions. Most commonly, it is used at concentration of 1-3% to make a solid agar medium. New Zealand agar has more gelling capacity than the Japanese agar. Agar is available as fibres (shreds) or as powders.For preparing agar in Petri plates, 3% agar (by weight) is added to the broth and autoclaved, when the medium is at ~50oC, it is poured on to sterile Petri plates and allowed to set. For preparing agar containing media in test-tubes, the culture medium is mixed with 3% agar and heated with stirring to melt. This ensures that all the tubes get equal amounts of agar. These tubes can then be sterilized by autoclaving. Semi-solid mediaReducing the amount of agar to 0.2-0.5% renders a medium semi-solid. Such media are fairly soft and are useful in demonstrating bacterial motility and separating motile from non-motile strains (U-tube and Cragies tube). Certain transport media such as Stuarts and Amies media are semi-solid in consistency. Hugh & Leifsons oxidation fermentation test medium as well as mannitol motility medium are also semi-solid. Biphasic mediaSometimes, a culture system comprises of both liquid and solid medium in the same bottle. This is known as biphasic medium (Castaneda system for blood culture). The inoculum is added to the liquid medium and when subcultures are to be made, the bottle is simply tilted to allow the liquid to flow over the solid medium. This obviates the need for frequent opening of the culture bottle to subculture.Preparation and storage:

Care must be taken to adjust the pH of the medium before autoclaving. Various pH indicators that are in use include phenol red, neutral red, bromothymol blue, bromocresol purple etc. Dehydrated media are commercially available and must be reconstituted as per manufacturers recommendation. Most culture media are sterililized by autoclaving. Certain media that contain heat labile components like glucose, antibiotics, urea, serum, blood are not autoclaved. These components are filtered and may be added separately after the medium is autoclaved. Certain highly selective media such as Wilson and Blairs medium and TCBS agar need not be sterilized. It is imperative that a representation from each lot be tested for performance and contamination before use. Once prepared, media may be held at 4-5oC in the refrigerator for 1-2 weeks. Certain liquid media in screw capped bottles or tubes or cotton plugged can be held at room temperature for weeks.Classification based on nutritional component:Media can be classified as simple, complex and synthetic (or defined). While most of the nutritional components are constant across various media, some bacteria need extra nutrients. Simple Media : Those bacteria that are able to grow with minimal requirements are said to non-fastidious and those that require extra nutrients are said to be fastidious. Simple media such as peptone water, nutrient agar can support most non-fastidious bacteria. Complex media such as blood agar have ingredients whose exact components are difficult to estimate. Synthetic or defined media such as Davis & Mingioli medium are specially prepared media for research purposes where the composition of every component is well known.

Classification based on functional use or application:These include basal media, enriched media, selective/enrichment media, indicator/differential media, transport media and holding media.

Basal Medium : Basal media are basically simple media that supports most non-fastidious bacteria. Peptone water, nutrient broth and nutrient agar considered basal medium

Enriched Medium : Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes them enriched media. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Blood agar, chocolate agar, Loefflers serum slope etc are few of the enriched media.Example : Blood agaris preparing by adding 5-10% (by volume) to a basal medium such as nutrient agar or other blood agar bases. Since blood can not be sterilized, it has to be collected aseptically from the animal. Animals have to be bled and the blood is collected in sterile containers with anticoagulant or glass beads. While sheep blood is preferred, blood from rabbit, horse and ox can also be collected. Human blood must be avoided since it may contain inhibitory substances including antibiotics. After the blood agar base is autoclaved, blood is added to the medium at temperature just above the solidifying point of agar. The mixture is then poured on to the plates and allowed to solidify. Blood agar is useful in demonstrating hemolytic properties of certain bacteria. Two major types of hemolysis are often seen on blood agar; beta and alpha hemolysis. Beta hemolysis is the complete lysis of RBC resulting in clearing around the colonies whereas alpha hemolysis is the partial lysis of RBC resulting in greenish discolouration around the colonies. Gamma hemolysis is a misnomer and it indicates non-hemolytic colonies.Chocolate agaris also known as heated blood agar or lysed blood agar. The procedure is similar to that of blood agar preparation except that the blood is added while the molten blood agar base is still hot. This lyses the blood cells and releases their contents into the medium. This process turns the medium brown, hence the name. This medium is especially useful in growing Hemophilus and Neisseria.Serum for medium can be obtained from animal blood but must be filtered through membrane or seitz filter before use. Selective and enrichment mediaare designed to inhibit unwanted commensal or contaminating bacteria and help to recover pathogen from a mixture of bacteria. While selective media are agar based, enrichment media are liquid in consistency. Both these media serve the same purpose. Any agar media can be made selective by addition of certain inhibitory agents that dont affect the pathogen. Various approaches to make a medium selective include addition of antibiotics, dyes, chemicals, alteration of pH or a combination of these. Thayer Martin Agar used to recover N.gonorrhoeae contains Vancomycin, Colistin and Nystatin. Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contain 10% NaCl. Potassium tellurite medium used to recover C.diphtheriae contains 0.04% Potassium tellurite. McConkeys Agar used for Enterobacteriaceae members contains Bile salt that inhibits most gram positive bacteria. Pseudosel Agar (Cetrimide Agar) used to recover P.aeruginosa contains cetrimide. Crystal Violet Blood Agar used to recover S.pyogenes contains 0.0002% crystal violet. Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by incorporating Malachite green. Wilson & Blairs Agar for recovering S.typhi is rendered selective by the addition of dye Brilliant green. Selective media such as TCBS Agar and Monsurs Tellurite Taurocholate Gelatin Agar used for isolating V. cholerae from fecal specimens have elevated pH (8.5-5.6), which inhibits most other bacteria. Enrichment mediaare liquid media that also serves to inhibit commensals in the clinical specimen. Selenite F broth, tetrathionate broth and alkaline peptone water are used to recover pathogens from fecal specimens.

Differential/Indicator media:Certain media are designed in such a way that different bacteria can be recognized on the basis of their colony colour. Various approaches include incorporation of dyes, metabolic substrates etc, so that those bacteria that utilize them appear as differently coloured colonies. Such media are called differential media or indicator media. When a particular carbohydrate is incorporated into a medium and a mixture of bacteria inoculated on it, only that bacterium that can ferment it produces acid. This change in pH is detected by using a pH indicator incorporated in the medium and the bacterium that can ferment the sugar appears in a different colour. This approach is used in MacConkeys agar, CLED agar, TCBS agar, XLD agar etc. MacConkeys agar is the most commonly used media to culture and identify gram negative bacilli (especially enterobacteriaceae members). It contains bile salts (selective agent), lactose (sugar), peptone and neutral red (pH indicator), agar and water. Those bacteria that can ferment lactose produce pink coloured colonies where non-lactose fermenting colonies produce colourless colonies. Similarly, Vibrio cholerae produces yellow coloured colonies on sucrose containing TCBS medium.Reduction of potassium tellurite to metallic tellurium by Corynebacterium diphtheriae results in production of black coloured colonies on PT agar. Production of H2S by Salmonella typhi results in production of black coloured colonies on Wilson & Blairs medium. Enterococcus fecalis produces black coloured colonies on bile esculin agar due to reduction of esculin to esculetin. Detection of hemolysis on blood agar can be considered as an indicator property of Blood agar. Transport media:Clinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth of contaminating organisms or commensals. This can be achieved by using transport media. Such media prevent drying (desiccation) of specimen, maintain the pathogen to commensal ratio and inhibit overgrowth of unwanted bacteria. Some of these media (Stuarts & Amies) are semi-solid in consistency. Addition of charcoal serves to neutralize inhibitory factors. Cary Blair medium and Venkatraman Ramakrishnan medium are used to transport feces from suspected cholera patients. Sachs buffered glycerol saline is used to transport feces from patients suspected to be suffering from bacillary dysentery. Pikes medium is used to transport streptococci from throat specimens.Anaerobic media:Anaerobic bacteria need special media for growth because they need low oxygen content, reduced oxidation reduction potential and extra nutrients.Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin K. Such media may also have to be reduced by physical or chemical means. Boiling the medium serves to expel any dissolved oxygen. Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings can render a medium reduced. Robertson cooked meat that is commonly used to grow Clostridium spps medium contain a 2.5 cm column of bullock heart meat and 15 ml of nutrient broth. Before use the medium must be boiled in water bath to expel any dissolved oxygen and then sealed with sterile liquid paraffin. Thioglycollate broth contains sodium thioglycollate, glucose, cystine, yeast extract and casein hydrolysate. Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated in the medium. Under reduced condition, methylene blue is colourless.

Isolation of Pure CultureThough microorganisms are generally found in nature (air, soil and water) as mixed populations to study the specific role played by a specific microorganism in its environment, one must isolate the same in pure culture. Pure culture involves not only isolation of individual microorganisms from a mixed population, but also the maintenance of such individuals and their progenies in artificial media, where no other microorganisms find way to grow.However, it is not easy to isolate the individual microorganisms from natural habitats and grow them under imposed laboratory conditions. For this, great deal of laboratory manipulation is required.Several methods for obtaining pure cultures are in use. Some common methods are in everyday-use by a majority of microbiologists, while the others are methods used for special purposes.Common Methods of isolation of pure culturePure culture of microorganisms that form discrete colonies on solid media, e.g., yeasts, most bacteria, many other microfungi, and unicellular microalgae, may be most commonly obtained by plating methods such as streak plate method, pour plate method and spread plate method.But, the microbes that have not yet been successfully cultivated on solid media and are cultivable only in liquid media are generally isolated by serial dilution method.1) Streak Plate Method This method is used most commonly to isolate pure cultures of bacteria. A small amount of mixed culture is placed on the tip of an inoculation loop/needle and is streaked across the surface of the agar medium. The successive streaks "thin out" the inoculums sufficiently and the microorganisms are separated from each other. It is usually advisable to streak out a second plate by the same loop/needle without reinoculation. These plates are incubated to allow the growth of colonies. The key principle of this method is that, by streaking, a dilution gradient is established across the face of the Petri plate as bacterial cells are deposited on the agar surface. Because of this dilution gradient, confluent growth does not take place on that part of the medium where few bacterial cells are depositedVarious methods of streaking

Presumably, each colony is the progeny of a single microbial cell thus representing a clone of pure culture. Such isolated colonies are picked up separately using sterile inoculating loop/ needle and restreaked onto fresh media to ensure purity.2) Pour Plate Method This method involves plating of diluted samples mixed with melted agar medium. The main principle is to dilute the inoculum in successive tubes containing liquefied agar medium so as to permit a thorough distribution of bacterial cells within the medium. Here, the mixed culture of bacteria is diluted directly in tubes containing melted agar medium maintained in the liquid state at a temperature of 42-45C (agar solidifies below 42C).The bacteria and the melted medium are mixed well. The contents of each tube are poured into separate Petri plates, allowed to solidify, and then incubated. When bacterial colonies develop, one finds that isolated colonies develop both within the agar medium (subsurface colonies) and on the medium (surface colonies). These isolated colonies are then picked up by inoculation loop and streaked onto another Petri plate to ensure purity. Pour plate method has certain disadvantages as follows: (i) the picking up of subsurface colonies needs digging them out of the agar medium thus interfering with other colonies, and (ii the microbes being isolated must be able to withstand temporary exposure to the 42-45 temperature of the liquid agar medium; therefore this technique proves unsuitable for the isolation of psychrophilic microorganisms.However, the pour plate method, in addition to its use in isolating pure cultures, is also used for determining the number of viable bacterial cells present in a culture.Pour Plate MethodA.Media/dilutionB.Pouring of the plate; andC.Colony development after incubation. Control consists of the sterilized plating medium alone

The isolated colonies are picked up and transferred onto fresh medium to ensure purity. In contrast to pour plate method, only surface colonies develop in this method and the microorganisms are not required to withstand the temperature of the melted agar medium.3) Spread Plate Method In this method the mixed culture of microorganisms is not diluted in the melted agar medium (unlike the pour plate method); it is rather diluted in a series of tubes containing sterile liquid, usually, water or physiological saline. A drop of so diluted liquid from each tube is placed on the centre of an agar plate and spread evenly over the surface by means of a sterilized bent-glass-rod. The medium is now incubated. When the colonies develop on the agar medium plates, it is found that there are some plates in which well-isolated colonies grow. This happens as a result of separation of individual microorganisms by spreading over the drop of diluted liquid on the medium of the plate. Serial Dilution MethodAs stated earlier, this method is commonly used to obtain pure cultures of those microorganisms that have not yet been successfully cultivated on solid media and grow only in liquid media. A microorganism that predominates in a mixed culture can be isolated in pure form by a series of dilutions. Spread plate method

The inoculum is subjected to serial dilution in a sterile liquid medium, and a large number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution. The aim of this dilution is to inoculate a series of tubes with a microbial suspension so dilute that there are some tubes showing growth of only one individual microbe. Example,if we have a culture containing 10 ml of liquid medium, containing 1,000 microorganisms i.e., 100 microorganisms/ml of the liquid medium.Serial dilution method

If we take out 1 ml of this medium and mix it with 9 ml of fresh sterile liquid medium, we would then have 100 microorganisms in 10 ml or 10 microorganisms/ ml. If we add 1 ml of this suspension to another 9 ml. of fresh sterile liquid medium, each ml would now contain a single microorganism. If this tube shows any microbial growth, there is a very high probability that this growth has resulted from the introduction of a single microorganism in the medium and represents the pure culture of that microorganism.Special Methods of Isolation on of Pure Culture1.Single Cell Isolation methodsAn individual cell of the required kind is picked out by this method from the mixed cultureand is permitted to grow. The following two methods are in use.(i)Capillary pipette methodSeveral small drops of a suitably diluted culture medium are put on a sterile glass-coverslip by a sterile pipette drawn to a capillary. One then examines each drop under the microscope until one finds such a drop, which contains only one microorganism. This drop is removed with a sterile capillary pipette to fresh medium. The individual microorganism present in the drop starts multiplying to yield a pure culture.(ii)Micromanipulator methodMicromanipulators have been built, which permit one to pick out a single cell from a mixed culture. This instrument is used in conjunction with a microscope to pick a single cell (particularly bacterial cell) from a hanging drop preparation. The micro-manipulator has micrometer adjustments by means of which its micropipette can be moved right and left, forward, and backward, and up and down. A series of hanging drops of a diluted culture are placed on a special sterile coverslip by a micropipette.Now a hanging drop is searched, which contains only a single microorganism cell. This cell is drawn into the micropipette by gentle suction and then transferred to a large drop of sterile medium on another sterile coverslip. When the number of cells increases in that drop as a result of multiplication, the drop is transferred to a culture tube having suitable medium. This yields a pure culture of the required microorganism.The advantages of this method are that one can be reasonably sure that the cultures come from a single cell and one can obtain strains with in the species. The disadvantages are that the equipment is expensive, its manipulation is very tedious, and it requires a skilled operator. This is the reason why this method is reserved for use in highly specialized studies.2.Enrichment Culture Method

Generally, it is used to isolate those microorganisms, which are present in relatively small numbers or that have slow growth rates compared to the other species present in the mixed culture. The enrichment culture strategy provides a specially designed cultural environment by incorporating a specific nutrient in the medium and by modifying the physical conditions of the incubation. The medium of known composition and specific condition of incubation favors the growth of desired microorganisms but, is unsuitable for the growth of other types of microorganisms.Proof of Purity of CulturesAssuming that one has isolated a pure culture, how does one establish that it is pure? A pure culture is one in which the cells are all of one kind, i.e., demonstrate "likeness". Hence, the proof of purity of cultures consists of demonstrating the "likeness" of microorganisms in the culture. It is based on certain criteria as follows:1.The microorganisms look alike microscopically and stain in the same fashion.2. When plated, all the colonies formed look alike.3. Streaks, stabs, etc. are uniform.4. Several isolated colonies perform identically, i.e., ferment the same sugars, and so on.Capillary method for obtaining a single microbial cell

Maintenance and Preservation of Pure CulturesOnce a microorganism has been isolated and grown in pure culture, it becomes necessary to maintain the viability and purity of the microorganism by keeping the pure cultures free from contamination. Normally in laboratories, the pure cultures are transferred periodically onto or into a fresh medium (subculturing) to allow continuous growth and viability of microorganisms. The transfer is always subject to aseptic conditions to avoid contamination.Since repeated sub culturing is time consuming, it becomes difficult to maintain a large number of pure cultures successfully for a long time. In addition, there is a risk of genetic changes as well as contamination. Therefore, it is now being replaced by some modern methods that do not need frequent subculturing. These methods include refrigeration, paraffin method, cryopreservation, and lyophilization (freeze drying).RefrigerationPure cultures can be successfully stored at 0-4C either in refrigerators or in cold-rooms. This method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the metabolic activities of the microorganisms are greatly slowed down but not stopped. Thus their growth continues slowly, nutrients are utilized and waste products released in medium. This results in, finally, the death of the microbes after sometime.Paraffin MethodThis is a simple and most economical method of maintaining pure cultures of bacteria and fungi. In this method, sterile liquid paraffin in poured over the slant (slope) of culture and stored upright at room temperature. The layer of paraffin ensures anaerobic conditions and prevents dehydration of the medium. This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture is preserved for several years.CryopreservationCryopreservation (i.e., freezing in liquid nitrogen at -196C) helps survival of pure cultures for long storage times. In this method, the microorganisms of culture are rapidly frozen in liquid nitrogen at -196C in the presence of stabilizing agents such as glycerol that prevent the formation of ice crystals and promote cell survival.Lyophilization(Freeze-Drying)In this method, the culture is rapidly frozen at a very low temperature (-70C) and then dehydrated by vacuum. Under these conditions, the microbial cells are dehydrated and their metabolic activities are stopped; as a result, the microbes go into dormant state and retain viability for years. Lyophilized or freeze-dried pure cultures and then sealed and stored in the dark at 4C in refrigerators. Freeze-drying method is the most frequently used technique by culture collection centers.