Preparation of Microbial Media

Aim:To prepare microbial media for growth and isolation of bacteria.

Theory:Much of the study of microbiology depends on the ability togrow and maintain microorganisms in the laboratory, and this ispossible only if suitable culture media are available. A culturemedium is a solid or liquid preparation used to grow, transport,and store microorganisms. To be effective, the medium mustcontain all the nutrients the microorganism requires for growth.Specialized media are essential in the isolation and identificationof microorganisms, the testing of antibiotic sensitivities,water and food analysis, industrial microbiology, and other activities.Although all microorganisms need sources of energy,carbon, nitrogen, phosphorus, sulphur, and various minerals, theprecise composition of a satisfactory medium will depend onthe species one is trying to cultivate because nutritional requirementsvary so greatly. Knowledge of a microorganismsnormal habitat often is useful in selecting an appropriate culturemedium because its nutrient requirements reflect its natural surroundings.Frequently a medium is used to select and grow specificmicroorganisms or to help identify a particular species. In such cases the function of the medium also will determine itscomposition.

Synthetic or Defined MediaSome microorganisms, particularly photolithotrophic autotrophs such as cyanobacteria and eukaryotic algae, can be grown on relatively simple media containing CO2as a carbon source (often added as sodium carbonate or bicarbonate), nitrate or ammonia as a nitrogen source, sulfate, phosphate, and a variety of minerals. Such a medium in which all components are known is a defined medium or synthetic medium. Many chemoorganotrophic heterotrophs also can be grown in defined media with glucose as a carbon source and an ammonium salt as a nitrogen source. Not all defined media are as simple as the examples in but may be constructed from dozens of components. Defined media are used widely in research, as it is often desirable toknow what the experimental microorganism is metabolizing.

Complex MediaMedia that contain some ingredients of unknown chemical composition are complex media. Such media are very useful, as a single complex medium may be sufficiently rich and complete to meet the nutritional requirements of many different microorganisms. In addition, complex media often are needed because the nutritional requirements of a particular microorganism are unknown, and thus a defined medium cannot be constructed. This is the situation with many fastidious bacteria, some of which may even require a medium containing blood or serum. Complex media contain undefined components like peptones, meat extract, and yeast extract. Peptones are protein hydrolysates prepared by partial proteolytic digestion of meat, casein, soya meal, gelatin, and other protein sources. They serve as sources of carbon, energy, and nitrogen. Beef extract and yeast extract areaqueous extracts of lean beef and brewers yeast, respectively. Beef extract contains amino acids, peptides, nucleotides, organicacids, vitamins, and minerals. Yeast extract is an excellent source of B vitamins as well as nitrogen and carbon compounds. Three commonly used complex media are (1) nutrient broth, (2) tryptic soy broth, and (3) MacConkey agar.If a solid medium is needed for surface cultivation of microorganisms, liquid media can be solidified with the addition of 1.0 to 2.0% agar; most commonly 1.5% is used. Agar is a sulfated polymer composed mainly of D-galactose, 3,6-anhydro-L-galactose, and D-glucuronic acid. It is usually extracted from red algae. Agar is well suited as a solidifying agent because after it has been melted in boiling water, it can be cooled to about 40 to 42C before hardening and will not melt again until the temperature rises to about 80 to 90C. Agar is also an excellent hardening agent because most microorganisms cannot degrade it. Other solidifying agents are sometimes employed. For example, silica gel is used to grow autotrophic bacteria on solid media in the absence of organic and to determine carbon sources for heterotrophic bacteria by supplementing the medium with various organic compounds.

Types of MediaMedia such as tryptic soy broth and tryptic soy agar are called general purpose media because they support the growth of many microorganisms. Blood and other special nutrients may be added to general purpose media to encourage the growth of fastidious heterotrophs. These specially fortified media (e.g., blood agar) are called enriched media.Selective media:It favors the growth of particular microorganisms. Bile salts or dyes like basic fuchsin and crystal violet favour the growth of gram-negative bacteria by inhibiting the growth of gram-positive bacteria without affecting gram-negative organisms. Endo agar, eosin methylene blue agar, and MacConkey agar three media widely used for the detection of E. coli and related bacteria in water supplies and elsewhere, contain dyesthat suppress gram-positive bacterial growth. MacConkey agaralso contains bile salts. Bacteria also may be selected by incubationwith nutrients that they specifically can use. A medium containingonly cellulose as a carbon and energy source is quite effectivein the isolation of cellulose-digesting bacteria. The possibilities for selection are endless, and there are dozens of specialselective media in use.Differential media are media that distinguish between differentgroups of bacteria and even permit tentative identificationof microorganisms based on their biological characteristics.Blood agar is both a differential medium and an enriched one. It distinguishes between hemolytic and nonhemolytic bacteria. Hemolytic bacteria (e.g., many streptococci and staphylococci isolatedfrom throats) produce clear zones around their colonies becauseof red blood cell destruction. MacConkey agar is both differential and selective. Since it contains lactose and neutral reddye, lactose-fermenting colonies appear pink to red in color and are easily distinguished from colonies of non-fermenters.

Thus the media prepared for growth of micro-organisms are:1. MacConkeys Agar:It is a selective and differential medium for cultivation of enteric micro organism from a variety of clinical specimens. The selective action for this medium is attributed to neutral red and bile salt which are inhibitory to more species of Gram positive bacteria. Gram negative bacteria usually grow well on the medium and can be differentiated by their ability to ferment the lactose.Lactose fermenting strains grow as pink colonies and may be surrounded by zone of a acid precipitated bile salt. The pink colour is due to production of acid by lactose absorption of neutral red and subsequent colour change of a dye when the pH of the medium falls below 6.8. Lactose non-fermenting strains such as shigella and salmonella are colourless and transparent and they do alter the appearance of the medium.

COMPOSITION:Peptone 20 gm/litSodium Taurocholate 5 gm/litLactose 10 gm/litSodium chloride 5 gm/litNeutral red 2% (w/v) in 50% ethanolDistilled water 1000 mlpH 7.2

ORGANISMS USED FOR MacConkeys Agar: Enterobacter aerogenes pink colony Escherishia coli pink colony Klebsiella pneumoniae pink colony Proteus species colourless colony Salmonella species colourless colony Shigella species colourless colony Staphulococcus aureus GETS INHIBITED

Incubation period: 370C for 1-2 days

2. Potato dextrose agar:PDA is common microbiological growth media made from potato infusion and dextrose. It is most widely used medium for growing fungi and bacteria which attack living plants or decaying dead plant matter.Potato infusion can be made by boiling 200gms unpeeled slice potatoes in 1 litre distilled water for 30 minutes and then decanting or staining the broth through cheesecloth. Distilled water is added such that the total volume of suspension is 1 litre. 20gms dextrose and 20gms agar powder is then added and the medium is then sterilised by autoclaving at 15 pounds per square inch (100kpa) for 15 minutes. Common organisms cultured on PDA are yeasts such as Candida albicans and Saccaromyces cerevisiae and molds such as A. niger.PDA is a general medium for yeasts and molds that can be supplemented with acid or antibiotic to inhibit bacterial growth .It is recommended for plate count methods. The nutritionally rich base potato infusion encourages mold sporulation and pigment production is some decmatophytes.

PRINCIPLE of the procedure:PDA is composed of dehydrated potato infusion and dextrose that encourage luxuriant fungal growth- agar is added as a solidifying agent. 10% Factaric acid is used to lower the pH of this medium.

COMPOSITION:Potatoes peeled and sliced 200 gmsDextrose 20 gmsDistilled water 1000 mlAgar 40 gms

Boil the potatoes for 30 minutes in water. Decant out the extract and make up the volume to 1 litre with water then add the agar constituents.

Result obtained would be:Yeasts will grow as creamy to white colonies Molds will grow as filamentous colonies of various colours.

Limitation of the procedure:Due to nutitional variation some strains may be encountered grow poorly or fail to grow on this medium.

Incubation period:(22-25)0C/ (30-32)0C for 2-7 days or longer

3. Luria Bertani medium:Luria Bertani medium subsequently became the medium of choice for growth of E.coliand other related enteric species.

COMPOSITION:Peptone 10gmsYeast extract10gmsNaCl10gms in 950ml deionized water Adjust pH of medium to 7.0 using 1N NaOH and bring volume upto 1 Litre. Autoclave on liquid cycle for 20 minutes at 15 psi.Allow solution to cool to 550C and add antibiotic if needed(50g/ml of ampicillin). Store at room temperature or 40C.

LB medium is a rich medium that is commonly used to culture members of the enterobacteriaceae as well as for coliphage plaque assays. LB is used extensively in rDNA work and other molecular biology procedures. Often an antibiotic is added to the medium for the selection of cells that contain a specific genetic element such as plasmid and transposon,or a gene disruption via an antibiotic resistance cassette.It is a nutrient rich media commonly used to culture bacteria in the laboratory. The addition of agar to LB results in the formation of a gel that bacteria can grow on;as they are unable to digest the agar but can gather nutrition from the LB within. The addition of an antibiotic to this gel allows the selection of only those bacteria with the specific antibiotic resistance usually conferred by a plasmid carrying the antibiotic resistant gene.LB media formulation have been an industry standard for the cultivation of E.coli. These media have been widely used in molecular microbiology application for the preparation of plasmid DNA and recombinant proteins.It continues to be one of the most common media used for maintaining and cultivating laboratory recombinant strains of E.coli for physiological studies. Peptides and peptones are provided by tryptone, vitamins and certain trace elements are provided by yeast extract.Sodium ions for transport and osmotic balance are provided by sodium chloride. Tryptone is used to provide essential amino acids to growing bacteria and yeast extract is used to provide organic compound helpful for bacterial growth.

25x M9 minimal media:M9 medium is a minimal growth medium used for bacterial cultures. It has the advantage of being cheap and has very low auto fluorescence and has very low absorbance.M9 medium can be supplemented to produce higher growth rates or to allow growth of strains that require additives. Eg: thiamine and casamino acids.

COMPOSITION: (gms/lit)25x M9 salt solutionNa2HPO4 188KH2PO4 75 +800ml D/W =1 litreNH4Cl 25NaCl 12.5

1M Cacl21M MgSO40.1% Thiamine 20% Glucose

The above solution was autoclaved at 121oC for 15 minutes. To prepare the M9 medium the individual components were mixed aseptically in the following order.

For 500ml M9 medium: 1X 2X

D/w 400ml 1M CaCl2 0.05ml 0.1ml1M MgSO4 5.0ml 10ml0.1% Thiamine 5ml 10ml25XM9 salt 20ml 40mlGlucose 5ml 10ml

ALGAE AGAR:Agar-agar and gelatine are products made primarily from the algae Geladium gracelaria (red seaweeds) best known as solidifying components of bacteriological culture media. Agar is isolated from algae as amorphous and translucent product sold as powder, flakes or bricks. Agar is insoluble in cold water. It absorbs as much as 20 times its own weight it dissolves readily in boiling water, a dilute solution is still liquid at 420C but solidifies at 370C into a firm gel. In the natural state agar occurs as a complex cell wall constituent containing a complex carbohydrate with sulfate and calcium.

ProtozoaEntaemoeba media:Composition: gm/litreLiver infusion from 272.00 gmsProteose peptone 5.50 gmsSodium beta glycerophosphate 3.00Sodium chloride 2.70Agar 11.00Final pH at 25o 7+ 0.2ProcedureSuspense 33gms in 1000ml of d/wHeat to boilingDispense in tubes and sterilise by autoclavingAllow tubes to solidify in slanted position, cover about half of the slant with fresh sterile horse serum saline mixture (1:6) and add a 5mm loopful of rice powder which has been sterilise in an oven at 160oC for 1 hr.

Reference:Hi manual Media and microbiology journal, 2nd editionIntroduction to Microbiology, Prescott, MacGraw Hill production, 2002