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Techniques in Microbiology: Bacteriological Media

World Health Organization

The role of suitable quality culture media for cultivation of microorganisms cannot be over emphasised. On it depends the very success of isolation of aetiological agents. Only in exceptional cases, can an organism be identified on the basis of its morphological characteristics alone.

Types of media

Bacteriological media can be broadly sub-divided into four categories.

1. Ordinary culture media

These are routinely employed in a laboratory e.g. nutrient broth, nutrient agar, infusion broth and lysate media.

2. Enriched media

Certain organisms do not grow on ordinary nutrient media. They require growth- promoting ingredients such as blood, glucose, serum, egg, etc. The media containing ingredients which enhance their growth-promoting qualities are enriched media e.g. blood agar, chocolate agar and Loeffler medium.

3. Enrichment media

Enrichment media are liquid media containing chemical constituents which inhibit some normal flora and allow pathogens which may be present in very small number in the specimen, to grow unhampered and thus enriching them. Isolated colonies of these organisms may be obtained by subculturing onto solid media. An example of enrichment media is selenite broth used for primary isolation of enteric bacteria.

4. Differential and selective media

Differential media have got some chemical constituents which characterize different bacteria by their special colonial appearances in the culture e.g. MacConkey agar contains lactose as a substrate and neutral red as an indicator. Bacteria fermenting lactose produce acid and this will change the colour of the indicator and thus the colonies will turn red. The red lactose fermenting colonies can be differentiated from the pale non-lactose fermenting colonies.

Selective media will selectively permit the growth of pathogens and inhibit the commensals. In addition, it may differentiate the pathogen from commensals that grow by the colour and opacity of the colonies e.g. blood tellurite medium for C.diphtheriae.

In addition, transport media are also frequently used to sustain the viability of organisms when a clinical specimen is to be transported from the periphery to laboratory. The transport medium prevents the outgrowth of contaminants during transit and sustains the pathogen. Cary and Blair and Stuart media are two examples of this group of media.

Preparation of media and checking of pH

Presently, a wide range of culture media are available commercially in the form of dehydrated media. These media are simply reconstituted by weighing the required quantities and by adding distilled water, as per the manufacturer’s instructions.

The pH determination can be conveniently done with the use of Lovibond comparator with phenol red indicator disc.

• Take two clean test tubes and add 5 ml of the medium to each of the tubes. One serves as a blank while phenol red indicator is added to the other tube.
• Compare the colour of the medium with the phenol red indicator at the appropriate pH marking.
• Add N/10 NaOH or N/10 HCl, drop by drop till the colour of the medium matches the colour of the
• disc at the required pH reading.
• Calculate the volume of the NaOH or HCL of 1/10 strength for 5 ml of the medium to get the required pH.
• Based on the calculation, the volume of 1N NaOH or IN HCl required for the total volume of medium can be calculated and added.
• Check the pH of the medium once again before use.

The quantity of agar given in the formulae of media may have to be changed depending upon the quality of agar used. The concentration varies from batch to batch and should be such that will produce a sufficiently firm surface on solidification. This can be tested by streaking with inoculating wire.

In some laboratories media are prepared by individual measurement of ingredients and then mixing the same. Hence the method of preparation is given likewise:

Nutrient broth

Meat extract 10.0 gm
Peptone 10.0 gm
Sodium chloride 5.0 gm
Distilled water 1000 ml

Mix the ingredients and dissolve them by heating in a steamer. When cool, adjust the pH to 7.5-7.6.

Nutrient agar

To the ingredients as in nutrient broth, add 15 gm agar per litre. Dissolve the agar in nutrient broth and sterilize by autoclaving at 121°C for 15 minutes. Prepare plates and slopes as required.

Glucose broth

Nutrient broth 900 ml
Glucose (10% solution) 100 ml

• Dissolve 9 gm glucose in distilled water and sterilize by tyndallisation.
• Add l00 ml of the glucose solution to 900 ml of sterile nutrient broth.
• Dispense 60 ml each in 100 ml pre-sterilized culture bottles.
• Sterilize by open steaming at l00°C for one hour.

Blood agar

Nutrient agar 100 ml
Sheep blood (defibrinated) 10 ml

• Melt the sterile nutrient agar by steaming, cool to 45°C.
• Add required amount of sheep blood aseptically with constant shaking.
• Mix the blood with molten nutrient agar thoroughly but gently, avoiding froth formation.
• Immediately pour into petri dishes or test tubes and allow to set.

Chocolate agar

The ingredients are essentially the same as in blood agar.

• Melt the sterile nutrient agar by steaming and cool to about 75°C.
• Add blood to the molten nutrient agar and allow to remain at 75°C after gently mixing till it is chocolate brown in colour.
• Pour in petri dishes or test tubes for slopes as desired.

XLD agar

Xylose 3.5 gm
1 — lysine 5.0 gm
Lactose 7.5 gm
Sucrose 7.5 gm
Sodium chloride 5.0 gm
Yeast extract 3.0 gm
Sodium desoxycholate 2.5 gm
Sodium thiosulphate 6.8 gm
Ferric ammonium citrate 0.8 gm
Phenol red 0.08 gm
Agar agar 15.0 gm
Water 1000 ml

Weigh the ingredients into a flask and add distilled water. Mix the contents well and steam it for 15 minutes (do not autoclave). Cool to 56°C and pour in plates.

Buffered glycerol saline

Glycerol 300 ml
Sodium chloride 4.2 gm
Disodium hydrogen phosphate 10.0 gm
Na2 H PO4 Anhydrous 15.0 gm
Phenol red aqueous solution 0.02 per cent 15.0 ml
Water 700 ml

• Dissolve NaCl in water and add glycerol.
• Add disodium hydrogen phosphate to dissolve.
• Add phenol red and adjust pH to 8.4.
• Distribute 6 ml in universal containers (screw -capped bottles of 30 ml capacity). Autoclave at 115°C for
• 15 minutes.

Loeffler serum medium

Nutrient broth 100 ml
Serum (sheep or horse or ox) 300 ml
Glucose 1.0 gm

• Dissolve glucose in nutrient broth and sterilize at 121°C for 15 minutes.
• Add serum aseptically.
• Mix thoroughly but gently, avoiding froth formation.
• Distribute in sterile test tubes or quarter ounce screw-cap bottles.
• Inspissate the medium in a slanting position in a water inspissator at 82°C for two hours.
• In the absence of an inspissator, the medium may be coagulated by standing over the top of a steam sterilizer for 6-7 minutes.

Blood tellurite agar

Agar base

Meat extract 5.0 gm
Peptone 10.0 gm
Sodium chloride 5.0 gm
Agar 25.0 gm|
Water 1000 ml

Dissolve the ingredients and adjust the pH to 7.6. Distribute in 100 ml quantities in a bottle and autoclave at 121°C for 15 minutes.

Glycerolated blood tellurite mixture

Sterile defibrinated sheep blood 14 ml
Sterile glycerol 6 ml
Sterile potassium tellurite solution
(1% in water) 4 ml

• Sterilize the glycerol in hot air oven at 160°C for 60 minutes and the tellurite solution by autoclaving at
• 115°C for 20 minutes. Mix the ingredients in a sterile flask, incubate for 1-2 hrs. at 37°C, then refrigerate.
• Haemolysis is complete after 24 hrs. The mixture keeps well in a refrigerator. One per cent solution of
• good quality tellurite is sufficient but 2% of some batches may be required.

Preparation of complete medium

Glycerolated blood tellurite mixture 24 ml
Agar base 100 ml

Melt the agar, cool to 45°C, add blood and tellurite and pour in sterile petri dishes.

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