Media for Biochemical Test Part - 2

Media for Biochemical Test Part - 2

Homo and Heterofermentation - Differentiation

Tomato juice-gelatin medium

Composition: 

Gelatin	120.0 g
Yeast hydrolysate	2.5 g
Tomato juice	100.0 g
Glucose	50.0 g
Distilled water	1000 ml
PH	7.0

Preparation: Sterilize glucose separately in 500 ml of water by filtration and remaining medium at 121°C for 30 minutes. Mix both the solutions aseptically.

Use: For differentiating homofermentative and heterofer-mentative lactic acid bacteria. Inoculate the tubes with culture. Mix it properly, chill the tubes in cold water to solidity the gelatin. Then seal with about I inch of melted nutrient agar. Incubate for 2-5 days at the temperature indicated and examine for gas production.

Hippurate Hydrolysis

Sodium hippurate broth

Composition:

 

Peptone	5.0 g
Beef extract	3.0 g
Sodium hippurate	10.0 g
Distilled water	1000 ml

 

Preparation: Dissolve the ingradients in distilled water. Distribute in tubes. Sterilize in autoclave at 121°C for min.

Use: To detect hippurate hydrolysis. Incubated broth is centrifuged and 0.8 ml clear supernate is taken into a kahn tube. 0.4 ml of ferric chloride solution [12g FeC136H2O dissolved in 100 ml of dilute (5.4 ml concentrated HC1 to 94.6 ml distilled water) HCI] is added in a kahn tube. Mixture is allowed to stand with occasional shaking for 10 minutes.

Positive test: A heavy cloudy, precipitate persisting after 10 minutes.

Negative test: Clearing of the initial precipitate within 10 minutes.

Hydrogen Sulfide Production

A. SIM agar (Sulfide indole motility agar)

Composition:

Peptone	30.0 g
Beef extract	3.0 g
Ferrous ammonium sulphate	0.2 g
Sodium thiosulphate	0.025 g
Agar	4.0 g
Distilled water	1000 ml
pH	7.2

 

Preparation: Dissolve all components indistilled water. Adjust the pH. Disribute in the tubes. Sterilize the medium at 121°C for 20 min. in the autoclave.

Use: This medium is used for detection of H,S, indole and motility. H2O producing organism cause blackening of the medium. H2S reacts with ferrous ammonium sulphate resulting black insoluble compound. ferrous sulphide. Indole production can be tested by the method prescribed in the illdole test. Motility is indicated by diffused growth from the inoculation point.

 

B. Peptone water 1

Composition:

Peptone	20.0 g
Sodium chloride	5.0 g
Distilled water	1000 ml
PH	7.2

 

Preparation: Dissolve all components in warm water. Adjust the pH. Autoclave at 121°C for 15 min.

Use: Used to test hydrogen sulphide production ability of bacteria. Inoculate the tube of peptone water by given organism. Dip the filter. paper in saturated solution of lead acetate. Dry the filter paper, sterilize and keep hanging in the inoculated tube of peptone water with the help of cotton plug. Incubate the tube.

Positive test: Filter paper turns black.

Negative test: No change in colour of filter paper.

C. Kligler's iron agar I

Composition:

Meat extract	3.0 g
Yeast extract	3.0 g
Peptone	20.0 g
Lactose	10.0 g
Glucose	1.0 g
Sodium chloride	5.0.g
Ferrio citrate	0.3 g
Sodium thiosulphate	0.3 g
Phenol red	0.05 g
Agar	12.0 g
Distilled water	1000 ml
PH	7.4

 

Preparation: Dissolve the ingredients in water. Dispense in about 5 min amounts in test tubes and sterilize them at 121°C for 15 minutes. Prepare slants with a butt.

Use: To detect H,S production and fermentation of lactose and glucose. Interpret the result like TSI agar.

D. Lysine iron agar

Composition:

Yeast extract	3.0 g
Peptone	5.0 g
L-lysine	10.0 g
Dextrose	1.0 g
Sodium thisulphate	0.04 g
Ferric ammonium citrate	0.5 g
Bromocresol purple	0.02 g
Agar	20.0 g
Distilled water	1000 ml

 

Preparation: Dissolve the ingredients by boiling in water. Dispense into tubes. Sterilize at 121°C for 20 minutes. Prepare slants with deep butts.

Use: To test production of H2O and lysine decarboxylase by Proteus.

(a) The production is detected by blackening of the medium.

(b) The lysine decarboxylase production is detected by development of red colour around the colony.

Indole Test

Durham's peptone water (Fermentation basal medium)

Composition: (See peptone water in hydrogen sulphide production test)

Use:

1. This medium is used chiefly as the basic for carbohydrate fermentation media.

2. It is also used fortesting the indole formation ability. Inoculate the tube with given bacteria. After incubation add few drops of xylene. Shake the tube vigourously. Add 2 to 3 drops of kovac's reagent along tlie side of the tube.

Note: 1. Use of tryptone water instead of peptone gives good results in indole formation.

2. All commercial peptones do not contain sufficient tryptophan for good indole production. Therefore the suitable brand of peptone must be selected for this purpose.

Lysozyme Sensitivity Test

Lysozyme broth

Composition:

A.   Glycerol broth

Peptone	5.0 g
Beef extract	3.0 g
Glycerol	70.0 ml
Distilled Water	1000 ml

Add all components in distilled water. Sterilize at 1210C for 20 minutes.

B.  Lysozyme solution

Composition:

Lysozyme	0.1 g
0.01 N HCI	100.0 ml

Add lysozyme to 0.01N HCI. Mix. Filter sterilize. This solution can be stored at 4°C for 7 days.

Preparation of complete medium

Lysozyme solution 0.5 ml

Glycerol broth 100 ml

Add aseptically and distribute in sterile tubes of 5 ml each.

Use: For differentiation of genera of actinomycetes based on sensitivity to lysozyme sensitivity actinomycetes do not grou in lysozyme broth. Growth can be compared by inoculating in glycerol broth.

e.g. Nocardia asteroids -- not sensitive to lysozyme

Streptomyces griseus -- sensitive to Iysozyme.

Lactate Fermentation

Lactate agar

Composition:

Trypticase	20.0 g
Yeast extract	5.0 g
Sodium lactate	12.0 g
Agar	25.0 g
Distilled water	1000 ml
pH	6.8

 

Preparation: Dissolve all components in distilled water except agar. Adjust the pH. Add Agar. Distribute in the tubes. Steam sterilize the medium at 1210C for 20 min.

Use: To test lactate fermentation ability of bacteria. Stab inoculates the tubes. Incubate. Lactate ferernentation is detected by gas production.

 

Lecithinase Production

A. Tween phosphate buffered substrate medium

Composition:

Phosphate buffer pH	7.0 /40.0 ml
Tween 80 (Polyoxyethylene sorbitainmonooleate)	0.2 ml
Neutral red(1 g/litre)	0.8 ml
pH	6.8 - 7.2

Preparation: Add neutral red solution to phosphate buffer and mix. Add tween 80 and mix gently. Distribute in 5 ml amounts in tubes and sterilize at 1210C for 20 minutes.

Use: It is used in tween80 hydrolysis test to detect lecithinase production.

 

B. Egg yolk agar

Composition:

1. Melted nutrient agar (sterile) 85 ml

2. Egg yolk suspension l5 ml

Preparation of egg yolk suspension:

Take fresh egg (less than four days old). Wash with water by brush and a plain alkaline soap. Rinse in running water for 10 min. Dry the egg by sprinkling methylated spirit and burning it off. Crack the, tapered end of egg with sterile knife. Separate the egg white from egg yolk. Collect the egg yolk in sterile container aseptically.

Preparation of complete medium:

Melt tlie agar. cool to 55°C and add tlie egg yolk. Pour plates.

Use: To test lecithinase activity of bacteria. Lecithinase production is shown by wide zones of opalescence around colonies more intense and larger than the zones caused by lipolysis.

C. TweenTM 80 hydrolysis

Composition :

Peptone	10.0 g
NaCl	5.0 g
CaCI2	0.1 g
TweenTM 80	10.0 ml
Agar	20.0 g
Distilled water	1000 ml
pH	7.2

 

Preparation: Autoclave at 1210C for 20 min.

Use: For testing the microorganisms for hydrolyzing TweenTM80 Colonies hydroluzing TweenTM80 are surrounded by an opaque zone.

Lipolysis Test

A. Fat agar

Composition:

Sterile melted nutrient agar 100 ml

Sterile melted butter (unsalted) 10 ml

Preparation: Mix thoroughly and pour plates.

Use: To study lipolytic activity of bacteria. Inoculate the centre of the solidified fat agar plate with bacterial suspension. Incubate for 5-7 days at given temperature. Flood the plates with a saturated aqueous solution of' CuSO4. After 20 minutes pour off the excess

solution. Bright greenish blue insoluble copper soap is formed with the fatty acids around the colony.

B. Egg yolk agar

composition:

(See lecithinase production)

Use: Used to test lipolytic activity of bacteria. Flood the date with copper sulphate solution. After 20 minutes pour off the excess solution. Bright greenish blue insoluble copper soap is formed with the fatty acids around the colony.

C. Tributyrin hydrolysis method

Composition:

Peptone	5.0 g
Yeast extract	3.0 g
Tributyrin (Glyceryl tributyrate)	10.0 g
Agar	20.0 g
Distilled water	1000 ml
pH	7.5

 

Preparation: The medium is prepared such that the tributyrin forms A stable emulsion in the nutrient agar and adjust the pH

Use: To test hydrolysis of tributyrin. An emulsion of microdroplets of the fat, tributyrin, in a solid medium makes it opaque. Lipolytic organisms remove the opacity by converting the fat to water soluble butyric acid.