Media for Biochemical Test Part-1

Amino acid Decarboxylation Test

A. Moeller's medium

Composition:

Peptone	5.0 g
Meat extracts	5.0 g
D --- Glucose	5.0 g
Pyridoxal	0.005 g
Bromcresol purple ( 1 in 500 solution)	5.0 ml
Cresol red ( 1 in 500 solution)	2.5 ml
Distilled water	1000 ml
PH 6.0	6.0

 

Preparation: Dissolve all in gradients except indicators in water and adjust the pH. Add indicators. Add 1 per cent amino acid (di or mono) Hydrochloride whose decarboxylation is to be tested. (L-lysine, L-ornithine or. L-arginine hydrochloride). Distribute 1 ml in each tube.

Add sterile liquid paraffin to provide a layer about 5 mm think above the medium. Autoclave at 1210 C for 15 min.

Use: To test amino acid decarboxylation ability of bacteria. Inoculate the tubes and incubate. After incubation positive test is indicated by violet colour of the medium. The control remains yellow. 

B. Falkow’s medium:-

Composition:

Peptone	5.0 g
Yeast extract	3.0 g
D - Glocose	1.0 g
0.2% bromocresol purple	10 ml
Amino acid (L-LysineiL-ornithinek-arginine)	5.0 ml
Distilled water	1000 ml
PH	6.7

Preparation: Dissolve all ingredients in water except bromocresol purple. Adjust the pH. Add the indicator. Autoclave at 12 1 O C for 15 min.

Use: To test amino acid decarboxylation ability of bacteria. The medium first becomes yellow due to acid production. Later violet colour of the medium appears due to decarboxylation. The control remains yellow. 

Amylase Production

Starch agar

Composition:

Starch (soluble)	20.0 g
Peptone	5.0 g
Beef extract	3.0 g
Agar	20.0 g
Distilled water	1000 ml
pH	7.0

Preparation: Dissolve all in-gradients in distilled water. Adjust the pH. Add agar. Steam sterilize the medium for one hour at 100°C.

Use: To study the starch hydrolysis by microgranisms. After incubation, flood the plate with Gram's iodine. Amyl lytic colony will be surrounded by clear zone against purple coloured background. 

Casein Hydrolysis

Milk agar

Composition:

Nutrient agar (sterile)	875.0 ml
Skimmed milk (sterile)	125.0 ml

Preparation: Sterilize the nutrient agar and milk separately at 121°C for 30 minutes. Cool the agar at 50°C. Add cooled milk aseptically in the melted nutrient agar.

Use: To set casein hydrolyzing ability of microorganisms. Casein hydrolytic colonies will develop clear zones around the colony.

Chitin Hydrolyse

Composition:

Chitin	4.0 g
K2HPO4	0.7 g
MgSO4.7H2O	0.5 g
KH2PO4	0.3 g
FeSO4.7H2G	0.01 g
MnCl2.4H2O	0.001 g
ZnSO4.7H2O	0.001 g
Agar	20.0 g
Distilled water	1000 ml
pH	7.0

Preparation: Autoclave the medium 1210C for 20 minutes.

Use: For the isolation of chitin lytic bacteria.

Note: Adjust the pH 8.0 and use the medium for isolation of actinomycetes.

Citrate Utilization Test

A. Citrate broth (Koser's modified)

Composition:

NaCl	5.0 g
MgSO4.7H2O	0.2 g
NH4H2PO4	1.0 g
KH2PO4	1.0 g
Na3C6H5O7.2H2O (Sodium citrate)	5.0  g
Distilled water	1000 ml
pH	6.8

Preparation: Dissolve all ingredients in distilled water. Adjust the pH. Distribute in the tubes and steam sterilize at 12 1 "C for 15 minutes.

Use: To test the ability of an organism to utilize citrate as the sole carbon and energy source for growth. Positive test is indicated by the presence of turbidity in the broth after incubation. Observe the turbidity by comparing uninoculated control tube.

B. Citrate agar

(Simmon's citrate medium)

Composition:

Koser's (modified) broth	1000 ml
Bromothymol blue (0.2%)	400 ml
Agar	20.0 g

Preparation: Add the indicator in the broth. Dissolve agar. Distribute in the tubes, steam sterilize at 12 1°C for 15 min. and allow to set as slants.

Use: To test the ability of an organism to utilize citrate as the sole carbon and energy source for growth. Positive test is indicated by the blue colour of medium after incubation. Colour of original medium is green.

DNase test

DNase agar

Composition:

Casein (pancreatic digest)	15.0 g
NaCl	5.0 g
Soybean meal (papaic digest)	5.0 g
Deoxyribonucleic acid	2.0 g
Agar	20.0 g
Distilled water	1000 ml
PH	7.3

Preparation: Add all comporients in distilled water by gentle heating. Steam sterilize at 1180C for 15 min. Pour plates.

Use: Spot inoculate the medium heavily by suspension of test organism. Incubate for 24 hrs. flood the plate with IN HCI. A clear halo around the colony indicate positive test. 1 N HCI precipitates unchanged deoxyribonucleic acid. 

Esculin Hydrolysis

Bile esculin medium

Composition:

Peptone	5.0 g
Beef extract	3.0 g
Bile (oxgall)	40.0 g
Esculin	1.0 g
Ferric citrate	15.0 g
Agar	15.0 g
Distilled water	1000 ml
PH	7.0

Preparation: Dissolve all ingredients in distilled water. Distribute into tubes, sterilize in autoclave at 12 1 OC for 15 min. and prepare slants.

Use: To set esculine hydrolyzing ability of bacteria. Esculin hydrolysis is indicated by causing the slant to blacken after incubation.

Note: This test is usually applied for indentifiecation of group D streptococci. Both enterococcal and non enterococcal species of group D are able to hydrolyse esculin.

Eijkman Test

MacConkey's broth 

Composition:

Peptone	5.0 g
Sodium taurocholate	3.0 g
Neutral red solution (2% in 50%ethanol)	40.0 g
Lactose (10% aqu. solution)	1.0 g
Distilled water	1000 ml
PH	7.5

Use: This test in used for identification of Escherichia coli. Inaculate MacConkey's broth with test organism. Incubate at 44* 0.2OC for 24 hrs. E. coli produce gas at this temperature.

Gelatin hydrolysis (Gelatin liqueefaction test)

Gelation agar:

Composition:

Gelatin	4.0 g
Glucose	0.05 g
KH2PO4	0.5 g
K2HPO4	1.5 g
Nutrient agar (melted)	1000 ml

Preparation: Dissolve the phosphates, gelatin and glucose in melted nutrient agar slowly for even distribution of the gelatin. Steam sterilize in a flask at 121⁰C for 30 min. Prepare plates.

Use: To test gelatin hydrolyzing ability of microorganisms. Flood the plate containing growth of the microorganisms with Frazier's solution. Gelatin hydrolysis is indicated by clear zone around colony against opaque background.

Gluconate Test

Composition:

Peptone	1.5 g
Yeast extract	1.0 g
K2HPO4	1.0 g
Potassium gluconate	40.0 g
Distilled water	1000 ml
pH	7.0

Preparation: Dissolve all components in water. Adjust the pH. Distribute in 1 rnl quantities in each tube and steam sterilize at 121°C for 15 min.

Use: used to test the ability of an organism to oxidize gluconates to the 2 keto-gluconate. Inoculate the tube with bacterial suspension and incubate at 370C for 48 hours. Then add 1 ml of qualitative benedict's solution and heat the tube for 5-10 minutes. Observe the change in colours. Benedict's reagents is blue in colour. Positive test is indicated by development of green to orange coloured precipitate.