APPENDIX I

Diluents employed for the preparation of standard antibiotic solutions

Antibiotic

Initial Solvent

Final Diluent

Cephalexin

B1

B1

Gentamicin

B2

B2

DMF

B4

Nystatin

(Dimethyl formamide)
Rifampicin

Methanol

B1

Streptomycin

Water

Water

Tetracycline

0.1N HCl

B3

Penicillin G

B1

B1

Kanamycin

Water

Water

B1

B2

K2HPO4

2.0g

K2HPO4

16.73g

KH2PO4

8.0g

KH2PO4

0.593g

Distilled Water

1.0 litre

Distilled Water

1.0 litre

pH

6.00.1

pH

8.00.1

B3

B4

KH2PO4

13.61g

K2HPO4

20.0g

Distilled Water

1.0 litre

KH2PO4

80.0g

pH

4.50.1

Distilled Water

1.0 litre

pH

6.00.1

163

APPENDIX II
All media were sterilized at 1210C for 20 minutes unless otherwise stated.
Antibiotics & B-vitamins were sterilized by filtration.
1. Potassium tellurite agar medium:
Potassium tellurite

0.1g

Peptone

5.0g

Yeast extract

2.5g

Agar

20.0g

Distilled water

1.0 litre

pH

6.4

Soluble starch

10.0g

Casein (Vitamin Free)

0.3g

KNO3

2.0g

MgSO47H2O

2.0g

K2HPO4

2.0g

MgSO47H2O

0.05g

CaCO3

0.02g

FeSO47H2O

0.01g

Agar

20.0g

Distilled water

1.0 litre

Nystatin

50.0mg

Benzyl Penicillin

0.8mg

2. Starch-casein agar medium :

164

3. Half-strength nutrient agar medium:

Oxoid nutrient agar granules

14.0g

Agar

10.0g

Distilled Water

1.0 litre

pH

7 to 7.2

Nystatin

50.0mg

Benzyl penicillin

0.8mg

Bacto-Tryptone (Difco)

5.0g

Bacto-Yeast extract (Difco)

3.0g

Distilled Water

1.0 litre

pH

7-7.2

Tripticase

15.0gm

Phytone

5.0gm

Sodium Chloride

5.0gm

Agar

15.0gm

Distilled Water

1.0 litre

4. ISP-1: Tryptone-yeast extract broth:

5. Tripticase Soyagar medium:

6. ISP-2: Yeast extract-malt extract agar (YEME agar):

Bacto-Yeast extract (Difco)

4.0g

Bacto-Malt extract (Difco)

10.0g

Bacto-Dextrose (Difco)

4.0g

Distilled Water

1.0 litre

pH

7.3

Bacto-Agar

20.0g

165

7. ISP-3: Oatmeal Agar:

Oatmeal

20.0g

Agar

18.0g

20g Oatmeal was cooked or steamed in 1000 ml Distilled Water for 20

minutes filtered through cheese cloth. Distilled water was added to restore
volume of filtrate to 1000 ml.
Trace salts solution

1.0ml

Agar

18.0g

pH

7.2

8. ISP-4: Inorganic salts-starch agar:

Solution I: Soluble starch 10.0g was added to small amount of cold
distilled water and prepared a paste and made up to volume of 500 ml.
Solution II:
K2HPO4 (anhydrous basis)

1.0g

MgSO47H2O

1.0g

NaCl

1.0g

(NH4)2 SO4

2.0g

CaCO3

2.0g

Distilled water

500ml

Trace Salts solution

1.0ml

pH

7.0 & 7.4

Mixed with starch suspension and salts solution.

Agar (Difco)

166

20.0g

9. ISP-5: Glycerol-asparagine agar:

L-asparagine (anhydrous basis)

1.0g

Glycerol

10.0g

K2HPO4 (anhydrous basis)

1.0g

Distilled Water

1.0 litre

Trace Salts Solution

1.0ml

pH

7.0-7.4

Agar

20.0g

10. ISP-6: Peptone-yeast extract-iron agar:

Bacto-Peptone iron agar,

36.0g

Bacto-Yast extract (Difco)

1.0g

Distilled water

1.0 litre

pH

7.0 7.2

dehydrated (Difco)

Bacto-peptone-iron agar dehydrated contained the following ingredients.

When reconstituted as 36.58g/litre of water:
Bacto-peptone

15.0g

Proteose peptone (Difco)

5.0g

Ferric ammonium citrate

0.5g

Dipotassium hydrogen phosphate

1.0 gm

Sodium thiosulphate

0.08g

Bacto-agar

15.0g

(K2HPO4)

167

11. ISP-9: Carbon utilization medium:

A) Sterile carbon source
B) Pridham and Gottilieb trace salts (only 1ml of this solution was used per litre
of final medium)
CuSO4 5H2O

0.64g

FeSO4 7H2O

0.11g

MnCl2 4H2O

0.79g

ZnSO4 7H2O

0.15g

Distilled water

100.0ml

C) Basal mineral salts agar (analytical reagent grade chemicals were used)
(NH4)2 SO4

2.64g

KH2PO4 (anhydrous)

2.38g

K2HPO4 3H2O

5.65g

MgSO4 7H2O

1.00g

Pridham and Gottlieb trace salts (B)

1.0ml

Distilled water

1.0litre

pH

6.8 7.0

Agar (Difco)

15.0g

D) Complete medium
The sterile basal agar medium (C) was cooled to 600C and sterile carbon
source was aseptically added to give a concentration of approximately 1%.

168

12. Gelatin agar:

Peptone

5.0g

Beef extract

3.0g

Gelatin Powder

4.0g

Agar

15.0g

Distilled water

1.0litre

pH

7.0

13. Skimmed milk:

Raw milk was centrifuged at 2000 rpm for 2 to 3 times until no fat is
observed on the surface. It is sterilized by steaming for 20 minutes on 3
successive days.
14. Milk casein agar:
Peptone

1.0g

Agar

20.0g

Sterile skimmed milk (10%):

100.0ml

Distilled water

1.0 litre

pH

7.6

Beef extract

3.0g

Starch soluble

2.0g

Agar

20.0g

Distilled water

1.0 litre

pH

7.6

15. Starch agar:

169

16. Nitrate broth:

Meat extract

3.0g

Peptone

5.0g

Potassium nitrate

1.0g

Distilled water

1.0 litre

pH

7.2

Meat extract

3.0gm

Peptone

5.0gm

Sodium chloride

5.0gm

Agar

15gm

Distilled water

1000ml

Yeast extract

1.0g

Beef extract

1.0g

Nz-amine A

2.0g

Agar

15.0g

Glucose

10.0g

Distilled water

1.0 litre

pH

7.3

17. Nutrient agar:

18. Benetts agar medium:

19. Potato dextrose agar (PDA):

Potatoes (peeled & sliced) :

200gm

Glucose (Dextrose)

20gm

Agar

20gm

Distilled water

1000ml
170

20. MR-VP broth:

Glucose

5.0gm

Peptone

5.0gm

Di-Potassium hydrogen

5.0gm

1.0 litre

Peptone

1.0gm

NaCl

5.0gm

K2HPO4

2.0gm

Phosphate
Distilled Water
21. Urea broth:

Phenolred (1:500mg Soln) :

6ml

Agar

20.0

10ml

Glucose (10% Sterile

Solution)

Urea (20% Sterile Solution): 100ml

Distilled water

1000ml

22. Simmon Citrate agar medium:

Magnesium Sulphate

0.2gm

Ammonium dihydrogen phosphate :

1.0gm

di-potassium hydrogen phosphate :

1.0gm

Sodium Citrate

2.0gm

Sodium Chloride

5.0gm

Bromothmnol blue

0.08gm

Distilled water

1.0 litre

171

23. Luria broth:

Pancreatic digest of casein

1.0gm

NaCl

5.0gm

Yeast extract

5.0gm

Glucose

10.0gm

Distilled water

1.0 litre

Dextrose

20gm

Peptone

10gm

Agar

15gm

Distilled water

1 litre

pH

5.6

24. Sabourauds dextrose agar

172

APPENDIX III
Details of the test organisms employed
A) Gram-positive bacteria:
B. megaterium

Bacillus megaterium NCIM 2054

B.subtilis

Bacillus subtilis NCIM 2063

Staph.aureus

Staphylococcus aureus NCIM 2079

B) Gram-negative bacteria:
E.coli

Escherichia coli NCIM 2685

Ps.fluorescens

Pseudomonas fluorescens NCIM 2639

P.vulgaris

Proteus vulgaris NCIM 2027

K. pneumoniae

Klebsiella pneumoniae NCIM 5082

C) Fungi:
A.niger

Aspergillus niger NCIM 1222

173