Cultivation Media Bacteria Part- 2

Cultivation Media 

Bacteria Part- 2

Azotobacter Medium

Nitrogen free glucose broth

Composition:

K2HPO4	1.0 g
MgSO4.7H2O	0.2 g
FeSO4.7H2O	0.05 g
CaCl2.2H2O	0.1 g
Na2MoO4.2H2O	0.001 g
Glucose	10.0 g
Distilled water to	1000 ml

Preparation: If this medium is to be used immediately, sterilization is not necessary. When it must be stored for long time, it should be sterilized.

If it is to be sterilized, the glucose should be sterilized separately in 100 ml of water at 115⁰C for 15 min. The remainder of the medium is steam sterilized at 121°C for 20 min.

After sterilization, the two solutions are mixed aseptically and dispensed in to sterilized bottles.

Use: Used for isolation of Azotobacter.

Nitrogen free glucose agar

Add 2.5% agar in above broth. Prepare the medium in similar manner.

Ashby's nitrogen free medium-modified

Composition:

Mannitol	15.0 g
MgSO4.7H2O	0.2 g
K2HPO4	0.2 g
Ferric chloride (10% aqueous solution)	0.5 ml
Molybdenum trioxide (10% aqueous solution)	1 drop
Agar	15.0 g
Distilled water	1000 ml
pH	7.2

Prepamtion: Steam sterilize at 121⁰C for 20 minutes.

Use: Used for the isolation of Azotobacter. Colonies appear flat, soft, milky and mucoid.

Ashby's mannitol agar

Composition:

Mannitol	20.0 g
K2HPO4	0.2 g
MgSO4.7H2O	0.2 g
NaCl	0.2 g
K2CO4	0.1 g
CaCO3	5.0 g
Agar	20.0 g
Distilled water to	1000 ml
pH	7.2

Preparation: Sterile CaCO₃ should be added after steam sterilization of remainder medium at 121⁰C for 20 minutes.

Use: Used for isolation of Azotobacter.

Burk's nitrogen free medium

Composition:

Burk's salt	1.3 g
Fe Mo mixture	1.0 ml
Sucrose	20.0 g
Distilled water	1000 ml

Burk's salt

MgSO4	20.0 g
K2HPO4	80.0 g
KH2PO4	20.0 g
CaSO4	13.0 g

          Fe Mo mixture

FeCl3	1.45 g
Na2NoO4	0.253 g
Distilled water to	1000 ml

Prepnration: Steam sterilize the medium at 11 5°C for 20 minutes.

Use: Used for isolation of Azotobactor.

Jensen's medium

Composition:

Sucrose	20.0 g
K2HPO4	1.0 g
MgSO4.7H2O	0.5 g
NaCl	0.5 g
FeSO4	0.1 g
Na2MoO4	0.005 g
CaCO3	2.0 g
Agar	20.0 g
Distilled water	1000 ml
pH	7.0

Preparation: Add all components except CaCO₃ and agar. Adjust the pH. Add agar. Sterilize at 115°C for 30 min. (not sterilize if freshly used). Add sterile CaCO₃ in melted sterile agar medium.

Use: Used for isolation of Azotobacter.

Azospirillum Semisolid Medium

Composition:

K2HPO4	0.1 g
KH2PO4	0.4 g
MgSO4	0.2 g
NaCl	0.1 g
CaCl2	0.02 g
FeCl2	0.01 g
Na2MoO4	0.002 g
Sodium malate	5.0 g
Bromothymol blue (0.05% Ehanol)	5.0 ml
Agar	2.25 g
Distilled water to	1000 ml
pH	6.8

Preparation: Steam sterilize the medium in autoclave at 121°C for 20 min.

Use: Used for cultivation of Azospirillum.

Azospirillum Medium

Composition:

K2HPO4	0.50 g
MgSO4.7H2O	0.10 g
NaCl	0.02 g
MnSO4.H2O	0.01 g
KOH	4.00 g
FeSO4.7H2O	0.05 g
Na2MoO4	0.002 g
CaCl2	0.01 g
Malic acid	5.0 g
Distilled water to	1000 ml
pH	6.0 : 7.0

Preparation: Steam sterilize the medium in autoclave at 121°C for 20 min.

Use: Used for cultivation of Azospirillum.

Bdellovibrio Medium

(Cation - supplemented yeast extract, peptone, Na acetate and cysteine agar1YPSC agar)

Composition:

Bacto yeast extract	0.1 g
Bacto peptone	0.1 g
Sodium acetate trihydrate	0.5 g
L-Cysteine hydrochloride	5.0 mg
CaCl2	0.002 .M
MgSO4	0.003 M
Distilled water	1000 ml
pH	7.6

Preparation: Autoclave at 121⁰C for 15 minutes.

Use: It is a medium of choice for plaque counts in most combinations of Bdellovibrio strains and associate bacterial strains

Beijerinckia Medium

(Becking's medium)

Composition:

Socrose	20.0 g
KH2PO4	0.8 g
K2HPO4	0.2 g
MgSO4.7H2O	0.5 g
FeCl3	0.1 g
Na2MoO4	0.005 g
Agar	20.0 g
Distilled water to	1000 ml
pH	6.5

Preparation: Steam sterilize at 121⁰C for 20 minutes.

Use: Used for isolation of Beijerinckia.

Bioluminescent Bacteria Medium

Basal medium

Composition:

(Tris) Hydrozyrnethyl amino methane	12.1 g
NH4Cl	1.0 g
K2HPO4	0.075 g
FeSO4.7H2O	0.028 g
Artificial sea water	1000 ml
(NaCl -23.4 g, KCl 1.5 g, MgSO4.7H2O -24.6 g, CaCl2 -2.9 g, D.W. -1 litre)
pH	7.2

Biochemicals should be prepared in the above basal medium as follows

Amino acids         0.5%

Sugars         1%

Preparation: Dissolve Tris. Adjust pH. Then dissolve remaining ingredients. Steam sterilize at 12 1 "C for 15 minutes.

Use: Basal medium is used for cultivation of bioluminescent bacteria.

Note: By adding 2% agar, prepare a solid medium.

Bordet Gengou medium

Composition:

Horse blood (defibrinated)	50.0 ml
Potato slices	125.0 g
Sodium chloride	4.5 g
Glycerol	10.0 ml
Proteose peptone	10.0 g
Agar	22.5 g
Distilled water to	1000 ml
pH	7.0

Preparation: Boil the slices in 250 ml water. Dissolve agar in 750 ml water and add to potato decoction, glycerol and peptone. Adjust the pH. Sterilize in autoclave at 115°C for 10 minutes. Add sterile defibrinated horse blood to the cooled agar. Mix and pour into sterile

Use: It is used for isolation of Bordetella.

Chacol blood agar

Charcol agar base

Composition:

Beef extract	10.0 g
Strach	10.0 g
Peptone	10.0 g
Charcoal (Bacteriological grade)	4.0 g
Yeast extract	3.5 g
Agar	15.0 g
Distilled water to	1000 ml
pH	7.4

Adjust the pH before addition of charcoal. Steam sterilize the medium 121°C for 20 min.

Complete medium

Charcoal agar base	100.0 ml
Horse blood (sterile, defibrinated)	10.0 ml
Penicillin (100 i.u./ml)	0.3 ml

Cool the charcoal agar base at 50°C. Add other ingredients aseptically. Pour plates.

Use: It is a selective medium of Bordetella pertussis.

Note: Some peptone brands are inhibitory to Bordetella, hence may be omitted from the medium.

Jones and Morgan medium

Composition:

Peptone	10.0 g
Agar	20.0 g
NaCl	5.0 g
Meat extract	5.0 g
Distilled water to	1000 ml
Agar	15.0 g
Distilled water to	1000 ml
pH	7.4

Steam sterilize the medium at 121⁰C for 20 min.

Complete medium

Basal medium Sterile inactivated	100 ml
Horse blood (sterile, inactivated	10.0 ml
Glucose solution 25% (sterile)	4.0 ml
Bacitracin solution (2000 units/ml in sterile water)	1.25 ml
Bacitracin solution (2000 units/ml in sterile water)	0.12 ml
Bacitracin solution (2000 units/ml in sterile water)	1.0 ml

(Dissolve in acetone dilute with sterile water)

Preparation: Cool the sterilized basal medium at 55⁰C. Add remaining solutions aseptically. Pour plates.

Use: Used for cultivation of Brucella.

Filde's eatract

Composition:

Defibrinated sheep blood	50.0 ml
Hydrochloric acid	6.0 ml
Pepsin	8.0 g
Normal saline	100 ml

Preparation: Heat at 65⁰C for 2 hrs. Add 20% NaOH, until a violet red colour develops with cresol red indicator. Then add pure HCL drop by until a definite red tint develops with phenol red.

Complete medium

Fildes extract	5.0 ml
Melted nutrient agar (Sterile)	100 ml

Use: Used for cultivation of Brucella.

Brucella selected medium

Composition:

Infusion from beef heart	500.0 g
Tryptone	10.0 g
NaCl	5.0 g
Glucose	2.5 g
Gelatin	1.0 g
Sheep blood	100.0 ml
Agar	15.0 g
Antibiotic solution	10.0 ml
Distilled water to	1000 ml
pH	7.4

 

Antibiotic solution

cycloheximide	1.0 g
Bacitracin	250,000 u
Circulin	250,000 u
Polymyxin B	100,000 u
Distilled water	100 ml

Filter sterilize the antibiotic solution.

Preparation: Add all components except blood and antibiotic solution in distilled water. Steam sterilizer at 121⁰C for 20 minutes.

Use: Used for selective isolation Brucella species.