Cultivation Media Bacteria Part- 2
Cultivation Media
Bacteria Part- 2
- Azotobacter Medium
- Nitrogen free glucose broth
- Ashby's nitrogen free medium-modified
- Ashby's mannitol agar
- Burk's nitrogen free medium
- Jensen's medium
- Azospirillum Semisolid Medium
- Azospirillum Medium
- Bdellovibrio Medium
- Beijerinckia Medium
- Bioluminescent Bacteria Medium
- Bordet Gengou medium
- Chacol blood agar
- Jones and Morgan medium
- Filde's eatract
- Brucella selected medium
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
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 |
|
|
|
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.
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.
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.
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