Cultivation Media For Bacteria
Cultivation
Media
1.
Bacteria
Actinomycetes medium
(Bennet’s agar)
Composition:
|
Glucose |
10.0
g |
|
Casein |
2.0
g |
|
Yeast
extract |
1.0
g |
|
Beef
extract |
1.0
g |
|
Agar |
20.0
g |
|
Distilled
Water |
1000
ml |
|
pH |
7.3
|
Preparation: Autoclave
the medium at 1210C for 20 min.
Use: Used for the cultivation of actinomycetes, specially Streptomyces species. This medium enhances sporulation.
Dextrose tryptone agar
Composition:
|
Glucose |
10.0
g |
|
Tryptone |
5.0
g |
|
K2HPO4 |
0.5
g |
|
NaCl |
0.5
g |
|
FeSO4.7H2O |
0.1
g |
|
Agar |
20.0 g |
|
Distilled
water |
1000
ml |
|
pH |
7.2
|
Use: Used for isolation of actinomycetes.
Glycerol yeast extract agar
Composition:
|
Glycerol |
10.0
g |
|
K2HPO4 |
1.0
g |
|
L-aspargine |
1.0
g |
|
Agar |
20.0
g |
|
Trace
salt solution(see oatmeal agar) |
1.0
ml |
|
Distilled
water |
1000
ml |
|
pH |
7.4
|
Preparation: Steam
sterilize the medium at 12 1°C for 20 min.
Use: Used for isolation of actinomycetes.
Oatmeal agar
Composition:
|
Oatmeal |
20.0
g |
|
Agar |
20.0
g |
|
Trace
salt solution |
1.0
ml |
|
Distilled
water |
1000
ml |
|
pH |
7.2
|
Pridham and Gottlieb trace salt solution
Composition:
|
CuSO4.5H2O |
0.64
g |
|
FeSO4.7H2O |
0.11
g |
|
MnCL2.4H2O |
0.79
|
|
ZnSO4.7H2O |
0.15
g |
|
Distilled
water |
1000
ml |
Preparation: Cook
the oatmenl in 1 lit. water for 20 min. Filter through cloth. Restore volume of
filtrate to 1 litre. Add I ml of trace salt solution. Adjust the pH. Add agar.
Steam sterilize the medium at 121°C for 20 min.
Potato-carrot agar
Composition:
|
Diced
potato |
150.0
g |
|
Diced
carrot |
30.0
g |
|
Tap
water |
1000
ml |
|
pH |
6.5
|
Preparation: Cook
the potato and carrot in 1 litre of boiling tap Mater for 30 min. Filter
through muslin and adjust the volume to 1 Litre. Adjust the pH and add agar.
Steam sterilize the medium at 121°C for 20 min.
Use: Used
for isolation of actinomycetes.
Carbon utilization medium-Shirling and Gottlieb
Composition:
|
(NH4)2SO4 |
2.64
g |
|
KH2PO4 |
2.38
g |
|
K,HP04 |
5.65
g |
|
MgS04.7H2O |
1.0
g |
|
Trace
salt solution |
1.00
ml |
|
Agar |
20.0
g |
|
Carbon
source(Sterilize separately by filtration) |
10.0
g |
|
Distilled
water |
1000
ml |
|
pH |
7.2
|
Preparation: Steam
sterilize the medium at 1210C for 20 min. Add sterile
carbon source solution after sterilization in the medium.
Use: Used to test carbon utilization ability of actinomycetes, important in identification.
Synthetic agar
Composition:
|
K2KPO4 |
1.0
g |
|
MgSO4.7H2O |
0.5
g |
|
KCl |
0.5
g |
|
FeSO4.7H2O |
0.01
g |
|
NaNO3 |
2.0
g |
|
Glycerol |
30.0
g |
|
Agar |
20.0
g |
|
Distilled
water |
1000
ml |
|
pH |
7.2
|
Preparation: Steam
sterilize the medium at 121°C for 20 min.
Use: Used for isolation of actinomycetes.
Yeast glucose agar
Composition:
|
Yeast
extract |
10.0
g |
|
Glucose |
10.0
g |
|
Agar |
20.0
g |
|
Distilled
water |
1000
ml |
|
pH |
7.2
|
Preparation: Steam
sterilize the medium at 121°C for 20 minutes.
Use: Used for isolation of actinomycetes.
Yeast extract-malt extract agar)
Composition:
|
Yeast
extract |
4.0
g |
|
Malt
extract |
10.0
g |
|
Glucose
|
4.0
g |
|
Agar
|
20.0
g |
|
Distilled
water |
1000 ml |
|
pH
|
7.3 |
Preparation:
Steam sterilize the medium at 1210C
for 20 minutes.
Use: Used for isolation of actinomycetes.
Actinomyces species
Composition:
|
Glycero
l5.0 |
15.0
g |
|
Sodium
propionate 4.0 |
4.0
g |
|
Sodium
caseinate 2.0 |
2.0
g |
|
K2HPO4 0.5
|
0.5
g |
|
Asparagine
0.1 |
0.1
g |
|
MgS04.7H2O
0.1 |
0.1
g |
|
FeS04.7H2O
0.001 |
0.001 |
|
Distilled water |
1000ml |
Acetobactor Medium
Composition:
|
Yeast extract |
10.0 g |
|
Glucose |
3.0 g |
|
CaCO3 |
10.0 g |
|
Agar |
20.0 g |
|
pH |
7.1 |
Preparation: Autoclave at 1210C for 20 min. Mix CaCO3 b: shaking the medium before pouring.
Use: Used for isolation of
Acetobacter and Gluconobacter.
Hoyer's medium
Composition:
|
(NH4)2SO4 |
0.1 g |
|
K2HPO4 |
0.01 g |
|
KH2PO4 |
0.09 g |
|
MgS04.7H2O |
0.025 g |
|
FeCl3.6H2O |
0.002 g |
|
Ethanol (15% v/v) |
20 ml |
|
Distilled water |
80 ml |
Preparation: Dissolve the salts in 80 ml of distilled water. Dispense as 4 ml aliquots in to test tubes and autoclave at 1210C. Ethanol may then be added as sterile filtered solution at the rate of 1 ml per tube.
Use: Used to cultivate Acetobacter. Acetobacter grow slowly, may take up to 14 days to develop.
Acetobacter broth
Compostion:
|
Peptone |
3.0 g |
|
Glucose |
18.0
g |
|
(NH4)2SO4 |
1.0 g |
|
Yeast extract |
2.0 g |
|
Solution A |
5 ml |
|
Solution B |
5 ml |
|
Distilled water |
1000 ml |
Solution A
|
K2HP04 |
50.0 g |
|
KH2PO4 |
50.0 g |
|
Distilled water |
500ml |
Solution B
|
MgSO4.7H2O |
20.0 g |
|
NaCl |
1.0 g |
|
FeSO4 |
1.0 g |
|
MnSO4 |
1.0 g |
|
Conc. HCL |
1.0 ml |
|
Distilled water |
500 ml |
Preparation: Steam sterilize at 1210C for 20 minutes.
Use: Used for cultivation of Acetobacter.
Acholeplasma Medium
Composition:
|
Papaic digest of soyabean meal |
10.0 g |
|
PPLO broth without crystal violet |
900.0 ml |
|
Fresh yeast extract solution (25.0 g Baker's yeast/100 ml water) |
100.0 ml |
|
Agar |
3.0 g |
|
pH |
7.8 |
PPLO broth without crystal violet
|
Beef heart, infusion
from |
225.0 g |
|
Peptone |
9.0 g |
|
NaCl |
4.5 g |
Preparation: Autoclave at 1210C for 20 min.
Use: Used for
cultivation of Acholeplasma.
Alcaligenes Medium
Composition:
|
K2HPO4 |
7.32 g |
|
CaCl2.2H2O |
0.014 g |
|
MgSO4.7H2O |
0.002 g |
|
FeSO4.7H2O |
0.04 g |
|
Ammonium tartarate |
4.6 g |
|
KH2PO4 |
1.09 g |
|
Distilled water |
1000 ml |
|
pH |
7.5 |
Preparation: Autoclave the medium at 1210C for 20 min.
Use: Used
for cultivation of Achromobacter and Alcaligenes speck.
Agrobacterium Isolation Medium
Composition:
|
Mannitol |
10.0 g |
|
Sodium nitrate |
4.0 g |
|
Magnesium
chloride |
2.0 g |
|
Calcium
propionate |
1.2 g |
|
Magnesium
phosphate. |
0.2 g |
|
Magnesium
sulphate |
0.1 g |
|
Sodium
bicarbonate |
0.075 g |
|
Magnesium
carbonate |
0.075 g |
|
Agar |
20.0 g |
|
Distilled
water |
1000 ml |
|
pH |
7.2 |
.
Preparation: Autoclave the constituents at 115°C for 20 min. Allow to cool to 50-550C and aseptically add the following:
|
Berberine |
275
ppm |
|
Sodium
selenit |
100
ppm |
|
Penicillin
G |
60
ppm |
|
Streptomycin
sulphate |
30
ppm |
|
Cyclohexamide
(85-100% active) |
250
ppm |
|
Tryothicin |
1
ppm |
|
Bacitracin
(65 units/mg) |
100
ppm |
Ammonification Medium
Composition:
|
KH2PO4 |
3.00 g |
|
KCl |
0.20 g |
|
MgSO4.7H2O |
0.20 g |
|
NaCl |
0.20 g |
|
CaSO4 |
0.10 g |
|
FeSO4 |
0.01 g |
|
Peptone |
10.00 g |
|
Distilled water |
1000 ml |
Preparation:
Steam sterilize the medium at 1210C
for 20 minutes.
Use:
Used to isolate ammonifying bacteria.
Bacteria are inoculated into tubes containing 5 ml of the above medium. After
10 days of incubation production of ammonias is testd by means of Nessler’s
reagent.
Anaerobic Bacteria Medium
Cooked meat broth (Robertson)
Composition:
Cooked
meat filtrate
|
Fresh bullocks heart |
500.0 g |
|
Water |
500 ml |
|
Sodium hydroxide (1 N NaOH) |
1.5 ml |
Mince the heart. Add in alkaline water Simmer for 20 min. Filter through muslin cloth. Dry minced meat, on filter paper partially.
Complete broth:-
|
Cooked meat
filtrate |
500 ml |
|
Peptone |
2.5 g |
|
NaCl |
1.25 g |
|
pH |
7.8 |
Preparation: Steam at 100°C for 20 min. Add 1 ml pure HCL and filter. Adjust the pH. Steam again at 100°C for 30 min. Adjust the reaction to pH 7.8.
Complete medium
Place the meat pieces in tubes to a depth of about 2.5 cm. Cover these pieces with about 10 ml broth. Autoclave at 1210C for 20 min.
Use: Used for isolation of anaerobic bacteria.
Note:
Before inoculation boil the tube vigorously in water bath to drive away
oxygen. Cool and inoculate. Put a layer of liquid paraffin if essential.
Anaerobic Broth:-
Clostridium:-
Composition:
|
Casein |
20.0 g |
|
Glucose |
10.0 g |
|
NaCl |
5.0 g |
|
Sodium thioglycollate
Sodium formaldehyde |
2.0 g |
|
Sulfoxylate |
1.0 g |
|
Methylene blue |
2.0 mg |
|
Distilled water |
1000 ml |
|
pH |
7.2 |
|
Sulfoxylate |
1.0 g |
Preparation: Distribute the broth in to tubes. Autoclave the medium at 1210C for 20 min.
Use: For cultivation of anaerobic
microorganisms especially Clostridium.
Thioglycollate medium
Composition:
|
Yeast extract (water solube) |
5.0 g |
|
Casein hydrolysate (Pancreatic digest) |
15.0 g |
|
Glucose |
5.5 g |
|
L-cysteine |
0.5 g |
|
Agar |
0.75 g |
|
Sodium chloride |
2.5 g |
|
Sodium thioglycollate |
0.5 g |
|
Methylene blue |
1.0 ml |
|
Distilled water |
1000 ml |
|
pH |
7.1 |
Preparation: Dissolve the ingredients except thioglycollate and indicator in water by heating. Add thioglycollate and adjust the pH. Filter. Add indicator solution. Sterilize at 121°C for 15 min. Cool and use.
Use: Used for isolation of anaerobic bacteria.
Note:
1.
If
the upper third is blue in colour, anaerobic condition should be stored by
boiling again for few minutes and cooling rapidly.
2. Resazurin sodium solution 1 in 1000, may be used replacing methylene blue.
Anaerobic Bacteria Medium
(Lapage et
al., 1970)
Composition:
|
K2HOP4 |
0.5 g |
|
NH4CL |
1.0 g |
|
Na2SO4 |
1.0 g |
|
MgSl4.7H2O |
2.0 g |
|
CaCl2.2H2O |
0.1 g |
|
Yeast extract |
1.0 g |
|
Ferrous sulphate (see below) |
10.0 ml |
|
Reducing agent (see below) |
10.0 ml |
|
Sodium lactate (70%) |
3.5 g |
|
Resazuri n (see below) |
1.0 ml |
|
Distilled water |
1000 ml |
|
pH |
7.4 |
|
Gas N2 : C02 |
80 : 20 |
|
|
|
Ferrous sulphate
|
FeSO4, 7H2O |
0.5 g |
|
Distilled water |
100 ml |
Reducing agent
|
Ascorbic acid |
1.0 g |
|
Sodium thioglycollate |
1.0 g |
|
Distilled water |
100 ml |
Redox
indicator (Resazurin)
|
Resazurin |
0.10 g |
|
Distilled water to |
100 ml |
Use: Used for the cultivation of Desulfomonas, Desulfobacter Desulfotomaculum and Desuvovibrio.
cellulomonas species
(Bagnra et at. 1985)
Composition:
|
K2HPO4 |
2.21
g |
|
KH2PO4 |
1.5
g |
|
(NH4)2SO4 |
1.3
g |
|
MgCl2 |
0.1
g |
|
CaCl2 |
0.02
g |
|
FeS04.7H2O |
0.001
g |
|
Yeast
extract |
5.0
g |
|
NaHCO3 |
0.8
g |
|
Cellulose |
10.0
g |
|
Cysteine
hydrochloride |
0.5
g |
|
Resazurin |
1.0
ml |
|
Distilled
water to |
1000
ml |
|
pH |
7.4 |
|
Gas
|
Argon
or Nitrogen |
Use: Used
for cultivation of cellulomonas species.
clostridiurn, Bacteroides Butyrivibrio and Bacterium
(Hungate, 1950)
Composition:
|
K2HPO4 |
0.5 g |
|
KH2PO4 |
0.2 g |
|
(NH4)2SO4 |
0.5 g |
|
CaCl, |
0.05 g |
|
NaCl |
1.0 g |
|
MgSO4 |
0.05 g |
|
Rumen fluid (see below) |
150.0 ml |
|
NaHCO2 |
5.0 g |
|
Resazurin (see page 51) |
1.0 ml |
|
Cysteine hydrochloride |
0.5 g |
|
Cellulose |
10.0 g |
|
Distilled water to |
1000 ml |
|
pH 7.5 |
7.5 |
|
Gas N2 : CO2 |
80 : 20 |
Rumen
fluid
Take 2000 ml fresh rumen fluid. Allow to settle down coarse particles. Decant supenatant to 1000 ml. Add 4.0 g yeast extract to it, pass nitrogen and incubate at 370C for overnight. Then centrifuge at 8000 rpm for 10 min at 6 to 8OC. Collect the supernatant in screw – capped bottle, pass nitrogen and store in refrigerator or -20°C. Pass nitrogen
on every use. Fermenting slurry of anaerobic digester may be processed in similar manner and used in place of rumen fluid.
Use: Used for cultivation of clostridiurn, Bacteroides Butyrivibrio and Bacterium species.
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