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In this article we will discuss about the requirements and procedure for isolation of azotobacter species from soil.
Azotobacter is a free-living N2 fixer found in soil. They have relatively large size when observed under phase contrast microscope. They are peritrichous or has polar flagella and form thick-walled microcysts in some species. They are obligate aerobes and fix nitrogen when provided with a suitable carbohydrate or energy source.
Requirements:
1. Soil sample.
2. Ashby’s medium:
Dissolve mannitol, MgS04. 7H20, NaCl, K2S04 and CaC03 in 200 ml distilled water. Dissolve K2S04 separately in 100 ml distilled water (to prevent precipitation) in another flask. Mix both solutions and make up the volume to 1000 ml. Sterilize at 15 lbs (121°C) for 15 minutes and use.
3. 90 ml sterile water blanks.
4. Sterile graduated pipettes (5).
5. Sterile Petri dishes (4).
6. Magnetic shaker.
7. Bunsen flame.
8. Laminar clean air flow hood.
9. Inoculation loop.
10. Glass marking pencil.
11. Oven.
12. Balance.
13. Sieve 2 mm.
Procedure:
1. Pour Ashby’s medium into sterile Petri plates and allow them to solidify.
2. Sieve the soil through 2 mm sieve, weigh two 10 g samples, keep one sample in an oven over night at 150°C. Weigh this sample to find out the percentage of moisture in soil.
3. Add the other 10 g soil sample into the 90 ml water blank, shake for 20-25 minutes on the magnetic shaker.
4. Make serial dilutions of this sample though sterile water blanks as mentioned under bacteria.
5. Add 1 ml of each dilution on to the agar plates, rotate the plates for even spreading of inoculum and incubate at 28°C for 3-4 days.
Azotobacter colonies appear as flat, soft, mucoid and milky colonies.