Isolation of Azotobacter

 Isolation of Azotobacter 

Aim

To isolate Azotobacter species from soil

Principle

The free living bacteria having the ability to fix molecular nitrogen can be distinguished into obligate aerobic and facultative anaerobic organisms. Members of the genus Azotobacter of the family Azotobacteriaceae, are heterotrophic obligate aerobic nitogen fixers. Azotobacter species are Gram- negative, aerobic soil-dwelling bacteria. They are grouped into three genera namely Azotobacter, Beijerinckia and Derxia. Several species of Azotobacter are recognized in neutral and alkaline soils, in water, and in the rhizosphere of some plants. Azotobacter chroococcum, Azotobacter agilis, Azotobacter vinelandii are some common examples.

Azotobacter species are large, usually oval, but may appear in different shapes from rods to spheres, Gram negative. The size of the cells ranges from 2-10 µm long and 1-2 µm wide. They are typically polymorphic, i.e. of different sizes and shapes. In microscopic preparations, the cells can be dispersed or in irregular clusters or chains of varying lengths. In fresh cultures, cells are motile due to peritrichous flagella. Later, the cells lose their mobility, become almost spherical, and produce a thick layer of mucus, forming the cell capsule. Azotobacter are free-living in soil and water. The nitrogen fixing enzyme, nitrogenase is oxygen-sensitive. The high respiration rate of Azotobacter uses up free oxygen within the cells and protects the nitrogenase. Azotobacter species are relatively easy to isolate from soil by growing on nitrogen free media, where the bacteria are forced to use atmospheric nitrogen gas for cellular protein synthesis. Cell proteins are mineralized in soil after the death of the Azotobacter cells, enhancing the nitrogen availability of the crop plants.

Azotobacter species can be used as an important biofertilizer. Inoculation of soil or seeding with Azotobacter is effective in increasing crop yield. It is also known to synthesize biologically active substances such as vitamins, IAA, and Gibberellins in pure culture. Fungistatic properties against certain pathogenic fungi such as Alternaria, Fusarium species etc. is also reported. These attributes explain the observed effects of Azotobacter in improving seed germination and plant growth.

Materials Required

Soil sample, Jensen’s medium, 10 ml and 9 ml sterile water blanks, Sterile 1 ml pippete, sterile petridishes, Bunsen burner, glass marking pencil

Procedure

1. Jensen’s medium was prepared and sterilized by autoclaving at 121⁰C 15 lbs per inch² pressure for 15 min.

2. 1 gm of sieved soil was added to 10 ml sterile water blanks and shaken for 15-20 min.

3. Serial dilution was performed to obtain 10^-2, 10^-3. 10^-4, 10^-5 dilutions.

4. 1 ml aliquots of various dilutions were pipetted onto sterile petriplates and pour plated by adding molten medium at bearable warmth.

5. The plates were rotated for uniform distribution and incubated at 28⁰C for 3 days.

Observation

The plates were observed after 3 days incubation for the appearance of colonies on the agar surface.

Result

On Jensen’s medium, Azotobacter colonies appeared as milky and mucoid. On microscopic observation, short rods of Azotobacter were confirmed.