The isolation of Azotobacter can be done by the soil dilution plating method. In the experiment 10grams of soil samples are transferred to 100 ml sterile distilled water and it is mixed thoroughly by continuous shaking the flask for five minutes. Then serial dilution of the suspensions is made using the sterile distilled water. Then a nitrogen free agar media specific for Azotobacter is prepared and poured into the sterile petri plates. Then 0.1ml samples from the appropriate dilution are spread evenly over cooled agar medium of the petri plates. These plates are then incubated at 30°C in an incubator for 3-4 days. The colonies begin to develop which appears as flat, soft, milky and mucoid on agar plates.
The isolation of the Azotobacter from the soil can also be done direct method. That is lumps of soil are spread on N-free agar medium and the plates are incubated at 28°C. Here also the colonies develop after three days of incubation.