Purification of Azospirillum

Azospirillum :

• 1st isolated by Bejerinck in 1922.

• It is an aerobic/microaerophilic, helical/fibroid, motile and gram-negative bacteria.

• It is an associative symbiotic N fixing bacteria.

• Not like other N-fixers , it benefits non-leguminous crops also.

Materials required :

1. Freshly collected plant roots (preferably tender roots of paddy).

2. Sterile water blanks.

3. Dobereiner's N-free malic acid media (pH 6.6 TO 7).

4. 0.1% mercuric chloride or 70% ethanol (disinfector).

5. Sterile petri dish.

Dobereiner's N-free malic acid media composition :

1. Malic acid 5g

2. Potassium hydroxide 4g

3. Dipotassium hydrogen phosphate 0.6g

4. Potassium dihydrogen phosphate 0.4g

5. Manganese sulphate 0.01g

6. Magnesium sulphate 0.05g

7. Ferrous sulphate 0.5g

8. Sodium chloride 0.02g

9. Sodium molybdate 0.002g

10. Distilled water 1L

11. Bromo thymol blue (0.5% alcoholic solution - 2ml/L media)

12. Agar - 1.75 to 2 g (semi-solid media) / 20g (solid media)

Purification

• Media is prepared and sterilized using autoclave.

• The solid media is poured in petri plates inside the laminar air-flow chamber.

• After the media solidifies , the culture from the slands are streaked in the media.

• These are incubated for 31 degree C for 72 hours.

• After 3-4 days , the purified colony is obtained in the last streak.

Note : Daily observation for contamination should be done.