Isolation of RHIZOBIUM

Isolation of RHIZOBIUM from soil / root nodules

Rhizobium are gram negative and aerobic bacteria that symbiotically form nodules with roots of leguminous plants. Rhizobia grow well on YEMA (yeast extract mannitol agar) and produce whitish colonies. Most of them produce extracellular polysaccharides. To confirm the isolates plant infection test is carried out to observe nodulation. From 750 genera and 19000 species of leguminous plant about 15 % plants have been examined for nodulation and less than 0.5% have been studied for their symbiotic relationship with nodule bacteria. They fix atmospheric nitrogen and render it into combined forms resulting in high amount of proteins in roots. The proteins are transported along the plants and also secreted in rhizosphere region. In recent years, rhizomes are used as biofertilizers for selected crops.

Requirements

1. Root nodules
2. YEM (yeast extract mannitol) agar medium
3. Test tube with nylon mesh
4. Petri dishes
5. 0.1% acidification HgCl2 (1g HgCl2, 5ml conc. HCl, 1 litre distilled water)
6. Sterile tap water
7. Nichrome blade
8. Plates containing YEM agar medium

Procedure

1. Procure healthy root nodules of a young leguminous plant by cutting with a blade.
2. Wash the nodules thoroughly first with tap water and then with sterile distilled water keeping over the nylon mesh under aseptic conditions so as to remove contaminants and adhering soil particles.
3. Thereafter, Immerse them in 0.1% acidification HgCl2 for 5 minutes.
4. Transfer nodules in a sterile beaker containing 10ml of 95% ethanol and wait for 2-3 minutes.
5. Wash the nodules thoroughly for 5 times with sterile tap water, and blot dry by using sterile blotting paper.
6. Aseptically crush the nodules with glass rod or dissect the nodules by using Nichole blade and prepare dilutions.
7. Pour 1 ml suspension on YEM agar plates.
8. Incubate the inoculated plates at 28 C for 48 hours. Thereafter, observe the bacterial colonies which are gummy, translucent or white opaque.
9. Pick up a discrete colony and streak on a second YEM agar plates for better separation.

Reference

Dr. R. C. Dubey – practical microbiology