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BACKGROUND
Rhizobium lives in soil and in the roots of legumes and non-leguminous plants. Bacteria exclusively depends on the root exudates that facilitates the growth of the rhizobial cells. Rhizobium are known to secrete some extracellular polysaccharides that assist to find the soil particles.
So, the basic objective of this experiment is to isolate the rhizobium bacteria from the root nodules of the leguminous plants.
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REQUIREMENTS
Sample: Legume Plant roots
Chemicals: 70% ethanol
0.1% mercurous chloride solution
Miscellaneous: Sterile distilled water
Pipettes
YEMA plates
Test Tubes
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PROCEDURE
Collect the root nodules
Carefully uproot some leguminous plants and then wash the root under running water for cleaning it from the soil particles. Usually the colour varies from brown to pink, based on the state of pigment present in them. It is always better to pick healthy, not broken pink nodules.1
Surface sterilization of the root nodules
Take a sterilizing agent and surface sterilize the nodules. After that wash the root nodules for removing any trace sterilizing agent prior to isolation. 0.1% mercuric chloride or 3-5% hydrogen peroxide can be used as sterilizing agent. For complete sterilization immerse the nodules in the sterilizing agent for about 4-5 minutes and then wash it with distilled water. After that again wash it with 70% ethanol and again with sterile water.2
Isolation
Wash the nodules in small aliquots of distilled water. Prepare YEMA plates and autoclave it. Then prepare 10-fold dilution of the nodular extract by taking 1 gm of the nodular extract and add it to 10ml of distilled water. Then mix it well for getting the nodular extract suspension.3 Then similarly, 1ml of the nodular extract is added to 9ml of sterile water for making dilution 10-2 and similarly repeat up to 10-8. Then 0.1 ml of suspension from 10-3 to 10-4 nodular extract are inoculated to sterilized YEMA plates by spread plate method. Then incubate the plate in incubator at 37oC for about 4-7 days.4
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CONCLUSION
Rhizobium colonies are seen as large, mucoid and elevated colonies.
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REFERENCES
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Deka AK, Azad P. Isolation of Rhizobial strains cultural and Biochemical Characteristics. Legume Res. 2006;29(3):209-12.
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Kayode AAA, Kayode OT. Some medicinal values of Telfairia occidentalis: A review. Am J Biochem Mol Biol. 2011;1:30-8.
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Bamshaiye OM, Adegbol JA, Bamishaiye EI. Bambara groundnut: an Under-Utilized Nut in Africa. Adv Agricul Biotechnol. 2011;1:60-72.
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Wikipedia, “Peanut,” Available at: https://en.wikipedia.org/wiki/Peanut. Accessed 16 July 2018.
