Isolation of symbiotic N2-fixing bacteria from root nodules from leguminous plants

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Rhizobia are the bacterium that infects the roots of leguminous plants and form nodules where it fixes nitrogen. Living bacteria are isolated from the root nodules for identifying the bacteria. Its identification is important for studying the symbiotic properties like host range specificity and nodules competitiveness. The process of isolation mainly includes handling of each nodule by sterilizing the surface of the nodule and then disrupting the nodule to release the bacteria. The disruption is carried out by cutting, stabbing and crushing each nodule.

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Samples:      Root of leguminous plants

Chemicals: Glycerol

Sodium hypochlorite

Mannitol broth


YM agar plate


Others:       Forceps

Microfuge tube

Vortex mixture

Microtitre plate

Multiple crushers

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First step is to store and sterilize the nodules. Rinse the root of the plants in tap water for removing any adhering soil.1 Then remove at least 5-10 nodules with the help of forceps and place it in microfuge tube with 1.3ml of glycerol. Then agitate this microfuge tube vigorously with the help of a vortex mixture for about 5s and then store it at -4oC.2 After 1-10 weeks thaw the tubes, thaw the tubes and then transfer the nodules to a tube having 1.3 ml of 2% sodium hypochlorite. Agitate the tube with the help of vortex mixer for about 1 min and then transfer the nodules to 15 ml of sterile water.3 Now take a 96- well microtitre plate and fill it with 120 tzl of yeast mannitol broth in every well and place the nodules in it. Now take a multiple crusher that is designed for being used with a 96 well microtitre plate.4 The apparatus is rinsed with acetone for removing any coating of manufacturers. Then dip it in ethanol, sterilize it with flame and position it and rock it gently for crushing the nodules.5 Those nodules that miss crushing in apparatus were crushed with the help of flame sterilized aluminium rod. Now carry the bacterial suspensions into YM agar plate.6 Then to plates having indicator antibiotics or the dye calcofluor. In such a way place isolate of half of the microtitre plate on agar plate.7

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This method can also be used by modifying for other technique of modification like identifying by phage sensitivity, hybridization to labelled DNA probes and binding of fluorescent antibodies.

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  1. El Hassan, G. A., Hernandez, B. S. and Focht, D. D. (1986) Comparison of Hup trait and intrinsic antibiotic resistance for assessing rhizobial competitiveness axenically and in soil. Appl. Environ. Microbiol. 51, 546-551.
  2. Brewin, N. J., Wood, E. A. and Young, J. P. W. (1983) Contribution of the symbiotic plasmid to the competitiveness of Rhizobium leguminosarurn. J. Gen. Microbial. 129, 2973-2977.
  3. Bromfield, E. S. P. (1984) Variation in preference for Rhizobium meliloti within and between Medicago sativa cultivars grown in soil. Appl. Environ. Microbial. 48, 1231-1236.
  4. Handelsman, J., Ugalde, R. and Brill, W. (1984) Rhizobium meliloti competitiveness and the alfalfa agglutinin. J. Bacterial. 157, 703-707.
  5. Gault, R.R., Byrne, P.T. and Brockwell, J. (1973) Apparatus for surface sterilization of individual legume root nodules. Lab. Pract. 22, 292-294.
  6. Lieberman, M.T., Zablotowicz, R.M. and Davis-Omholt, N.P. (1986) Improved method for typing Bradyrhizobiurn japonicum in soybean nodules. Appl. Environ. Microbiol. 51, 715-719.
  7. Beynon, J. L. and Josey, D. P. (1980) Demonstration of heterogeneity in a natural population of Rhizobium phaseoli using variation in intrinsic antibiotic resistance. J. Gen. Microbial. 118, 437-442. 8. Wacek, T. J. and Brill, W. J. (1976) Simple, rapid assay for screening nitrogen-fixing ability in soybean. Crop Sci. 16, 519-523.