Isolation of Rhizobium

 Isolation of Rhizobium 

Aim

To isolate Rhizobium species from root nodules of legumes

Principle

Nitrogen fixation is the phase of the nitrogen cycle during which enzymatically competent microorganisms (with nitrogenase) convert atmospheric nitrogen into nitrogenous compounds. This process replenishes the soil with usable nitrogen that is rapidly removed by plant use, denitrification and leaching. Nitrogen fixation is mediated by two microbial systems. One consists of non-symbiotic free living microorganisms such as members of Azotobacter, Beijerinckia, Clostridium and Cyanobacteria, which are capable of using Nitrogen gas as their nitrogen source. The second system involve symbiotic microbial forms such as members of Rhizobium, which following infection grow in a nodule in the root of leguminous plants. A mutually benefited association is established in which nutrients in the plant sap is used by Rhizobium as it fixes atmospheric nitrogen to ammonia which can be assimilated by the plants into various proteins. Rhizobium finds application as biofertilizer in recent times.

Rhizobium species are generally cultured aerobically at 25-30⁰C in Yeast extract mannitol agar (YEMA) medium. Fast growing Rhizobia develop colonies in 3-6 days and slow growing Rhizobia in 7-10 days. Congo Red is used as a dye to enable differentiation of Rhizobium species from other bacteria. Rhizobium appear as pale coloured colonies while soil bacteria such as Agrobacterium species and others absorb the red dye. YEMA is widely used for the cultivation of Agrobacterium, Rhizobium and other soil microorganisms. It contains mannitol as a carbon source and yeast extract as a source of readily available amino acids, vitamin B complex and growth factors for Rhizobia. Congo red. Colonies of Rhizobia stand out as colourless, translucent, glistening and elevated, with entire margins

Rhizobium can fix atmospheric nitrogen only in root nodules of legumes where it is in the bacteroid stage of its life cycle. It possesses the entire complement of genes for nitrogen fixation, which are normally latent and become active only under special conditions. Rhizobium makes nitrogen available to the plant and in turn, the bacteria derive nutrients from the tissues of the plants. A crushed leguminous root nodule may be used as the source of a stained slide preparation. Rhizobium appear as bacteroids within the nodule, and are distinguished by their pleomorphism, wherein they appear in a variety of shapes, such as x, y, t, v etc.

Materials Required

Freshly picked leguminous plants with root nodules as source of Rhizobium, Yeast extract mannitol agar (YEMA) plates (pH 6.8-7), 0.1% acidified mercuric chloride, 95% ethanol, methylene blue, crystal violet, safranin, Gram’s Iodine

Equipments – Bunsen burner, inoculating loop, glass slide, staining tray, glassware marking pencil, blade, glass rod.

Procedure

1.      Healthy root nodules of a young leguminous plants were obtained by cutting with a blade

2.      The nodules were washed thoroughly first with tap water, then with sterile distilled water, under aseptic conditions so as to remove contaminants and adhering soil particles

3.      Thereafter the nodules were immersed into 0.1% acidified mercuric chloride for 5 minutes

4.      The nodules were then transferred into a sterile beaker containing 10 ml of 95% ethanol and kept for 2-3 minutes and blot dried

5.      The nodules were finally washed for 5 minutes with sterile tap water using sterile blotting paper

6.      Nodules were aseptically crushed with glass rod

7.      The nodules were streaked on the YEMA medium incorporated with Congo Red

8.      Plates were incubated in an inverted position at 28⁰C for 48-72 hours

Microscopic Observation

1.      A root nodule was thoroughly rinsed and crushed between two slides.

2.      A loopful of the material from the nodule was used for preparing smears on 2 slides

3.      Gram staining was done

4.      The slides were observed under the microscope

Observation and Result

            On YEMA plates, growth was obtained on 2-3 days of the incubation.  Rhizobium appeared as gummy cream coloured colonies as it does not absorb Congo red dye. Agrobacteria and other soil bacteria appeared red since they take up the dye strongly.

On microscopic observation, Rhizobia appeared as Gram negative, non-motile, rod shaped bacteria

 

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