To observe the rhizobium bacteria in root nodules of legume plants.

PRINCIPLE : Rhizobium is known to survive a soil and in roots of legumes as well as non leguminous plant roots. Bacteria depends on soil exudates. No single component in the root exudates have special role in stimulating the growth. Rhizobium secretes extracellular polysaccharides which helps to bind soil particles.

REQUIREMENTS :

Groundnut, Sterile petriplates, YEMA medium, mercuric chloride, 70 % ethylalcohol.

PROCEDURE :

1. Collection of Root Nodules :

Leguminous plants are carefully uprooted and the root system is washed under running water to remove the adhering soil particles. The colour of nodules varies from brown to pink depending on the state of the pigment present in them for the experiment. Healthy unbroken pink nodules are selected.

2. Surface sterilisation of Legume Root Nodule :

The nodules must be surface sterilised by sterilising agent and the sterilising agent must be washed of from nodules before they are used for isolation. The sterilising agent used for isolation is 0.1% mercuric chloride or 3-5 % hydrogen peroxide. The nucleus are immersed n sterilising agent for 4-5 min an are washed in 70% ethyl alcohol followed by washing with sterile distilled water.

3. Observation of Rhizobium by Root Sectioning :

The surface sterilised root nodule is cross sectioned and a thin section is selected. It is placed on a clean glass slide and treated with saffranin for 2-3 min. The stained section is mounted by coverslip in glycerin solution and observed under microscope.

OBSERVATION :

Pink coloured, rod shaped bacteria are observed.