NODULE SAMPLING
Isolating Rhizobium species, a group of bacteria that form symbiotic
relationships with leguminous plants, involves a series of steps, beginning
with the collection of root nodules. Here's a simplified guide to isolating
Rhizobium spp. through nodule sampling:
1. Nodule Sampling:
Select healthy leguminous plants that are likely to have
Rhizobium nodules. These are typically found on the roots of
plants like beans, peas, clovers, and other legumes.
Carefully dig up the plant, ensuring to keep the root system
intact to avoid losing nodules.
Wash the root system gently under running water to remove soil
and debris, taking care not to detach the nodules.
2. Surface Sterilization:
Once the nodules are visible, excise them from the roots using
sterile scissors or a scalpel.
Surface-sterilize the nodules to eliminate any contaminating
microorganisms. This can be done by immersing the nodules in a
disinfectant solution (like 70-95% ethanol) for a brief period,
followed by immersion in a sodium hypochlorite solution (1-3%
available chlorine) for several minutes.
Rinse the nodules several times in sterile distilled water to
remove any traces of the sterilizing agents.
3. Isolation of Rhizobium:
In a sterile environment (e.g., a laminar flow hood), crush the
sterilized nodule using a sterile pestle and mortar or a similar
tool to release the bacteria into a sterile solution.
Spread a small amount of the resulting suspension onto the
surface of a suitable agar medium designed for Rhizobium
growth (such as yeast extract-mannitol agar with Congo red
dye).
Incubate the plates at an appropriate temperature (usually
around 28°C) for several days to allow colonies to develop.
4. Colony Selection and Purification:
After incubation, select distinct colonies that exhibit
characteristics typical of Rhizobium (such as mucoid texture and
pink or white color).
Subculture these colonies onto fresh agar plates to obtain pure
cultures. This may need to be repeated to ensure the culture is
pure.
5. Identification and Confirmation:
Perform further tests on the purified isolates to confirm their
identity as Rhizobium spp. These tests can include microscopic
