Isolation of Rhizobium from Root Nodule

Rhizobium is a gram negative bacteria which is present in symbiotic relationship with roots of leguminous crops, it fixes atmospheric nitrogen for the plant and in return the plant provides shelter and food. There are several different species of rhizobacteria and each legume crop has its own compatible species which can effectively fix nitrogen in association with that particular crop. This is also known as cross inoculation in rhizobium

Materials required

• Root of leguminous crop having vigorous root nodules
• 1:1000 HgCl₂ Solution
• 75% alcohol
• Sterile petri plates
• Sterile Test tube
• Sterile Forceps
• Sterile Scalpel
• Sterile Distilled Water
• Sterile Glass Rod
• Sterile Blade
• Culture Media :- Congo red Yeast Extract Mannitol Agar

Composition of Congo Red Yeast Extract Mannitol Agar Media

Procedure

• Uproot vigorously growing legume crop preferably of the flowering stage and collect the roots having a bunch of nodules
• Wash the collected roots thoroughly with water and remove the
• Cut off pink colored nodules using a blade leaving small portion of root attached to the nodules
• Place the nodules in a Sterile petri plate containing HgCl₂ and dip the root nodules, agitate with sterile forceps for 4-5 minutes
• Transfer the nodules in a separate petri plate containing 75% alcohol, using sterile forceps and agitate for 2 minutes
• Transfer it to another petri plate containing water to remove remaining alcohol from the surface, repeat 2-3 times.
• Collect the sterilized root nodules in a test tube containing 1 ml water and crush them with the help of sterilized glass rod
• Mix the root exudates with water and transfer two loopful of exudate in another test tube containing 1 ml water
• Perform serial dilution upto the concentration of 10⁶ to 10⁸
• Transfer 1 ml of diluted root exudate to a petri plate and pour Congo red Yeast extract Mannitol Agar medium
• Incubate for 4-5 days. After incubation you will see 2 different types of colonies, one which takes red color and another white
• The colony which takes up red color is of azotobacter and white one is of rhizobium
• Transfer the rhizobium colonies on a slant

Precautions

• Always use sterilized equipment for isolation as it eliminates the risk of contamination
• Do not uproot young and immature plants as they don’t have well developed nodules
• Use sterile water while washing and preparation of media
• Pour the media in petri plate before it starts solidifying
• Perform the procedure under sterile environment.