Nexus Research Group - Rhizobium bacteria isolation

Partners for Life

A particular type of bacteria invades and lives inside clover root cells...and both are better off for it!

Rhizobium bacteria in root nodules of leguminous plants are estimated to carry out 50-70% of the world biological nitrogen fixation, reducing approximately 20 million tonnes of atmospheric nitrogen to ammonia. They provide fertilliser for free! It is also non-polluting compared to the manufacture of urea or the risk of fertiliser run-off into rivers and lakes. Find out more about these amazing microbes - isolate your own!

Isolation of Nitrogen fixing Rhizobium bacteria:

Obtain an intact root system from a healthy White Clover plant. Rinse off the dirt until you can clearly see any root nodules present.

Sit a pair of tweezers and a glass rod (with a blunt flat end) in a beaker of 70% alcohol to sterilise them. Always replace these into the alcohol in between manipulations later on.
Cut off sections of root no more than one cm long that contains a large healthy (pink) root nodule.
Immerse the 5 or 6 root sections you have obtained in 1% chlorine (bleach) solution in a sterile plastic petri dish for 15 minutes. Keep the lid on the petri dish after each manipulation of the root nodules from now on.
Pour off the solution. Cover the root nodules with about 20 ml of 70% ethanol, replace the lid, and swirl gently for about 1 minute.
Pour off the solution. Cover the root nodules with sterile water, replace the lid, and swirl gently for about one minute. Repeat this rinsing step three more times. Remember to replace the lid between washings!
Pour off the last rinse water and sterilise the tweezers by removing from the alcohol and passing through a bunsen flame. The alcohol will burn off. Allow the tweezers to cool for a few seconds.
Pick up one of the root nodules and transfer to a drop of sterile water in a petri dish. Flame the glass rod (and allow to cool) and crush the nodule in the drop of water. This should have released the nitrogen fixing Rhizobium bacteria into the drop of sterile water.
Heat sterilise a microbiological loop by flaming for a few seconds to a cherry red colour. Allow to cool for a few seconds. Streak plate onto Yeast Mannitol agar (YMA) or Tryptone Yeast Extract agar (TYA) the crushed root nodule.
Repeat steps 8 and 9 for the other root nodules. This should ensure that you will be successful with at least one of the nodules!
Invert the agar plates before Incubating at 20 to 25oC for at least 3 to 5 days. If growing Rhizobium on YMA you should see gloopy pale coloured colonies. If you do a gram stain from these colonies and examine under the light microscope, you should have a gram negative rod-shaped organism.

Did you succeed in isolating this very important non-pathigenic organism?

Try the next experiment that looks at the plant/bacteria symbiosis and the benefit to the plant.

For those interested in soil pH, this species of Rhizobium prefer a soil pH of no lower than 5.5.

What do you think would happen to the Rhizobium, and hence the plant, in more acidic soils?

70% ethanol solution is made by mixing 70 ml ethanol with 30 ml water.
Sterile water can be prepared in a pressure cooker (15 minutes at 15 lbs pressure)
Pre-sterilised plastic petri dishes are available from most biomedical supply companies. An alternative is to use glass petri dishes with matching lids that can be sterilised by baking in an oven at 250oC for 1 hour.
YMA and TYA recipes to follow here soon...

FIND OUT MORE:-

Find out more about Rhizobium bacteria here...
What does the word "symbiotic" mean? What other organisms live in a symbiotic relationship?
What disadvantages are there in making fertiliser in a factory and adding this to pasture compared to the process carried out by Rhizobium bacteria?
Learn about other microbes here...