Rhizobium Bacterial Culture: Preparation and Use

After reading this article you will learn about the preparation and use of rhizobium bacterial culture.

Preparation of Rhizobium Bacterial Culture:

A vigorously growing leguminous plant that has been grown for the time on the field is carefully uprooted. The root is carefully washed with sterilized water. The nodules are then carefully detached from the roots by cutting a portion of the root on either side of it.

The nodules are then carefully washed with sterilized water and kept immersed in 0.1 per cent mercuric chloride solution for five minutes. They are washed with alcohol first and then with water six times. Then a little sterilized water is added to the test tube containing the nodules, which are crushed with a sterilized glass rod to release the rhizobium bacteria to the sterilized water.

Now this rhizobium bacteria is ready for culturing on the yeast extract mannitol-agar medium which is prepared as follows: Take 20gms of agar, 10gms of mannitol, 0.5gms of di-potassium hydrogen phosphate, 0.2gm of magnesium sulphate, 0.1gm of sodium chloride, 3gms of powered calcium carbonate, 1gm of yeast extract and 1000 ml of distilled water in an Erlenmeyer flask, plug its mouth with cotton wool and sterilize it in the autoclave, at a pressure of 15 pounds per square inch for 15 minutes.

Then pour it into a few sterilized test tubes. Then a little water containing the rhizobium bacteria is added to each of these test tubes with the help of sterilized platinum loop. Thoroughly mix the bacteria with the culture medium by gently rotating the test tubes.

Then pour the contents into one of these test tubes containing the bacteria in agar medium into a sterilized Petridis. Pour a little of the sterilized agar medium to these empty test tubes containing drops of agar medium containing the bacteria.

Shake it gently and pour its contents into another sterilized petri dish. Maintain the temperature of the agar medium at 45°C and spread the contents of the petri dish uniformly over it, by gently rotating it in a clockwise manner.

When the agar medium has solidified, invert it. Put the Petridis and the test tube containing the bacteria in a slanting position in the incubator at a temperature of 30°C. Usually bacterial colonies appear after four to five days. They resemble shining water drops.

Preparation of Rhizobium Bacterial Culture in Soil:

Thoroughly mix 1000gms of soil, 10gms of sugarcane powder, 10gms of legume hay powder, 0.9gms of potassium sulphate, and 120 ml water. The soil is low in lime; also add 0.5gms of powdered calcium carbonate to it. Put 400gms of the above mixture in each tin. Then sterilize tins containing the mixture for 2 hours, at 9kgs pressure for two consecutive days and again on the fourth day.

Take 150 ml of the yeast extract-mannitol-medium in a 500 ml conical flask; plug it with cotton wool and sterilize it at 6.8kgs of pressure for half an hour. This sterilized medium is then inoculated with the rhizobium bacterial culture from the slant of one test tube where the rhizobium bacteria have grown for 24 hours.

Shake the flask in the shaker for 24 to 72 hours, depending upon the generation time of the organisms. Then add 100 ml of the bacterial culture from the flask to the soil contained in the tin. Mix it thoroughly with the soil. Incubate the soil in the tin for a week at 28°C.

Use of this Bacterial Culture in Soil:

Dissolve 150gms of gur (raw indigenous sugar) in 1000 ml of water. Boil for half an hour and the cool. Add one tin-full of the soil culture to this gur solution and mix thoroughly. This gur solution containing the soil culture is then thoroughly mixed with the seed meant for sowing one hectare of land. The seeds are then dried in the shaker for an hour and shown in this field.

It India, the nitrogen content of the soil was increased when the seeds of the leguminous crops that had been inoculated with the Rhizobium bacteria of the concerned cross inoculation group were sown.

Fox example, the agricultural scientists of the Indian Agricultural Research Institute have found that the nitrogen content of the soil increased by 120 kg per hectare per year when barseem (Trifoliom alexandrinum) which had been inoculated with the Rhizobium bacteria of the concerned cross-inoculation group (Rhizobium trifolii), was grown.