Experiments on the isolation of aerobic autochthonous nitrogen-fixing bacteria in Asubelia medium

Summary

Ashby's (Ashby) nitrogen-free medium is a lack of binding state inorganic or organic nitrogen source of the medium, can make autochthonous nitrogen-fixing bacteria in the guarantee of carbon and inorganic nutrient supply, the use of free nitrogen in the air to synthesize their own need for nitrogen-containing organic matter, and other types of microorganisms do not have autochthonous nitrogen-fixing ability, so as to achieve enrichment and separation of the effect.

Operation method

selective cultivation method

Principle

Selective culture is a kind of culture designed according to the special nutritional requirements of certain microorganisms or their resistance to certain chemical and physical factors, which has the function of making the disadvantageous bacteria in the mixed bacteria develop into the advantageous bacteria, so it is widely used in the field of strain screening. Ashby's (Ashby) nitrogen-free medium is a kind of medium lacking in bound inorganic or organic nitrogen sources, which can effectively isolate autochthonous nitrogen-fixing bacteria from soil. The principle of selection of Ashby's nitrogen-free medium is that autochthonous nitrogen-fixing bacteria can synthesize their own nitrogenous organic matter using free nitrogen in the air under the guarantee of carbon source (mannitol or glucose) and inorganic nutrient supply, while other types of microorganisms do not have autochthonous nitrogen-fixing ability, so it achieves the effect of selective enrichment.

Materials and Instruments

Asube's nitrogen-free medium Soil
Water Mannitol KH2PO4 NaCl CaSO4-2H2O CaCO3 Distilled water Agar powder
Petri dishes Straws Mantles Inoculation rings Triangular flasks Slides

Move

I. Collection of soil samples

A slightly dry soil sample of about 1 g from 10 cm depth and 20 cm depth was taken with a small spatula and crushed properly;
II. Preparing the tablet

Melt the Asube's medium by heating, and pour several plates until it cools down to about 50 ℃, and then cool it down for use;
III. Cardboard cloth soil

Pour the soil sample onto a roughly 15 cm x 15 cm sized piece of cardboard, shake it a few times, and gently pour away the larger particles;
IV. Soil inoculation

Put the soil-soaked side of the cardboard towards the plate and cover it, and then gently flick it several times by hand. Then remove the paper and put the lid on the plate;
V. Holding culture

The petri dish plates were inverted and incubated at a constant temperature of 28 ℃ for 4-7 days;
VI. Observation of colonies

Any colony that appears large, slimy, translucent and white, brown or dark brown in color is generally an aerobic nitrogen fixing bacteria.
VII. Separation of pure breeds

Strain purification can be carried out by plate scribing method.

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