Microbiological soil analysis

Soil samples should be taken using cores with a diameter of about 10 mm to a depth of 10-20 cm. It is best to sterilize cores in boiling water (100 ° C) first.

Samples should be placed in sterile Petri dishes , which must then be sealed. ½ the weight part of each sample is usually taken for chemical analysis (studying the concentration of nitrogen-containing ions), the rest goes for microbiological analysis.

The microbiological activity of soils can be determined by the protease activity of soil-growing microorganisms , and the protease activity of soils is determined by the activity of exoenzymes of soil microorganisms and depends on their abundance and activity. Exoenzymes are enzymes secreted by microorganisms into the environment that break down proteins, polysaccharides and lipids.

In Petri dishes with soil samples, you need to place a lighted film cut in 2-3 frames, previously soaked in water for 10 minutes. Water should be pre-sterilized by boiling for 10 minutes (100 ° C). It is also better to place one fragment of the film in an empty sterile Petri dish (control) in order to compare the obtained results with the control at the end of the experiment and minimize the risk of errors.

Next, soil samples must be kept at room temperature (20 ° C) for 9 days. Then the film should be carefully removed and washed under running water, then dried. After that, you need to calculate the percentage of "eaten" emulsion layer on a palette. The higher the percentage of destruction of the emulsion layer, the higher the exoenzymatic activity of the soil.

A specific feature of Gram staining is the unequal ratio of various microbes to dyes of the triphenylmethane group: gentian violet, methyl violet , and crystal violet. Microbes included in the group of gram-positive give a strong connection with the specified dyes and iodine . Stained microbes do not discolor when exposed to alcohol , as a result of which, with additional fuchsin staining, gram-positive microbes do not change the initially accepted violet color. Gram-negative microorganisms form, with gentian, crystal, or methyl violet and iodine, a compound that is easily degraded by the action of alcohol, as a result of which they become discolored and then stained with fuchsin, becoming red. The ratio of microorganisms to Gram staining is of great diagnostic value.

Microbial Culture Preparation

A small amount of soil sample should be sown in Petri dishes with algal agar using a microbiological loop, then seeded Petri dishes should be kept at room temperature for 9 days. It is also better to do a control sowing from the air in order to later compare the colonies of microorganisms grown in different Petri dishes and to know which microorganisms could get out of the air, and not from the soil.

Smear preparation

Place a drop of distilled water on a clean, fat-free glass slide, then use a microbiological loop to add a small amount of the microbial culture grown on agar. Next, you need to make strokes and fix them on a flame. It is better to do 2-3 strokes for each soil sample and one control smear.

Microorganism staining

Gram microorganisms should be stained as follows:

• The smear, fixed on fire, must be painted through filter paper with the main dye, a solution of the main carbolic crystal violet. Staining should last 1-2 minutes.
• Next, the paper should be removed, the excess paint should be drained and the Lugol solution should be poured for 1-2 minutes before the blackening of the preparation.
• Next, Lugol's solution must again be drained, a glass slide to discolor the smear immersed several times in a glass with alcohol; the bleaching process is considered complete when violet-colored trickles of liquid no longer separate from the smear.
• Then the drug should be thoroughly washed with tap water and refilled with an alcohol-water solution of fuchsin for two minutes.
• At the end, the smear should be washed with water and dried.

The result of staining: gram - positive bacteria are stained with the main paint in dark purple color, gram-negative bacteria, perceiving additional color, acquire a bright crimson color.

• Nitrogen exchange of the soil

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• Labinskaya A. S. Microbiology with the technique of microbiological research. - M .: Medicine, 1972. - 480 p.