Nutritive medium for extraction of lactobacteria

FIELD: biotechnologies.

SUBSTANCE: nutritive medium includes lactoserum, yeast autolysate, acetic acid 70%, agar and cabbage brew at the specified component ratio.

EFFECT: invention allows increasing selectivity of nutritive medium and simplifying its production.

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The invention relates to Microbiology and can be used in the study of microflora of animals and bacteriological diagnosis of intestinal dysbacteriosis.

Flora closed cavities of humans and animals are represented by different species of bacteria, isolation and identification is difficult, routine work, and to significantly reduce the time of the studies used selective environment.

For isolation of lactobacilli - the main representatives of symbiotic microflora proposed a number of mediums.

Known nutrient medium for isolation of lactic acid bacteria (Abrosimova N.A., Kushnaryov MV Nutrient medium for isolation of lactic acid bacteria // Laboratory business, 1991, No. 3, P.78-79) which includes, g/l:

Salt solution comprises, wt.%:

In this environment, grow well in various lactic acid bacteria, it has selective properties, but its disadvantage is the complexity and inaccessibility of a number of ingredients.

Known nutrient medium for isolation of lactic acid bacteria (U.S. Pat. Of the Russian Federation No. 2202609), which contains, g/l:

In this environment grow well lactobacilli, the indication of the colonies which is carried out by melting of chalk under the colony. However, this environment does not have selective properties, and it can grow any bacteria, concerns E. coli or enterococci, which can also produce acid and discolor the environment under the colonies. It is not transparent, which complicates the description of grown colonies. In addition, not all laboratories are available hydrolyzate molozivnyi caseino-whey mass.

Known framework for selection of lactic acid bacteria (U.S. Pat. Of the Russian Federation No. 2213779), the composition of which is presented in the following form, g/l:

The environment has a pronounced selectivity, prepared without sterilization. However, it has a purple color, which makes IP is the study colonies in transmitted light, the number of components of expensive and difficult to access.

Known for simple, easily reproducible culture medium for isolation of lactic acid bacteria (Kvasnikov H., Nesterenko O.A. Lactic acid bacteria and their use. - M.: Nauka, 1975. - P.60-61, prototype). This environment consists of, in wt.%:

The disadvantage of this environment is weak growth and selective properties, resulting in her equally grow bacteria of other genera, which can inhibit the growth of lactic acid bacteria, especially in low concentrations in the studied material.

Object of the present invention to provide a simple to prepare and economical nutrient medium for isolation of lactic acid bacteria from the feces of animals.

This object is achieved in that the proposed nourishing the environment for selection of lactobacilli, including cabbage broth, glucose, agar, characterized in that as an additional power sources and selectivity contains whey, yeast autolysate and acetic acid in the following ratio of components, wt.%:

The whey was received in the laboratory in 1 liter heated to 60-70°C milk with a fat content of 2.5% with constant stirring, was added 2.5-3 ml of 70% acetic acid (essences), after yearning clot casein was separated and the serum was filtered through a cotton-gauze filter and used for cooking environment. Unused balance whey can be saved, exposing his autoclaving at 115°C for 20 minutes

Yeast autolysate received Nigrosines (Microbiological diagnosis of diseases agricultural the state animal. M, GOS., in C.-H. literature, 1952. - S).

Cabbage broth was prepared by Kvasnikova (Kvasnikov H., Nesterenko O.A. Lactic acid bacteria and their use. - M.: Nauka, 1975. - P.65).

Taken components of the nutrient medium are mixed in a flask and bring to a boil and boil for 2-3 minutes to fully melt the agar, filtered through a cotton-gauze filter, poured into vials and autoclave 20 min at 0.5 ATM, after cooling medium to 45-50°C in her make acetic acid and poured into Petri dishes. Ready to use environment transparent, light brown in color, with a pleasant dairy plant with acetic tinged scent, with a pH of 5.3 to 5.5.

The novelty of the claimed proposal is that developed a nutrient medium that has marked selective properties against lactobacilli, where they give good growth and form typical colonies 1.5-2 mm. Selective properties of the environment due to contained in its composition acetic acid, to which lactic acid bacteria are resistant, while other bacteria (staphylococci, enterobacteria, found bacilli and others) to this substance sensitive.

Examples of specific receiving nutrient medium for isolation of lactobacilli.

As the test strains used Lactobacillus plantarum, Escherichia coli and Staphylococcus aureus isolated from F. the potassium calves. The subjects of culture separately cultivated on agar media at 37°C for 24 h, after which the optical turbidity standard of No. 10 was prepared 1 billion a suspension of each test strain. After this method tenfold serial dilutions in saline increased the concentration of microorganisms to 1000 microbial cells in 1 ml Of this dilution was chosen as 0.1 ml was made in 3 Petri dishes with nutrient medium. Crops were incubated in anemostat using gas-generating packages "Anaerogas within 24 h at 37°C, after which take into account the number of grown colonies.

Example 1. The test strains were grown on nutrient medium containing, wt.%: whey 7,0; yeast autolysate 7,0; glucose 1,7; acetic acid 70% 0,10; agar 1,4; cabbage broth - the rest. With this ratio of ingredients, the number of colonies of lactobacilli averaged 25, Escherichia 54 and Staphylococcus 33. The environment has a soft texture, which makes it difficult to work with a bacteriological loop.

Example 2. The test strains were grown on nutrient medium containing, wt.%: whey 8,0; yeast autolysate 8,0; glucose 1,8; acetic acid 70% 0,12; agar 1,6; cabbage broth - the rest. With this ratio of ingredients, the number of grown colonies of lactobacilli averaged 43, Escherichia 24, staphylococci is 17. The environment also has a soft texture.

Example 3. The test strains were grown on nutrient medium containing, wt.%: whey 9,0; yeast autolysate 9,0; glucose 1,9; acetic acid 70% 0,14; agar 1,8; cabbage broth - the rest. With this ratio of ingredients, the number of grown colonies of lactobacilli averaged 56, Escherichia 0 and 0 Staphylococcus. The environment has a dense texture and does not scarificial when working bacteriological loop.

Example 4. The test strains were grown on nutrient medium containing, wt.%: whey 10.0 g; yeast autolysate 10,0; glucose 2,0; acetic acid 70% 0,16; agar 2,0; cabbage broth - the rest. With this ratio of ingredients, the number of grown colonies of lactobacilli averaged 62, Escherichia 0 and 0 Staphylococcus. The environment has a tight elastic consistency and not scarificial when working bacteriological loop.

Example 5. The test strains were grown on nutrient medium containing, wt.%: whey 11,0; yeast autolysate 11,0; glucose 2,1; acetic acid 70% 0,18; agar 2,2; cabbage broth - the rest. With this ratio of ingredients, the number of grown colonies of lactobacilli averaged 46, Escherichia 0 and 0 Staphylococcus. Despite the good growth of lactobacilli on the environment, the grown colonies smaller than option the environments in examples 3 and 4. The environment has a tight elastic consistency and not scarificial when working bacteriological loop.

Example 6. The test strains were grown on nutrient medium containing, wt.%: whey 12,0; yeast autolysate 12,0; glucose 2,2; acetic acid 70% 0,20; agar 2,4; cabbage broth - the rest. With this ratio of ingredients, the number of grown colonies of lactobacilli averaged 23, Escherichia 0 and 0 Staphylococcus. In this version of the environment the growth of lactobacilli more slowed down, and the emerging colonies are much smaller (0.5 to 0.7 mm)than in variants environments in examples 3 and 4, on which the test strain laktobakterii formed colonies by size of 1-2 mm

Example 7. Researched faeces from the 30-day calf. This saline solution made tenfold dilution of 1 g of faeces to the value of 10^-9. From tubes with dilution 10^-3, 10^-5, 10^-7and 10^-9selected 0.1 ml of the suspension and planted in a nutrient medium containing, wt.%: whey 9,0; yeast autolysate 9,0; glucose 1,9; acetic acid 70% 0,14; agar 1,8; cabbage broth - the rest. After 24 h incubation at 37°C under elevated CO₂take into account the growth of bacteria. From breeding 10^-3on the environment has grown 265 colonies that morphologically corresponded to the colonies of lactobacilli, when is microscopii these were gram-positive Bacillus. Colonies typical of enterobacteria, staphylococci, bacilli, was not. From breeding 10^-5rose 96 colonies, typical colonies of lactic acid bacteria, microscopic examination of colonies established that they were formed by gram-positive Bacillus. Colonies typical of enterobacteria, staphylococci, bacilli, was not. From breeding 10^-7and 10^-9bacterial growth was completely absent. Therefore, in the faeces of calves number of lactobacilli was a 9.6·10⁶colony forming units in 1 year

Example 8. Researched faeces from the 30-day calf. This saline solution made tenfold dilution of 1 g of faeces to the value of 10^-9. From tubes with dilution 10^-3, 10^-5, 10^-7and 10^-9selected 0.1 ml of the suspension and planted in a nutrient medium containing, wt.%: whey 10.0 g; yeast autolysate 10,0; glucose 2,0; acetic acid 70% 0,16; agar 2,0; cabbage broth - the rest. After 24 h incubation at 37°C under elevated CO₂take into account the growth of bacteria. From breeding 10^-3on the environment has increased 215 colonies that morphologically corresponded to the colonies of lactic acid bacteria, microscopic examination of these were gram-positive Bacillus. Colonies typical of enterobacteria, staphylococci, bacilli, was not. From breeding 10^-5rose 63 to onii, typical colonies of lactic acid bacteria, microscopic examination of colonies established that they were formed by gram-positive Bacillus. Colonies typical of enterobacteria, staphylococci, bacilli, was not.

From breeding 10^-7and 10^-9bacterial growth was completely absent. Therefore, in the faeces of calves number of lactobacilli was of 6.3×10⁶colony forming units in 1 year

Thus, the obtained results allow to determine the optimal ratio of ingredients of the nutrient medium for isolation of lactobacilli, namely whey 9,0-10%; yeast autolysate 9,0-10%; glucose 1,9-2,0%; acetic acid 70% 0,14-0,16%; agar 1,8-2,0%; cabbage broth - the rest. This environment provides a good growth of lactobacilli in the complete inhibition of growth of Escherichia, Staphylococcus and other bacteria, and therefore can be used as selective in the selection of lactic acid bacteria from animals.

Nutrient medium for isolation of lactobacilli, including cabbage broth, glucose, agar, characterized in that as an additional power sources and selectivity contains whey, yeast autolysate and acetic acid in the following ratio, wt.%: