RU2288950C1 - Liquid nutrient medium for culturing anthrax microorganism and closely related saprophytes - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to a method for preparing microbiological nutrient media used in culturing anthrax microorganism and closely related spore-forming saprophytes (Bacillus subtilis, Bacillus mesentericus) in process of manufacturing anthrax vaccines, anthraxin, biotherapeutic preparations and for science-research aims. Proposed nutrient medium comprises refining molasses, tap water, sodium chloride and yeast extract. Invention provides simplifying method for preparing nutrient medium and decreasing its cost.

EFFECT: improved preparing method of medium.

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Description

The invention relates to biotechnology, in particular to the production of culture media for the cultivation of anthrax microbe and closely related spore-forming saprophytes (Bacillus subtilis, Bacillus mesentericus) in the production of anthrax vaccines, anthraxin, biotherapeutic drugs, as well as for research purposes.

A nutrient medium of the following composition is known, g / l: 1% peptone, 0.5% x. including sodium chloride, meat water - up to 1 l, pH 7.3 ± 0.1 (Reference book on microbiological and virological research methods. Edited by M.O. Birger, M .: Medicine, 1982, p. 48).

The disadvantage of this environment is the use of expensive ingredients.

Closest to the proposed invention is a nutrient medium of the Hottinger broth, including, g / l: beef hydrolyzate diluted with distilled water to 1 liter with a total nitrogen content of 250-300 mg% and amine nitrogen 30-130 mg%; sodium chloride 5.0; sodium dihydrogen phosphate 0.3, pH 7.4-7.6 (Polyak M.S., Sukharevich V.I., Sukharevich M.E. Nutrient media for medical microbiology. St. Petersburg, 2003, p. 56). The disadvantages of this environment are the high cost and complicated cooking technology, due to the fact that the basis of the environment is products of animal origin.

The purpose of the present invention is to select high-quality cheap basis of nutrient media of plant origin, which would provide a cumulative effect and reduce its cost, as well as simplifying the method of preparation.

This goal is achieved in that the nutrient basis of the proposed nutrient medium is refined molasses, sodium chloride in tap water with the addition of a stimulating additive to the liquid medium, and the yeast extract is used as a stimulating additive, in the following ratio of ingredients, g / l:

Molasses 20,0 Yeast extract 10.0 Sodium chloride 5,0 Tap water rest

Refined molasses (OST 18-233-75) in its composition contains at least 45% sucrose. Unlike the prototype, the proposed environment provides cumulative properties, optimal conditions and low cost.

The use of molasses with yeast extract in tap water as a nutrient base is a hallmark of the proposed nutrient medium.

The method is illustrated by the following example.

The nutrient medium is prepared as follows: the molasses is dissolved in tap water, yeast extract, sodium chloride are added, heated to 60 ° C, the pH is adjusted to 7.7-8.2 with 20% sodium hydroxide, boiled for 5 minutes with stirring. The precipitate formed is filtered through a fabric filter and filter paper. Set the pH to 7.2 ± 0.1 with hydrochloric acid in a dilution of 1: 1. The prepared medium is poured into bacteriological tubes of 10.0 ± 0.1 ml and sterilized in an autoclave at 0-5 atm for 30 minutes.

According to the guidelines for controlling biological media, M., 1980, microbial suspensions of the tested strains of Bacillus anthracis 81/1, Bacillus anthracis STI-1 and saprophytic spore-forming strains of Bacillus subtilis 3 and Bacillus mesentericus 1024 are prepared, with a concentration of 10 optical units industry standard turbidity for CCA GISK them. L.A. Tarasovich, then tenfold dilutions in buffered saline in a volume of 4.5 ml are adjusted to a dilution in 1 ml of 100 m.k. From this dilution, suspension of cultures is sown in 0.1 ml sterile bacteriological pipette 3 tubes of experimental and control environments. As the latter used a pre-tested nutrient medium (liquid) Hottinger broth for the cultivation of microorganisms, pH 7.3 ± 0.1. 1 tube of each of the media was left inoculated. Crops were incubated at 37 ° C for 22-24 hours. The growth properties of the nutrient medium were assessed visually: for anthrax strains 81/1 and STI-1, a characteristic growth in the form of a flocculent precipitate (cotton ball) should be observed at the bottom of the tube with transparency of the supernatant liquids with inoculation from each dilution, whereas for saprophytic strains, growth should be observed in the form of uniform turbidity of the broth. After 24 hours of incubation at 37 ° C, the growth of all test strains on the experimental medium was more pronounced. Droplets were prepared from cultures grown on both media, stained according to Graham and microscopic. The morphology of vegetative cells was characteristic of these strains. To study the morphology of colonies from diurnal cultures grown on experimental and control media, 0.1 ml of inoculation was performed on plates with Hottinger agar, pH 7.3 ± 0.1. Regardless of the growth medium, colony morphology was typical of all strains.

Example 1

Test strains were grown on a nutrient medium (liquid) containing, g / l: refined molasses 18.0; yeast extract 9.0; sodium chloride 4.0; tap water - the rest. With this ratio of ingredients, anthrax strains 81/1 and STI-1 grew in the form of a small lump of cotton wool at the bottom of the tube in transparent broth, and for the saprophytic strains Bacillus subtilis 3 and Bacillus mesentericus 1024, a slight uniform turbidity of the medium was observed.

Example 2

Test strains were grown on a nutrient medium (liquid) containing, g / l: refined molasses 20.0; yeast extract 10.0; sodium chloride 5.0; tap water - the rest. With this ratio of ingredients, a characteristic growth of anthrax strains 81/1 and STI-1 was observed in the form of a pronounced lump of cotton wool at the bottom of the tube and a clear liquid above it, and for saprophytic strains Bacillus subtilis 3 and Bacillus mesentericus 1024, a significantly pronounced uniform turbidity of the medium was observed.

Example 3

Test strains were grown on a nutrient medium (liquid) containing, g / l: refined molasses 22.0; yeast extract 11.0; sodium chloride 6.0; tap water - the rest. With this ratio of ingredients, anthrax strains 81/1 and STI-1 grew in the form of a small cotton ball on the bottom of the tube and a clear liquid above it, and for spore-forming saprophytic strains Bacillus subtilis 3 and Bacillus mesentericus 1024, a slight uniform turbidity of the medium was observed.

The results obtained allowed us to determine the optimal version of the nutrient medium (liquid) based on refined treacle (example 2).

Thus, based on the foregoing, we can say about the obvious advantages of the proposed nutrient medium, consisting of refined molasses 20 g / l, sodium chloride 5 g / l, yeast extract 10 g / l, tap water - the rest. The nutrient medium is technically easy to prepare (without a preliminary stage of preparation of the base). The proposed nutrient medium is cheaper, as the raw material is used (refined molasses with yeast extract) and distilled water is not required for its preparation - the medium is prepared using tap water.

Claims (1)

Nutrient medium (liquid) for the cultivation of anthrax microbe and closely related saprophytes, containing a nutrient base, sodium chloride and water, characterized in that it additionally contains yeast extract as a growth promoter, refined molasses as water base, water - tap water, and the following ingredients, g / l: Molasses 20,0 Yeast extract 10.0 Sodium chloride 5,0 Tap water Rest