RU2303630C1 - Dense nutrient medium for cholera vibrio cultivation - Google Patents

Abstract

FIELD: biotechnology, medicine.

SUBSTANCE: claimed medium contains microbiological agar, ferment soybean hydrolyzate containing 0,08 % of amine nitrogen, sodium chloride, disubstituted sodium phosphate, sodium sulfite, and distilled water.

EFFECT: nutrient medium for cholera vibrio cultivation with improved quality and decreased cost.

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Description

The invention relates to microbiology, namely to obtain nutrient media that create optimal conditions for the isolation and cultivation of cholera vibrio.

In a bacteriological study of cholera, a nutrient medium is used, including, g / l: agar-agar 20.0; beef hydrolyzate containing amine nitrogen, 0-12%, sodium chloride 5.0; 20% sodium hydroxide 0.002; distilled water up to 1 liter (Handbook of microbiological and virological research methods Birger M.O., 1972, p. 53.)

The disadvantage of this environment is its high cost.

Closest to the proposed nutrient medium is a medium for the cultivation of cholera vibrio, containing, g / l: pancreatic hydrolyzate of casein 4.0; pancreatic hydrolysis of feed yeast 4.0, as a nutritional base - sodium chloride 5.0; disubstituted sodium phosphate 0.4; sodium pyrosulphate (metabisulphite) 0.5; agar 14.0 and distilled water to a liter (Handbook of microbiological culture media. Edited by M. Medzhidov Makhachkala, 1989, p. 66.)

The disadvantage of this environment is its high cost, the complexity of the preparation.

The aim of the invention is to obtain high-quality, cheap nutrient medium for the cultivation of cholera vibrio.

The essence of the invention lies in the fact that the nutrient medium contains a nutrient base - an enzymatic hydrolyzate of soybeans and a stimulating additive - sodium sulfite in the following ratio of ingredients, g / l:

Microbiological agar 11.0-13-0 Enzymatic Soya Bean Hydrolyzate 60.0-160.0 Sodium Chloride 4.0-6.0 2-substituted sodium phosphate 3.0-5.0 Sodium Sulphate 0.3-0-5 Distilled water rest

Soy is used as a raw material, the fruits of which - beans contain an average of 40% protein, in addition, soy is rich in trace elements and vitamins. As is known (Y.P. Yupta, 1987), soy proteins are characterized by an increased content of glutamic and aspartic acids, which supply macroergic bonds. Soy proteins also contain a large number of essential amino acids such as lysine, leucine, arginine. There is also information about the ability of leucine, as branched chain amino acids, to initiate protein synthesis (E.I. Chazov, X. S. Morgan (USA), 1989).

The preparation of a nutrient base for growing microorganisms is as follows. Soy fruits - beans in an amount of 0.5 kg are soaked five times in 5 l of hot water for 1 day, and the last infusion with beans is boiled for 40 minutes. Then the infusion is poured into a 10 liter cylinder, cooled, the beans are minced with a meat grinder and placed in the infusion. The cattle pancreas is added at the rate of 20.0 per 1 liter, the pH is adjusted to 8.2 with a 20% sodium hydroxide solution in an amount of 3 ml, and 1% chloroform is added. Hydrolysis is carried out in a heat chamber at a temperature of 37 ° C for 3 days. For the first 2 hours, the mixture is stirred every 15 minutes, 5 at a later time; the mixture is stirred every 6 hours. The level of amine nitrogen, which is 0.6 ± 0.05% daily, is measured daily.

A nutrient medium based on soybean enzymatic hydrolyzate for growing microorganisms is prepared as follows. Microbiological agar 12.0; enzymatic hydrolyzate from soybeans, diluted with distilled water to an amine nitrogen reading of 0.08%, 110 ml, sodium chloride 5.0; 2-substituted sodium phosphate 4.0; distilled water up to 1 liter.

The medium is boiled until the ingredients are completely dissolved, filtered through a cotton-gauze filter, then the pH is adjusted to 7.8 ± 0.1 using a 20% sodium hydroxide solution, and a stimulant is added: sodium sulfide at a concentration of 0.4 g / l. The prepared medium is poured into 0.5 l bottles of 400.0 ± 10.0 ml and sterilized in an autoclave at 0.5 atm. 30 minutes. According to the guidelines for determining the quality of nutrient media for growing cholera vibrio (Instructions for bacteriological monitoring of diagnostic nutrient media for cholera vibrio), a culture of avirulent strain NAG - vibrio R-9741 is prepared. Why culture stored on semi-liquid agar, seeded in 1% peptone water or Marten broth (pH 7.6-7.8). After 18–20 h of growth, smooth HAT vibrio R-9741 colonies were selected from agar and transferred to agar plates (2-passage). The culture is grown for 3-6 hours at 37 ° C, after which it is used for control. A suspension of bw is prepared from the daily culture, equal to 5 units according to the optical turbidity standard according to the GISK named after L.A. Tarasevich. Then, serial ten-fold dilutions in physiological saline in a volume of 4.5 ml are adjusted to a content of 1 ml of 1000 and 100 MK From these dilutions, culture suspensions are seeded with 0.1 ml sterile bacteriological pipette of 3 Petri dishes of the experimental and control medium. As the latter, a pre-tested high-quality nutritious alkaline agar is used. Cultivation is carried out at 37 ° C. A solid nutrient medium is considered suitable if, when sowing 100 and 10 m.k. after 12 hours of cultivation, the test strain grows at least 30 and 1 colonies with a diameter of 1 mm on all plates, respectively.

Example 1. The test strain was grown on a nutrient medium containing, g / l: microbiological agar 11.0; soybean enzymatic hydrodysate (beans) 60.0; sodium chloride 4.0; 2-substituted sodium phosphate 3.0; sodium sulfite 0.3; distilled water up to 1 liter. With this ratio of ingredients, the number of colonies grown on dense agar plates for cholera vibrio at a sowing dose of 100 and 10 m.k. amounted to 29 and 1 colonies with a diameter of 1.0 mm, respectively.

Example 2. The test strain was grown on a nutrient medium containing, g / l: microbiological agar 12.0; soy enzymatic hadrolyzate (beans) - 110.0; sodium chloride 5.0; 2-substituted sodium phosphate 4.0; sodium sulfide 0.4; distilled water up to 1 liter. With this ratio of ingredients, the number of colonies grown on dense agar plates for cholera vibrio at a sowing dose of 100 and 10 m.k. amounted to respectively 45 and 4 colonies with a diameter of 1.5 mm.

Example 3. The test strain was grown on a nutrient medium containing, g / l: microbiological agar 13.0; soybean enzymatic hydrolyzate (beans) 160.0; sodium chloride 6.0; 2-substituted sodium phosphate 5.0; sodium sulfite 0.5; distilled water up to 1 liter. With this ratio of ingredients, the number of colonies grown on dense agar plates for cholera vibrio at a sowing dose of 100 and 10 mk amounted to 31 and 1 colonies with a diameter of 1.0 mm respectively.

Thus, the claimed nutrient medium is dense for the cultivation of cholera vibrio (example 2), prepared on the basis of an enzymatic hydrolyzate of soy (beans), can be used for the cultivation of cholera vibrio as cost-effective.

Claims (1)

A nutrient medium that is dense for the cultivation of cholera vibrio, containing a nutrient base, sodium chloride, sodium phosphate disubstituted, microbiological agar and distilled water, characterized in that it additionally contains sodium sulfide as a stimulating additive, and an enzymatic hydrolyzate of soybeans as a nutritional base with an amino nitrogen content of 0.08% in the following ratio of components, g / l: microbiological agar  11.0-13.0 soybean enzymatic hydrolyzate with an amine nitrogen content of 0.08% 60.0-160.0 sodium chloride  4.0-6.0 disubstituted sodium phosphate 3.0-5.0 sodium sulfite  0.3-0.5 distilled water  rest