RU2479630C1 - Nutrient medium for detection of l-forms of mycobacteria - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: nutrient medium contains monosubstituted potassium phosphate, magnesium sulphate, sodium phosphate, iron citrate, glycocol, monosubstituted sodium glutamate, glycerine, agar, potato broth, whey of cattle and distilled water at specified ratios.

EFFECT: invention makes it possible to increase yield of biomass of L-forms of mycobacteria and to reduce time of biomass accumulation.

1 tbl

Description

 The invention relates to veterinary microbiology and relates to nutrient media for obtaining L-forms of mycobacteria from pathological material, provides a reduction in growth time and increase the yield of biomass of L-forms of mycobacteria.

A dense nutrient medium is known for isolating and cultivating mycobacterium tuberculosis from animal biomaterial containing chicken eggs, malachite green, potassium phosphate disubstituted, sodium citrate, magnesium sulfate, peptone, glycerin, tetra or tape, or hexadecane, distilled water ["Nutrient medium for cultivation of mycobacteria "RU 2410425 C1, C12N 1/20, C12R 1/32, 01/27/2011].

However, this medium is not suitable for the cultivation of L-forms, since they require a semi-liquid nutrient medium that provides the osmotic conditions necessary for their growth.

The closest analogue, i.e. The prototype for isolation and cultivation of L-forms of Mycobacterium tuberculosis is a semi-liquid Dorozhkova medium, which contains, wt.%:

potassium phosphate - 0.026-0.035;

sodium phosphate - 0.25-0.33;

magnesium sulfate - 0.05-0.07;

sodium citrate - 0.35-0.47;

iron ammonium citrate - 0.005-0.007;

asparagine - 0.41-0.56;

glycerin - 7.44-9.28;

agar - 0.25-0.3;

distilled water - the rest;

additives to one liter of medium: sucrose 10; cattle blood serum 10.

[Manual on the diagnosis of animal tuberculosis / Ministry of Agriculture of the Russian Federation Department of Veterinary Medicine 11/18/2002, p.17-16].

However, the known environment does not allow to isolate in a short time from the pathological material of the L-culture of mycobacteria, necessary for experiments to study the characteristics of the infectious process caused by them. In addition, it does not provide sufficient biomass accumulation.

The objective of the invention is to create a medium for isolation from pathological material of the L-culture of mycobacteria, which allows to reduce the growth time and increase the biomass yield of L-forms of mycobacteria.

To solve this problem, an environment is proposed consisting of the following components, wt.%:

- potassium phosphate monosubstituted - 0,026-0,035;

- magnesium sulfate - 0.05-0.07;

- sodium phosphate - 0.25-0.33;

- iron citric acid - 0.005-0.007;

- glycocol - 0.35-0.41;

- monosubstituted glutamine sodium - 0.1-0.2;

- glycerin - 0.3-0.4;

- agar - 0.3-0.4;

- distilled water - the rest;

additionally enriched with potato broth 9-12% and blood serum of cattle 10-20%, which creates the necessary buffering and plays a significant role in metabolism.

The effectiveness of the applied medium for the isolation of L-forms of mycobacteria, which allows to reduce the growth time and increase the biomass yield of L-forms of mycobacteria, was tested on three versions of the test media.

The nutrient medium for the isolation of L-forms of mycobacteria "Test A" is composed of the following components, g / l:

monosubstituted potassium phosphate - 1.5;

magnesium sulfate - 0.5;

sodium phosphate - 2.5;

iron citrate - 0.05;

glycocol - 1;

glycerin - 40;

agar-agar - 3;

distilled water ml up to 1000;

ingredients in the indicated order are dissolved in distilled water, each ingredient after dissolving the previous one, agar is added, filtered through a cotton-gauze filter. Sterilized in an autoclave at 1 atmosphere, 120 ° C, 30 minutes. A few days before sowing, the medium is heated in a water bath to a temperature of 40-45 ° C and sterile potato broth 20% and blood serum of cattle 10% are added as a growth stimulant. The medium from the flask is poured into test tubes of 6 ml, checked for sterility for two days.

The nutrient medium for the isolation of L-forms of mycobacteria "Test B" is composed of the following components, g / l:

monosubstituted potassium phosphate - 1.5;

magnesium sulfate - 0.5;

sodium phosphate - 2.5;

iron citrate - 0.05;

glycocol - 1;

monosubstituted glutamine sodium - 5;

glycerin - 40;

agar-agar - 3;

distilled water ml up to 1000;

the ingredients in the indicated order are dissolved in distilled water, each ingredient after dissolving the previous one, agar is added, filtered through a cotton-gauze filter. Sterilized in an autoclave at 1 atmosphere, 120 ° C, 30 minutes. A few days before sowing, the medium is heated in a water bath to a temperature of 40-45 ° C and sterile potato broth 20% and blood serum of cattle 10% are added as a growth stimulant. The medium from the flask is poured into 6 ml tubes, checked for sterility for two days.

The nutrient medium for the isolation of L-forms of mycobacteria "Test B" is composed of the following components, g / l:

monosubstituted potassium phosphate - 1.5;

magnesium sulfate - 0.5;

sodium phosphate - 2.5;

iron citrate - 0.05;

glycocol - 2;

monosubstituted glutamine sodium - 10;

glycerin - 40;

agar-agar - 3;

distilled water ml up to 1000;

the ingredients in the indicated order are dissolved in distilled water, each ingredient after dissolving the previous one, agar is added, filtered through a cotton-gauze filter. Sterilized in an autoclave at 1 atmosphere, 120 ° C, 30 minutes. A few days before sowing, the medium is heated in a water bath to a temperature of 40-45 ° C and sterile potato broth 20% and blood serum of cattle 10% are added as a growth stimulant. The medium from the flask is poured into 6 ml tubes, checked for sterility for two days.

The results obtained by comparing the three options for the nutrient medium are presented in table 1.

Comparative analysis showed that, unlike Test A and B, the use of the proposed nutrient medium of Test B allows to reduce the time of primary growth of L-forms of mycobacteria by 2–3 times, to increase the biomass accumulation rate by 2 times, necessary for further study of the characteristics of the infection process caused by them , and also reduce the time of work.

The proposed environment is available for production and meets modern requirements when working with infectious material, safe and effective when working.

Information sources

1. "Nutrient medium for the cultivation of mycobacteria" RU 2410425 C1, C12N 1/20, C12R 1/32, 01/27/2011

2. Manual for the diagnosis of animal tuberculosis / Ministry of Agriculture of the Russian Federation Department of Veterinary November 18, 2002 p.17-16.

Additional sources of information

1. "The method of reversing the L-forms of mycobacteria" RU 2275422 C2, C12N 1/20, C12R 1/32, 04/27/2006

2. "Nutrient medium for the isolation and cultivation of L-forms of Mycobacterium tuberculosis" RU 2242511 C2, C12N 1/20, C12Q 1/04, 12/20/2004

3. "The method of species differentiation of mycobacterium tuberculosis" RU 2428484 C2, C12Q 1/04, C12N 1/20 20, 12.12.2010

Claims (1)

Nutrient medium for detecting L-forms of mycobacteria from pathological material, containing cattle blood serum, agar, characterized in that it additionally contains monosodium glutamine monosubstituted, glycol and, as a growth promoter, sterile potato broth in the following ratio of components, wt.% :
potassium phosphoric acid monosubstituted 0.026-0.035 magnesium sulfate 0.05-0.07 sodium phosphoric acid 0.25-0.33 citric acid iron 0.005-0.007 glycocol 0.35-0.41 monosubstituted glutamine sodium 0.1-0.2 glycerol 0.3-0.4 agar agar 0.3-0.4 potato broth 9-12 cattle serum 10-20 distilled water rest