Nutrient medium for detection of l-forms of mycobacteria

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

 

The invention relates to veterinary Microbiology and relates to culture media to obtain the L-forms of mycobacteria from pathological material, provides a reduction in terms of growth and increased biomass yield of L-forms of mycobacteria.

Known dense nutrient medium for the selection of the biomaterial animals of Mycobacterium tuberculosis and their cultivation, containing eggs, malachite green, potassium phosphate dvuhkamernyi, sodium citrate, magnesium sulfate, peptone, glycerol, Tetra or tape, or hexadecan, distilled water ["Nutrient medium for cultivation of mycobacteria" EN 2410425 C1, C12N 1/20, C12R 1/32, 27.01.2011,].

However, this environment is not suitable for cultivation of L-forms, as they require semi-solid nutrient medium, providing the necessary for their growth osmotic conditions.

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

potassium phosphate was 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 are;

agar - 0,25-0,3;

distilled water - other;

additives to the od of the WMD liter of medium: sucrose 10; the blood serum of cattle 10.

[Counsel for the diagnosis of tuberculosis of animals / the Ministry of agriculture of the Russian Federation Department of veterinary 18.11.2002, p.17-16].

However, the known environment doesn't allow for a fast time of pathological material L culture mycobacteria required for experiments on the study of the characteristics caused by an infectious process. In addition, it does not provide a sufficient accumulation of biomass.

The objective of the invention is to provide a framework for the allocation of pathological material L culture of mycobacteria, which allows to reduce terms of growth and increase biomass yield of L-forms of mycobacteria.

To solve the problem proposed environment consisting of the following components, wt%:

- potassium phosphate one-deputizing was 0.026-0,035;

- magnesium sulfate - 0,05-0,07;

- sodium phosphate - 0,25-0,33;

iron citrate - 0,005-0,007;

- glycocol - 0,35-0,41;

- sodium glutamic one-deputizing - 0,1-0,2;

- glycerin - 0,3-0,4;

agar - 0,3-0,4;

- distilled water - other;

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

The effectiveness of the enclosed environment to highlight the L-forms of mycobacteria, which allows reduced the ü terms of growth and increase biomass yield of L-forms of mycobacteria, tested on three different test environments.

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

potassium phosphate one-deputizing - 1,5;

sulphate magnesium - 0,5;

sodium phosphate - 2,5;

iron citrate - 0,05;

glycocol - 1;

glycerin - 40;

agar-agar - 3;

distilled water ml to 1000;

the ingredients in the above order dissolved in distilled water, each ingredient after the dissolution of the previous one, add agar, filtered through a cotton-gauze filter. Sterilize in an autoclave at 1 atmosphere, 120°C, 30 minutes. A few days before planting medium is heated on a water bath to a temperature of 40-45°C and as a growth stimulant add sterile potato broth 20% and the serum of cattle 10%. The environment of the flask are poured into a test tube, 6 ml, tested for sterility for two days.

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

potassium phosphate one-deputizing - 1,5;

sulphate magnesium - 0,5;

sodium phosphate - 2,5;

iron citrate - 0,05;

glycocol - 1;

sodium glutamic one-deputizing - 5;

glycerin - 40;

agar-agar - 3;

distilled water ml to 1000;

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

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

potassium phosphate one-deputizing - 1,5;

sulphate magnesium - 0,5;

sodium phosphate - 2,5;

iron citrate - 0,05;

glycocol - 2;

sodium glutamic one-deputizing - 10;

glycerin - 40;

agar-agar - 3;

distilled water ml to 1000;

the ingredients in the above order dissolved in distilled water, each ingredient after the dissolution of the previous one, add agar, filtered through a cotton-gauze filter. Sterilize in an autoclave at 1 atmosphere, 120°C, 30 minutes. A few days before planting medium is heated on a water bath to a temperature of 40-45°C and as a growth stimulant add sterile potato broth 20% and the serum of cattle 10%. The environment of the flask are poured into the tube 6 is l, check for sterility for two days.

The results obtained when comparing the three variants of the nutrient medium are presented in table 1.

Comparative analysis showed that in contrast to the Test a and B using the proposed nutrient medium under Test In allows to reduce terms of the primary growth of L-forms of mycobacteria in 2-3 times, to increase the intensity of accumulation of biomass in 2 times necessary for further study of the features caused by the infection and reduce the time for the operation.

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

Sources of information

1. "Nutrient medium for cultivation of mycobacteria" EN 2410425 C1, C12N 1/20, C12R 1/32, 27.01.2011,

2. Counsel for the diagnosis of tuberculosis of animals / the Ministry of agriculture of the Russian Federation Department of veterinary 18.11.2002, p.17-16.

Additional sources of information

1. "The way the reversion of L-forms of mycobacteria" EN 2275422 C2, C12N 1/20, C12R 1/32, 27.04.2006,

2. "Nutrient medium for the isolation and cultivation of L-forms of Mycobacterium tuberculosis" EN 2242511 C2, C12N 1/20, C12Q 1/04, 20.12.2004,

3. "The way the species differentiation of Mycobacterium tuberculosis" EN 2428484 C2, C12Q 1/04, C12N 1/20 20, 12.2010,

Nutrient medium on the I detection of L-forms of mycobacteria from pathological material, containing the serum of cattle, agar, characterized in that it further contains sodium glutamic one-deputizing, glycocol and as a growth promoter - sterile potato broth in the following ratio, wt.%:

potassium phosphoric acid one-deputizing0,026-0,035
sulfuric acid magnesium0,05-0,07
sodium phosphoric acid0,25-0,33
iron citric acid0,005-0,007
glycocol0,35-0,41
sodium glutamic one-deputizing0,1-0,2
glycerin0,3-0,4
agar-agar0,3-0,4
potato broth9-12
serum cattle10-20
distilled waterrest



 

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