RU2332452C2 - Composition for obtaining nutrient medium for recovery and cultivation of mycobacteria - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: composition includes components at the following ratio: chicken eggs 400.0-500.0 g, chicken egg yolks 90.0-100.0 g, milk 140.0-160.0 g, potato decoction 140.0-160.0 ml, 2% basic green solution 10.0-12.0 g, disubstituted potassium phosphate 0.14-0.16 g, sodium citrate 0.14-0.16 g, magnesium sulphate 0.14-0.16 g, peptone 0.8-1.0 g, glycerin 3.2-4.0 g, alkane 15.3-30.6 g, distilled water up to 140.0-160.0 ml, 10% solution of Lucevita phythopreparations 0.25-0.5 ml.

EFFECT: possibility to recover mycobacteria with attenuated viability.

1 tbl, 1 dwg

Description

The invention relates to veterinary microbiology and can be used for laboratory diagnosis of tuberculosis.

A dense nutrient medium is known for isolating animals from biomaterial and cultivating mycobacterium tuberculosis (FAST-ZL), containing chicken eggs, egg yolks, milk, potato broth, glycerin, peptone, malachite green, a solution of mineral salts and one of alkanes with a chain length from C _14- C ₁₆ (1).

However, the known medium does not allow the detection of mycobacteria with reduced viability.

The purpose of the invention is to increase the excretion of mycobacterium tuberculosis during sowing of samples of biological material and environmental objects.

This is achieved by the fact that before the sowing of Fast-3L, an additional 10% sterile solution of the phytopreparation Lutsevita in a dose of 0.25-0.5 ml per tube containing 4-5 ml of medium is additionally introduced as a stimulator of mycobacteria growth. The phytopreparation is manufactured in CJSC Lutsevita registration number 004836.P.643.09.2002.

Phytomedication “Luzovita” is biologically active on the basis of alfalfa extract obtained by treating it with an extractant containing a mixture of salts of Mn, Co, Cr, V, Zn, Fe, Cu, Ti, and Mo.

Phytomedication “Lucevoita” contains up to 20 vital amino acids, B, E, K vitamins, macro- and microelements, carboxylic (succinic, oxalic) and phenol carboxylic acids, proteins, carbohydrates, saponins, isoflavones (2).

“Lyutsovita”, added to the Fast-ZL nutrient medium, has a beneficial effect on the growth and reproduction of mycobacteria, especially pathogenic ones, which have reduced biochemical activity, and they multiply much more slowly, contain less enzymes and growth substances. The addition of biologically active additives to the nutrient media allows enriching their composition with proteins, vitamins, macro- and microelements, which are necessary for building cellular material and increasing the enzymatic activity of mycobacteria.

Example

It was tested the effect of various doses of "Lyutsovita" on the excretion of mycobacteria from various objects.

For this purpose, nutrient media were prepared in the following ratio of components, g:

Eggs 400.0-500.0 Chicken Egg Yolks 90.0-100.0 Milk ml 140.0-160.0 Potato broth, ml 140.0-160.0 2% solution of malachite green 10.0-12.5 Saline solution: Potassium Disubstituted Potassium Phosphate 0.14-0.16 Sodium Citrate 0.14-0.16 Magnesium sulfate 0.14-0.16 Peptone 0.8-1.0 Glycerin 3.2-4.0 Alkan 15.3-30.6 Distilled water, ml Up to 140.0-160.0 10% solution of the phytopreparation "Lutsevita", ml 0.03 each; 0.12; 0.25; 0.5

Fast-ZL nutrient medium without phytopreparation was used as a control.

The studied material was lymph nodes, milk, and manure from cows that responded positively to mammalian PPD tuberculin. Pre-sowing treatment was carried out according to the method of A.P. Alikayeva. After plating, the tubes were placed in a thermostat and incubated at 38 ° C for 60 days. The research results are presented in the table.

The effect of various doses of the phytopreparation "Lucevoita" on the excretion of mycobacteria from biomaterial Test material Number of samples Growth on the fast-ZL nutrient medium the control with the addition of "Lyutsovita" qty % 0,03 0.12 0.25 0.5 qty % qty % qty % qty % The lymph nodes 9 6 66.7 four 44,4 8 88.8 9 one hundred 9 one hundred Milk 26 eleven 42.3 8 30.8 16 61.5 25 88.5 24 92.3 Manure 25 eleven 44.0 5 19.2 10 38.5 13 50,0 23 92.0

The experimental data show that the addition of phytopreparation to the medium at a dose of 0.03 ml does not affect the increase in excretion of mycobacteria. With the addition of Luceutovita in a dose of 0.12 ml, the excretion of mycobacteria from milk increases to 61.5% and from lymph nodes to 88.8%. The highest results in all cases were obtained with the addition of Lutsevita in a dose of 0.25-0.5 ml. The growth of cultures of mycobacteria is observed in 88.5-100.0% of cases.

Thus, the proposed nutrient medium, which additionally contains a biologically active additive of the phytopreparation “Lutsevita” at a dose of 0.25-0.5 ml per tube, helps to increase the excretion of mycobacteria when sowing samples of biomaterial (lymph nodes, milk) and environmental objects (feed) , manure), which allows us to conclude that the proposed solutions meet the criteria of novelty, inventive step, industrial applicability.

Information sources

1. A.S. No. 2059728 C12Q 1/04. Nutrient medium for the isolation of animal biomaterial and the cultivation of Mycobacterium tuberculosis. Author: Hodun L.M., Taller L.A. and etc.

2. RF patent No. 2176918 SZ. Biologically active drug based on alfalfa extract and method for its production. Author: Grishchenko V.V., Zholnin A.V., Kamynin A.V. and etc.

Claims (1)

Composition for preparing a nutrient medium for the isolation and cultivation of mycobacteria, containing chicken eggs, egg yolks, milk, potato broth, peptone, glycerin, potassium phosphate disubstituted, sodium citric acid, magnesium sulfate, malachite green, alkane and distilled water, characterized in that it additionally contains a 10% solution of the phytopreparation "Lutsevita" in the following ratio of components, g: Eggs 400.0-500.0 Chicken Egg Yolks 90.0-100.0 Milk ml 140.0-160.0 Potato broth, ml 140.0-160.0 2% solution of malachite green 10.0-12.5 Potassium Phosphoric Acid Disubstituted 0.14-0.16 Sodium citric acid 0.14-0.16 Magnesium Sulfuric Acid 0.14-0.16 Peptone 0.8-1.0 Glycerol 3.2-4.0 Alkan 15.3-30.6 Distilled water, ml Up to 140.0-160.0 10% solution of the phytopreparation "Lutsevita", ml 0.25-0.5