RU2320716C2 - Solid nutrient medium for mycobacterium culturing - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to a nutrient medium containing potassium hydrogen phosphate, magnesium sulfate, L-asparagine, glycerol, citric acid, iron ammonium citrate, distilled water, sodium pyruvate, agar, humivit and chicken yolk egg aqueous solution (1:1). Invention allows decreasing cost of the nutrient medium and to simplify carrying out bacteriological researches. Invention can be used in investigation of mycobacterium tuberculosis.

EFFECT: valuable properties of nutrient medium.

4 tbl, 4 ex

Description

The invention relates to microbiological research, relates to a nutrient medium for the cultivation of mycobacteria and can be used to characterize mycobacteria by their biological activity.

Known dense nutrient medium for growing primary cultures of mycobacteria, consisting of egg mass and salt base: Levenshtein-Jensen, Gelberg, Petraniani, environment Finn-2 and Mordovian / 1 /. The manufacturing technology of these media provides for their coagulation, which excludes the possibility of their use as a component of a two-layer medium for studying the biological activity of mycobacteria.

The purpose of the invention is a dense nutrient medium for the cultivation of mycobacteria, suitable for use as a layered component of bilayer media.

This goal is achieved in that the nutrient medium containing the salt base and the egg base contains L-asparagine, potassium disubstituted potassium, magnesium sulfate, citric acid, ammonium citric acid, sodium pyruvate, glycerin, agar, humivite and distilled water as the salt base. and as an egg base - an aqueous solution of chicken yolk (1: 1) in the following ratio of components:

L-asparagine 4.0 g Cadmium phosphate disubstituted 0.5 g magnesium sulfate 0.5 g lemon acid 2.0 g ammonium citrate 0.05 g pyruvate sodium 0.5 g glycerol 40.0 g agar 30.0 g gumivit 90.0-95.0 ml aqueous solution of chicken yolk (1: 1) 250.0 ml distilled water up to 1000.0 ml

Gumivit - an alkaline hydrolyzate of lowland peat varieties (manufactured by Agroprom LLC) is a highly dispersed sol of sodium salts of a complex of humic acids - humic, humatemeanic and fulvic acids / 2 /. The solution of salts of humic acids consists of long fragmentary chains with various functional groups, the most important of which are carboxylic (COOH) and phenolic (OH), capable of forming chelate complexes with trace elements, which ensures their high exchange capacity and transport to the cell of microorganisms / 3, 4 /. Gumivit is a liquid of dark brown to black color with a specific sweetish odor, due to which the finished medium has an intensely brown color, against which even small colonies are clearly visible in the initial growth phase.

The essence of the method is illustrated by examples.

Example 1. To prepare the medium in a chemically pure sterile flask with a capacity of 2.0 l, 300.0 ml of distilled water was poured, heated in a water bath to 75-85 ° C. 2.0 g of citric acid, 4.0 g of L-asparagine, 0.5 g of magnesium sulfate, 0.5 g of potassium phosphate disubstituted, 0.05 g of ammonium citric acid, 0.5 g of sodium pyruvate, 40 were successively dissolved in water , 0 g of glycerol, 90.0 ml of humivit and 30.0 g of agar. Each component was added after complete dissolution of the previous one. After all components were added, the total volume was adjusted to 750.0 ml with sterile distilled water. The pH was adjusted to 7.1-7.2 by the addition of 1N. NaOH or 1 N. HCl. The solution was autoclaved for 30 minutes at 1 atm. Fresh chicken eggs were washed well in running water, wiped with 96 ° ethanol, broken under sterile conditions, and the protein was separated from the yolk. In a sterile flask, yolk and distilled water were mixed in a ratio of 1: 1 and 250.0 ml of the resulting solution, added to the molten agar medium, mixed, poured into 3.0 ml tubes and mowed.

Example 2. Table 1 shows the options for a dense nutrient medium in accordance with the invention.

Table 1 The composition of different options for dense nutrient media Structure New Environment Option 1st 2nd 3rd 4th L-asparagine, g 4.0 4.0 4.0 4.0 Potassium phosphate disubstituted, g 0.5 0.5 0.5 0.5 Magnesium sulfate, g 0.5 0.5 0.5 0.5 Citric acid, g 2.0 2.0 2.0 2.0 Ammonium iron citrate, g 0.05 0.05 0.05 0.05 Pyruvate sodium, g 0.5 0.5 0.5 0.5 Glycerin, g 40,0 40,0 40,0 40,0 Agar, g 30,0 30,0 30,0 30,0 Humivit, ml 85.0 90.0 95.0 100.0 An aqueous solution of chicken yolk (1: 1), ml 250,0 250,0 50,0 250,0 Distilled water, ml up to 1000.0 up to 1000.0 up to 1000.0 up to 1000.0

Example 3. To determine the effectiveness of a dense nutrient medium for the cultivation of mycobacteria, reference strains of mycobacteria M. bovis (pieces Vallee and BCG), M. tuberculosis (pieces Academia, H ₃₇ Ra), M.avium (pieces HIS, 44) were used , 659), M.smegmatis (pcs. 53), M.phlei. All strains were stored on Levenshtein-Jensen medium in a refrigerator at a temperature of + 4 ° C. The crops were sown by applying one drop of a suspension of mycobacteria containing 30-60 CFU when plated on Levenshtein-Jensen medium and on all compared medium variants in accordance with the invention. Sowing of each strain was carried out in 3-5 tubes. When assessing the growth of cultures, the timing of the appearance of primary colonies, the growth rate (± weak, + medium, ++ plentiful) were taken into account and cultural-morphological properties were studied. Control is the dense nutrient medium of Levenshtein-Jensen. The results obtained (tables 2 and 3) showed that the optimal options are 2 and 3 of a dense nutrient medium in accordance with the invention, providing earlier terms for the appearance of primary colonies and a higher growth rate relative to option 1 and comparable with those on the Levenshtein-Jensen medium . The increase in the amount of humivitis (option 4 of a dense nutrient medium in accordance with the invention) did not reduce the time of growth and did not increase its intensity, therefore, it was impractical. The cultural-morphological properties and color of the grown colonies of the strains did not differ from those in the initial medium.

table 2 The timing of the appearance of the primary growth of mycobacteria on media of different variants Strains Dates of growth, days The environment in accordance with the invention Wednesday of Levenshtein - Jensen Option 1 Option 2 Option 3 Option 4 M.bovis (pc. Vallee) fifteen 13 12 12 fourteen M.bovis (pc. BCG) fifteen fifteen fifteen fifteen fifteen M.tuberculosis (pc. Academia) 13 eleven 10 10 9 M.tuberculosis (pc. H ₃₇ Ra) 13 eleven 10 10 9 M.avium (PCHIS) 9 7 7 7 7 M.avium (pcs. 44) 12 10 9 9 7 M.avium (pcs. 659) eleven 10 10 10 8 M.phlei four 3 2 2 2 M.smegmatis (pcs. 53) four 3 2 2 2

Table 3  The massive growth of mycobacteria on different media Wednesday Mycobacterial strains M.bovis Vallee M.bovis BCG M.tub. Academia M.tub. H ₃₇ Ra M.avium GISK M.avium 44 M.avium 659 M.phlei M.smegmatis 53 Massive growth 3 weeks growth 1 week growth Option 1 ± ± ± + ± ± ± + + Option 2 + + + ++ + + + ++ ++ Option 3 + + + ++ ++ + ++ ++ ++ Option 4 + + + ++ ++ + ++ ++ ++ Wednesday of Levenshtein Jensen + + + ++ ++ + ++ ++ ++

Example 4. Table 4 shows the results of a study of the antagonistic activity of the probiotic "Kolibacterin" in relation to mycobacteria using the medium in accordance with the invention, which was layered on seeded with probiotic strains of meat-peptone agar.

Table 4 Antagonistic activity of the probiotic "Kolibacterin" in relation to mycobacteria The dose of colibacterin, CFU / ml suspension, when sowing 10 μl Growth rate M.tuberculos is H ₃₇ Ra M.tuberculos is Academia M.bovis Vallee M. avium 44 M.smegmatis 53 1-5 × 10 ³ + + + + + 1-5 × 10 ⁴ + + + + + 1-5 × 10 ⁵ ± ± ± ± + 1-5 × 10 ⁶ ± ± ± - + 1-5 × 10 ⁷ ± - - - + 1-5 × 10 ⁸ - - - - ± Note: + average growth, ± weak growth, - lack of growth

Studies have confirmed the effectiveness of a dense nutrient medium in accordance with the invention for the cultivation of mycobacteria (the timing of the appearance of primary growth and growth rate are comparable to those on traditionally used media). The reversibility of the medium allows its use as a layered component of a two-layer nutrient medium in the study of the biological properties of mycobacteria. The claimed dense nutrient medium is characterized by low cost and manufacturability, and its use simplifies bacteriological studies by visualizing the initial growth of colonies.

Information sources

1. Vasiliev V.N. Mycobacteriosis and mycosis of the lungs. - Sofia, 1971. - S.146-175.

2. Grishin G.I., Slinina K.N. The gift of nature - gumivit.// Practician. - 2004. - No. 3-4. - S.80-82.

3. Levinsky B.V. All about humates. - Irkutsk: IP Markov S.E., 1999 .-- 198 p.

4. Sorokina N.F. The physiology of the activity of peat oxidation products. // New processes and products of peat processing. - Minsk: Nauka, 1982. - S.109-115.

Claims (1)

A dense nutrient medium for the cultivation of mycobacteria, containing salt and egg bases, characterized in that it contains L-asparagine, potassium phosphoric disubstituted, sulfuric acid magnesium, citric acid, citric acid ammonium iron, sodium pyruvate as a salt base glycerin, agar, gumivit and distilled water, and as an egg base - an aqueous solution of chicken yolk (1: 1) in the following ratio of components: L-asparagine 4.0 g disubstituted phosphoric acid potassium 0.5 g magnesium sulfate 0.5 g lemon acid 2.0 g citric acid ammonia iron 0.05 g pyruvate sodium 0.5 g glycerol 40.0 g agar 30.0 g gumivit 90.0-95.0 ml aqueous solution of chicken yolk (1: 1) 250.0 ml distilled water up to 1000.0 ml