RU2315812C1 - Nutrient medium for isolation and culturing mycobacteria from biological material - Google Patents

Abstract

FIELD: biotechnology, medicine, microbiology.

SUBSTANCE: invention proposes nutrient medium that comprises as the nutrient base the following components: hen yolk egg aqueous solution, potassium hydrogen phosphate, magnesium sulfate, L-asparagine, glycerol, malachite green, citric acid, ferrous ammonium citrate, humivite, agar and distilled water. Invention provides increasing the growth rate of tuberculosis mycobacteria and to enhance purity in their indication from biological material. Invention can be used in bacteriological diagnosis of tuberculosis.

EFFECT: improved and valuable properties of nutrient medium.

3 tbl, 3 ex

Description

The invention relates to microbiology, relates to a nutrient medium for isolation from pathological material and cultivation of mycobacteria and can be used in bacteriological diagnosis of tuberculosis.

The bacteriological method, in particular the sowing of biological material on special nutrient media, allows not only to obtain a pure culture of mycobacteria, but also to identify them, to determine virulence, biological and biochemical properties.

In bacteriological practice, dense nutrient media of Levenshtein-Jensen, Gelberg, Petraniani, Finn-2 and FAST-3L / 1 / are most often used, which provide comparable indicators of growth rate and intensity, as well as seeding of mycobacteria from pathological material.

As a preferred analogue, we consider the Levenshtein-Jensen egg medium containing a nutritious egg base, disubstituted potassium phosphate, magnesium sulfate, magnesium citrate, L-asparagine, chemically pure glycerin, malachite green, and distilled water. The Levenshtein-Jensen medium provides the best results in the cultivation of bovine and human species of mycobacteria / 2 /.

The purpose of the invention is to increase the growth rate and frequency of indication of mycobacteria from biological material.

The goal is achieved in that the nutrient medium containing the nutrient base of eggs, disubstituted potassium phosphate, magnesium sulfate, ammonium citrate, L-asparagine, chemically pure glycerin, malachite green and distilled water, additionally contains citric acid, ammonium citric acid iron , gumivit and agar, and as a nutritious basis of eggs - an aqueous solution of chicken yolk (1: 1) in the following ratio of components:

potassium phosphate disubstituted 0.5 g magnesium sulfate 0.5 g L-asparagine 4.0 g chemically pure glycerin 40.0 g malachite green 20.0 ml lemon acid 2.0 g ammonium citrate 0.05 g gumivit 90.0-95.0 ml agar 30.0 g aqueous solution of chicken yolk 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 / 3 /. 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 / 4, 5 /. 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 invention 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, 0.5 g of magnesium sulfate, 0.5 g of disubstituted potassium phosphate, 0.05 g of ammonium citrate, 20.0 ml of malachite green, 40.0 g of chemically pure glycerol, 90 were dissolved successively in water , 0 ml of humivitis 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 adding 1 N 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. The yolk was mixed in a sterile flask and distilled water in a ratio of 1: 1 and 250.0 ml of the resulting solution was added to the molten agar medium, mixed, poured into 3 ml tubes and beveled.

Example 2. For a comparative study of the information content of the four variants of the environment in accordance with the invention, differing in the content of humivitis, and the environments of Levenshtein-Jensen, Finn-2, Gelberg, FAST-3L and Petraniani used reference strains of M. bovis mycobacteria (Vallee, Ravenal BCG), M. tuberculosis (Academia pcs.), M.avium (GIS pcs 659), M.smegmatis (4 pcs.), M.fortuitum and biological material from cattle that were not responsive to tuberculin with characteristic lesions in the organs and lymph nodes. All strains were stored on Levenshtein-Jensen medium in a refrigerator at a temperature of + 4 ° C. Biological material was pretreated according to the method of A.P. Alikayeva. Crops of crops were produced by applying one drop of a suspension of mycobacteria containing 30-60 CFU, when sowing on known media and on all compared medium options 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 was taken into account and cultural and morphological properties were studied. The growth rate was studied by sowing the culture of M. bovis (urr.Vallee) on all analyzed media. The results are presented in tables 1 and 2.

Table 1. The growth rate of different species of mycobacteria on solid nutrient media Culture medium The growth rate, days. M. bovis pc Vallee M. bovis pc. Ravenal M. bovis BCG M. tuberculosis pc. Academia M.avium pc. M.avium pcs. 659 M. smegmatis piece 4 M.fortuitum Cattle Biomaterial Levenshtein-Jensen 13-15 14-16 16-17 13-15 10-12 11-13 3-4 6-7 29-33 Finn 2 13-15 14-16 16-17 13-15 10-11 11-12 3-4 5-8 29-33 Gelberg 17-19 17-19 20-21 17-19 11-12 13-13 2-3 7 33-37 FAST-3L 16-18 16-18 18-19 15-17 10 eleven 3 6 30-34 Petraniani 19-21 19-21 19-21 16-18 10-12 11-13 4-5 9-10 36-40 The claimed medium containing humivit, ml h option 1 - 85.0 14-16 17-19 20-21 17-19 12-14 13-15 4-5 eleven 35-39 option 2 - 90.0 11-12 14-16 16-17 13-15 10-11 10-11 2 6 26-28 option 3 - 95.0 11-12 12-14 16-17 12-13 10-11 10-11 2 5 26-28 option 4 - 100.0 11-12 12-14 16-17 12-13 10-11 10-11 2 5 26-28

Table 2. The growth rate of M. bovis (pieces Vallee) on solid nutrient media Culture medium Day 7 9 10 eleven 12 13 fourteen fifteen 16 24 Levenshtein-Jensen - - - - - + + ++ ++ +++ Finn 2 - - - - - + + ++ ++ +++ Gelberg - - - - - - ++ ++ ++ +++ FAST-3L - - - - - - + ++ +++ +++ Petraniani - - = = - - + ++ +++ +++ The claimed environment with the content of gumivita, ml
option 1 - 85.0 option 2 - 90.0 - - - - ++ ++ ++ ++ +++ +++ option 3 - 95.0 - - - + ++ ++ ++ +++ +++ ++++ option 4 - 100.0 - - - + ++ ++ ++ +++ +++ ++++ Note: + - single colonies; ++ - 10-20 colonies: +++ - 21-50 colonies; ++++ - solid growth

According to the rate of primary growth of pure cultures on solid nutrient media, the best indicators were observed on the claimed medium with a content of gumivita 90.0-100.0 ml (table 1). Given that an increase in the amount of humivitis above 95.0 ml (option 4) did not affect the growth rate, options 2 and 3 with the content of humivit 90.0 and 95.0 ml, respectively, were recommended for practical use. The culture of M. bovis on the medium of Levenshtein-Jensen, Gelberg and in accordance with the invention grew in the form of separate, small, smooth, spherical, ivory colonies; on Finn-2, FAST-3L and Petraniani - more often in the form of dry wrinkled colonies. M. tuberculosis on the same media most often grew in the form of small, nodular-like, wrinkled, dry, ivory-colored colonies, and M.avium, M.fortuitum and M. smegmatis in the form of soft, mucous, grayish-white colonies. On the FAST-3L medium, the M.avium culture grew sparse - single colonies. According to the indicator of the growth rate of M. bovis culture, the most informative was the environment in accordance with the invention (options 2-4), in which intensive growth of cultures (++) manifested itself in the 12th, significant accumulation of bacterial mass in the 15th, and solid colony growth - on the 24th day (table 2). The growth rate of the culture of mycobacteria was significantly lower on Gelberg, FAST-3L and Petraniani media.

Example 3. To determine the seeding rate of mycobacteria, 12 samples of biomaterial from cattle with characteristic lesions of internal organs and lymph nodes were examined. When crops were prepared, a common homogeneous sample was prepared by mixing and thoroughly homogenizing all 12 separate samples. After processing the material according to the Alikayev method, samples were sown in 40 test tubes of each medium — Levenshtein-Jensen, Finn-2, Gelberg, FAST-3L, Petraniani, in accordance with the invention (with a content of gumivita 95.0 ml). The results are presented in table 3.

Table 3. Inoculation of cultures of mycobacteria from biomaterial from cattle on different nutrient media Culture medium The number of crops, test tubes Crop growth The growth of extraneous microflora test tubes % test tubes % Levenshtein-Jensen 40 23 57 2 5 Finn 2 40 22 54 one 2 Gelberg 40 eleven 27.5 3 7.5 FAST-3L 40 eleven 27.5 four 10.0 Petraniani 40 5 12.5 5 12.5 in accordance with the invention 40 thirty 73 one 2

According to the indicator of inoculation of mycobacteria from biomaterial from animals, the most informative were the media in accordance with the invention, of Levenshtein-Jensen and Finn-2 - 73, 57 and 54%, respectively (table 3). The smallest growth of extraneous microflora was observed on the media in accordance with the invention and Finn-2 - 2%. Inoculation of mycobacteria on other media was 2-4 times lower.

The main characteristics of the known solid nutrient media and environments in accordance with the invention, confirmed by the research results:

Levenshtein-Jensen - standard unified, industrial production, fine-tuning in the conditions of bacteriological laboratories. The nature of the growth of crops corresponds to the morphological properties of different types of mycobacteria;

Gelberg - the quality of the environment depends on the qualifications and experience of a specialist, often takes a pale green color, which makes it difficult to view colonies. Growth corresponds to the basic classical characteristics of mycobacterial cultures;

Finn-2 - the composition includes a scarce and expensive glycocol, with respect to seeding and growth rate at the initial indication of mycobacteria, it is comparable to the Levenshtein-Jensen medium;

Petraniani - in terms of informativeness, speed and intensity of growth of mycobacteria, it is inferior to all other media, but it allows the detection of mycobacteria from environmental objects because of the ability to hold pH 7.0 for a long time;

FAST-3L - standard, industrial production, in terms of growth properties not inferior to Gelberg's environment. The cultures grown on it, do not give growth when reseeding to other nutrient media;

in accordance with the invention, it is technologically advanced in preparation, in terms of information, speed and intensity of growth and inoculation of mycobacteria, it surpasses tested media, including Levenshtein-Jensen. Highly effective for the accumulation of biomass of primary cultures of mycobacteria. The nature of the growth of crops corresponds to the morphological properties of different types of mycobacteria. Intensively brown color of the medium allows detecting small colonies in the initial stage of growth.

Sources of information taken into account:

1. Tuberculosis of farm animals / Ed. Shishkova V.P., Urbana V.P. - M .: Agropromizdat, 1991. - S.120-121.

2. RU 2268298 C1, 01.20.2006.

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

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

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

Claims (1)

A nutrient medium for the isolation from biological material and cultivation of mycobacteria, containing a nutrient base from eggs, potassium phosphate disubstituted, magnesium sulfate, L-asparagine, glycerin, malachite green and distilled water, characterized in that it additionally contains citric acid, ammonium citric acid, humivit and agar, and as a nutrient base it contains an aqueous solution of chicken yolk in the following ratio of components: disubstituted potassium phosphate 0.5 g magnesium sulfate 0.5 g L-asparagine 4.0 g glycerol 40.0 g malachite green 20.0 ml lemon acid 2.0 g ammonium citrate 0.05 g gumivit 90.0-95.0 ml agar 30.0 g aqueous solution of chicken yolk 250.0 ml distilled water up to 1000.0 ml