RU2242511C2 - Nutritive medium for isolating and cultivating tubercular mycobacterial l-forms - Google Patents

Abstract

FIELD: veterinary microbiology.

SUBSTANCE: the suggested medium additionally contains saturated hydrocarbon at chain length being C14-C17, isoniazide, as for saline foundation it is represented by Hanks' buffer solution. The innovation provides shortened terms for the growth and increased output of mycobacterial L-forms' biomass.

EFFECT: higher efficiency.

3 ex, 2 tbl

Description

The invention relates to veterinary microbiology and relates to nutrient media for the experimental production and cultivation of L-forms of Mycobacterium tuberculosis.

A dense nutrient medium is known for isolating tuberculosis mycobacteria from the biomaterial of animals and cultivating mycobacteria containing chicken eggs, egg yolks, milk, potato broth, malachite green, potassium phosphate disubstituted, sodium citrate, magnesium sulfate, peptone, glycerin, tetra, or penta, or hexadecane, distilled water [1].

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

The closest analogue is the semi-liquid medium in Dorozhkovaya’s modification for growing L-forms of mycobacterium tuberculosis, containing: a nitrogen source, sucrose, native blood serum of cattle or horses, sodium citrate, ammonium citrate, potassium phosphate, sodium disodium, magnesium sulfate, , agar-agar and water [2].

However, the known environment does not allow to obtain in laboratory conditions the experimental L-culture of mycobacteria, necessary for experiments to study the characteristics of the infectious process caused by them. In addition, it does not ensure the rapid growth of L-forms of Mycobacterium tuberculosis and sufficient biomass accumulation, and the presence of impurities in the salt base has a toxic effect on the cell.

The objective of the invention is: to reduce the growth time and increase the biomass yield of L-forms of mycobacteria, which is achieved by the fact that in the proposed environment the salt base (Hanks ready standard solution) is characterized by the absence of toxic ions, creates the necessary buffering and plays a significant role in metabolism. For its preparation, pre-purified reagents and deionized water are used.

The salt base (Hanks solution) is prepared from two basic solutions “A” and “B”.

The basic solution “A”, g:

Sodium Chloride 160.0

Potassium chloride 8.0

Calcium Chloride Anhydrous 2.8

Magnesium Sulfate 4.0

Water, ml Up to 1000

Sterilize by autoclaving for 20 min 1.5 atm.

The main solution of “B”, g:

Sodium bicarbonate 7.0

Disubstituted Sodium Phosphate 1.2

Monosubstituted Potassium Phosphate 1.2

Glucose 20

Water, ml Up to 1000

Solution “B” is sterilized by filtration through EC ₂ plates in a Seitz filter.

To 900 ml of distilled water add 50 ml of solution “A”, autoclave and aseptically add 50 ml of solution “B”. Set the pH to 7.2 - 7.3 by adding sterilized by filtration of a 1.5% solution of sodium bicarbonate.

As a growth promoter, the nutrient medium contains one of the saturated hydrocarbons with a chain length of C ₁₄ -C ₁₇ . Limit hydrocarbons with a chain length of C ₁₄ -C ₁₇ at a dose of 0.1-0.2 ml per tube have a growth-promoting effect, actively affect the metabolic processes in the microbial cell, activate carbohydrate synthesis, are an energy source and plastic material, lowering the dose does not growth-stimulating effect, and increasing the dose leads to a decrease in the growth rate, therefore this amount (0.1-0.2 ml) is optimal for achieving the effect.

As an L-transforming agent, the nutrient medium contains isoniazid, which affects the protein synthesis of mycobacterial cells, resulting in a violation of the integrity of the bacterial cell wall and the formation of L-forms.

Pathogenic mycobacteria are biochemically less active, as evidenced by their slow reproduction and the need for a certain complex of nutrients. They contain less enzymes and growth substances, therefore they are more sensitive to antibacterial drugs.

The presence of native cattle or horse blood serum is required. It possesses buffering properties, and also has a stimulating effect on the reproduction and growth of L-forms, since proteins and a number of vitamins firmly connected with them enter the blood serum.

Example 1

The effect of different doses of isoniazid on cultures of M. bovis pcs was tested. 8 and M. avium pcs. 9. Cultures were seeded on a test nutrient semi-fluid medium. The results of the study are presented in table 1.

When conducting microscopy of smears, we noted that in test tubes containing 0.5 μg / ml of isoniazid, both cultures were represented by typical cells (97-100%).

At a concentration of 1 μg / ml in a bovine species culture, typical cells were absent, and the altered forms were represented by cocciform and vacuolated cells (37-43%) granular rods and spherical bodies (10-20%). M. avium pcs. 9 in this case was also represented by typical cells (93%).

With a 2-fold increase in concentration, typical M. bovis cells 8 - were absent, altered forms were observed in the form of cocciform cells (55%), in test tubes with M. avium pcs. 9, a large number of typical cells was noted (68%), but altered forms, mainly granular rods (23%), were already observed.

With an increase in the content to 5 μg / ml, M. bovis 8 growth was absent, and M. avium 9 grew in the form of a mixed culture: typical 60% and altered 40%. And only with an increase in the concentration of isoniazid to 10 μg / ml in the culture of M. avium 9 - typical cells were not observed.

Thus, we experimentally established that isoniazid has a L-transforming effect on pathogenic mycobacteria at a dose of 1 μg / ml of medium. Mycobacteria of the avium species and atypical mycobacteria synthesize more growth substances and vitamins, therefore, a chemotherapeutic substance acts on them in higher concentrations (10 μg / ml). Therefore, isoniazid in the proposed environment is used in a dose of 1 to 10 μg / ml, which is effective to achieve this goal.

Cooking medium. A portion of agar-agar (0.3-0.4 g) is diluted (heated in a water bath at 50-60 ° C) in 50 ml of Hanks solution, after which 9-12 ml of 200% sucrose, hydrocarbon are added (1, 1-2.2 ml), isoniazid (1-10 μg / ml), bring the volume of the medium to 100 ml with Hanks solution and autoclave at 0.5 atm. 30 minutes. The nutrient medium is poured into 9 ml into a test tube and kept in an incubator at 37 ° C for 2-3 days to check for sterility. Store the medium in well-corked tubes at 0 - (+5) ° C for 3-4 weeks. Before sowing, native blood serum of cattle or horse (10-20 ml) is added.

The proposed nutrient medium differs from the known one in that it additionally contains a saturated hydrocarbon with a long chain of C ₁₄ -C ₁₇ , isoniazid, and the salt base is represented by Hanks buffer solution in the following ratio of components:

Agar-agar, g 0.3-0.4

Native blood serum

cattle

or horse, ml 10-20

Sucrose 200%, ml 9-12

Saturated hydrocarbon

with a chain length of C ₁₄ -C ₁₇ , ml 1.1-2.2

Isoniazid, mcg / ml 1-10

Salt base - buffer

Hanks solution, ml Up to 100

Example 2

To prepare 100 ml of culture medium, the following ingredients are taken:

Agar-agar, g 0.3

Sucrose 200%, ml 9

Saturated hydrocarbon

with chain length C ₁₄ -C ₁₇ , ml 1.1

Native blood serum

cattle

or horse, ml 10

Isoniazid, mcg / ml 1

Salt base - buffer

Hanks solution, ml Up to 100

A portion of agar-agar is diluted in a salt base, after which 200% sucrose, hydrocarbon, isoniazid are added and autoclaved at 0.5 atm for 30 minutes. After cooling, native cattle or horse serum is added. The nutrient medium is poured into 9 ml in a test tube, kept in a thermostat at 37 ° C for 2-3 days to check for sterility. Store the medium in well-corked tubes at 0 - (+5) ° C for 3-4 weeks.

Example 3

Prepare the medium in accordance with example 2, but the components are taken in the following quantities:

Agar-agar, g 0.4

Sucrose 200%, ml 12

Saturated hydrocarbon

with chain length C ₁₄ -C ₁₇ , 2.2 ml

Native blood serum

cattle

or horse, ml 20

Isoniazid, mcg / ml 10

Salt base - buffer

Hanks solution, ml Up to 100

The test results of the culture media prepared in examples 1 and 2 for the cultivation of L-forms of various types of mycobacteria are presented in table 2.

Comparative analysis showed that, unlike the prototype, the use of the proposed nutrient medium allows to accelerate the growth of L-forms by 2-3 times, to increase the intensity of biomass accumulation by 2 times.

Thus, the use of a new environment ensures the achievement of the purpose of the invention, which allows us to conclude that the proposed solution meets the criterion of “positive effect”.

The combination of distinctive features meets the criteria of “novelty” and “inventive step”.

Sources of information

1. A.S. No. 2059728, C 12 Q 1/04. Nutrient medium for isolation of animal biomaterial and cultivation of Mycobacterium tuberculosis. Authors: Hodun L.M., Taller L.A. and etc.

2. Dorozhkova I.R., Kochemasova Z.N., Dykhno M.M. Isolation of L-forms of Mycobacterium tuberculosis from pathological material / Methodical recommendations, M., 1984.

Claims (1)

Nutrient medium for isolation and cultivation of L-forms of Mycobacterium tuberculosis, containing sucrose, native blood serum of cattle or horses, agar-agar, salt base, characterized in that it additionally contains a saturated hydrocarbon with a chain length of C ₁₄ -C ₁₇ , isoniazid , and as a salt base - Hanks buffer solution in the following ratio of components: Agar-agar, g 0.3-0.4 Native large blood serum cattle or horses, ml 10-20 Sucrose 200%, ml 9-12 Saturated hydrocarbon with chain length C ₁₄ -C ₁₇ , ml 1.1-2.2 Isoniazid, mcg / ml 1-10 Salt base - buffer solution Hanks, ml Up to 100