RU2328526C1 - Method for revealing cattle tuberculosis mycobacteria - Google Patents

Abstract

FIELD: bioengineering.

SUBSTANCE: method for revealing micobacteria provides the preparation of diagnostic material. The prepared diagnostic material is seeded into the solid medium which contains the mixture of the standard meat infusion agar and vaseline oil. The micobacteria are incubated in thermostat. After the incubation, the results are recorded in 24-48-72-96 hours.

EFFECT: timelines for revealing micobacteria are reduced

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Description

The invention relates to veterinary and medical microbiology, and in particular to cultural methods for detecting mycobacteria of the M. tuberculosis complex, and can be used in anti-tuberculosis measures in research and production laboratories of medical and veterinary profile.

Traditionally, cultural studies of diagnostic material are carried out on dense egg and semisynthetic liquid media, since tuberculosis mycobacteria are demanding on the composition of nutrient media and grow only on special ones containing the necessary set of salts and amino acids. This distinguishes them from a number of other mycobacteria, less demanding of nutrient media and growing on standard meat-peptone agar.

There is a known cultural method for detecting mycobacteria by direct seeding, in which, to obtain pure cultures of mycobacteria, the contaminated or biomaterial of each test sample after preliminary treatment is seeded in 10 test tubes with selective dense Levenshtein-Jensen egg medium and at the same time 10 test tubes in at least two of the following selective media: Gelberg, Petraniani, Finn-2, Mordovian, Novaya, Fast-3. During the incubation process, the results of the growth of mycobacteria are recorded weekly (Manual on the diagnosis of tuberculosis. Approved by the Department of Veterinary Medicine, November 18, 2002, p. 6, pp. 26-29).

The known method of detecting mycobacteria in pure form by direct seeding on selective nutrient media from any biomaterials due to the peculiarity of their biological properties has a number of significant disadvantages:

- macroscopic growth of mycobacteria is detected extremely late, in the period from three weeks to three months;

- accumulation of biomass in the first generation is meager;

- in addition, with a slight contamination by the mycobacteria of the studied biomaterial or in the case of their weakened viability, colonies of these microorganisms visible to the naked eye may not appear at all;

- methods of direct sowing on selective dense egg culture media are not informative enough, time-consuming, ineffective, require a significant investment of time and money.

The objective of the invention is the development of an effective highly informative method for detecting mycobacteria, which reduces the time of diagnosis of tuberculosis and reduces the complexity and material costs of bacteriological research.

The technical result is the creation of favorable conditions for life and the effective growth of mycobacterium tuberculosis in a nutrient medium based on standard meat and peptone agar, reducing the incubation time for diagnostic material to 4 days.

The technical result is achieved by the fact that in the method for the detection of mycobacterium tuberculosis in cattle, which involves preparing diagnostic material, sowing it on a solid nutrient medium, incubating and recording the results, a mixture of standard meat peptone agar and liquid paraffin is used as the nutrient medium in the following ratio of components, wt .%: meat-peptone agar - 95%, liquid paraffin - 5%, and the results are recorded in 24-48-72-96 hours.

The essence of the invention lies in the fact that liquid paraffin (C ₅ H ₁₂ - C ₁₆ H ₂₄ ) enriches the standard meat-and-agar hydrogen and carbon. Possessing pronounced adsorption properties with respect to mycobacteria, the oil acts as a stimulant that provides the necessary acceleration to significantly reduce the time of their growth. After preparation and solidification of the mixture, the oil is on the outer surface of the nutrient medium and favorable conditions are created for the multiplication and growth of colonies of mycobacteria in the surface layer, including due to aeration. The selected ratio of vaseline oil (5%) and MPA (95%) in the nutrient medium was established experimentally and is optimal for high purity of mycobacteria indication during sowing of pathological materials, increasing the rate of their isolation, growth and biomass accumulation.

To implement the method, meat-peptone vaseline agar is used as a nutrient medium, which is a mixture of standard meat-peptone agar containing 2-3% agar-agar, ph-7.4-7.6, and sterile paraffin oil in a ratio of components, wt.%: vaseline oil - 5, meat-peptone agar - 95. To prepare it for molten meat-peptone agar (MPA), stirring vigorously, add sterile vaseline oil. After thorough mixing and shaking, the mixture is poured into large bacteriological tubes and Petri dishes, sterilized in an autoclave at 120 ° C, at 1 atm for 30 minutes. After sterilization, hot tubes with the obtained nutrient medium were laid obliquely at an angle of 5-6 °, and Petri dishes were left on a flat table surface, and then they were cooled at room temperature. When the medium solidifies, a dense, moist surface is formed. 1 ml of a suspension of prepared diagnostic material prepared in sterile physiological saline at a concentration of 2 billion microbial bodies / ml according to the optical bacterial standard is added to it. Diagnostic material for crops is used after pretreatment, including grinding, thorough grinding with sterile sand, treatment with 6% sulfuric acid solution (1: 4 ratio), centrifugation for 15 min at 3000 rpm, double washing of the precipitate with sterile saline and preparation of the suspension by adding sterile physiological saline (1: 4) to the washed precipitate.

Incubation is carried out in a thermostat at a temperature of 37-38 ° C. To identify the initial growth of mycobacteria, crops are examined after 24-48-72-96 hours. The selected primary cultures are studied and described:

- timing of detection of primary growth;

- nature of growth (individual colonies, their accumulation, continuous growth);

- colony characteristics (size, shape, surface, relief, constitution, emulsifiability, pigmentation);

- tinctorial properties of mycobacteria in smears stained according to Zil-Nielsen.

For a comparative study of the specificity of the method, it was tested. Seven mycobacterial strains obtained from the Museum of the Federal State Institution of the All-Russian State Research Institute for Standardization and Certification of Veterinary Medicines (Mycobacterium tuberculosis No. 192, Mycobacterium bovis No. 14, Mycobacterium avium No. 961-97, Mycobacterium ihtracellularaeer No. 13, -S, Mycobacterium fortuitum No. 342 and Mycobacterium phlei No. 6-78); 15 samples of biomaterial (mediastinal lymph nodes with changes typical of tuberculosis), taken in a meat factory during the slaughter of tuberculin-positive cattle from a tuberculosis-poor farm in the Voronezh region, and 9 samples of an unsterile mixture of land and manure from a tuberculosis-free cattle farm, which laboratory conditions of the department artificially contaminated with suspensions of 4 museum strains of mycobacteria: M. bovis-14, M. tuberculosis - 192, M. avium - 961-97, M. phlei-6-78. For this purpose, 0.5 gram samples were prepared from each sample, to which 10 ml of suspension prepared in sterile physiological saline at a concentration of 2 billion microbial bodies / ml according to the optical bacterial standard were added in parallel.

Museum strains of mycobacteria to obtain bacterial mass were grown on selective Levenshtein-Jensen medium for 3-4 weeks. The purity of the culture was checked visually and by studying smears stained according to Ziehl-Nielsen.

To identify the primary cultures of mycobacteria, two solid media were used: standard, selective egg medium, Levenshtein-Jensen and meat-peptone vaseline agar (MPVA).

Biomaterials prepared for the study were simultaneously sown on both nutrient media. Crops were incubated in a thermostat at a temperature of 37-38 ° C for 30-45 days.

To identify the initial growth of mycobacteria, crops were examined every day and the results were recorded.

On the meat-peptone vaseline agar, the appearance of the initial growth of pathogenic mycobacteria - M. tuberculosis, M. bovis:

- in the crops of museum strains observed after 24-36-48 hours;

- in crops of biomaterials (from animals and environmental objects of livestock farms artificially contaminated with these mycobacteria) - after 48-72-96 hours;

For M.avium - intracellularae, M.phlei - (in all cases) after 24-36-48 hours.

In addition to the accelerated growth of all types of mycobacteria, an abundance of colonies on the surface of the nutrient medium and intensive accumulation of their bacterial mass were noted.

The research results are presented in the table, from which it can be seen that the speed and growth rate of different species of mycobacteria depends on the method of culture isolation, which indicates the specificity of the method in accordance with the invention.

The time ranges for identifying primary cultures and the intensity of their growth during these periods significantly reflect the difference in the methods for isolating pathogenic and non-pathogenic mycobacteria in pure form from various pathological materials.

Using the proposed method for the detection of mycobacterium tuberculosis can significantly reduce the time of diagnostic tests, in a very short time to get a significant amount of bacterial mass in the first generation, which is of great importance in the manufacture of biological products (antigens, allergens).

The proposed method is expressed, has significant resolution and allows you to effectively, in the shortest possible time, with the lowest cost of money and labor, to obtain mycobacterium cultures in a pure form, which creates the basis for the timely and successful implementation of a complex of anti-TB measures.

Table Types of Mycobacteria A method for detecting mycobacterium tuberculosis Known (on Levenshtein-Jensen culture medium) Nutrient peat agar + petrolatum Growth rate (days) growth rate Growth rate (hour) growth rate M.bovis-14 24-30 + 24-48 +++ M. tuberculosis-192 23-28 + 24-48 +++ M.avium-961-97 8-10 + 24-48 +++ M.intracellularae-3 8-10 + 24-48 +++ M.scrofixlaceum-13-s 9-10 + 24-48 +++ M.fortuitum-342 9-11 ++ 24-48 +++ M.phlei-6-78 5-6 24-48 +++ Biomaterial from cattle 23-30 48-72-96 +++ Biomaterial from the environment artificially contaminated 25-30.8-12 48-72-96 +++ Note: growth rate:
(+) - single colonies
(++) - 10-15 colonies
(+++) - from 15-20 or more colonies

Claims (1)

A method for detecting mycobacterium tuberculosis in cattle, which involves preparing diagnostic material, sowing it on a solid nutrient medium, incubating and recording the results, characterized in that a mixture of standard meat peptone agar and liquid paraffin is used as the nutrient medium in the following ratio of components, wt.%: meat peptone agar 95 Vaseline oil 5, and the registration of the results is carried out after 24-48-72-96 hours