RU2402781C1 - Method of pre-inoculation processing of samples collected from environment objects for release of microbacteria - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: milled samples of biological material are poured with physiological solution for 24 hours at room temperature, filtered through wadding, centrifuged for 20 min at 3-4 thousand revolutions per minute. After that 1.5% solution of lauryl sulphate per 1.5% solution of caustic soda is added to sediment, kept for 20 minutes, centrifuged for 20 minutes at 3-4 thousand revolutions per minute, sediment is washed with physiological solution 2-3 times and inoculated on nutrient medium.

EFFECT: application of method allows to optimise pre-inoculation processing of biomaterial, and increase efficiency of bacteriological diagnostics of tuberculosis.

1 ex, 1 tbl

Description

The invention relates to veterinary medicine and medicine, in particular to the detection of Mycobacterium tuberculosis in samples taken from environmental objects.

Presowing treatment of the test material for the isolation of mycobacteria is based on the fact that the substances used for these purposes should have a bactericidal effect on the concomitant microflora, while not significantly affecting the viability of mycobacteria. The detection of such substances is one of the urgent tasks of veterinary and humane medicine, as well as for microbiology in general.

For these purposes, in the practice of microbiological laboratories, sulfuric solutions are currently used (A.F. Korzhinskaya, 1926; K.K. Kresling, 1929; A.N. Makkaveyskaya, 1956), hydrochloric (Yu.A. Yudenich, 1928), oxalic acids (method of Gon, Levenshtein-Sumiosi, method of A.P. Alikayeva, 1971), caustic soda (flotation method of O.V. Martov, 1971), calcium hypochlorite and other solutions.

The disadvantages of these methods include the destructive, to some extent, the effect of acids and alkalis on mycobacteria. However, the percentage of pollution remains high, especially when processing samples taken from environmental objects. Solutions of acids in low concentrations for processing the material studied for the presence of mycobacteria do not ensure its release from the accompanying microflora.

Detergents used in medical practice for processing the material are unsuitable, since in 70-87.5% of cases they do not suppress the growth of concomitant microflora in samples taken from environmental objects and in homogenates of biomaterial obtained from an animal.

It should be noted that when processing the material with a 5% solution of oxalic acid without subsequent washing with physiological saline, Bannikova recommends B.N. (1980), the growth of mycobacteria on Levenshtein-Jensen, Finn II media does not occur, since the pH of the medium in it varies below 6.8.

Of the above methods, the claimed invention closest in technical essence to the claimed invention is a method for processing samples from environmental objects with a 2-4% sodium hydroxide solution, followed by washing twice with sterile physiological saline.

The test sample was ground in a mortar with a pestle along with small glass, poured with 4% sodium hydroxide solution, kept for 30 minutes. Then centrifuged for 20 minutes at 3-4 thousand revolutions per minute. The supernatant was discarded and washed 2 times with sterile saline. The precipitate was plated on Wednesday Finn II.

The disadvantages of these methods include the low incidence of mycobacteria and a high percentage of seedlings of concomitant microflora in nutrient media. The experiments showed insufficient disposal of the homogenate from concomitant microflora. In addition, the frequency of mycobacterial isolation during pre-treatment of samples with this method was low. The experiments also showed that caustic soda did not sufficiently reduce the viscosity of the homogenate, and therefore crops on nutrient media became difficult, did not completely cover the entire surface of the medium, and, probably, mycobacteria, which are in a state covered by mucus, do not find contact with the nutrient medium.

The aim of the invention was to increase the efficiency of bacteriological diagnosis of tuberculosis by optimizing the conditions of pre-treatment of biomaterial with a solution that has a high bactericidal effect on the concomitant microflora and the lowest possible effect on mycobacteria.

This goal is achieved by processing samples taken from environmental objects with a 1.5% solution of lauryl sulfate in a 1.5% solution of sodium hydroxide. The homogenate was pretreated according to the following recipe: samples taken from environmental objects were crushed and poured with physiological saline. It was left for 24 hours at room temperature, then it was filtered through cotton moistened with physiological saline, and the filtrate was centrifuged for 20 minutes at 3-4 thousand rpm. A 1.5% solution of lauryl sulfate in a 1.5% solution of sodium hydroxide was added to the precipitate, held for 20 minutes, centrifuged for another 20 minutes at 3-4 thousand rpm. Under sterile conditions, the supernatant was discarded, the precipitate was washed with saline 2-3 times. From the precipitate obtained, seeding was made on the surface of the nutrient medium by uniform rubbing.

Comparative analysis with the prototype allows us to conclude that the claimed composition of the solution differs from the prototype in the introduction of new components, namely sodium lauryl sulfate and sodium hydroxide. Thus, the claimed technical solution meets the criterion of "novelty." An analysis of the known methods used for the preliminary processing of materials for the isolation of mycobacteria showed that the components used are known. However, their use alone does not provide the properties that they exhibit in the claimed solution, namely, a significant expansion of the bactericidal and bacteriostatic ability on the concomitant microflora (due to the detergent effect of lauryl sulfate) and sparing on mycobacteria. Thus, this composition of the components gives the solution new properties, which allows us to conclude that the proposed solution meets the criterion of "significant differences"

In the samples taken from environmental objects, where bird-type mycobacteria or BCG were introduced, the preliminary processing of which was carried out by the proposed method, the initial culture was always found.

A bacteriological study of both samples of environmental objects and pathological material by the proposed method is available to each veterinary laboratory and is quite acceptable for research in the practice of medical institutions.

During the experimental verification, the effect of sodium lauryl sulfate solutions on sodium hydroxide solution in different concentrations was determined: 3% sodium lauryl sulfate solution in 1, 2, 3% sodium hydroxide solutions; 1.5% solution of sodium lauryl sulfate in 1.5 and 2% sodium hydroxide solution at exposures of 30, 40, 60 minutes and 3 hours. The test procedure was as follows: bird mycobacteria were introduced into a sample taken from an environmental object, treated with test solutions in various concentrations and exposures. The following properties were studied and described in the selected primary cultures:

- timing of detection of primary growth;

- nature of growth;

- growth rate (weak, moderate, good, plentiful).

According to the results of repeated studies, the best results were obtained when treated with a 1.5% solution of lauryl sulfate in a 1.5% solution of sodium hydroxide. In this case, primary growth was detected 5-6 days earlier. Moreover, the growth was continuous and more plentiful. In our experience, the exposure did not play a special role, since lauryl sulfate turned out to be much more sparing even with a 3-hour exposure. This is very important when handling heavily contaminated materials.

In the following experiments, a 1.5% solution of lauryl sulfate was tested on a 1.5% sodium hydroxide solution at an exposure time of 40 minutes for presowing treatment of samples taken from environmental objects. These studies were conducted comparatively. The control was presowing treatment with 10% sulfuric acid solution at an exposure of 20 minutes and 4% sodium hydroxide solution at an exposure of 30 minutes (table). We examined 47 samples (scrapings from feeding troughs, from the floor, from the walls), feed (hay, straw) taken from a tuberculosis-poor farm. 11 cultures were identified: 2 - paratuberculosis, 1 - avian species and 8 - atypical mycobacteria.

Thus, the studies showed that when presowing the processing of environmental samples for the isolation of mycobacteria, the most effective is a 1.5% solution of sodium lauryl sulfate in a 1.5% solution of sodium hydroxide at an exposure time of 40 minutes.

Information sources

1. Schurevsky V.E., Kosenko V.I., Tazhgaliev N.M. The use of various chemicals to treat pathological material and their effect on the sowing rate of mycobacteria. / Bulletin VIEV. - 1987. - V. 64. - S.15-19.

2. Kolychev M.N., Shlygin I.V. The importance of direct bacteriological quality control of disinfection in a complex of measures to combat cattle tuberculosis. / Tr. Omsk Vet. Institute. - S. 54-55.

3. Bannikova V.N. Improving the methods of bacteriological examination of pathological material for tuberculosis. / Thes. doc. scientific conf. Omsk, 1980. - B.4, - S.63-64.

4. Kolychev N. characterization of mycobacteria isolated from objects of animal husbandry and livestock farms. / Sat nt Sib. NIVI. - 1976. - S. 47-55.

Claims (1)

The method of presowing treatment of samples taken from environmental objects for the isolation of mycobacteria, which consists in exposing them to a solution of caustic soda, characterized in that the crushed samples are poured with physiological saline for 24 hours at room temperature, filtered through cotton, centrifuged for 20 minutes at 3 4 thousand revolutions per minute, a 1.5% solution of lauryl sulfate in a 1.5% sodium hydroxide solution is added to the precipitate, incubated for 20 minutes, centrifuged for 20 minutes at 3-4 thousand revolutions per minute, the precipitate is 2-3 times washed with saline and affairs ayut sowing on a nutrient medium.