RU2559523C1 - Method of pre-seeding treatment of pathological material for extraction of nocardioformal actinomycetes - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method of pre-seeding treatment of pathological material for extraction the nocardioformal of actinomycetes provides crushing of pathological material with the subsequent mixing with the disinfectant Septustin with the concentration 0.1% in the volume ratio 1:2 during 180 minutes at the room temperature with subsequent double washing with sterile physiological solution within 15 minutes.

EFFECT: method improvement.

4 tbl, 5 ex

Description

The invention relates to bacteriological research, relates to a method for processing pathological material and can be used to isolate nocardioform actinomycetes. The method is based on presowing treatment of pathological material and sowing on nutrient media.

According to the modern classification of microorganisms in group 22 (nocardioform actinomycetes), the genera Nocardia and Rhodococcus were determined [1].

The genera Nocardia and Rhodococcus (nocardioform actinomycetes), as well as the genera Corynebacteriwn and Mycobacterium, have type IV cell walls that contain mycolic acids, common antigens, have acid resistance to varying degrees, and other common features. The close composition and structure of chemical components determines no less close biological properties. DNA-RNA hybridization studies have established close genetic relationships between fast-growing mycobacteria, nocardia and rhodococci, and the SAB coefficient is M.phlei and R. spp. so high (0.72) that they are characterized as distantly related species.

The effectiveness of bacteriological research largely depends on the preliminary processing of pathological material.

In the practice of microbiological laboratories, sulfuric solutions are used for presowing treatment of pathological material for the detection of mycobacteria (A.F. Korzhinskaya, 1926; K.K. Kresling, 1929; A.N. Makkaveyskaya, 1956, method A.P. Alikaeva, 1971), hydrochloric acid (Yu.A. Yudenich, 1928), oxalic acid (Gon, Levenshtein-Sumiosi method), caustic soda (flotation method OV Martova, 1971), calcium hypochlorite and other solutions [2, 3]. The disadvantages of these methods include the destructive effect of acids and alkalis on nocardioform actinomycetes, corynebacteria and weakly acid-resistant mycobacteria.

The purpose of the invention is a method for processing pathological material for the isolation of nocardioform actinomycetes (nocardia and rhodococcus).

This goal is achieved by pre-sowing treatment of pathological material with a disinfectant "Septustine".

Septutin disinfectant (manufacturer of Uralstinol BIO LLC, Russia) is recommended for disinfection of veterinary surveillance facilities. This preparation from the group of cationic surfactants contains catamine AB as a disinfectant, as well as isopropyl alcohol, nonionic surfactant, sodium bicarbonate and bromphenol blue [4], has a wide spectrum of action against pathogens of bacterial infectious diseases (including tuberculosis), viral and fungal etiology .

The essence of the method consists in the presowing treatment of pathological material with an aqueous solution of the Septutin disinfectant at a concentration of 0.1% by mixing in a volume ratio of 1: 2 for 180 minutes at room temperature, followed by washing twice with sterile physiological saline for 15 minutes.

Example 1. The effect of the disinfectant "Septustine" depending on the concentration and exposure was determined on cultures of microorganisms obtained from GISK them. L.A. Tarasevich and isolated from pathological material (Rhodococcus equi No. 12). The results are presented in table 1.

As can be seen from table 1, a 0.1% aqueous solution of the Septutin disinfectant at an exposure of 15 minutes had a bactericidal effect on all microorganisms, with an increase in exposure to 30 minutes on E.coli and Staph. aureus, and with an increase in concentration to 0.25% and with an exposure of 30 minutes - on Rhodococcus equi. The bactericidal effect of the Septutin disinfectant on all nocardioform actinomycetes was observed at a concentration of 0.25% and an exposure of 60 minutes.

Example 2. Biomaterial (liver, spleen, lungs, kidneys, heart) from white mice infected with R. equi culture was crushed in a sterile mortar with sterile scissors, the pieces were poured with a solution of the disinfectant Septupine 0.1, and 0.25% concentration in a volume ratio of 1: 2 and left for 15, 30, 60, 120 and 180 minutes at room temperature. Then the supernatant was drained and washed with sterile saline twice for 15 minutes, mixed well and drained, and the pieces were ground with sand with a sterile pestle. The resulting suspension was seeded on a Levenshtein-Jensen nutrient medium, the crops were incubated for 15 days at + 37 ° C. Analysis was carried out daily. The results are presented in table 2.

Example 3. A cultural study of pathological material (pieces of organs with changes characteristic of tuberculosis) from cows with a positive reaction to PPD-tuberculin for mammals from a tuberculosis-free farm was carried out. The pathological material was crushed in a sterile mortar with sterile scissors, the pieces were poured with a solution of the Septutin disinfectant 0.1% concentration in a volume ratio of 1: 2 and left for 180 minutes at room temperature. Then the supernatant was drained and washed with sterile saline twice for 15 minutes, mixed well and drained, and the pieces were ground with sand with a sterile pestle. From the resulting suspension, crops were plated on Levenshtein-Jensen nutrient medium and placed in a thermostat. On the second day of cultivation at a temperature of + 37 ° C, growth was noted in the form of large mucous membranes without surface mycelium of whitish-yellow colonies. Zil-Nielsen stains showed non-acid-resistant microorganisms, polymorphic cells - rods and cocci, which were identified by gas-liquid chromatography (pronounced peak of tuberculostearic acid, low molecular weight esters of fatty acids with the number of carbon atoms from 12 to 20 and the absence of high molecular weight mycolic acids) as smears .

Example 4. Conducted a cultural study of pathological material (lymph nodes, liver, lungs) from guinea pigs infected with Nocardia asteroides. The pathological material was crushed in a sterile mortar with sterile scissors, the pieces were poured with a solution of the Septutin disinfectant 0.1, 0.25 and 0.5% concentration in a volume ratio of 1: 2 and left for 30, 60, 120 and 180 minutes at room temperature . Then the supernatant was drained and washed with sterile saline twice for 15 minutes, mixed well and drained, and the pieces were ground with sand with a sterile pestle. The resulting suspension was seeded on a Levenshtein-Jensen medium and incubated at + 37 ° C. Analysis was carried out daily. Primary growth was observed after 3-4 days. The results are presented in table 3.

Example 5. Conducted a cultural study of biomaterial (mesenteric, pharyngeal, submandibular, lymph nodes, liver, lungs) from five cows with a positive reaction to PPD-tuberculin for mammals by the method in accordance with the invention. When browsing crops, crop isolation and crop cleanliness were taken into account. A.P. method was chosen as a control. Alikayeva. The results are presented in table 4.

As can be seen from table 4, the optimal parameters (detectability of nocardioform actinomycetes,% seedlings) were observed when presowing the treatment of pathological material with an aqueous solution of the Septutin disinfectant 0.1% concentration with an exposure of 180 minutes. With an increase in concentration and exposure, nocardioform actimecites were not detected. During pre-sowing treatment according to A.P. Alikayeva (1971) no nocardioform actinomycetes were detected.

Studies have confirmed that the treatment of pathological material with an aqueous solution of Septutin disinfectant 0.1% concentration with an exposure of 180 minutes inhibits the development of extraneous microflora (spore rod-shaped forms, molds), increases the cleaning efficiency, reduces the likelihood of extraneous microflora sprouts, does not affect the intensity and nature of the growth of nocardioform actinomycetes, which indicates the absence of the inhibitory effect of a disinfectant with the stated parameter x.

Information sources

1. Hoult D. et al. Determinant of the bacteria Bergey. 2 volumes. - M .: Mir, 1967.

2. Dolzhansky V.M. et al. Modern methods of laboratory diagnosis of tuberculosis: Method. recommendations. - M., 1992.

3. Kassich, Yu.Ya. Animal tuberculosis and measures to combat it. - Kiev, 1990.

4. Manual on the use of the drug "Septustine" for the disinfection of objects of veterinary surveillance. Uralstinol. - BIO. - 2002.

Claims (1)

A method for pre-sowing treatment of pathological material for the isolation of nocardioform actinomycetes, including exposure to a disinfectant, characterized in that the disinfectant "Septutin" is used as a disinfectant, and the treatment is carried out by mixing an aqueous solution of the disinfectant "Septutin" with a concentration of 0.1% and pathological material in a volume ratio of 1: 2 for 180 minutes at room temperature, followed by twice washing for 15 minutes with sterile washing isiological solution.