RU2401862C2 - Method of isolating enterococcus from fresh pathological material - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: without grinding, a sample of freshly collected pathological material is put into an accumulation medium - guanosine monophosphate broth with addition of 1% glucose and then cultured in a temperature-controlled cabinet at 37°C for 24 hours. Seeding is then carried out on a Petri dish with agar medium with addition of 5% erythrocytes and on enterococcus agar. The inoculations are kept in a temperature-controlled cabinet for 24-48 hours at 37°C. Samples which failed to grow in aerobic conditions are further inoculated on a Petri dish with agar medium with addition of 5% erythrocytes and then put into an anaerobic jar for 24 hours at 37°C. The inoculations are examined under a microscope and microorganisms are identified from the results using traditional methods.

EFFECT: higher isolation rate of enterococcus in anaerobic conditions at 31,2-36% and possibility of detecting presence of microorganisms in clinical material which do not grow in aerobic conditions.

1 tbl

Description

The invention relates to medical and veterinary microbiology and can be used to diagnose enterococci.

Known methods for the isolation of enterococci, based on the fact that they belong to facultative anaerobic microorganisms and in practice, resort to direct seeding on simple nutrient media, or first on the medium with the addition of 5% sheep erythrocytes, and only then on selective nutrient media. Cultivation is carried out in a thermostat (37 ° C) in air. (Medical Microbiology, edited by Acad. RAMS V.I. Pokrovsky, Prof. O.K. Pozdeeva. GEOTAR MEDICINE, M., 1999, p.206-207).

However, the known methods have the following disadvantages: with direct seeding on simple nutrient media, enterococci cannot always be distinguished due to the small number of microorganisms in the test sample or due to a violation of the physiological and growth properties of enterococci, which are in adverse conditions for them.

Of the known technical solutions, the closest in technical essence to the claimed object is the Order of the Ministry of Health of the USSR No. 535 dated April 22, 1985 "On the unification of microbiological (bacteriological) research methods used in clinical diagnostic laboratories of medical institutions, Moscow, 1985 g. In accordance with this document, the test material is seeded, using the line technique of sowing, into half a Petri dish with blood agar, then sowing in sugar broth.

Liquid samples delivered to the laboratory (pus, exudate, contents of tubovar formations, amniotic fluid) are inoculated with 0.1 ml of solid nutrient medium, grinding the material on the surface of the medium with a spatula. Use blood agar, Endo medium. In addition, sowing is carried out in sugar broth.

Pieces of tissue are crushed, observing sterility, in a micromixer of tissues, or ground in a mortar with sand, adding nutrient broth or saline. The resulting suspension is seeded on several cups with solid nutrient media, as well as in sugar broth.

Crops are incubated in an incubator at a temperature of 37 ° C, viewing daily. When growth appears on solid media, colonies of different morphology are counted, taking into account their ratio.

When the broth is clouded, smears are made on glass (Gram stain) and, in accordance with the results of microscopy, seeding is performed on solid nutrient media (blood agar, yolk-salt agar, Endo medium). Then carry out species identification of sensitivity to antibacterial drugs.

In the absence of growth on all nutrient media within 72 hours, a conclusion is issued on the absence of microflora in the samples.

This method has a number of disadvantages, namely, when using the indicated technique for sowing tissue pieces at the time of grinding, the contamination of the sample with extraneous microflora is very likely, the cultivation conditions of crops at 37 ° C in air do not allow long-term parasitizing in the absence of atmospheric oxygen and acquiring the ability to grow conditions of anaerostat enterococcus.

The aim of the invention is to increase the seeding rate of enterococci from pathological material.

This goal is achieved by the fact that pathological material (dense pieces of organs are not crushed) is placed immediately in the accumulation medium (nutrient broth for the cultivation of microorganisms - GMF - broth with the addition of 1% glucose), followed by cultivation in an thermostat at T 37 ° C for 24 hours. Then we do seeding on solid nutrient media and place in a thermostat for 24-48 hours. Samples that did not grow under aerobic conditions are additionally seeded on MPA with 5% sheep erythrocytes and placed in an anaerostat for 24 hours at T 37 ° C.

The essence of the method: freshly harvested pathological material without grinding is placed in the accumulation medium, cultivated in a T 37 ° C thermostat for 24 hours. Then, seeding is carried out on Petri dishes with MPA with the addition of 5% red blood cells and on enterococcagar. Crops are kept in the thermostat for 24-48 hours at 37 ° C. Samples that did not grow under aerobic conditions are additionally seeded on Petri dishes with MPA with the addition of 5% red blood cells and placed in an anaerostat for 24 hours at 37 ° C. Crops are examined, microscopy is carried out, and microorganisms are identified by its results according to generally accepted methods.

The results of the study are presented in table No. 1.

Comparative evaluation of different diagnostic methods.

Table No. p / p Total material examined by the bacteriological method The number of positive finds in accordance with 535 Order The number of additionally identified by the proposed method Additionally identified by the proposed method,% one. 50 samples of embryo tissue taken from women with non-developing pregnancy in a gynecological hospital 16 5 31.2% 2. 50 smears of the cervical canal of the cervix uteri of cows with gynecological diseases 29th eighteen 36%

The technique for sowing clinical material from female genital organs in accordance with the 535 Order involves the cultivation of microorganisms only under aerobic conditions. Cultivation of crops under strict anaerobic conditions allows additionally detecting from 31.2 to 36% of enterococcal cultures, which is associated with the conditions of the ecosystem in which these microorganisms parasitize. Microaerophilic or severe anaerobic conditions are normal in the cervical canal and uterus of the macroorganism, which creates the prerequisites for the adaptation of enterococci to similar conditions and suggests their observance when isolating enterococci from clinical material.

Inoculation of enterococci under anaerobic conditions increases from 31.2 to 36% and allows you to detect the presence of microorganisms in the clinical material that do not give growth under aerobic conditions.

Claims (1)

A method for isolating enterococci from freshly taken pathological material, characterized in that a sample of the pathological material is directly placed in a GMF broth with 1% glucose added, followed by cultivation in an incubator at 37 ° C for 24 hours, then seeding on solid nutrient media spilled in Petri dishes - MPA with the addition of 5% sheep and enterococcagar erythrocytes, crops are kept in an thermostat for 24-48 hours, samples that did not grow under aerobic conditions are additionally seeded on MPA with Niemi 5% sheep erythrocytes and placed in Anaerobic for 24 hours at 37 ° C.