UA65968A - Method for assessing sensitivity of clinical bacterial isolates to disinfectants - Google Patents

Abstract

The method for assessing the sensitivity of the clinical bacterial isolates to the disinfectants comprises preparing the bacterial suspension containing 2 billions of the bacterial bodies, exposing the suspension to the disinfectant solution, transferring the bacteria to the solid nutrient medium, and incubating Petri dishes seeded with bacteria at the temperature of 37 DEGREE c for 24 h. The solid nutrient medium in Petri dishes is divided into the sectors with different neutralizing substances. The absence of the colonies upon incubation points to the sensitivity of the bacteria to the disinfectants being assayed.

Description

The proposed method refers to the field of medicine, namely to microbiology.

A well-known method of determining the sensitivity (resistance) of microorganisms to antiseptic (disinfectant) agents, developed at the Department of Microbiology of the Minsk State Medical Institute (O.P. Krasilnikov, Handbook of antiseptics // Minsk: Vyishzhyshaya shkola-1995. - 3670.)

The method of determining the sensitivity of microorganisms to disinfectants is the closest to the one proposed by us (N.S. Morozova, N.N. Kibardina, PP. G. Voloshenko. Laboratory quality control of disinfection measures in treatment and prevention facilities // Methodological recommendations. -

Kharkiv Institute of Microbiology and Immunology named after Mechnikova I.I. - 1989. - 25 p.), which are carried out as follows: a 2 milliard suspension is prepared from the daily culture of microorganisms, which is tested for sensitivity to the disinfectant, which is used to prepare test objects according to the generally accepted method. Test objects are filled with disinfectant (at the rate of 0.5 ml per test object), after every 5 minutes for 2 hours, two test objects are removed from the disinfectant solution, successively washed in sodium hyposulfite and sterile water before adding to a liquid nutrient medium (meat-peptone broth). After incubation of test tubes with meat-peptone broth and test objects in a thermostat for 24 hours, the sensitivity of the culture to the disinfectant is assessed by the presence (absence) of its growth in the liquid nutrient medium.

The disadvantages of the known method are: 1) the laboriousness of the research process and the difficult implementation of some of its stages (necessity of almost simultaneous transfer of test objects to different test tubes using a loop); 2) the method requires a large amount of working time, the use of a large number of laboratory dishes, reagents and nutrient medium; 3) during the stay of the test objects in the disinfectant and successive washing in a solution of sodium hyposulfite and sterile water before introduction into the meat-peptone broth, the dose of the test culture on the test object decreases, which reduces the degree of accuracy of determining the sensitivity .

These shortcomings of the method are the reason for its difficult implementation, and therefore, its introduction into the daily work of bacteriological laboratories that carry out quality control of disinfection measures in medical and preventive institutions.

The invention is based on the task of developing a method for determining the sensitivity of clinical strains of microorganisms to disinfectants by improving what is known, achieving simplification of its implementation, reducing labor and time consumption, and ensuring an increase in the degree of accuracy of determining the sensitivity of microorganisms to disinfectants.

The task is solved by creating a method for determining the sensitivity of clinical strains of microorganisms to disinfectants, which includes the production of a 2 billion suspension of microorganisms, exposure of microorganisms to a disinfectant solution, hanging on a nutrient medium and holding in a thermostat for 24 hours at 37"C, which, according to the invention, differs in that a suspension of microorganisms is exposed to the action of a disinfectant, they are sown on the surface of a solid nutrient medium with a neutralizer, placed in a Petri dish and divided into sectors, and the sensitivity of microorganisms to disinfectants is judged by the presence or absence of colonies on the surface of the nutrient medium of the corresponding sectors.

The method is carried out as follows: a Petri dish with meat-peptone agar and a neutralizer is divided into 6 sectors (according to the number of crops). A 2 milliard suspension is prepared from the daily culture of microorganisms in an isotonic solution of sodium chloride and a disinfectant. After a certain exposure, 0.4 ml of the suspension is transferred from a test tube with a disinfectant with a micropipette and distributed with a bacteriological loop on the surface of the corresponding sector of the Petri dish. The number of inoculations (and, accordingly, the sectors on the Petri dish) may vary, but we consider it expedient to carry out Z of inoculations before the exposure established for this disinfectant and 2 inoculations after the established exposure. If the set exposure is 60 minutes, then sowing is carried out after 5(10), 20, 40, 60, 90 minutes, if the exposure is set for 30 minutes, then sowing is carried out after 5, 15, 20, 30, 60 minutes.

The control should be inoculation on the appropriate sector of the cup with 0.4 ml of the suspension of the investigated microorganisms in a physiological solution of sodium chloride. The cup with crops is placed in a thermostat for 24 hours at 37"C. After a day, the results of the study are recorded and evaluated.

The lack of growth of microorganisms on the corresponding sector of meat-peptone agar indicates the death of microorganisms in the disinfectant at this exposure.

The presence of growth of microorganisms on the sector of meat-peptone agar indicates the insensitivity of microorganisms to the disinfectant at this exposure.

We consider microorganisms highly sensitive to this disinfectant if they do not grow on meat-peptone agar after exposure to the disinfectant for 5-10 minutes.

Sensitive - in the absence of growth on meat-peptone agar after exposure established for this disinfectant.

Insensitive - in the presence of growth on meat-peptone agar after exposure established for this disinfectant.

The method proposed by us differs from the known method of determining the sensitivity of microorganisms to disinfectants as follows: 1) the disinfectant is not exposed to the test object, but the suspension of microorganisms, therefore the stage of preparation of test objects is omitted; 2) after exposure to the disinfectant, microorganisms are seeded on the surface of a solid nutrient medium, and not in a liquid nutrient medium, as when using the prototype method;

C) the solid nutrient medium on which microorganisms are inoculated contains sodium hyposulfite or another neutralizer (depending on the disinfectant), which eliminates our method from the time-consuming step of washing the test objects in sodium hyposulfite and sterile water before inoculation on the nutrient medium; 4) the number of inoculations of microorganisms on the nutrient medium when using our method is less: from the inoculation before the exposure established for this disinfectant, 2 inoculations after the established exposure and 1 control, in contrast to 24 inoculations when performing the prototype method; 5) the sensitivity of microorganisms to the disinfectant is judged not by the presence (absence) of turbidity of the meat-peptone broth, but by the presence (absence) of colonies of microorganisms on the corresponding sectors of the meat-peptone agar, that is, the results of the study are more obvious than when performing prototype method.

Example:

It is necessary to determine the sensitivity of the isolated culture of staphylococcus to 395 chloramine solution. The Petri dish is divided into 6 sectors. A 2 milliard suspension of staphylococcus is prepared in a physiological solution of sodium chloride and a 395% solution of chloramine. Exposure time of 30 minutes is set for this disinfectant. After 5, 15, 20, 30, 60 minutes, 0.4 ml suspension of staphylococcus in a chloramine solution and saline solution are inoculated onto the corresponding sectors of the Petri dish (control inoculation). A day after the incubation of the cups in the thermostat, the results of the study are recorded. Culture growth is present on the sector corresponding to exposure in the disinfectant for 5 minutes and on the control sector. There is no growth of staphylococcus in the remaining sectors. Therefore, the isolated culture of staphylococcus is sensitive to 395 chloramine solution.

Conclusion: based on the above, the positive effect of using the method proposed by us for determining the sensitivity of clinical strains of microorganisms to disinfectants consists in simplifying the implementation, reducing the cost of working time, laboratory dishes, reagents, increasing the degree of accuracy of determining sensitivity, and increasing the visibility of research results.

Claims (1)

The method of determining the sensitivity of clinical strains of microorganisms to disinfectants, which includes the production of a 2-billion suspension of microorganisms, its exposure in a disinfectant solution, hanging on a nutrient medium and exposure in a thermostat for 24 hours at 37"C, which is characterized by the fact that the action of the disinfectant is exposed suspension of microorganisms, their seeding is carried out on the surface of a solid nutrient medium with a neutralizer, placed in a Petri dish and divided into sectors, and the sensitivity of microorganisms to disinfectants is judged by the presence or absence of colonies on the surface of the solid nutrient medium of the corresponding sectors.