RU2378363C1 - Method for determining microorganism sensitivity to disinfectant (versions) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: suspension of microorganism culture is prepared. The prepared suspension is mixed in a test tube with a disinfectant or when applied on a surface, the prepared suspension is kept for drying and covered with a disinfectant. After the disinfectant added, the prepared mixture is exposed during the period of time common for said disinfectant. Thereafter, a neutraliser of the disinfectant is added in a test tube or applied on the surface. Then the prepared suspension is inoculated on a solid nutrient medium, and when applied on the surface, exposition is followed with washing the prepared mixture with a tampon from the surface to inoculate the prepared mixture by means of a tampon on a solid nutrient medium. The microorganism sensitivity to the disinfectant is shown by growth of microorganisms on the nutrient medium up to 300 CFU/ml. Thus, growth up to 100 CFU/ml shows incomplete bactericidal action, and growth more than 300 CFU/ml indicate stability of microorganisms to the disinfectant.

EFFECT: possibility for fast quantitative estimation of degree of formed stability of microorganisms to disinfectants.

2 ex

Description

The invention relates to medicine, namely epidemiology, microbiology, disinfection, and can be used to assess the sensitivity (resistance) of microorganisms to disinfectants.

The development of the invention is caused by the need to create simplified methods for determining the sensitivity (stability) of microorganisms to disinfectants, available for conducting in bacteriological laboratories of medical facilities and federal health institutions. The relevance of this invention lies in the need to conduct studies of cultures of microorganisms isolated from patients from objects of the external environment of hospitals, including hospital strains, and foci of infectious diseases for sensitivity to disinfectants. The study of the sensitivity (resistance) of the microbial flora to disinfectants is one of the most important stages in the characterization of hospital and non-hospital strains. The formation of resistance to the applied disinfectants among hospital microflora is one of the serious problems of practical public health. In the medical literature of recent years, cases of insufficient effectiveness of the use of disinfectants have been repeatedly described, as a result of which the pathogenic microflora could not only persist for a long time on environmental objects, but even accumulate in ready-made disinfectants, which dramatically reduces the effectiveness of anti-epidemic measures and helps to maintain a high level of morbidity . The determination of sensitivity (stability) is necessary to optimize the disinfection regimen and timely change of disinfectants to more effective against resistant microflora.

Currently, for assessing the sensitivity of microorganisms to disinfectants, for example, the method of accelerated determination of bacterial resistance to disinfectants is used (Methodological recommendations for the accelerated determination of bacterial resistance to disinfectants. Approved by the head of the state sanitary and epidemiological surveillance department of the Ministry of Health of Russia A.A. Monisov January 10, 2000 for No. 1100-26-0-117).

The method is based on the use of a colored nutrient medium that changes color under the influence of the vital activity of propagating bacteria. Effective disinfectant concentrations prevent the accumulation of bacteria and preserve the color of the medium.

This analogue has several disadvantages:

- the recommended special color culture medium is not on sale, and the recipe is not presented in the methodological recommendations;

- the need for preliminary testing of cultures of microorganisms for the possibility of applying the described method for determining resistance;

- this method does not allow to limit the exposure time of the disinfectant (exposure);

- the possibility of distortion of the color of the nutrient medium under the influence of disinfectants;

- the impossibility of using an additional accounting feature - turbidity of the environment - to evaluate the results of the use of those disinfectants that, when added to a colored nutrient medium, form pronounced opalescence;

- subjectivity of the assessment and interpretation of the results (visual assessment of color changes, turbidity of the nutrient medium);

- the need to use special equipment (apparatuses such as Multiscan) for an objective assessment of the results;

- the impossibility of quantifying the results (the method is qualitative, not quantitative);

- the impossibility of assessing the degree of sensitivity (stability) of the studied microorganisms to disinfectants (determining only the minimum disinfectant concentration);

- the complexity / impossibility of isolation and identification of cultures of microorganisms resistant to disinfectants grown on a nutrient medium after exposure to a disinfectant (non-compliance with such a requirement as cultivation on solid nutrient media), and as a result, the lack of quality control of the grown cultures of microorganisms;

- the complexity of the widespread introduction of this method in the practical activities of bacteriological laboratories;

- the complexity and cost of research.

Also known is the "Method for determining and indicators of the sensitivity (resistance) of bacteria to disinfectants" (Krasilnikov AP, Zmushko LS, Adarchenko AA // Disinfection and sterilization: Prospects for development. - Volgograd, 1983. - p. 61-63).

The technique is based on determining the effectiveness of disinfection of carriers (pins of the stamp-replicator) contaminated with suspensions of the tested cultures of microorganisms by sequentially immersing them in solutions of disinfectants and neutralizers and plating on solid nutrient media by pressing the terminal pads of the pins to the surface of the medium on bacteriological plates. Evaluation of the results is carried out according to the presence or absence of bacterial growth in the areas of inoculated prints. The disadvantages of this technique include:

- the inability to provide deposition on the surface of the pins of a standard number of microorganisms from bacterial suspension in connection with the different adhesive ability of microorganisms;

- drying the bacterial suspension on the surface of the pins can lead to partial or complete death of microorganisms;

- the possibility of partial or complete washing of microorganisms from the surface of the pins when immersed in solutions of disinfectants and neutralizers;

- all of the above leads to the impossibility of quantitative quantitative seeding of bacteria on a nutrient medium;

- the proposed exposure time of disinfectants to the tested bacterial cultures (10 minutes) does not take into account the recommended exposure disinfection;

- the impossibility of simultaneously testing several disinfectants with various recommended disinfection modes;

- the impossibility of assessing the degree of sensitivity, the lack of gradation of the results of the study;

- during the study, the components are in a different state (the tested microorganisms are dried, and the disinfectants are in solution), which may affect the quality of the results, since the effect of disinfectants on the microflora in the solution and on the surface manifests itself in different ways;

- the complexity of the technique, the need for special equipment (stainless steel replicating dies with 50 pins; support rings for drying contaminated replicators), conducting research in an aseptic room and, as a result, the difficulty of introducing into the daily activities of medical facilities.

The objective of the invention is to simplify the method while reducing the risk of death of microorganisms, as well as providing the ability to quantify the sensitivity of microorganisms to disinfectants.

The goal is achieved as a result of 2 variants of the proposed method for determining the sensitivity of microorganisms to a disinfectant: option No. 1 - when the agent is in solution, option No. 2 - when the agent is on the surface. The goal in the method according to option No. 1 is achieved by the fact that a suspension of the microorganism culture is prepared according to the turbidity standard of 5 Units, then 0.1 ml of this suspension is mixed in a test tube with 0.9 ml of a disinfectant and the resulting mixture is exposed for a time known for this disinfectant, as the time required for the manifestation of the disinfecting effect, then add 0.5 ml of the neutralizer, take 0.1 ml of the resulting mixture and plated on a solid nutrient medium, the sensitivity of microorganisms to the disinfectant the agent is judged with the growth of microorganisms on a nutrient medium up to 300 CFU / ml, while with growth up to 100 CFU / ml, an incomplete bactericidal effect is judged, with an increase of 100-300 CFU / ml - about a sub-bactericidal effect, with an increase of more than 300 CFU / ml - About the resistance of microorganisms to a disinfectant.

The goal in the method according to option No. 2 is achieved by the fact that a suspension of the microorganism culture is prepared according to the turbidity standard of 5 Units, then 0.1 ml of this suspension is applied to the sterile surface of the test object with an area of 10 × 10 cm and distributed with a sterile spatula over the entire square area, after the microbial suspension is dried, 0.9 ml of the disinfectant is applied to the surface and distributed on the surface of the square with a sterile spatula, the resulting mixture is exposed for a time known for this disinfectant as Name, which is necessary for the manifestation of the disinfecting effect, then 0.5 ml of the disinfectant neutralizer is applied to the surface of the test object, and after exposure of the neutralizer, the mixture is washed off the surface of the test object with a sterile swab, the same swab immediately sown 0.1 ml of the mixture on a solid nutrient medium, the sensitivity of microorganisms to a disinfectant is judged with the growth of microorganisms on a nutrient medium up to 300 CFU / ml, while with growth up to 100 CFU / ml, an incomplete bactericidal effect is judged, with an increase of 100-300 CFU E / ml - about subbactericidal action, with growth of more than 300 CFU / ml - about the resistance of microorganisms to a disinfectant. The need to use the definition of quantitative distinguishing features in the proposed methods provides the ability to quantify the sensitivity. This is justified by the following calculations performed by the authors.

A suspension of microorganisms is prepared according to the turbidity standard of 5 Units (this means that 1 ml of suspension contains 5 × 10 ⁸ microbial cells), 0.1 ml of such suspension is taken (this volume contains 5 × 10 ⁷ microbial cells). Then, 0.9 ml of disinfectant is added to the microbial suspension (thus, the total volume of the mixture is 1 ml and contains 5 × 10 ⁷ microbial cells). 0.5 ml of neutralizer is added (thus, the total volume of the mixture is already 1.5 ml and contains 5 × 10 ⁷ microbial cells).

From the resulting mixture with a volume of 1.5 ml is taken 0.1 ml (for seeding on a nutrient medium). This volume (0.1 ml) contains 15 times less microbial cells than 1.5 ml (i.e. 0.3 × 10 ⁷ - this is the amount we sow on a nutrient medium).

A disinfectant is considered effective if it kills 99.99% of microorganisms (in accordance with the “Methods of testing disinfectants to assess their safety and effectiveness” developed by the Scientific Research Institute of Disinfectology of the Ministry of Health of Russia, approved by the Chief State Sanitary Doctor of the Russian Federation - Moscow, 1998), thus, growth of 0.01% of microorganisms is allowed. To do this, we calculated how many microorganisms survived from those that we sowed on a nutrient medium:

0.3 × 10 ⁷ - 100%

x - 0.01%

;

;

X = 300 CFU

Thus, growth of up to 300 CFU is allowed. Therefore, with the growth of microorganisms after the action of the disinfectant up to 300 CFU, we are talking about sensitivity to this disinfectant, the growth of more than 300 CFU is about stability.

Nevertheless, we cannot ignore the growth of less than 300 CFU, a number of clinical trials have been conducted that have proved that the presence of such growth is one of the stages in the formation of resistance of microorganisms to disinfectants.

The proposed method according to option No. 1 is as follows.

I. Preparation of bacterial suspension

1. Culture of microorganisms * grown in solid nutrient medium at a temperature of 37 ° C for 18-24 hours, rinse with 0.9% sterile isotonic sodium chloride solution.

2. Bring microbial suspensions of each type to 5 units (5 × 10 ⁸ ) according to the turbidity standard for optical standardization of bacteria (GISK named after L. Tarasevich).

II. Preparation of disinfectant solutions

1. Solutions are prepared according to the active substance in sterile distilled water.

2. Disinfectants (DS) are periodically tested for activity of the active substance.

III. Conducting research

1. Disinfectant solutions (0.9 ml) are poured into sterile tubes with a rubber stopper.

2. Add 0.1 ml of microbial suspension into test tubes with disinfectant solutions and mix by shaking for several seconds.

3. After the action of the disinfectant, in the exposure indicated in the methodological recommendations for the use of the preparation (or necessary for the experiment), add 0.5 ml of the neutralizer solution ** and mix with shaking.

4. Sown on solid nutrient medium (MPA) in 0.1 ml of the mixture.

5. In parallel with the experiment, the controls are:

1) Monitoring the viability of the microorganism

2) Disinfectant control without adding culture

3) Monitoring the completeness of neutralization of the disinfectant

IV. Profitability Analysis

Results are taken into account after 24 hours according to the number of colonies grown on the Petri dish. In the absence of growth, the cups are left in the thermostat for up to 2 days. The grown colonies are subjected to microscopy.

V. Evaluation of the results

Cultures are considered sensitive in the absence of growth or with growth of not more than 300 CFU / ml, which corresponds to the required DS efficiency (99.99% of microorganisms die under the influence of a disinfectant).

1. With a growth of less than 300 CFU / ml, the strain is considered sensitive: sensitive cultures are evaluated according to the degree of sensitivity:

1) full sensitivity - in the absence of growth;

2) incomplete sensitivity - in the presence of growth:

100-300 CFU / ml - the disinfectant has a subbactericidal effect;

up to 100 CFU / ml - incomplete bactericidal effect.

2. The strain is resistant to this disinfectant in the studied mode with an increase of 300 CFU / ml or more.

The multiplicity of the experiment should be at least three (subject to the same results). If necessary, statistical processing - at least eight.

* investigated the culture of microorganisms isolated from clinical material, environmental objects, test microorganisms (E. coli pcs. 1257, S.aureus pcs. 906)

** to neutralize the antimicrobial action of disinfectants from various chemical groups, the following neutralizers are used:

- for agents from the group of oxidizing agents (halide-containing, oxidizing agents, agents containing peracetic acid, ozone) - 0.5-1% sodium thiosulfate solutions;

- for aldehyde and phenol-containing products - water; a universal converter containing tween-80 - 3%, saponin - 3%, histidine - 0.1% and cysteine - 0.1%;

- for cationic surfactants - 0.1-1.0% solutions of sulfonol or 0.5-1.0% solutions of solfonol with 10% skim milk.

The proposed method for option No. 2 is as follows.

I. Preparation of bacterial suspension

3. Culture of microorganisms * grown in solid nutrient medium at a temperature of 37 ° C for 18-24 hours, rinse with 0.9% sterile isotonic sodium chloride solution.

4. Bring microbial suspensions of each type to 5 units (5 × 10 ⁸ ) according to the turbidity standard for optical standardization of bacteria (GISK named after L. Tarasevich).

II. Preparation of disinfectant solutions

3. Solutions are prepared according to the active substance in sterile distilled water.

4. Disinfectants (DS) are periodically tested for activity of the active substance.

III. Conducting research

6. On the sterile surface of the test object ** with an area of 10 × 10 cm, microbial suspension is measured in an amount of 0.1 ml and distributed with a sterile spatula over the entire area of the square.

7. After the microbial suspension is dried, 0.9 ml of the disinfectant solution is gradually applied to the surface and distributed on the surface of the square with a sterile spatula. The disinfectant is used in the concentrations indicated in the guidelines for the use of the product (or necessary for the experiment).

8. After exposure to the desired exposure, a 0.5 ml neutralizer *** is applied to the test objects and rubbed evenly on the surface. The exposure of the disinfectant is determined based on the guidelines or objectives of the experiment. Catalyst exposure is standard.

9. Wash off with a sterile swab from the surface of the test object.

10. Sowing on plates with nutrient medium is carried out immediately with the same swab (0.1 ml each).

11. In parallel with the experiment, the controls are:

1) Monitoring the viability of the microorganism

2) Contamination control of the test object

3) Control the sterility of the test object

4) Disinfectant control without adding culture

5) Monitoring the completeness of neutralization of the disinfectant

IV. Profitability Analysis

Results are taken into account after 24 hours according to the number of colonies grown on the Petri dish. In the absence of growth, the cups are left in the thermostat for up to 2 days.

The grown colonies are subjected to microscopy.

V. Evaluation of the results

Cultures are considered sensitive in the absence of growth or with growth of not more than 300 CFU / ml, which corresponds to the required DS efficiency (99.99% of microorganisms die under the influence of a disinfectant).

2. If the growth is less than 300 CFU / ml, the strain is considered sensitive: sensitive cultures are evaluated according to the degree of sensitivity:

1) full sensitivity - in the absence of growth;

2) incomplete sensitivity - in the presence of growth:

100-300 CFU / ml - the disinfectant has a subbactericidal effect;

up to 100 CFU / ml - incomplete bactericidal effect.

3. The strain is resistant to this disinfectant in the studied mode with an increase of 300 CFU / ml or more.

The multiplicity of the experiment should be at least three (subject to the same results). If necessary, statistical processing - at least eight.

* studied the culture of microorganisms isolated from clinical material, environmental objects, test microorganisms (E. coli pcs. 1257, S.aureus pcs. 906)

** as test objects you can use metal surfaces, oilcloth, plastic, ceramic tiles, wooden surfaces, painted with paints, etc.

*** to neutralize the antimicrobial action of disinfectants from various chemical groups, the following neutralizers are used:

- for agents from the group of oxidizing agents (halide-containing, oxidizing agents, agents containing peracetic acid, ozone) - 0.5-1% sodium thiosulfate solutions;

- for aldehyde and phenol-containing products - water; a universal converter containing tween-80 - 3%, saponin - 3%, histidine - 0.1% and cysteine - 0.1%;

- for cationic surfactants - 0.1-1.0% solutions of sulfonol or 0.5-1.0% solutions of solfonol with 10% skim milk.

It should be noted that the distribution of the neutralizer and the subsequent collection of the flush for sowing is essential. In the proposed method, the neutralizer is applied evenly to the surface of the test object after the expiration of the exposure, then a wash is taken from the surface of the test object with a sterile swab (it absorbs 0.1 ml of solution) and the same swab is applied to the Petri dishes with nutrient medium. In known methods, flushing from the surface is carried out with sterile napkins moistened with a neutralizer, which are then beaten off in containers with saline solution with glass beads for a certain time, from which a certain amount of the mixture is then taken, followed by seeding on a nutrient medium; in addition, the technique of sequentially immersing carriers (pins of the stamp-replicator) contaminated with suspensions of microorganism cultures in a disinfectant and then in a neutralizer followed by seeding on solid nutrient media by pressing the end pads of the pins to the surface of the medium on Petri dishes is used.

Thus, taking the wash off the surface of the test object with a sterile swab and then plating it on a nutrient medium seems convenient from a practical point of view, in addition, 0.1 ml of the solution is contained on a standard swab, which corresponds to the amount of sown mixture of option No. 1 of our proposed method, which allows you to compare the results of both variants of the method with each other.

In addition, grinding (rather than spraying, not irrigation) with a sterile spatula of suspensions of microorganisms, disinfectant solutions and neutralizers on the surface of the test object is close to the practical conditions of use of the disinfectant, because medical facilities use the method of wiping with disinfectant solutions of various surfaces, while spraying or irrigation with solutions of disinfectants is rarely used in practice.

The advantages of the invention:

1) there is no need for preliminary testing of cultures of microorganisms for the possibility of applying the proposed method;

2) limiting the time of exposure of the disinfectant to the studied culture of microorganisms through the use of disinfectant neutralizers, which allows studies to be carried out taking into account the recommended disinfection regimes;

3) the possibility of simultaneous testing of several disinfectants with various recommended disinfection modes;

4) the standard methodology (measured application of disinfectant solutions, suspension of microorganisms and solutions of neutralizers, as well as quantitative seeding on nutrient media);

5) the ability to identify microorganisms grown on solid nutrient media;

6) quantitative assessment of the results (counting the number of colonies grown);

7) gradation of sensitivity (determination of the degree of sensitivity of the tested cultures of microorganisms to disinfectants);

8) the present invention is a micromethod that allows you to work with small amounts of disinfectants (measured in milligrams);

9) the simplicity and cost-effectiveness of the methodology, which makes it possible to widely use it in various bacteriological laboratories.

Examples of specific performance are given in the form of test reports.

Example No. 1

Test report of the action of Javelide-Solid on the infected surface with cultures isolated from the external environment.

The sensitivity of 17 cultures isolated from the external environment of the hospital to the disinfectant "Javel-Solid" on various surfaces was studied.

The action of the disinfectant was studied according to its own methodology for determining the sensitivity (stability) of microorganisms to disinfectants in the treatment of various surfaces.

Study progress

1. Preparation of microbial suspensions from the studied cultures grown in solid nutrient medium at a temperature of 37 ° C during the day. Suspensions are prepared on a 0.9% sterile isotonic sodium chloride solution according to a turbidity standard of 5 units. (i.e., 5 × 10 ⁸ microbial cells are contained in 1 ml of solution).

2. Preparation of a working solution of the disinfectant "Javel-Solid" on sterile distilled water in a bactericidal concentration of 0.015%.

3. On the sterile surfaces of test objects with an area of 10 × 10 cm, a microbial suspension is applied in an amount of 0.1 ml and distributed over the surface with a sterile spatula over the entire surface of the square.

After drying the suspension, 0.9 ml of disinfectant is applied to the surface and rubbed in a similar way.

As test surfaces were used:

- oilcloth,

- metal

- glass

- textiles

- tile

- plastic

- a wall painted with oil paint.

The action of the disinfectant on the oilcloth was carried out with cultures No. 267 (S.epidermidis), 304 (S.epidermidis), 309 (S.epidermidis), 314 (S.epidermidis), 315 (S.epidermidis);

on metal - with cultures No. 268 (S.epidermidis), 281 (S.epidermidis), 286 (S.epidermidis), 308 (S.epidermidis);

on glass - with culture No. 311 (S.aureus);

on textiles - with culture No. 312 (S.epidermidis);

on tile - with culture No. 316 (S.epidermidis);

on plastic, with cultures No. 285 (S.epidermidis), 288 (S.epidermidis), 296 (S.epidermidis), 302 (S.epidermidis);

on a wall painted with oil paint, with culture No. 310 (S.aureus). The interaction time of disinfectants with a microbial suspension was 60 minutes (the exposure is indicated in the methodological recommendations for the use of this disinfectant), after completion of exposure, 0.5 ml of a neutralizer (sodium thiosulfate solution) is measuredly applied to the test objects and evenly distributed over the surface for several seconds . After that, the surface of the test object was washed with a sterile swab, followed by seeding on Petri dishes with nutrient medium (the same swab). In parallel, control is set:

- crop viability,

- disinfectant without adding culture,

- the completeness of the neutralization of the disinfectant,

- contamination of the test object,

- sterility of the test object.

4. The results were taken into account after 24 hours of incubation in a thermostat at t = 37 ° C. Microscopy of grown crops was carried out.

The following results were obtained:

When processed with Javelide-Solid 0.015% at an exposure of 60 minutes, the efficiency indicator was 100% (no growth on Petri dishes) on oilcloth, metal, glass, textiles, tile, plastic infected with cultures No. 267 (S.epidermidis), No. 304 (S .epidermidis), No. 309 (S.epidermidis), No. 314 (S.epidermidis), No. 315 (S.epidermidis), No. 268 (S.epidermidis), No. 281 (S.epidermidis), No. 286 (S.epidermidis ), No. 308 (S.epidermidis), No. 311 (S.aureus), No. 312 (S.epidermidis), No. 316 (S.epidermidis), No. 285 (S.epidermidis), No. 288 (S.epidermidis), No. 296 (S.epidermidis), No. 302 (S.epidermidis).

Treatment of a wall painted with oil paint infected with culture No. 310 (S.aureus) proved to be ineffective (up to 100 CFU growth on Petri dishes was detected - incomplete bactericidal effect of the disinfectant).

Example No. 2

Test report on the effects of disinfectant solutions on Salmonella agona.

Salmonella agona cultures (No. 784, 823, 824), isolated from patients, were delivered to the problematic scientific laboratory of microbiology of the Research Institute of PFM GOU VPO NizhGMA from the State Health Institution “Dzerzhinsky Specialized Orphanage No. 2”, isolated from patients, for conducting studies on sensitivity to disinfectants that are used in this institution (disinfectants "Purjawel" and "Sulfochlorantin" were provided).

The study of the action of drugs was carried out according to our own methodology for determining the sensitivity (resistance) of microorganisms to disinfectants.

Study progress

1. Preparation of microbial suspensions from S. agona cultures grown in solid nutrient medium at a temperature of 37 ° C for a day. The suspension is prepared on a 0.9% sterile isotonic sodium chloride solution according to the turbidity standard of 5 units. (i.e., 5 × 10 ⁸ microbial cells are contained in 1 ml of solution).

2. Preparation of working solutions of disinfectants on sterile distilled water in bactericidal concentrations. Disinfectants were used in the following concentrations: Purjavel 0.015%, Sulfochlorantin 0.2%, Chloramine 1%.

3. Disinfectant solutions are poured into glass tubes with rubber stoppers in measured quantities (0.9 ml each), after which a microbial suspension (0.1 ml) is introduced into the tubes and mixed by shaking for several seconds. The interaction time of disinfectants with a microbial suspension was 60 minutes (the exposure is indicated in the methodological recommendations for using these disinfectants), after completion of the exposure, 0.5 ml of a neutralizer (sodium thiosulfate solutions) is added to the mixture and mixed by shaking for several seconds, after which it is sown 0.1 ml of the resulting mixture in Petri dishes with a dense nutrient medium (meat-peptone agar).

In parallel, the controls are set:

- viability of S. agona,

- disinfectants without adding culture,

- the completeness of the neutralization of the disinfectant.

4. The results were taken into account after 24 hours of incubation in a thermostat at t = 37 ° C. Microscopy of grown crops was carried out.

The following results were obtained:

1. Cultures of Salmonella agona (No. 784, 823) were sensitive to all disinfectants presented (lack of growth).

2. The culture of Salmonella agona No. 824 showed incomplete sensitivity to the agent “Sulfochlorantin” 0.2% with an exposure of 60 minutes (growth up to 100 CFU on Petri dishes - incomplete bactericidal effect).

Claims (2)

1. A method for determining the sensitivity of microorganisms to a disinfectant when it is in solution, comprising exposing a mixture of a suspension of microorganism culture with a disinfectant, adding a disinfectant neutralizer, plating suspensions on a solid nutrient medium, followed by assessing the growth of microorganisms on a nutrient medium, characterized in that the suspension microorganism cultures are prepared according to the turbidity standard 5 U, then 0.1 ml of this suspension is mixed in a test tube with 0.9 ml of a disinfectant and conducting exposure of the resulting mixture over a period of time known for a given disinfectant as the time required for the disinfecting effect to appear, then add 0.5 ml of a neutralizer, take 0.1 ml of the resulting mixture and plated on a solid nutrient medium, judge the sensitivity of microorganisms to the disinfectant with the growth of microorganisms on a nutrient medium up to 300 CFU / ml, while with growth up to 100 CFU / ml, an incomplete bactericidal effect is judged, with an increase of 100-300 CFU / ml, a sub-bactericidal effect is judged, with an increase of more than 300 CFU E / ml - about the resistance of microorganisms to a disinfectant. 2. A method for determining the sensitivity of microorganisms to a disinfectant when exposed to surfaces, comprising exposing a mixture of a suspension of microorganism culture with a disinfectant, adding a disinfectant neutralizer, plating suspensions on a solid nutrient medium, followed by assessing the growth of microorganisms on a nutrient medium, characterized in that the suspension microorganism cultures are prepared according to the standard turbidity of 5 Units, then 0.1 ml of this suspension is applied to the sterile surface of the test object with an area of 10 × 10 cm and spread with a sterile spatula over the entire area of the square, after drying the microbial suspension, 0.9 ml of the disinfectant is applied to the surface and distributed over the square with a sterile spatula, the resulting mixture is exposed for the time known for this disinfectant as the time required for manifestations of a disinfecting effect, then 0.5 ml of a disinfectant neutralizer is applied to the surface of the test object, and after exposure to the neutralizer, the mixture is washed off the surface t st-object with a sterile swab, the same swab immediately sown 0.1 ml of the mixture on a solid nutrient medium, the sensitivity of microorganisms to a disinfectant is judged with the growth of microorganisms on a nutrient medium up to 300 CFU / ml, while with growth up to 100 CFU / ml they judge incomplete bactericidal action, with an increase of 100-300 CFU / ml - about a sub-bactericidal effect, with an increase of more than 300 CFU / ml - about the resistance of microorganisms to a disinfectant.