RU2409679C1 - Method of bacterial disinfectant resistance test - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: there are prepared a solution of a disinfectant, a bacterial culture and a nutrient medium with a chromaticity indicator which is presented with a redox-indicator - 2,3,5-triphenyltetrazolium chloride. The prepared disinfectant solution is added with a bacterial culture, and the produced mixture exposed for 15-30 minutes is transferred in wells containing a nutrient medium and a preliminary added and dried disinfectant neutraliser. It is kept for 30 minutes and transferred in the wells containing a preliminary dried nutrient medium with redox chromaticity indicator. The tray is placed in a thermostat at temperature 37°C for 4-24 h, and the results are estimated by discoloration of the medium. The medium colour changed from initial yellow to bright-pink testifies to bacteria increase, and the disinfectant is considered to be uneffective.

EFFECT: higher effectiveness and enhancement of the method.

3 dwg, 1 ex

Description

The invention relates to medicine, in particular to microbiology.

A known method for determining the sensitivity (resistance) of bacteria to disinfectants [1]. The method consists in the fact that suspension of bacteria with a density of 2 × 10 ⁹ CFU / ml contaminate the pins of the stamp-replicator and dried under aseptic conditions in air at room temperature. Then the pins are disinfected in the holes of the replicator punch into which the test disinfectant solution is introduced. After a 10-minute exposure in a disinfectant, the pins are transferred to the wells of another replicator stamp filled with a neutralizer corresponding to the tested disinfectant and held there for 10 minutes. Control is provided by using distilled water instead of a disinfectant. After the expiration of the exposure time, the experimental and control replicators (pins) are removed from the neutralizer and the seed is replicated onto AGV medium or other nutrient medium on Petri dishes. Cups with crops are incubated for 48 hours at 37 ° C and take into account the presence or absence of bacterial growth in the areas of inoculated fingerprints, considering the culture resistant to the disinfectant test solution in the presence of growth in the inoculated areas.

The disadvantages of this method.

1. The use of rather sophisticated equipment - replicator dies, which should be several: for microbial cultures, for disinfectant solutions and for a neutralizer solution.

2. The implementation of the method in 2 stages: in the stamps-replicators and on a nutrient medium.

The disadvantage of this method is the duration (2 stages) and the need to use non-standard equipment not manufactured by the industry.

The closest in technical essence and the achieved result is an accelerated and simplified method for determining the antibacterial activity of disinfectants [2]. The method is based on the use of a nutrient medium with a pH indicator that changes color under the influence of the vital activity (metabolism) of propagating bacteria. Effective concentrations of the disinfectant prevent the growth of bacteria, which does not lead to a discoloration of the medium.

Bromothymol blue is used as a pH indicator.

The study is carried out in sterile 96-well plastic disposable tablets with a well volume of 200.0 μl. 100.0 μl of culture medium with a pH indicator is added to all wells. In the first wells of each row, 100.0 μl of the initial dilution of the disinfectant solution diluted to the desired concentration equal to twice the concentration recommended for practice is added. Therefore, the initial dilution is the concentration that is set for practical use or lower if the disinfectant in the usual concentration adversely affects the color of the nutrient medium with a pH indicator.

50.0 μl of suspension of test bacteria (5.0 × 10 ⁷ microbial cells / ml) are added to wells with disinfectant dilutions and controls (except medium control). The tablets are covered with lids and placed in a thermostat with a temperature optimal for this type of bacteria.

The results are taken into account after thermostating by changing the initial color of the nutrient medium and the appearance of turbidity. As bacteria grow in a nutrient medium, they lower the pH, which entails a change in the color of the medium: an effective disinfectant should prevent color conversion, and an ineffective one, on the contrary, should cause it.

The authors of this method themselves note that some disinfectants in working concentrations by themselves can distort the color intensity of the nutrient medium, therefore, convincing changes should be considered the results when the color of the nutrient medium has become clearly yellow. In addition, not all types of bacteria change the color of the medium to yellow. So, when Pseudomonas is introduced into the nutrient medium after 3 hours, the color of the medium turns blue, which indicates not a decrease, but an increase in pH.

Given the disadvantages noted above, the authors of this method offer all disinfectants in assessing the effectiveness of this method to divide into 3 groups, guided by the pH value: acidic, neutral and alkaline. Acidic preparations are characterized by the fact that immediately after adding to a nutrient medium with a pH indicator, its color changes to yellow. Such preparations can be tested only starting from those concentrations that do not distort the initial color of the nutrient medium. Similarly, alkaline preparations are difficult to use due to the fact that after addition they change the color of the nutrient medium with a pH indicator to light blue.

Difficulties also arise with some disinfectants, which by themselves change the color of the nutrient medium, for example, Septabic, which is used in concentrations of 0.2-2.0%, in concentrations of 1.0-2.0% it discolours the nutrient medium. and at concentrations of 0.5% it distorts its original color. Therefore, the effectiveness of "Septic" in this way can be estimated at its concentrations below 0.5%.

Thus, the disadvantages of this method are the low efficiency and limitations of its application.

The task of the invention is to increase the efficiency of the method and expand the functionality.

The problem is achieved in that in a method for determining the resistance of bacteria to disinfectants, including the preparation of a disinfectant solution, a bacterial culture and a nutrient medium with a color indicator, followed by a bacterial culture in the disinfectant in the wells of the plate, and temperature control of the mixture with visual determination of the color of the nutrient medium through 4-24 hours, a redox indicator 2,3,5-triphenyltetrazolium chloride is used as an indicator of color, and after exposure for 15-30 minutes, bact ble culture disinfectant solution resulting mixture was transferred to a previously introduced into the wells and the plates are dried in a neutralizer disinfectants, maintained it for 30 minutes and transferred to wells containing pre-dried in their growth medium with a redox indicator.

The essence of the proposed method consists in the use of a redox indicator of 2,3,5-triphenyltetrazolium chloride with a concentration of 0.02% as a indicator that changes the color of the nutrient medium under the influence of the metabolism of propagating bacteria, and in the use of dried and ready to use Ingredients: a universal neutralizer of disinfectants and a standard nutrient medium (trypcase-soy broth) with a redox indicator located in the wells of the tablet.

The redox indicator 2,3,5-triphenyltetrazolium chloride (empirical formula C ₁₉ H ₁₅ N ₄ Cl) is an oxidizing agent, colorless, and when electrons are attached, red formazone is reduced to a stable substance [3]. It relates to tetrazolium salts, which are used in calorimetric determinations, tests in the food and pharmaceutical industries, in detecting changes in living bodies, in studies of metabolism and metabolism.

The invention is illustrated by drawings, where in Fig.1 shows a tablet (a device for determining the resistance of bacteria to disinfectants) before the experiment with the following conventions:

- пустые лунки для внесения дезинфектантов в различных концентрациях (последний ряд лунок используется для выполнения контролей и заполняется стерильной водопроводной водой, на которой разводятся дезинфектанты);

- empty wells for introducing disinfectants in various concentrations (the last row of holes is used to perform controls and is filled with sterile tap water, on which disinfectants are diluted);

- лунки с сухим нейтрализатором дезинфектантов;

- wells with a dry disinfectant neutralizer;

- лунки с сухой питательной средой и редокс-индикатором.

- wells with a dry nutrient medium and a redox indicator.

Figure 2 presents a tablet with one of the markup options in the study of two cultures of bacteria and three different disinfectants with the following conventions:

Sectors I-VI - experimental - rows of holes 1-9, sector K - control - rows of holes 10-12. The letter P denotes the wells where the working concentrations of disinfectants are introduced during the study, 1/2, 1/4 and 1/8 - dilutions of disinfectants from the working concentration, the letters KS indicate the control of the environment, KK ₁ - control of the first culture, KK ₂ - control of the second culture, KH ₁ and KH ₂ - control of the first and second culture, respectively, after exposure in the neutralizer.

Figure 3 is a variant of filling out a protocol for determining the resistance of bacteria to disinfectants (according to the example below).

Numeric and letter designations on the drawings along the lateral and upper edge of the tablet are given as a coordinate system that allows you to apply the sectoral principle of conducting research and recording results.

The inventive method is implemented using a tablet, which is a sterile plastic 96-well (12 rows of 8 wells with a volume of 200.0 μl) single-use tablet with a cover (figure 1). Four vertical rows of holes (1st, 4th, 7th, 10th) do not contain ingredients; four rows of holes located next to them (2nd, 5th, 8th, 11th) contain a universal disinfectant neutralizer in a dried state; four rows (3rd, 6th, 9th and 12th) contain nutrient medium pre-dried directly in the plate wells with a redox indicator. A universal neutralizer and nutrient medium with a redox indicator are introduced into the wells of the plates in advance, under aseptic conditions, in a volume of 100.0 μl and are dried. The tablets are stored in a sterile packaging in the refrigerator until they are used.

As a neutralizer, a mixture of the following composition is used: tween 80 - 3.0 g, cysteine - 0.1 g, histidine - 0.1 g, saponin - 3.0 g, buffer M / 15-10.0 ml; sterilization - 121 ° C for 15 minutes [four]. Trypcase-soy broth produced by bioMerieux with a redox indicator of TTX is used as a nutrient medium [3].

The sequence of operations in the method

1. Preparation of a bacterial suspension of the studied culture: 18-20-hour agar cultures of microorganisms are washed off with a sterile 0.85% sodium chloride solution with the addition of 20.0% horse serum or 0.1% albumin. The resulting bacterial suspension is thoroughly mixed, in the presence of flakes and uneven suspension - using a shaker, and standardized with the same solution to a density of 1 billion / ml according to the turbidity standard GISK them. L.A. Tarasevich or a similar standard Ms. Farnland.

2. Preparation of disinfectant solutions: disinfectant solutions, starting from the working concentration, are prepared in a series of test tubes using sterile tap water and in macro volumes (50-100 ml), which reduces the error in the concentration of the drug.

3. Preparation of the tablet (a device for determining the resistance of bacteria to disinfectants): the tablet is removed from the package and perform “marking” on its cover, taking into account the chosen version of the experiment. One of the layout options presented in figure 2.

4. In the rows of holes with a dried neutralizer (2nd, 5th, 8th and 11th) and a nutrient medium with a redox indicator (3rd, 6th, 9th and 12th) 8 - A 100.0 μl sterile distilled water is pipetted into a channel pipette and the contents of the wells are mixed until completely dissolved. The role of the neutralizer is to stop the effect of the disinfectant on the bacterial culture.

5. In the rows of empty holes (1st, 4th and 7th rows) add 100.0 μl of disinfectants in working concentrations (the lowest concentration recommended by the instructions for use of the drug as a working one) and 1 / 2,1 / 4 and 1/8 from her; for example, Slavin is introduced into the wells of the first row A, B, C, D (sector I) and E, F, G, H (sector II), in the holes of the fourth row A, B, C, D (sector III) and E, F , G, H (sector IV) —increcept, in the holes of the seventh row (sector V, VI) - CDI (complex instrument disinfectant).

6. In wells with dilutions of disinfectants (1st, 4th and 7th rows) and empty wells of the 10th row with 100.0 μl of tap water (controls) add 20.0 μl of a suspension of the test bacterial culture, standardized up to 1 billion suspension.

7. After exposure of the bacterial culture in a disinfectant solution, 20.0 μl of each of the contents of the wells of the 1st, 4th and 7th rows are transferred with an 8-channel pipette into the neutralizer solution (wells of the rows of the 2nd, 5th and 8- d), as well as from the wells of the control sector: from the holes of the 10th row to the 11th row (growth control of the studied cultures) after exposure to the neutralizer (SC).

8. After a 30-minute exposure in a neutralizer, 20.0 μl of the contents of the wells of rows of the 2nd, 5th, 8th and control 11th are transferred to wells with nutrient medium and thoroughly mixed with multi-channel mixing.

9. Wells of the 10th, 11th and 12th rows are used to perform controls:

- growth control of the studied bacterial culture (CC);

- control the growth of the same culture after exposure to a neutralizer (KN);

- control of the nutrient medium with a redox indicator (CS).

10. The tablet is placed in a thermostat at 37 ° C for 18-24 hours, after which the results are evaluated.

The presence in the nutrient medium of a redox indicator allows you to register a change in the redox potential due to the multiplication of bacteria by a change in the color of the medium. The bactericidal action of the disinfectant causes the death of bacteria, as a result of which the original color of the medium is preserved.

A change in the color of the medium in the wells (3rd, 6th, 9th and 12th row in the QC and KN controls) from the initial yellow to bright pink indicates the growth of bacteria, the disinfectant is evaluated as ineffective, and the tested bacterial culture how sustainable.

Preservation of the initial yellow color in the control medium with an indicator (CS) and wells after exposure to the disinfectant at working and diluted concentrations indicates that there is no bacterial growth, the disinfectant is evaluated as effective, and the tested bacterial culture is sensitive.

The implementation of the proposed method is illustrated by the following example.

EXAMPLE

The resistance of 2 bacterial strains was studied: Pseudomonas aeruginosa (Ps. Aeruginosa No. 231G) and Echerichia coli (E. coli No. 375H) to 3 disinfectants (Slavin, Intracept and CDI).

The sequence of operations in the example

1. Preparation of a bacterial suspension of the studied cultures (P. aeruginosa and E. coli): to prepare a bacterial suspension, 18-20-hour agar cultures of these microorganisms are washed off with a sterile 0.85% sodium chloride solution in a volume of 5.0 ml with the addition of 20.0 % horse serum or 0.1% albumin. The resulting bacterial suspension is thoroughly mixed; in the presence of flakes, uneven suspension - using a shaker, and standardize with the same solution to a density of 1 billion / 1 ml according to the standard turbidity GISK them. L.A. Tarasevich or a similar standard Ms. Farnland.

2. Preparation of disinfectant solutions: disinfectant solutions, starting from the working concentration, are prepared in vials using sterile tap water in macro volumes (50 ml):

- to prepare a working concentration of slavin, 0.25 ml of slavin was added to 49.75 ml of sterile tap water;

- to prepare a similar solution of Intracept, to 1.0 ml of sterile tap water was added 1.0 ml of Intracept;

- for the preparation of CDI - to 49.5 ml of sterile tap water was added 0.5 ml of a commercial solution of CDI.

3. Preparation of a device for determining the resistance of bacteria (tablets):

- the tablet was removed from the package and performed its marking (marking) on the lid, taking into account the chosen version of the experiment (figure 2 and figure 3) while filling out the research protocol (figure 3). In sectors I, III, and V, Ps culture was investigated. aeruginosa, and sectors II, IV and VI - E. coli culture in sensitivity-resistance to slavin (sector I and II), intracept (sector III and IV) and CDI (sector V and VI). In sector K, experience controls were performed;

- in the wells of the plates with the dried ingredients (neutralizer - rows of holes 2, 5, 8, 11 and nutrient medium with a redox indicator - rows 3, 6, 9, 12) 100.0 μl of sterile distilled water was added with an 8-channel pipette and mixed the contents of the wells until completely dissolved.

4. The study:

- 100.0 μl of disinfectants in the working concentration (0.5% for Slavin, 2.0% for Intracept and 1.0% for CDI) were added to the rows of empty holes (1st, 4th, 7th) ) and 1/2, 1/4 and 1/8 of them, i.e. holes of the first row A, B, C, D (sector I) and E, F, G, H (sector II) were added slavins, holes of the 4th A, B, C, D (sector III) and E, F, G, H (sector IV) —increcept, wells of the 7th (sector V, VI) —CDI;

- in wells with disinfectants (1st, 4th, 7th rows) and empty wells of the 10th row (controls), which were previously added with 100.0 μl of tap water, 20.0 μl of the studied bacterial cultures were added standardized to 1 billion / ml: Ps. aeruginosa (sectors I, III, V and K) and E. coli (sectors II, IV, VI and K).

- after exposure (30 minutes for Slavin, 15 minutes for Intracept and CDI) of the bacterial culture in a solution of preparations of 20.0 μl from the contents of the wells of these rows (1st, 4th, 7th), as well as from the wells of the control sectors (10 rows) were transferred with an 8-channel pipette into the neutralizer solution (rows of the 2nd, 5th, 8th and 11th). In the 11th row, the growth control of the studied cultures was performed after exposure in a neutralizer (KN);

- after a 30-minute exposure in a neutralizer, 20.0 μl of the contents of the wells of the 2nd, 5th, 8th rows and the control of the 11th were transferred to wells with nutrient medium and mixed thoroughly;

- holes of the 10th, 11th and 12th rows were used to perform controls:

- KK ₁ - growth control Ps. aeruginosa (Pseudomonas aeruginosa) and KK ₂ - growth control of E. coli (Escherichia coli) after exposure to the neutralizer;

- KH ₁ and KH ₂ - growth control of the same cultures after exposure in the neutralizer;

- KS - control of the nutrient medium with a redox indicator.

5. Incubation of the tablet: after performing the above steps, the tablet was closed with a lid and placed in a thermostat at 37 ° C for 18-24 hours.

6. Consideration of results: the presence in the nutrient medium of a redox indicator allows you to register a change in the redox potential due to the multiplication of bacteria by a change in the color of the medium. The bactericidal action of the disinfectant causes the death of bacteria, as a result of which the original color of the medium is preserved. Analysis began with controls:

- growth control of bacterial cultures (KK ₁ and KK ₂ ; KH ₁ and KH ₂ ): the color of the culture medium changed, turned pink, which indicates the viability and active metabolism of the studied cultures;

- control of the nutrient medium (CS): the color of the nutrient medium with the indicator has not changed (remained yellow), indicating the absence of its contamination and / or spontaneous response of the indicator.

Taking into account the results of the experiment by example: a change in the color of the nutrient medium to pink is indicated in the protocol by a “+” sign. When taking into account the results of the experiment, a change in the color of the nutrient medium was noted in all the wells (A, B, C, D) of the 3rd row of the 1st sector and the wells of C and D of the 6th row, which indicates the resistance of Pseudomonas aeruginosa to the working concentration of Slavin and 4 from the working concentration of the inject. A change in the color of the nutrient medium in wells G and H of the 3rd row and holes P of the 6th and 9th rows indicates the stability of E. coli to 1/4 of the working concentration of slavin and 1/8 of the insept and CDI.

Information sources

1. Gudkova E.I., Krasilnikov A.P. "Methodology for determining and indicators of the sensitivity (resistance) of bacteria to disinfectants" / Clinical laboratory diagnostics. - 1994, No. 6, p. 48-50.

2. Levi M.I., Suchkov Yu.G. “An accelerated and simplified method for determining the antibacterial activity of disinfectants” / Disinfection business. - 1999, No. 3. p.30-33.

3. R. Dawson and others. Reference biochemist. - Moscow: "World". 1991, p.302.

4. European Standart CEN / TC216 / HWG, No. 46 E.S. 7, Dec. 1992, Brussels.

Claims (1)

A method for determining the resistance of bacteria to disinfectants, including the preparation of a disinfectant solution, a bacterial culture and a nutrient medium with a color indicator and the subsequent introduction of a bacterial culture in a disinfectant located in the wells of the tablet, and temperature control of the mixture with visual determination of the color of the nutrient medium after 4-24 hours, characterized in that the redox indicator 2,3,5-triphenyltetrazolium chloride is used as a color indicator, and after exposure for 15-30 minutes the bacterial culture in solution D disinfectant resulting mixture was transferred to a previously introduced into the wells and the plates are dried in a neutralizer disinfectants, maintained it for 30 minutes and transferred to wells containing pre-dried in their growth medium with a redox indicator.

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