CS277170B6 - Method of quantitative "in vitro" susceptibility testing of bacteria and yeasts to antimicrobial agents - Google Patents

Abstract

The solution relates to a method for quantitative "in vitro" determination of the sensitivity of bacteria and yeasts in culture media and biological material to antimicrobial substances by indicating a change in the growth of a defined concentration and volume of a suspension of microorganisms in a liquid culture medium. The cultivation of microorganisms is carried out on an absorbent substrate in the form of one or more microdiscs in a limited closed space, with the microdiscs being separated from each other to prevent mixing of individual samples. The growth indication is carried out in various ways, e.g. using a redox indicator. The microdiscs are impregnated with a solution of an antimicrobial substance before use and the solvent is removed after impregnation.

Description

1 CS 277170 B6>

Technical field

The invention relates to a method for the quantitative "in vitro" determination of the susceptibility of bacteria and yeast in culture trays to biological material for antimicrobial agents.

Background Art

For "in vitro" susceptibility testing of microorganisms, "diffuse" or "dilution" methods are used in practice. In the diffusion method, a well is formed in a solid well in a petrihomisk with inoculated microorganisms, into which a solution with the exact amount of antimicrobial is sanitized. The antimicrobial material diffuses into the bar during incubation and creates a concentration gradient around the disc. The antimicrobial efficacious substance is evaluated based on the formation of a "barrier zone" of microorganisms around the well. In an alternative process, storage discs of inert material, usually filter paper, impregnated with an exact amount of antimicrobial material, which diffuses from the disc into the surroundings of the disc, are applied to the surface of the pad. Contact between the microorganism and the antimicrobial agent takes place in the culture medium, outside the disc. The disadvantages of the diffusion method are mainly that, even when quantifying the inhibition zone average, the evaluation provides only a qualitative result. Macroscopically detectable growth of microorganisms on the podium is required for evaluation, so the assay is evaluated after 24 hours. In addition, this method requires a large consumption of culture media and reagents.

In quantitative dilution methods, the defined amount of antimicrobial agent is most readily dissolved in a separate reaction vessel. In addition to the direct addition of the antimicrobial, the reservoirs of the absorbent material impregnated with the desired amount of antimicrobial to be placed in the culture vessel reaction vessels prior to assay are also used as its source. Upon elution of the antimicrobial from the disc into the pad, the disc itself will be removed from the stage, or the culture pod will be displaced by another measuring cup. The need to remove discs from the cultivation method is eliminated by the solution of GB 1057000, in which discs from a culture medium-soluble material are used. Thereafter, the test microorganism is added to the cans, cultivated and the growth evaluated.

The disadvantage of this method is that it requires a technologically demanding preparation of several under the different dilutions of the antimicrobial, and the storage of pre-prepared anti-microbial culture media is problematic; when the antimicrobial solution in the culture medium is not lyophilized prior to storage, its stability is limited, even when stored in a frozen state. Prior to testing, additional operations are required, e.g., dissolving and tempering frozen kits or lyophilizing below before testing, or mixing samples during dissolution. In the case of dosing the antimicrobial with carriers (tablets, disks) into the basic culture vessel in the reaction vessels just before the test, a large number of carriers are handled. The procedure is time consuming, less efficient and increases the risk of contamination below.

SUMMARY OF THE INVENTION

The above-mentioned disadvantages are eliminated by the method of quantitative "invitro" determination of the susceptibility of bacteria and yeast to antimicrobial agents according to the invention, by indicating the growth of defined concentration and volume of suspension of microorganisms in liquid culture medium at various concentrations of antimicrobials deposited on the absorbent substrate. The essence of the invention is that the cultivation of the microorganisms is carried out on an absorbent substrate in the form of one or more microspheres placed in a confined space, the microspheres being separated so as not to mix the individual samples. The growth of microorganisms takes place both on the surface of the microtitre and in its pores. Growth indications can be carried out in various ways, preferably by a redox indicator (e.g., a tetrazolium dye salt). The microspheres are impregnated with solutions with a defined amount of antimicrobial agent prior to use and the solvent removed (e.g., by drying). In particular, the advantage of the method of the invention is its ease of preparation of test kits with a large number of different dilutions of antimicrobial agents. The availability of ready kits is simple (lower storage temperature requirements); The dry-stored antimicrobial feedstocks allow longer use times and expand the selection of antimicrobials. The prepared test kit can be used directly without further operations (thawing, dissolving lyophilizate). In addition, there is a possibility of choosing a culture method directly in the preparation of a suspension of test microorganisms.

Due to the fact that the growth takes place in a minimal reaction volume (microtube), there is no need for the use of special reaction vessels and it is possible to work with extremely small amounts of reactions. This is associated with the reduction of claims for the disposal of infectious biological waste material. Example of the Invention Example

Quantitative determination of G-bacteria susceptibility to antibiotic netilmycin, cephalotin, ofloxacillin and tetracycline.

Attach microtubes from white filter paper to a non-wet plastic hydrophobic surface plate, which was prewashed first with 0.1 NHC1 and then twice with deionized water. The dimensions of the microspheres are chosen so that 4 rows of 10 microtens, each with an area of about 0.8 cm 2, separated by spaces without a filter paper width of 1.5 mm, are formed. Using an adhesive tape, a flat plastic cap with a hydrophobic surface is attached to the plate. The plate is sterilized with ethylene oxide.

After sterilization of the plate, 4 µl of the solution is examined under sterile conditions by micropipette for each microtube in distilled water so that the individual microtitre tubes contain 0.64, 0.32, 0.16, 0.08, 0 , 04, 0.02, 0.01, 0.005 and 0.000 μ9 of antibiotic. A suitable sodium azide solution is added to the last microtube in the row to control growth inhibition (the final content in the microtube is 100 µ9). The plate is placed in the dryer after addition of the solutions and dried for 30 minutes at 37 ° C. Thereafter, the lid is closed and the plate is sealed into a plastic sealed pouch.

In carrying out the method for determining the susceptibility of the present invention, it prepares a suspension of the bacterial strain under investigation (e.g., Escherichia coli in Mueller-Hinton broth) and adjusts the concentration of microorganisms to 1x10 6 / ml. Transfer each 10 µl microtitre tube of the prepared suspension through a tube applicator, cover the plate with a cap and seal hermetically in a sealed plastic bag to prevent evaporation of water from the microns. The plate is incubated at 37 ° C for 4-6 hours. Thereafter, 2 μΐ of the MTT color indicator (5 mg / ml 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenylthrazolium bromide in saline) is added to each micrometer by means of a tube applicator, the plate is placed in plastic bags and incubate for 30 minutes at 37 ° C.

After incubation, the plate is removed from the sachet and the growth of the microorganisms in individual microspheres is evaluated according to the color change of the indicator. The blue microsphere indicates the growth of the bacteria (not affected by the antimicrobial agent), unchanged staining of the antibacterial effect of the substance under investigation at the given concentration. According to the first microsphere with unchanged color in a number of decreasing concentrations of the test antibiotic, its minimum inhibitory concentration is determined.

Industrial applicability

The invention can be used in microbiological diagnostics (medical, veterinary, industrial and food).

Claims (2)

PATENT CLAIMS 1. A method for quantitatively in vitro determination of the susceptibility of bacteria and yeasts in culture media and biological material to antimicrobials by indicating a change in the growth of a defined concentration and volume of a microorganism suspension in a liquid culture medium at different concentrations of antimicrobials applied on an absorbent substrate. is carried out on an absorbent substrate in the form of one or more microtubes in a confined enclosure, the microdots being separated from one another so as not to mix the individual samples. 2. The method according to item 1, characterized in that after impregnation of the microtangles with the antimicrobial solution, the solvent is removed. End of document