CS197867B1 - Method for the identification of bacteria for diagnostic purposes - Google Patents

Description

The invention relates to a method for identifying bacteria for diagnostic purposes in microbiology. At present, diagnostics is performed in non-standard glass tubes with a non-standard set of tests with a non-standard evaluation, according to the choice of the appropriate workplace.

In addition, several types of identification of gram-negative fermenting rods are known. The PathoTec System (General Diagnostic Division, Warner-Lambert Company, New York, USA) consists of using 12 strips of filter paper impregnated with reagents for the following tests: Oxidase, beta-D.galactosidase, phenylalanine desaminase, indole production, nitrate reduction to nitrite, Voges- Proskauer reaction (production of acetoin), utilization of malonate, decarboxylase of lysine, production of urease, production of hydrogen sulfide and cleavage of esculin. The method, named Enterotube (Roche Diagnostics, Nutley, New Jersey, USA and Hoffmann-La Roche and Cie, Basle, Switzerland), uses special multi-chamber containers filled with solid soils for biochemical purposes and steel wire to inoculate these soils with one by pulling through the holes in the cell compartments. The main disadvantage is the high cost of using this system due to the complexity of the container and the use of agar media.

Another method, called the RB-System (Diagnostic Research, Roslyn, New York, USA), uses a smaller column of agar soil from two tubes, the larger of which contain sloped soil.

Larger is inoculated with bacteriological loop by puncture and surface. The second test tube is

197 867

197 887 inoculated with a puncture. They include tests for the disamination of phenylalanine, lactose cleavage, glucose cleavage, hydrogen sulfide production, lysine decarboxylation, omitin movement and decarboxylation. The relative disadvantage is the combination of soils and tests and the resulting sensitivity to strict adherence to the technological process. The API 20 system (Annalstab Products, Carle Flace, New York, USA) utilizes a molded strip of film of specially modeled cells containing lyophilized substrates for 20 tests. The chambers are inoculated with a pasteuric pipette of a suspension of bacteria, some of which are overlaid with paraffin oil, placed in plastic cases moistened with distilled water. Read in 18 hours by comparison with a table or diagnostic list. Finally, the Minitek System (BBL, Cockeysville, Maryland, USA) uses a plastic panel. with ten wells into which paper discs saturated with substrates are inserted. Inoculate with a suspension in a special broth with a mechanical micropipette and disposable spikes. The test report can be selected.

The disadvantages of the known methods are mainly the burden on the auxiliary staff during the washing, sterilization, assembly and supply of glass tubes, cleaning the racks and filling the tubes. Large volumes of media burden the laboratories' budget, the method of performing the tests is uneconomical. The tubes in the racks occupy space on the table and in the thermostats, and the energy consumption to heat the tubes and metal racks during incubation is quite high. The diagnostic procedure is not standardized; interpretation is possible over a wide range. This does not ensure continuity with other procedures, in particular the precise classification of antibiotic resistance data processing strains.

According to the invention, the above-mentioned drawbacks are overcome by a method of identifying bacteria for microbiology diagnostic purposes using microtests.

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It is based on inoculating the bacteria in separate plastic wells, which store the dried substrates for biochemical tests, then incubating them at 30 to 37 ° C for 18 to 42 hours, and then the results of the individual reactions with a fixed set of test results.

The advantages of the method according to the invention lie primarily in the possibility of using miniaturized tests, obtained according to the content of the information in the system and easy to implement, formulated for liquid soils and according to the degree of reproducibility. These assays are divided into two steps, but no more than three, performed sequentially. All factors were taken into account in the compilation, with the content of the information not being preferred. A further advantage is that the media are prepared in a dried state and dissolved in the inoculum during execution. The tests are carried out in plastic serological plates, which allow working in small volumes. Serological plates generally have eight rows of wells of 12 in a row. 12 or 6 wells are used for one examination. The wells are filled with liquid medium, which is dried either freeze-dried or at elevated temperature under vacuum or at normal atmospheric pressure. The filled plates are packaged in a plastic thermoplastic film or in a plastic transport bag. A total of 8 semi-solid plastic pipettes, sealed at the end, are provided with each plate. These pipettes have a calibrated mouth 86 to deposit droplets in either a 0.025 ml or 0.050 ml volume. Quality control is performed by a set of test strains. After inoculation, the plate is incubated in a humid atmosphere (in a plastic box) in a normal laboratory thermostat. Read with a simple 24-hour device against the diffuse light source. The test report allows you to link the interpretation of the results to a computer, or to build an identification list that provides the probability of identification for possible character combinations and to instruct further tests for the second step.

The process according to the invention is explained in more detail below with reference to an exemplary embodiment.

Example

Using a bacteriological platinum loop, a suspension was prepared from two or one colonies in saline in a small volume tube or vial. The concentration of microbes in the suspension was from 10 to 10 cells per ml. Pipette 0.1 ml of the suspension into each of the twelve wells containing the dried substrates. (Eight rows of 12 wells are placed on one plate so that eight strains can be inoculated at the same time.) The plate with inoculated wells was placed in a box filled with water vapor and incubated at 37 ° C for 24 hours. Then, about 10 µl of Kovacs's reagent for detecting indole was dropped into the fourth well, and about 9 µl of Barrit's reagents I and II for the detection of acetoin. It was viewed against the white area in sufficient light and found the following:

a) if the first hole is unchanged (hydrogen sulphide -), the second deep yellow (mannit +), the third purple (lysine +), the fourth with a red ring, or deep red (indole +), the fifth yellow (ornithine -), the sixth and seventh unchanged (urea citrate -), eighth bright yellow (ONPG +), ninth, tenth unchanged (VPT inosit -), eleventh unchanged (lipase -) and twelfth yellow (glucose +), determined by comparison with the diagnostic table Escherichia coli;

(b) if the first well is cloudy with a deep black precipitate, the second colored yellow, third, fourth fifth unchanged, sixth deep blue, seventh unchanged, eighth deep yellow, ninth, tenth, eleventh unchanged, twelfth bright yellow, the bacteria are identified Citrobacter freundii;

(c) if the first well is cloudy with a deep black precipitate, the second and third intact, the fourth with a red ring or deep red, the fifth and sixth intact, the seventh deep red or pink, the eighth, ninth, tenth and eleventh intact and the twelfth yellow , Proteus vulgaris is diagnosed.

Claims (1)

SUBJECT OF THE INVENTION Method for the identification of bacteria for diagnostic purposes in microbiology by microtests, characterized in that the bacteria to be determined are inoculated into separate wells in a plastic plate in which dried substrates for biochemical tests are stored, and then incubated at a temperature of 30 to 37 ° C for 18 to 42 hours, after which the results of the individual reactions are compared with a fixed set of microtest results * Printed by Moravian Printing Works,