JPS6196999A - Method of testing sensitivity of bacteria to medicine - Google Patents

Abstract

PURPOSE:A series of cultivation tubes is arranged in rows and filled with a culture medium containing a medicine whose concentration is diluted in a certain step in a series and the tubes are covered with a sheet to which a reaction material is set to places corresponding to individual cultivation tubes, then, the whole body is turned upside and down, when the reaction is over, to detect the growth of a microorganism by the change in color of the reaction material. CONSTITUTION:As shown in the figure, a series of tubes for cultivation 3 are set in rows, e.g., to a perforated plate 1 and an impermeating sheet 8, to which a reaction material containing an indication reagent or coloring reagent for the substrate or the product therefrom is sticked on the places corresponding to individual cultivation tubes 3, is bonded to the perforated plate 1. For example, a culture medium containing an antibiotic is placed in the cultivation tubes 3, a microorganism is inoculated to the medium, then cultivation is effected. After cultivation for a certain period, the sheet 8 is peeled off to observe the color change of the reaction material 7 to determine the MIC of the antibiotic.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a method for testing the drug susceptibility of bacteria to select an effective drug or to determine the antibacterial activity of the drug when conducting chemotherapy for infectious diseases. It is.

[Conventional technology]

In general, bacterial drug susceptibility testing methods are broadly divided into dilution methods and diffusion methods, but usually the minimum inhibitory concentration (Minimum Inhibitory Concentration) of the drug is
um Inhibitory Concentrati
on ; MIC) is determined by a dilution method. Dilution methods include the agar plate dilution method and the liquid medium dilution method. In both cases, a test bacterial solution is inoculated into a medium containing a drug at a certain concentration level, and after culturing for a certain period of time, a determination is made.

Conventionally, determinations are made using test tubes or petri dishes.
The number and properties of bacterial colonies that have developed on the medium are based on the bacterial growth itself, such as the color contrast and turbidity of the medium surface.In comparison with the control medium, a large number of test tubes, The test requires observation of petri dishes, etc., and uses a certain type of evaluation table, which is far from a simple operation and may lead to errors in the test results. Furthermore, drug susceptibility measuring machines that have been developed in recent years that use liquid media are not only expensive, but their operating methods are still only semi-automated, and ultimately require complicated operations. is the current situation.

[Problem that the invention seeks to solve]

The present invention provides a method for testing the drug susceptibility of bacteria, which allows the growth of bacteria to be accurately determined with simple operations without the drawbacks of the conventional drug susceptibility tests described above.

[Means for solving problems]

Currently, when fixing bacteria, bacterial species are identified based on various biochemical properties. Although mainly enzymatic reactions are involved, bacteria are inoculated into a medium containing a combination of a substrate and an indicator, positive or negative is determined by the change in color, and the properties are examined in more than 10 items. Identification up to this level has been carried out.

The present invention focuses on this identification theory, and uses the reaction of the bacteria to a certain type of substrate, that is, the increase in enzyme activity associated with the growth of small surfaces, as an indicator for making judgments in bacterial drug susceptibility tests, that is, confirming the growth of bacteria. The aim is to simplify, increase accuracy, and provide it at a low price. A specific method for this is to fill the culture tank of a bacterial culture container with a medium containing a certain dilution series of drugs, and add a substrate that is an indicator of bacterial growth before or after inoculating the test bacteria. After a certain period of reaction, by inverting the container, the presence or absence of bacterial growth can be detected by a change in color using a reaction material containing an indicator or coloring agent for the substrate or substrate decomposition products. .

The substrates include adonitol, arabinose, amidabrine, cellobiose, dulcimitol, fructose, glycerol, glycogen, α-methyl glycoside, N-acetyl glycosamine, 2-keto D-gluconic acid, galactose, inositol, inulin, Lactose, maltose, mannitol, mannose, melibiose, fructose, raffinose,
Ribose, salicin, sorbitol, starch, sucrose, trehalose, turanose, xylose, xylit, arginine, esculin, gelatin, 6-B
r-2 naphthyl α-D galactopyranoside, 2-naphthyl β-D galactopyranoside, naphthol MS-B
Iβ-D glucuronate, hisoplate, lysine, L-
leucyl 2-naphthylamide, orthonitrophenyl-β-D-galactopyranoside, 2-naphthyl phosphate, urea, hydrogen peroxide, citrate, malonate,
Examples include nitrate, pyruvate, tryptophan, peptone, thiosulfate, various tetrazolium derivatives, and various 4-methylumbelliferyl derivatives.

Indicators or coloring agents that are pre-contained in the reaction material include phenol red, bromothymol blue, bromcresol purple, methyl violet, thymol blue, neutral red, chlorophenol red, cresol red, phenolphthalein, resazurin, and ninhydrin. Examples include reagents such as ferric chloride, baraminodiethylaniline, sulfanilic acid, α-naphthylamine, potassium chloride, α-naphthol, and Kohasoku's reagent.

[Effect]

As a result of implementing the above measures, the drug susceptibility testing method of the present invention allows the growth of all bacterial species that are normally handled in clinical laboratories, including Enterobacteriaceae, Mycobacterium tuberculosis, and fungi, to be easily performed. It is now possible to make accurate judgments.

〔Example〕

Hereinafter, the configuration of the present invention will be explained in detail according to examples.

In the example, as shown in FIG. 1, a plurality of bacterial liquid inoculation tanks (2) whose upper parts communicate with each other are provided on one longitudinal side of a container body (1), and each is independent on the other side. A plurality of culture tanks (3) are provided, and these bacterial solution inoculation tanks (2) and culture tanks (3) are communicated by an undulating passageway (4), and a position above the culture tank (3) is provided. A covering film (6) with small holes (5) provided therein is pasted on the top surface of the container body (1), and the small holes (5) provided in this covering film (6) are attached to the upper surface of the container body (1).
For bacterial testing, a tape (8) with a reactive material (7) containing an indicator or coloring agent for the substrate or substrate decomposition products is attached to the top surface of the coating film (6) so as to cover the substrate. A culture container, as shown in FIG. 2, is a container body (1) with a large number of culture tanks (3) arranged in multiple rows, and these culture i! A culture for bacterial testing in which multiple rows of tape (8) each containing a reaction material (7) containing an indicator or a coloring agent for the substrate or substrate decomposition product are pasted above I (3). The testing method of the present invention was carried out using the container.

Therefore, regarding the drug susceptibility testing method using the liquid medium dilution method, we tested 10 strains of clinically isolated Mycobacterium tuberculosis.
I did an ICIC window. There are four types of anti-tuberculosis drugs: rifampicin, kanamycin, isoniacyto, and streptomycin, and 5V! Each time, the concentration after inoculating the bacterial solution was 10/-J',
5shi//%/, 2%/di, 12. Fire, 6.25/ly
3.1, 1.5624 ri/,,/, 0.
No. 7, (Metsu 1-)? , 0.3Qnme/zuinichi,
0.29. To〆/1-l pi, 0.1o/14/JT,
Middle br so that it is 12/degrees of Ol.

Adjusted with ok7H9 liquid medium. As a comparative method, a commercially available 1% Ogawa medium containing the above drug was used. Inoculation is carried out using the liquid medium dilution method, in which a bacterial solution adjusted to 0.2 Munck-Farland is added to the culture tank (3) at a concentration of 50% and the above-mentioned drug-containing 1.
% Ogawa medium is 1oo/A/! The pouring and culturing period is
It was kept at 37°C for 10 days in the liquid medium dilution method, and for 3 weeks in the drug-containing 1% Ogawa medium. For judgment, sensitivity and resistance were expressed as + and - based on the occurrence, number, and properties of conventional colonies in the 1% Ogawa medium containing drugs, and the method according to the present invention was used in the liquid medium dilution method. The detection method in this case examines the nitrate-reducing ability of Mycobacterium tuberculosis, and as a substrate, M150 sodium nitrate solution is used on the day of determination, and 100% of each culture tank (3) is used.
After adding J in portions and leaving it for 2 hours at 37°C, the container body (1) was overturned, and the presence or absence of growth was observed by the appearance of reddish-purple color on the reaction material (7) containing the coloring agent, and the MIC value was determined. The reaction material (
As for 7), a disk with a diameter of 8 fins was impregnated with 30 ml of a solution in which 2 g of sulfanilic acid, 2 g of N-1 naphthyl engineered ethylene diamine, and 20 g of tartaric acid were dissolved in 100 to 100 ml of distilled water, and then air-dried. .

MIC values of clinical isolates measured by the above procedure according to the method of the present invention and approximate MIC using drug-containing 1% Ogawa medium
The results of comparison with the values are shown in Table ■.

(The following is a blank space) [Effects of the Invention] Since the bacterial drug susceptibility testing method of the present invention is configured as described above, bacterial growth can be accurately detected with a simple operation by inverting the container body. It can be determined and has excellent effects.

In addition, in the present invention, since one drug is prepared in the same container in a constant concentration dilution series, operations such as inoculation are easy and accurate MIC values can be obtained, and growth can be confirmed easily. Since this method is based on the biochemical properties of bacteria rather than on the occurrence of colonies, it has the effect of reducing erroneous determination of MIC values.

Furthermore, in the present invention, since the indicator or coloring agent of the detection system is configured separately from the medium, storage stability and reagent stability are better than those that are mixed in the medium in advance. Furthermore, since the coloring operation is performed after the enzymatic reaction, it has the advantage that it is also possible to perform drug sensitivity tests on bacteria whose growth is inhibited by certain indicators or coloring agents.

In addition, in the present invention, it can be carried out not only for the original MIC value measurement performed with a dilution series of a constant concentration of a drug, but also for screening purposes by performing with a constant concentration of each of various antibacterial agents. Furthermore, it has the effect that it can have a function for identifying bacteria by appropriately combining an added substrate and a coloring system.

[Brief explanation of drawings]

FIG. 1 is a perspective view showing one embodiment of the culture container for bacterial testing used in the bacterial drug susceptibility testing method of the present invention, and FIG. 2 is a perspective view showing another embodiment of the culture container for bacterial testing.

Claims (1)

[Claims] 1. Using a bacterial culture container each equipped with a reaction material above a culture tank consisting of a plurality of independent concave portions, fill the culture tank with a medium containing a drug at a certain dilution series, and A substrate serving as an indicator of bacterial growth is added before or after inoculation of bacteria, and after reaction for a certain period of time, the bacterial culture container is overturned to immerse the bacterial liquid in the reaction material to determine the presence or absence of bacterial growth. A bacterial drug susceptibility testing method that detects by detecting changes in the color tone of wood.