JPH0310695A - Sensitivity measurement for strain - Google Patents

Abstract

PURPOSE:To accomplish the title measurement accurately and simply in such a way that, from the circumferential part of a laboratory dish filled with an agar-agar culture medium, plural culture solutions with the same number of bacteria are linearly applied toward the center, and a circular filter paper impregnated with a chemical is placed at the center, and then the lengths for bacteria non-growth after culture are measured. CONSTITUTION:From the circumferential part of a laboratory dish 1 provided with an agar-agar culture medium 2, plural culture solutions 6 with the same number of bacteria are linearly applied toward the center of the dish 1 followed by placing a circular filter paper 4 impregnated with a chemical at the center of said dish 1. Thence, the strain is put to culture; and after completion of the culture, the objective strain measurement is made with the lengths for bacteria non-growth from the edge of said filter paper 4 as indices. Alternatively, from said circumferential part, said plural culture solutions 6 are linearly applied in the same direction at regular intervals followed by placing, so as to cover the ends of these straight lines, a rectangular filter paper 7 impregnated with the chemical rectangularly to the above straight lines, thus accomplishing the objective sensitivity measurement in a similar way as described above.

Description

[Detailed Description of the Invention] [Summary] Regarding a method for measuring the susceptibility of bacterial strains to drugs, the purpose is to accurately and easily measure the susceptibility of multiple bacterial strains. After applying culture solutions of multiple species with the same number of bacteria in a straight line toward the center of the Petri dish, a circular filter paper impregnated with a drug is placed in the center of the Petri dish, and after culturing the bacterial strains, Sensitivity can be measured using the length of the end surface of a circular filter paper with no bacteria growing as an indicator, or culture solutions of multiple species with the same number of bacteria can be measured at regular intervals from the circumference of a Petri dish equipped with an agar medium. After applying it in a straight line in the same direction, a rectangular filter paper impregnated with the drug is placed in a direction perpendicular to the straight line, including at least the end of the straight line, and after culturing the strain, A method for measuring the susceptibility of a bacterial strain is constructed by measuring the susceptibility using the length from the end where no bacteria grow as an index.

[Industrial application field]

The present invention relates to a method for measuring the susceptibility of bacterial strains to drugs.

Some strains are susceptible to (kill) certain drugs
There are some that show resistance (survival).

The reason for this is that strains that produce enzymes that inactivate certain drugs in the body are resistant to them.
On the other hand, strains that are unable to produce such enzymes in their bodies, or that are unable to produce them due to some kind of mutation, are susceptible.

Here, the reasons why the body is unable to produce enzymes that inactivate drugs are: ■ Bacteria take in and take out substances through cell membranes, but drugs interfere with the permeability of cell membranes. ■ If the drug is a substance that causes mutations in the gene structure itself, ■ If the drug inhibits this synthetic pathway, where various enzymes are involved in the process of synthesizing proteins from genes. In the case of a substance like this, the following can be considered.

In recent years, the field of genetic recombination and biotechnology has shown rapid development, but it is necessary to have a host for genetically modified organisms that can be biologically contained, and from this point of view, bacteria that are highly sensitive to drugs are needed. is used.

For these reasons, there is a need for a technique that can accurately and easily measure sensitivity to drugs.

[Conventional technology]

Figure 3 shows the conventional method for measuring the sensitivity of bacteria to drugs, in which agar is added to the bacterial culture solution and the agar concentration is adjusted to 0.5 to 2.
After mixing the bacteria into a mixture of approximately 10% and 100%, pouring it into a Petri dish 1 and solidifying it, a circular filter paper 4 impregnated with a drug and having a diameter of 8 to 13+ nm is placed on top of the agar medium 2 containing the bacteria, and the bacteria are cultured. do.

In this way, the drug gradually oozes out from the circular filter paper 4 to the isotropic rod, creating a drug concentration gradient, but this prevents the cultivation of bacteria that are highly sensitive to the drug, and the growth of the bacteria. The diameter of the blocking zone 5 in which this is not performed becomes large.

Therefore, sensitivity has been measured using the size of this inhibition zone 5 as an index.

This method measures the susceptibility of a single bacterial strain by changing the concentration of the drug, and it has not been possible to accurately compare and measure the susceptibility of multiple bacterial strains to a single drug.

[Problem to be solved by the invention]

As described above, there has been a problem that the susceptibility of multiple bacterial strains to a specific drug cannot be measured under the same conditions, and therefore the susceptibility cannot be measured accurately.

[Means for solving the problem] The above problem can be solved by
After applying culture solutions of multiple species with the same number of bacteria in a straight line toward the center of the Petri dish, place a circular filter paper impregnated with a drug in the center of the Petri dish, and after culturing the bacterial strains, Sensitivity can be measured using the length of the edge where no bacteria grow as an indicator, or culture solutions of multiple species with the same number of seedlings can be placed at regular intervals from the circumference of a petri dish equipped with an agar medium. After coating in a straight line in the direction, a rectangular filter paper impregnated with the drug is placed in a direction perpendicular to the straight line, including at least the straight edge, and after culturing the bacterial strain, the bacteria grow from the edge of the filter paper. This problem can be solved by constructing a method for measuring the susceptibility of bacterial strains by measuring the susceptibility using the length of the strain as an indicator.

[Effect]

As a method for accurately measuring the susceptibility of multiple bacterial strains to a specific drug, the present invention involves preparing multiple types of culture fluids containing the same number of bacterial strains, and using a pipette or the like to pour this culture fluid onto an agar medium. Apply in a straight line at intervals of .

Next, a filter paper containing a drug is placed perpendicular to this linear culture solution.

In this way, the drug that has soaked into the filter paper gradually oozes out into the linear culture solution containing the agar medium, creating a drug concentration gradient.

Next, depending on the concentration of the bacterial strain, if the bacteria are grown to the limit where they can grow, the bacterial strain will grow and form colonies, but the growth of bacteria that are highly sensitive to the drug will be hindered.
Colony formation occurs only up to a distance measured from the filter paper.

In the present invention, an indicator bacterium whose sensitivity to a drug is already known is always included in the plurality of bacterial strains whose susceptibility is to be measured.
Sensitivity is determined quantitatively by comparing and measuring the length of non-growth of other bacteria using the length of non-growth of this strain as a standard value.

To do this, as shown in Figure 1, use a pipette or sterilizer to apply the culture solution 6 radially toward the center of the Petri dish 1 on which the agar medium 2 is placed, and then spread the drug into the center. As shown in Fig. 2, the culture solution 6 was applied linearly in the same direction on the agar medium 2 using a pipette or sterile tool at regular intervals. Afterwards, a method of placing a rectangular filter paper 7 impregnated with a drug so as to be perpendicular to this may be considered.

Then, measure the length from the end surface of the circular filter paper 4 or the rectangular filter paper 7 to the end of the culture solution where bacteria did not grow,
The magnitude of susceptibility can be determined accurately by determining the length and comparing it with the length for the indicator bacteria.

Note that since a large number of bacteria are applied onto the agar medium, there is a risk of alternate contamination of bacteria if care is not taken.

Therefore, specifically, the following steps are recommended.

■ Prepare an agar medium in a petri dish.

■ Use ink to draw the line on the back of the petri dish to apply the bacteria.

■ Mark the position where the circular or rectangular filter paper will be placed.

■ 108 cells/m from the circumference of the petri dish! Apply a certain amount of bacteria using a pipette or sterile instrument.

■ Place a circular or rectangular filter paper soaked with the chemical solution in place.

- After culturing day and night, measure the length of ungrown tissue.

〔Example〕

The following three types of drugs were used.

(1) MNNG (N-methyl-N'-
(abbreviation of nitro-N-nitrosoguanidine) (2) MMS (Methyl Methan 5u
(abbreviation of lfonate) (3) MC (Mitomi
cyn C) The following three types of Bacillus subtilis were used as bacterial strains.

(1) 1IWTs201 (Wild strain
te) )(2) )ITS112 (Recombination defective temperature range susceptible strain (rec Ts)) (3) HE302
(Recombination defective strain (rec E)) Radius 4511II1
Prepare a standard agar medium using a petri dish from No. 1, and add the above three types of Bacillus subtilis at 10' cells/mjl! Containing culture solution 0.1m
ff1 was applied linearly from the edge of the Petri dish to the center using a pipette.

Then, MNNG was at a concentration of 1 mg/ml and MC was 10
At a concentration of 0 μg/rgl, and MMC in its undiluted state, a round filter paper with a diameter of 8 ribs was placed in the center of a Petri dish and cultured overnight at a temperature of 37°C. The length of the missing Bacillus subtilis was measured from the edge of the circular filter paper.

Table 1 Table 1 shows the results, and Bacillus subtilis HWTS201
The susceptibility of the other two types of Bacillus subtilis was determined based on the length at which no growth occurred.

From the above, the sensitivity of Bacillus subtilis is H regardless of the type of drug.
It was found that the values increased in the order of WTS201→HTS112→HE302, and this result agreed with the result measured by the conventional method.

[Effects of the Invention] As described above, according to the present invention, the susceptibility of a large number of bacterial strains to drugs can be easily measured under the same conditions.

In addition, by measuring the susceptibility of bacterial strains with known susceptibilities, it is possible to quantitatively measure the susceptibility of various bacterial strains.

From the above, by carrying out the present invention, it becomes possible to rapidly measure the sensitivity of drug-sensitive strains necessary for gene recombination technology.

figure, FIG. 2 is a plan view showing another sensitivity measurement method according to the present invention; FIG. 3 is an explanatory diagram of a conventional sensitivity measurement method.

In the figure, 1 is a petri dish, 2 is an agar medium, 4 is a circular filter paper, 5 is an inhibition zone, and 6 is a culture solution.

[Brief explanation of the drawing]

FIG. 1 is a flat book showing the susceptibility measurement method according to the present invention.
= A set of hand drawings showing the sensitivity of the sensitivity pillow 1 Figure (construction drawing) August 1999: Hand drawings showing the sensitivity measurement method of image filtering
2 The feeling of the mouth of the drain 1 shell '1' ``Writing 51. B Moonfish 3 Figure

Claims (2)

[Claims] (1) From the circumference of a Petri dish equipped with an agar medium, culture solutions of multiple species with the same number of bacteria are applied in a straight line toward the center of the Petri dish, and then the drug is soaked into the center of the Petri dish. A method for measuring the susceptibility of a bacterial strain, the method comprising: placing a round filter paper, cultivating the strain, and then measuring the susceptibility using the length of the end surface of the round filter paper where no bacteria grow as an index. (2) Apply culture solutions of multiple species with the same number of bacteria in a straight line in the same direction at regular intervals from the circumference of a petri dish equipped with an agar medium, and then apply the culture solution to the circumference of the petri dish with an agar medium, and then apply the culture solution in a straight line in the same direction at regular intervals, and then ,
A rectangular filter paper impregnated with a drug is placed in a direction perpendicular to the straight line, and after culturing the bacterial strain, sensitivity is measured using the length of the filter paper where no bacteria grow as an indicator. A method for measuring the susceptibility of bacterial strains.