JPS60248168A - Plate for screening - Google Patents

Abstract

PURPOSE:A plate for screening capable of detecting easily, rapidly, accurately, and hygienically a mold capable of producing antibiotics, by a simple operation, obtained by supporting a hydrophilic high polymer layer containing a mold of microorganism, etc. on a substrate. CONSTITUTION:A hydrophilic high polymer layer containing at least one of a mold of microorganism (mold having sensitivity to antibiotic) and a spore of microorganism (e.g., mold, etc.), and, further a nutrient source (e.g., peptone, etc.) is formed on a substrate (e.g., polyurethane, etc.) by coating or immersion, to give the aimed plate.

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field) The present invention relates to a screening plate for detecting antibiotic-producing bacteria.

(Prior art) Antibiotic-producing bacteria can be detected by growing a colony of a single species or multiple species of test bacteria on a plate medium such as agar, and spraying antibiotic-susceptible bacteria onto this colony, or spraying antibiotic-susceptible bacteria onto this colony. Pour in the agar.

This is done by observing the presence or absence of growth of susceptible bacteria around the test bacteria. When the growth of susceptible bacteria is inhibited by antibiotics, inhibitors are formed around colonies of antibiotic-producing bacteria. The ability of a test bacterium to produce antibiotics can also be determined by inoculating a test bacterium onto an agar medium containing antibiotic-sensitive bacteria and observing the presence or absence of growth of the susceptible bacteria. In the conventional method, susceptible bacteria are sprayed onto colonies of test bacteria.

It is extremely difficult to uniformly spray an appropriate amount of susceptible bacteria onto colonies on a medium. If the amount of spray is insufficient, susceptible bacteria will not grow sufficiently even in the absence of antibiotics.

On the other hand, if the amount of spray is too large, colonies of test bacteria often float up and flow away. Since this method uses spraying, the susceptible bacteria used become aerosols and are dispersed into the air, contaminating the surrounding area.

This aerosol may be inhaled, which is undesirable from a sanitary standpoint. In the method of layering agar containing susceptible bacteria on test bacterial colonies and observing the growth status of susceptible bacteria, the agar is dissolved in water or buffer solution at high temperature and then heated to 50-55℃.
Cool and mix the susceptible bacteria. Since this agar suspension is poured in liquid form onto the test bacterial colony culture medium without solidifying, it is possible that the colony agar medium will partially dissolve due to the heat, causing the colonies in that area to rise and move. . Once the colony has migrated, even if an inhibition circle is observed on the upper medium, it is impossible to determine which colony is responsible for the inhibition circle. It is extremely difficult to pour the agar suspension onto the colonies without causing them to float, and requires a high degree of skill.

Furthermore, agar was poured into each sample and the temperature was 50 to 55 degrees.
It takes an extremely long time to cool to C and solidify.

Similarly, in the method of inoculating test bacteria into an agar medium containing susceptible bacteria, operations such as appropriately adjusting the temperature of the agar and mixing susceptible bacteria to prepare a medium are complicated and require a long time.

(Objective of the Invention) An object of the present invention is to provide a screening plate that allows antibiotic-producing bacteria to be detected easily, quickly, accurately, and hygienically with simple operations.

(Structure of the Invention) According to the present invention, antibiotic-producing bacteria can be easily detected by culturing a plate containing or coated with antibiotic-susceptible bacteria in close contact with a colony of test bacteria and observing the resulting inhibition circle. It was completed based on the inventor's knowledge. Therefore, (1) the screening plate of the present invention is a hydrophilic polymer layer having at least one of microbial cells and microbial spores; a molecular layer, whereby the above object is achieved.

The hydrophilic polymer layer of the screening plate of the present invention can retain microbial cells, microbial spores, nutrients, etc., and has the property of being able to elute them when it comes into contact with a medium such as agar.

Such a hydrophilic polymer is a compound that is water-containing and capable of forming a gel layer. Usually, a compound capable of supporting 90w8% or more of water is used. It is necessary that this hydrophilic polymer can be preserved while keeping the microorganisms contained therein alive. It is also desirable to have excellent transparency. Examples of such hydrophilic polymers include agar, sodium alginate, and
Examples include carrageenan, acrylamide, hydrophilic urethane, 7-da acrylate, polyvinyl alcohol, starch, and gelatin. Such compounds may be used after being crosslinked. In particular, agar, sodium alginate and carrageenan are preferably used.

Microorganisms contained in the hydrophilic polymer layer.

There are no particular limitations as long as the bacteria are sensitive to the desired antibiotic. Such susceptible bacteria may be microbial cells, microbial spores, or a mixture of both. Many of the bacteria that form spores are aerobic bacteria such as mold and Bacillus subtilis. Anaerobic bacteria such as Clostridium can also be used, but @
It is preferable to use aerobic bacteria since it is relatively difficult to maintain the pre-air condition. Susceptible bacteria usually contain 104 to 1 in the hydrophilic polymer layer.
07 pieces/m11. It is preferably contained at a rate of 105 to 106 pieces/mA. This number is such that when cultured in contact with a colony medium of the test bacteria, the bacteria grow uniformly throughout the medium and turn white.

If the number of susceptible bacteria is too small, viable bacteria will not spread uniformly throughout the medium, and the inhibition circle formed will be blurry and difficult to judge. Even if the number of susceptible bacteria is too large, it is difficult to determine whether the growth of susceptible bacteria is inhibited by antibiotics.

If the susceptible bacteria on the plate cannot grow on the growth medium of the test bacteria, it is necessary to pre-contain a nutrient source necessary for the growth of the susceptible bacteria on the plate. Such nutrient sources include, for example, peptone, meat extract, yeast extract, etc., depending on the type of susceptible bacteria and the type of culture medium for the test bacteria. These nutritional sources usually contain 0.1-5
．． It is added in a range of 0%.

The screening plate of the present invention usually has two types.

Although it is composed of a hydrophilic polymer layer, a support plate can be added to the component to support it if necessary. The support plate may be made of any material as long as it supports the hydrophilic polymer layer and maintains its shape until the polymer layer is brought into close contact with the colonies. Examples of materials for the support plate include polyurethane, polyamide, polycarbonate, acrylic resin, polypropylene, polyethylene terephthalate, and polyvinyl chloride. The thickness of the support plate is not particularly limited, but is usually about 0.5 to 1.0 nm. If the thickness of the support plate is too thin, it may function as a support plate, resulting in poor releasability from the hydrophilic polymer. If it is extremely thick, it will be inconvenient to handle. Even without the support plate, the function of the screening plate remains the same.

The screening plate of the present invention contains susceptible bacterial cells and spores of susceptible bacteria in the above-mentioned hydrophilic polymer solution.

It is prepared by adding a nutrient source and mixing to form a hydrophilic polymer layer. It goes without saying that the hydrophilic polymer solution must be sufficiently sterilized before being mixed with susceptible bacteria.

The hydrophilic polymer layer is usually formed to a certain thickness on a support plate, and its water content is 50% or more, preferably 70% or more.
% or more and 99.5% or less. A gel-like layer of hydrophilic polymer may be formed and a solution containing susceptible bacteria etc. may be applied to the surface. A gel-like layer of hydrophilic polymer may be immersed in a solution containing susceptible bacterial cells.

In a screening plate made of a hydrophilic polymer layer prepared by coating or dipping.

When this plate is placed in close contact with a colony of test bacteria, sufficient oxygen is not supplied to the susceptible bacteria in this close contact area. Therefore, if the susceptible bacteria are aerobic bacteria such as Bacillus subtilis or mold, it will be difficult for them to grow. If susceptible bacteria and their spores are dispersed and contained within the hydrophilic polymer layer, even if there is a lack of oxygen near the contact area with the culture medium, contact with the outside air of other hydrophilic polymer layers will be prevented. Susceptible bacteria that can receive air supply from the area can grow. therefore.

If the susceptible bacteria are aerobic, in order to prevent the susceptible bacteria from being unable to grow due to anaerobic conditions, apply the susceptible bacteria to the colony contacting side of the polymer layer, and apply a hydrophilic layer rather than f. It is preferable to mix it into the polymer layer.

The thickness of the hydrophilic polymer layer is sufficient as long as it can contain the necessary amount of susceptible bacteria and nutrients for growth.

Usually 1 to 5 dragons. The hydrophilic polymer layer may be in a hydrated state or in a dry state depending on the type and condition of susceptible bacteria. For example, when microbial cells such as Escherichia coli are used, the hydrophilic polymer layer is preferably in a water-containing state. When the hydrophilic polymer layer is in a water-containing state, if it also contains nutrients necessary for its growth, there is a risk that susceptible bacteria will grow before the plate is used for testing. It is necessary to use the product for a long time or to store it at a low temperature until use. When spores of microorganisms such as mold or Bacillus subtilis are used, the spores will not be killed even if the hydrophilic polymer layer is dried. In such cases, the plates can be dried and stored for long periods of time.

The above-mentioned screening plate is used after being cut into any shape and size. For example, it is cut to fit the size of a Petri dish for culture medium with a diameter of about 9 cm. The screening plate 1 has a hydrophilic polymer layer 4 and a support plate 6 for supporting the same, for example, as shown in FIG. As shown in FIG. 2, the screening plate 1 is stacked on top of the 70-knee 2 so that the hydrophilic polymer layer 4 is in contact with the culture medium 3 in which colonies 2 of the test bacteria have grown. If the susceptible bacteria are aerobic bacteria, the support plate 6 is peeled off and removed.

This is left at a predetermined temperature for a predetermined time to culture susceptible bacteria. The culture temperature and time are appropriately selected depending on the type of susceptible bacteria. Susceptible bacteria grow on the nutrient source in the plate or medium, spread throughout the medium, and appear white. When the test bacterium produces antibiotics, as shown in FIG. 3, a block of susceptible bacteria is observed around the test bacterium colony. In this way, antibiotic-producing bacteria can be detected with extremely simple operations.

If the susceptible bacteria are anaerobic bacteria, the support plate 6 does not need to be removed from the hydrophilic polymer layer 4. Support plate 6 in this case
If the plate is transparent, the resulting inhibition of susceptible bacteria can be observed through the support plate. The support plate may be opaque; in that case, the support plate is peeled off from the polymer layer with the hydrophilic polymer layer in close contact with the colony, or the inside of the block is exposed from the back side of the test bacterial colony growth medium. All you have to do is observe.

(Example) The present invention will be explained by examples.

3.0 g of agar powder was added to 100 ml of a 0.01M phosphate buffer solution with a pH of 7.0 and dissolved in a 100°C hot bath.

This was cooled to approximately 60°C, which is the temperature just before the agar solidifies, and 3 ml of Bacillus subtilis cell culture solution (5 x 10" spores/-l) that had been pure cultured in advance to form spores was added and mixed. This agar suspension was spread and gelled on a 200 μm thick polyester film to a thickness of 1 square inch.This was cut into a disk with a diameter of 85 m to obtain a screening plate.

Bacteria collected from soil were cultured on an agar medium to form colonies. The above screening plates were stacked so that the hydrophilic polymer layer was in close contact with the colonies.

The polyester film of the support plate was peeled off to allow air to be supplied to the bacterial cells, and the cells were left at 30° C. overnight. An inhibition zone where Bacillus subtilis does not grow was observed around the bacterial colony.

It was confirmed that the colony cells produced an antibiotic that has antibacterial activity against Bacillus subtilis.

The screening plate prepared in this example can be stored because it contains microbial spores.

When storing, take care to prevent microorganisms from growing during storage.
Preferably, no nutrients are included in the plate.

Step 1 Escherichia coli (E, coli) culture solution (50 mJ of physiological saline)
5 x 10 cells/mj 3 ml was added.

7.5g of acrylamide in the paste, N・N'' as a crosslinking agent
- 0.2 g of methylenebisacrylamide, 5% solution of β-dimethylaminopropionitrile as polymerization accelerator 5
m1. Then, 5 ml of a 2.5% solution of potassium belloxonisulfate (KzSzOa) as a polymerization initiator was added and mixed. This suspension was spread on a polypropylene film having a thickness of 200 μm so that the film had a thickness of 2 squares, and was left at 30° C. for 1 hour to obtain a sheet having a hydrophilic polymer layer. This sheet was cut out into a disc with a diameter of 85U to obtain a screening plate.

Using this screening plate, bacteria producing antibiotics having an antibacterial effect on E. coli were detected according to the method of Example 1.

In the screening plate prepared in this example, a nutrient source such as peptone, meat extract, yeast extract, etc. is added to the hydrophilic polymer layer as necessary.

(Effect of the invention) When using the screening plate of the present invention.

In this way, bacteria capable of producing a desired antibiotic can be detected quickly and accurately in a container with simple operations. Use of such a screening plate is preferable from a hygienic point of view, since bacteria susceptible to antibiotics will not be scattered around when antibiotic-producing bacteria are detected.

[Brief explanation of drawings]

FIG. 1 is a partial cross-sectional view of one embodiment of the screening plate of the present invention, FIG. 2 is a cross-sectional view showing the screening plate stacked on a medium in which test bacterial colonies have grown, and FIG. FIG. 2 is a plan view showing a state in which an inhibition circle is formed around a test bacterial colony by the screening plate. DESCRIPTION OF SYMBOLS 1... Screening plate, 2... Test bacterial colony, 3... Medium, 4... Hydrophilic polymer layer, 5...
... Blocking circle, 6... Support plate. Agent Patent Attorney Shusaku Yamamoto

Claims (1)

[Scope of Claims] 1. A hydrophilic polymer layer having at least one of microbial cells and microbial spores; or a hydrophilic polymer layer having at least one of microbial cells and spores and a nutrient source. Screening plate with 2. The plate according to claim 1, wherein the hydrophilic polymer layer is supported by a support plate.