JPH08280377A - Culturing apparatus - Google Patents

Abstract

PURPOSE: To provide a culturing apparatus having reservability, especially excellent in simplicity and culturing property in use, in a culturing apparatus of a microorganism. CONSTITUTION: In a microorganism culturing apparatus having a waterproof base material sheet 1, a waterproof and water vapor-impermeable cover sheet 2 to cover the base material sheet 1 and having a printed square grid pattern 3 and a layer of medium mixture at least on the base material sheet 1, one of the following three kinds of means is provided as a means for regulating the spread of a test solution to be inoculated to constant. (A) An outer frame layer 5 using a hydrophobic ink is placed on an inside face of the base material sheet 1 or the cover sheet 2, or on both inside faces. (B) A porous film-like filter 6 is placed on n inside face of the base material sheet 1 or the cover sheet 2. (C) An outer frame layer 5 is placed on an inside face of the base material sheet 1 or the cover sheet 2 and a porous film-like filter 6 having a shape corresponding to a region surrounded by the outer frame layer 5.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a culturing apparatus for microorganisms used for bacterial count inspection, and more particularly to a culturing apparatus which can be stored for a long period of time and is easy to use.

[0002]

2. Description of the Related Art As a method for measuring the number of microorganisms such as foods, the agar plate pour method has been conventionally used. In this method, a predetermined amount of agar medium, which had been sterilized by high-pressure steam and the temperature of which was about 45 ° C, and a separately prepared sample solution were each dispensed into a dish that had been sterilized in advance, and the mixture was allowed to pour well and left to stand. It is confirmed that the coagulation has been solidified, and the coagulation is put into an incubator or the like to culture, and after a predetermined time, the number of colonies that have developed in the petri dish is counted. However, in the case of this method, an autoclave for autoclaving the agar medium and an inspection room capable of performing a series of microbial tests aseptically are required, and the sampling and adjustment of the sample solution from the sampling of microorganisms is required. It is difficult for a beginner to accurately perform the operation of microbiological examination including pouring, pouring with a medium, culturing, and counting.

[0003] Therefore, as a result of research on a method for conducting a microbial test easily and without requiring a high degree of skill, a dry culture medium has been developed and used as one of the culture devices. Examples of such a culture device include Petri film plate [trade name of 3M] and BACcT [trade name of Shimakyu Pharmaceutical Co., Ltd.].

The former Petri film plate is composed of a waterproof flat plate and a cover film that overlaps with the flat plate. An adhesive is applied to the inner surfaces facing each other, and a cold water-soluble substance (gelling agent) is further applied. ) Powder and medium components or powder of cold water soluble substance (gelling agent) is sprayed, and then excess powder is shaken off to form a dry medium, which is further sterilized. It is a product. Then, the outline of the method of use is as follows. First, the cover film film laminated on the flat plate is lifted, and the test is performed on a dry medium composed of a cold water-soluble substance and a medium component formed on the flat plate via an adhesive layer. A predetermined amount of the liquid is inoculated, and after the inoculation, the cover film is lowered, and a plastic spreader is pressed on the cover film to spread the test liquid circularly and evenly to absorb the liquid. Since the gelling agent, which is a cold water-soluble substance, solidifies in about 1 minute, it is stored in the incubator as it is after being solidified, cultured at a predetermined temperature and time, and the number of generated colonies is counted. .

The latter BACcT has a waterproof flat plate and a transparent cover film that covers the flat plate, and the surface of the waterproof flat plate opposite to the cover film has a cold water-soluble gelling agent and a microbial culture medium. The mixture is coated, and the fibrous water-absorbent sheet is laminated on at least a part thereof, and the surface of the cover film opposite to the waterproof flat plate is a mixture of a cold water-soluble gelling agent and a microbial culture medium or cold water. It is a product obtained by coating a soluble gelling agent and sterilizing it. The method of using this BACcT is to lift a cover film covering a waterproof flat plate, and to test on a fibrous water-absorbent sheet laminated on a coating layer of a mixture of a cold water-soluble gelling agent and a microorganism culture medium on the waterproof flat plate. Inoculate a predetermined amount of liquid. As a result, the test liquid diffuses throughout the water-absorbent sheet due to the capillary phenomenon due to the overlapping of the fibers of the fibrous water-absorbent sheet, and the cold water-soluble gelling agent underneath absorbs water and swells and gels, and the microbial culture medium dissolves. . At this time, the fibers of the fibrous water absorbent sheet are wrapped in the gel or adhere to the gel. After that, the cover film is taken down, placed in an incubator, incubated at a predetermined temperature and for a certain period of time, and the number of colonies generated is counted.

[0006]

The culturing apparatus as described above is convenient in use because it is not necessary to prepare a Petri dish or a medium each time when culturing a microorganism. . However, in the case of the former Petri film plate, its method of use is to lift the cover film and place the test solution on a dry medium composed of a cold water-soluble substance and a medium component formed via an adhesive layer on the flat plate. After inoculating a predetermined amount and lowering the cover film, press down with a plastic spreader to spread the test liquid evenly, and leave the spreader on for about 1 minute to allow the cold water soluble substance to absorb water and gel. Is formed and at the same time the medium components are dissolved. Therefore, with one spreader, only one sample can be processed in one minute, and if the spreader shifts, the test liquid will spread further on the dry medium.

Further, in the case of BACcT, as described above, by dropping the test liquid onto the fibrous water-absorbent sheet on the waterproof flat plate side, the test liquid spreads throughout the sheet due to the capillary phenomenon between fibers, The swelling / gelling of the cold water-soluble gelling agent and the dissolution of the culture medium underneath it are performed. At this time, the fibers of the fibrous water absorbent sheet are wrapped in the gel or adhered to the gel. Therefore, although the spread area of the test liquid can be made substantially constant, the bacterial cells have entered the fibers of the fibrous water-absorbent sheet, and the growth of the cells proceeds in the sheet. For this reason,
The generated colonies are difficult to see, and there is a problem that counting becomes difficult, especially when bacterial cells grow close to each other.

The present invention has been made in view of the above problems, and an object of the present invention is that it has a preservative property as a culture device and the operation of the culture is simple, and particularly, the test liquid To provide a culturing apparatus for microorganisms, which can be spread uniformly over a certain area without using a special instrument during inoculation, can clearly discriminate the generated colonies, and has excellent culturing accuracy. is there.

[0009]

The above-mentioned problems can be solved by the present invention described below. That is, the invention of claim 1 has a waterproof base sheet and a cover sheet that covers the base sheet, and at least the layer of the culture medium mixture is provided on the base sheet. In the culture device, an outer frame layer made of hydrophobic ink is provided on the inner surface of the base material sheet that overlaps with the cover sheet, or on the inner surface of both the base material sheet and the cover sheet, in order to make the spread of the test liquid to be inoculated constant. It consists of a culture device characterized by the above.

The invention of claim 2 has a waterproof base sheet and a cover sheet covering the base sheet, and a microorganism mixture comprising a medium mixture layer provided on at least the base sheet. In the culturing device, a porous film filter for making the spread of the test liquid to be inoculated constant is provided on the inner surface of the overlapping base material sheets or the cover sheet.

The invention of claim 3 has a waterproof base sheet and a cover sheet covering the base sheet, and a layer of the medium mixture is provided on at least the base sheet. In an apparatus for culturing microorganisms, an outer frame layer made of a hydrophobic ink for uniformly spreading the test solution to be inoculated on the inner surface of the overlapping base sheet or cover sheet, and an outer frame inside the outer frame layer. And a porous film filter having a shape corresponding to a region surrounded by layers. In the present invention, the layer of the medium mixture is a layer comprising a water-absorbent resin, a nutrient component of one or more kinds of microorganisms, and a system in which a cold water-soluble substance (gelling agent) is further added, if necessary. Will be explained again in the example section.

[0012]

As described above, according to the first aspect of the present invention, the test liquid to be inoculated so that the inner surface of the base material sheet overlapping the cover sheet or both the inner surface of the base material sheet and the inner surface of the cover sheet are vertically aligned. An outer frame layer made of hydrophobic ink is provided to make the spread of the ink uniform. Therefore, at the time of culturing, the cover sheet is lifted, and a predetermined amount of the test liquid is dropped using a pipette or the like on the layer of the medium mixture surrounded by the outer frame layer made of the hydrophobic ink provided on the base material sheet, By simply lowering the cover sheet and pressing it lightly from above, the test liquid can be evenly spread over the entire area surrounded by the outer frame layer of the hydrophobic ink without using any special equipment, and the layer of the medium mixture can be used. Absorbs the water of the test liquid, swells and gels, and the nutrient components of the microorganism dissolve. As a result, since the microorganisms in the test liquid are uniformly dispersed and captured by the gel, they can be placed in an incubator as they are and cultured, and colonies that can be easily counted by culture can be formed.
The cover sheet has a function of preventing contamination by falling bacteria and the like during the culturing operation and a function of preventing evaporation of water such as gel.

Further, the invention of claim 2 is a culture device comprising a base material sheet and a cover sheet covering the base material sheet, and at least a layer of the medium mixture is provided on the base material sheet. The inner surface of the sheet or the inner surface of the cover sheet is provided with a porous film filter for making the spread of the test liquid to be inoculated constant. Here, when the porous film filter is provided on the inner surface of the substrate sheet, the cover sheet is lifted and a predetermined amount of the test liquid is dropped onto the porous film filter using a pipette or the like, and the cover is covered. By lowering the sheet and gently pressing from above, the test liquid is spread evenly over the entire area of the porous film filter, and water is absorbed by the lower medium mixture layer through the filter,
At the same time as forming the gel, the nutrient components of the microorganism are dissolved.
At this time, when a layer of the medium mixture is provided also on the cover sheet side, gel formation and dissolution of nutrient components are similarly performed on the filter. Then, by using a porous film-like filter having a pore size smaller than the size of the microorganisms dispersed in the test liquid, the microorganisms to be cultured are captured in a uniformly dispersed state on the entire surface of the filter. Therefore, colonies that can be easily counted can be formed by placing the culture device in an incubator and culturing as it is.

Further, when the porous film filter is provided on the inner surface of the cover sheet, the cover sheet is lifted up and the test liquid is dropped near the center of the medium mixture layer of the base sheet. After inoculation with the test solution, the cover sheet is lowered and gently pressed from above, so that the test solution is spread evenly over the entire area of the porous film filter, and The layer of the medium mixture absorbs water, swells and gels, and at the same time, the nutrient components dissolve. At this time, the microorganisms dispersed in the test liquid are
It is captured by the gel in a uniformly dispersed state. The thus prepared culture device can be placed in an incubator as it is and cultured, and colonies that can be easily counted can be formed as described above. In this case as well, the action of the cover sheet is similar to that described above.

The culturing apparatus according to the invention of claim 3 has a waterproof base sheet and a cover sheet covering the base sheet, and a layer of the medium mixture is provided on at least the base sheet. In an apparatus for culturing microorganisms, an outer frame layer made of hydrophobic ink is provided on the inner surface of the overlapping base material sheets or the cover sheet to make the spread of the test liquid to be inoculated constant, and the outer frame layer Inside, a porous film filter having a shape corresponding to the area surrounded by the outer frame layer is provided. In the culture device of this configuration, in the culture device of the present invention of claim 2, an outer frame layer made of a hydrophobic ink is added around the porous film filter provided on the inner surface of the substrate sheet or the cover sheet. The configuration is the same as that of the basic operation. However, by adding this outer frame layer, it becomes easier to spread the inoculated test liquid over the entire surface of the porous film filter, and as a result, the easiness of the culturing operation and the culturing accuracy are improved. Is what you get.

[0016]

The present invention will now be described in detail with reference to the drawings and examples. FIG. 1, FIG. 3 and FIG. 5 are schematic perspective views showing the configuration of an embodiment of the culture apparatus of the present invention. 2 is a cross-sectional view taken along the line AA showing the structure of the base material sheet portion of the culture device shown in FIG. 1, and FIG.
Is a cross-sectional view taken along the line BB of the culture device shown in FIG.
FIG. 6 is a sectional view taken along the line CC of the culture device shown in FIG.

The steps will be described in detail below. FIG.
Is formed by printing a grid print pattern 3 on the base material sheet 1 and providing a layer 4 of the medium mixture on it, and further by using a hydrophobic ink for making the spread of the test liquid to be inoculated on the layer 4 uniform. The outer frame layer 5 is provided to form the main body of the culture device, and the cover sheet 2 is joined to the end of the main part by an adhesive or the like. In addition, also on the inner surface of the cover sheet 2 (the surface on the side that overlaps with the base material sheet), a layer of the medium mixture or a layer of a water-absorbent resin,
An outer frame layer made of hydrophobic ink may be provided in that order, or only one of the three types of layers may be provided. When the outer frame layer made of the hydrophobic ink is also provided on the cover sheet 2 side, it is preferable that the outer frame layer 5 on the base sheet 1 is vertically aligned with the outer frame layer 5 on the base sheet 1 when the outer frame layer is placed on the cover sheet 2. Further, the cover sheet 2 can be used as a separate body without being joined to the base material sheet 1. However, it is preferable to bond them when inoculating the test solution and spreading it evenly in the outer frame layer, or from the viewpoint of preventing displacement or peeling in the subsequent culture process. 2 is a schematic cross-sectional view taken along the line AA showing the structure of the main body of the culture apparatus shown in FIG. 1, in which a grid printed pattern (not shown) is printed on the base material sheet 1, A layer 4 of the medium mixture is provided thereon, and an outer frame layer 5 made of a hydrophobic ink is further provided thereon. In this case, the outer frame layer 5 has a function of stopping the spread of the test liquid inoculated to the inside due to its hydrophobicity and thickness.

FIG. 3 is a schematic perspective view showing the constitution of another embodiment of the culture apparatus of the present invention, in which the grid printed pattern 3 is printed on the base material sheet 1 and the layer of the medium mixture is formed thereon. 4 is provided,
Furthermore, a porous film-like filter 6 for making the spread of the test liquid to be inoculated constant is provided thereon to form the main body of the culture device, and the cover sheet 2 is provided at the end thereof by bonding with an adhesive or the like. It has a different structure. In addition, in the case of this configuration, a layer of the medium mixture or a layer of the water absorbent resin may be provided on the inner surface of the cover sheet 2. In the case of the configuration shown in FIG. 3, the test liquid is inoculated on the porous film filter 6, the cover sheet 2 is lowered, and the cover film 2 is lightly pressed down, so that the entire surface of the porous film filter 6 is tested. The liquid can be spread. Further, the porous film filter 6 may be provided by being laminated on the inner surface on the cover sheet 2 side (the surface overlapping the base sheet), or may be simply sandwiched between the both. When the porous film filter 6 is sandwiched between the two, it is placed in the center of the medium mixture layer 4 of the main body at the time of use, and the test solution is inoculated on the cover sheet. It is possible to spread the test liquid on the entire upper surface of the porous film filter 6 by lowering 2 and gently pressing from above. In addition, the test solution is inoculated first on the layer 4 of the medium mixture of the main body,
Even if the porous film-like filter 6 is placed thereon, the test liquid can be spread over the entire lower surface of the porous film-like filter 6 by lowering the cover sheet 2 and lightly pressing it from above. Even in the above configuration, the cover sheet 2 does not have to be joined to the main body portion,
It may be used as a separate body. When the method of inoculating the test liquid on the porous film filter 6 is adopted, a layer of the medium mixture or a water absorbent resin layer may be provided on the inner surface of the cover sheet 2. In addition, FIG.
FIG. 3 is a schematic cross-sectional view taken along line BB showing the configuration of the main body of the culture device shown in FIG. It is provided with a porous film filter 6 thereon. In this case, the test liquid is inoculated on the porous film-like filter 6, the cover sheet 2 is lowered onto the porous film-like filter 6 and lightly pressed from above to spread the test liquid over the entire surface of the porous film-like filter. To be

FIG. 5 is a schematic perspective view showing the structure of still another embodiment of the culturing apparatus of the present invention, in which the grid printed pattern 3 is printed on the base material sheet 1 and the medium mixture is printed thereon. A layer 4 is provided, and an outer frame layer 5 made of a hydrophobic ink is further provided on the layer 4 to make the spread of the test liquid to be inoculated constant.
Inside the outer frame layer 5, a porous film filter 6 having a shape corresponding to the area surrounded by the outer frame layer 5 is provided to form a main body of the culture device, and the cover sheet 2 is bonded to the end thereof. It is joined by an agent. In the case of this configuration, the test solution is inoculated on the porous film filter 6, the cover sheet 2 is lowered, and the cover sheet 2 is lightly pressed so that the test solution is porous as in the case of FIG. Although it is spread on the entire upper surface of the high quality film-like filter 6, there is an outer frame layer 5 made of hydrophobic ink around it,
The spread of the test liquid can be more surely limited within this range. When the outer frame layer 5 and the porous film filter 6 made of the hydrophobic ink are provided on the base sheet side as described above, the medium mixture layer or the water-absorbing layer may be formed on the inner surface of the cover sheet 2 as necessary. A layer of resin can be provided. Further, in the case of the configuration of FIG. 5, the outer frame layer 5 and the porous film filter 6 made of the hydrophobic ink are
It may be provided on the cover sheet 2 side, in which case the test liquid is inoculated on the layer 4 of the medium mixture on the base material sheet 1, but the effect of making the spread of the test liquid constant is the same. Can be obtained. Further, the cover sheet 2 can also be used as a separate body in this case as in the case of FIGS. 1 and 3. 6 is a schematic cross-sectional view taken along line CC of the main body of the culture device shown in FIG. 5, in which a grid print pattern (not shown) is printed on the base material sheet 1, This is a structure in which a layer 4 of the medium mixture is provided on top of it, and an outer frame layer 5 made of a hydrophobic ink and a porous film filter 6 are further provided thereon.

The constituent materials of the culture apparatus of the present invention will be described below. First, the base material sheet 1 needs to have waterproofness, and for example, a plastic sheet of polyester, polyethylene, polypropylene, polystyrene, polycarbonate, polyvinyl chloride or the like can be used. When used as a culture device in combination with the cover sheet 2, at least one of them is preferably transparent in order to observe and count the grown colonies. Usually, since a transparent film is used for the cover sheet 2, the base material sheet 1 may be an uncolored transparent material, but a white colored material is preferable because it makes it easy to count the grown colonies. In addition to the plastic sheet, water-resistant paper can also be used. For example, paper coated with synthetic resin or extrusion-coated, or laminated with plastic film can be used.

The substrate sheet 1 is preferably flat without curling and the thickness thereof is not particularly limited, but is usually in the range of 50 to 500 μm, and 80 to 80 μm.
200 μm is more preferable. It is preferable to print the grid print pattern 3 on the base sheet 1 in advance with an ink that is insoluble in water and does not affect the growth of microorganisms, in order to facilitate the counting of colonies. The printing plate type is not particularly limited and may be any one, but gravure printing and the like are preferable because of the wide selection range of colorants, resins, solvents and the like. The size of the grid is 1c
About m square is appropriate.

The cover sheet 2 is waterproof and impermeable to water vapor, and at the same time, after culturing microorganisms, the cover sheet 2
It is preferable to be transparent because it is common to count colonies through, and for example, a plastic film such as polyester, polyethylene, polypropylene, polystyrene, polycarbonate, polyvinyl chloride, etc. as mentioned in the above-mentioned substrate sheet 1 is used. can do.
At the time of the culture test, the cover sheet 2 is lifted from the base material sheet 1 to drop the test liquid, so that the cover sheet 2 is preferably made of a material having a suitable flexibility and a thickness. Therefore, the thickness of the cover sheet 2 is preferably about 10 to 200 μm,
It is more preferably about 70 μm.

When the base material sheet 1 or the cover sheet 2 is provided with the layer 4 of the medium mixture or the layer of the water-absorbent resin by coating or the like, it is necessary to bond them appropriately. Pretreatment such as corona discharge treatment can be performed on the coated surface of the base sheet 1 or the cover sheet 2. In addition, it is preferable that the base sheet 1 and the cover sheet 2 have suitable gas permeability, mainly oxygen permeability, depending on the kind of microorganisms to be cultivated.

In the present invention, the layer 4 of the culture medium mixture provided on the base sheet 1 and the cover sheet 2 is, as described above, a water-absorbent resin, a nutritional component of one or more kinds of microorganisms, and optionally cold water. A layer made of a system to which a soluble substance (gelling agent) is added. For example, as a water-absorbent resin, it has good adhesion to the base sheet 1 and the cover sheet 2, and is a sample to be tested during culturing. It is preferable to use a resin capable of absorbing the water content of the liquid to form a gel suitable for culture and at the same time supplying the absorbed water to the microorganisms. Specifically, polyethylene oxide, electron beam cross-linking type polyacrylamide, carboxymethyl cellulose, etc. can be used. Among them, polyethylene oxide and electron beam cross-linking type polyacrylamide are preferable.

Examples of the nutrient components of the microorganisms include yeast extract, peptone, dipotassium hydrogen phosphate, glucose, lactose, sodium chloride and the like. Alternatively, more than one can be selected and mixed and used.

As the cold water-soluble substance (gelling agent), the above water-absorbent resin can be used, but as a function, it can quickly absorb the water of the test solution inoculated during the culture to form a gel. Preferred are carrageenan, guar gum, xantham gum, and mixtures thereof.

In addition, a dye capable of being metabolized by microorganisms can be added to the layer 4 of the medium mixture. Such dyes are metabolized by microorganisms that grow in the culture process,
Since the colonies are colored, it has the effect of making it very easy to count the number of colonies. Specific examples of such dyes include triphenyltetrazolium chloride (hereinafter referred to as T
TC), p-tolyl tetrazolium red, tetrazolium violet, petetryl tetrazolium blue and the like.

The method for providing the layer 4 of the medium mixture composed of the above materials on the base sheet 1 or the cover sheet 2 is not particularly limited, but it can be formed by the following method, for example. As a method by coating, the above water-absorbent resin and nutrient components of microorganisms, and, if necessary, a cold water-soluble substance, a dye is dissolved or suspended in water or an organic solvent,
After mixing and stirring at a predetermined ratio, the layer 4 of the culture medium mixture can be formed by applying by a known method such as roll coating, bar coating, air knife coating, comma coating, gravure coating, and heating and drying. For example, when the water-absorbent resin is polyethylene oxide, it can be applied as a toluene or ethyl alcohol solution in which other components are dispersed, and when electron beam cross-linking polyacrylamide is used, as an aqueous solution, Other components can be added to this and applied. In this case, after coating and drying, the resin is crosslinked by irradiating it with an electron beam of about 10 Mrad, so that the water of the test liquid is absorbed during the culture to form a good gel.

When it is desired to form the layer 4 of the medium mixture on the base sheet 1 by the above-mentioned method and the layer on the cover sheet 2 containing only the water-absorbing resin, only the solution of the water-absorbing resin is used on the cover sheet. 2 may be applied and dried alone, and if necessary, further irradiated with an electron beam to crosslink. Since electron beam irradiation also has a sterilizing effect, it is efficient because it can be used for both crosslinking and sterilization of the resin by performing this in the final step (for example, after packaging as a product). The coating amount of the layer 4 of the culture medium mixture or the layer of the water absorbent resin is preferably about ²⁰ to 200 g / m ^{2 in} terms of the coating amount when dried.
When the coating amount is 20 g / m ² or less, the ability of the test liquid to absorb water during culture is insufficient, which is not preferable. Further, when the coating amount is 200 g / m ² or more, there is no need for it, it takes time to dry, and the material is wasted. Depending on the drying capacity of the coating apparatus, particularly when an aqueous coating solution is used, when the coating cannot be completed by one coating, the coating can be performed in several times. The advantage of the coating method is that the coating amount can be stabilized, and the loss of material is small and the productivity is good.

In the second method, first, the water-absorbent resin and, if necessary, the dye are dissolved or suspended in an organic solvent or water at a predetermined ratio, and the solution is roll-coated, bar-coated, Air knife coat, comma coat,
It is applied by a known method such as gravure coating, and then, a mixture of a powdery microbial nutrient component and a powdery cold water-soluble substance in a predetermined ratio is uniformly sprayed on the undried coating film, and the After removing various powders with air or the like, the layer 4 of the medium mixture can be formed by drying. When a layer containing only the water-absorbent resin is provided, the solution of the water-absorbent resin may be applied by the same method as described above, and heated and dried as it is.
Also in this method, when electron beam cross-linking type polyacrylamide is used as the water-absorbing resin, it is irradiated with an electron beam of about 10 Mrad for both crosslinking and sterilization of the resin after drying and preferably packaging as a product. Is efficient. still,
The coating amount is as described above and will be omitted.

In the culturing apparatus of the present invention, the outer frame layer 5 made of a hydrophobic ink is provided in order to keep the spread area of the test liquid inoculated during the culture at a constant area surrounded by the outer frame layer 5. Since the test liquid is aqueous, a hydrophobic ink that can repel it is used. Therefore, as the ink used for the outer frame layer 5, most of the inks can be used unless they are water-soluble inks or hydrophilic inks. For example, polyurethane-based resins, polyamide-based resins, polyvinyl chloride-based resins, polyacrylate-based resins, polyester-based resins as binders, and polyolefin-based resins such as chlorinated polypropylene, and rubbers such as chlorinated rubber and cyclized rubber. The ink using the can be used. It is more preferable that these inks are water-repellent rather than simply hydrophobic, and from this point, the ink obtained by adding silicones or waxes to the above resins is further preferable. In addition, the ink used for the outer frame layer 5 is hydrophobic and water repellent, and at the same time, it is necessary to increase the thickness of the coating film. It is also effective to use as. The shape of the outer frame layer 5 formed using such an ink is not particularly limited and is free, but in order to spread the test liquid inoculated into the outer frame layer 5 uniformly in the frame, For example, a circular shape is easier to spread than a rectangular shape. Therefore, the outer frame layer 5 preferably has a circular inner shape, such as a donut shape. The size of the circle is generally about 5 cm in diameter. Moreover, the thickness (height) of the outer frame layer 5 is 5 to 500 μm.
A range of about m is preferable. When the thickness is 5 μm or less, it is not preferable because the test liquid tends to protrude from the outer frame layer 5 when the test liquid is spread, and when the thickness is 500 μm or more, the necessity is not so great, which is also disadvantageous in terms of manufacturing cost.

In the culture apparatus of the present invention, as another means for making the spread of the inoculated test liquid constant, it is provided on the medium mixture layer 4 of the base sheet 1 or on the inner surface of the cover sheet 2. Alternatively, the porous film filter 6 used by simply inserting it has a pore size of 0.20 to 0.45.
It is preferable to use a material that has many uniform and fine pores in the range of about μm and is easily wet with water. Specific examples of such a porous film filter 6 include known membrane filters, among which hydrophilic nitrocellulose type, cellulose acetate type, hydrophilic PTFE (polytetrafluoroethylene) type, etc. Membrane filters are preferred. The shape of the porous film filter 6 is preferably circular for the same reason as in the case of the outer frame layer 5, and the size thereof is generally about 5 cm in diameter. The thickness is usually 35 to 150 μm.
It is prepared stepwise within a range of about m, and any of them can be used. The porous film filter 6 is
The base sheet 1 may be provided so as to be fixed on the medium mixture layer 4 or on the inner surface side of the cover sheet 2, or may be simply inserted between the both. When fixing, when the substrate is a layer of medium mixture or a layer of water-absorbent resin,
A water-absorbent resin, a medium mixture, or a solution of a cold water-soluble substance can be used instead of the adhesive, and when the base is the base sheet or the cover sheet itself, the water-absorbent resin solution is used instead of the adhesive. Can be used. Also,
As shown in FIGS. 5 and 6, when the outer frame layer 5 made of hydrophobic ink and the porous film filter 6 are used together,
The thickness balance between the two is not particularly limited, but usually,
It is preferable that the thickness of the outer frame layer 5 is made slightly thicker than the thickness of the porous film-like filter 6 so as to prevent the test liquid from protruding when the test liquid is spread. It is preferable because it can be surely prevented and the spread area can be made constant.

Specific examples of the culture apparatus of the present invention will be given below to actually perform a culture test, and the performance thereof will be described in comparison with a pour method using a standard agar medium. [Example 1] A transparent biaxially stretched polyester film having a thickness of 100 µm (hereinafter referred to as PET film) [Lumirror (single-sided corona discharge treatment) manufactured by Toray Industries, Inc.] was used as a substrate sheet, and its corona discharge treated surface was used. After printing a grid pattern with a pitch of 10 mm by gravure printing, apply the layer mixture of the medium mixture adjusted to the following composition with a comma coater so that the dry coating amount will be 30 g / m ^2. Then, this was cut into a size of 10 × 10 cm square to prepare a sheet constituting a main body portion.

Composition of coating solution for medium mixture layer Water-absorbent resin 10% by weight toluene solution of polyethylene oxide [Aquacork Sumitomo Seika Chemicals, Ltd.] 1000 parts by weight Peptone 10 parts by weight yeast extract 5 parts by weight Glucose 2 parts by weight Dye TTC 0.1 parts by weight

Next, a PET film having a thickness of 25 μm [Lumirror (one-sided corona discharge treatment) manufactured by Toray Industries, Inc.] was used as a cover sheet, and polyethylene oxide [Aquacork] was used as a layer of a water-absorbent resin on the corona discharge-treated surface. Sumitomo Seika Chemicals Co., Ltd.] 10 wt% toluene solution was applied by a comma coater so that the dry coating amount would be 20 g / m ^2, and this was cut into a 10 × 10 cm square dimension to absorb water. A cover sheet having a layer of was prepared. In addition, a porous film-like filter for keeping the spread of the inoculated test solution constant is used as an attachment method, and a nitrocellulose-type membrane filter (ADV
A product having a thickness of 145 μm and a hole diameter of 0.45 μm was punched into a circular shape having a diameter of 48 mm to prepare for use. As described above, three sets were set as one set, five sets were prepared, and each set was sterilized by γ-rays to obtain the culture device of Example 1.

(Culture test) Preparation of bacterial solution : Escherichia coli was used as a strain and a liquid medium (NU
TRIENT BROTH) at 37 ° C. for 24 hours with shaking culture was diluted with sterile physiological saline to a bacterial count of 10 ² / ml to prepare a bacterial solution used for the culture test. 1 ml of each of the above bacterial solutions was inoculated with a sterilized pipette on the layer of the medium mixture of each substrate sheet of the 5 sets of the culture apparatus of Example 1, the membrane filter was placed thereon, and further on top of it. After placing the cover sheet with the surface provided with the layer of the water-absorbent resin facing downward, gently pressing the filter part from above to spread the test liquid over the entire filter part, and allowing it to stand for about 1 minute to form a gel The cells were placed in an incubator and incubated at 37 ° C for 48 hours.

Example 2 In the same manner as in Example 1, a base sheet provided with a layer of a grid printed pattern and a medium mixture and a cover sheet provided with a layer of a water absorbent resin were prepared. Next, in order to provide an outer frame layer of hydrophobic ink on the medium mixture layer of the base sheet, an inner diameter of 5 is obtained by screen printing.
Using a hydrophobic ink RAM (manufactured by Seiko Advance Co., Ltd.), a doughnut-shaped pattern of 0 mm and an outer diameter of 70 mm (width of the inked portion) was formed on the base sheet at a predetermined pitch (outer frame layer). Was printed to have a thickness of about 10 μm. Each of the substrate sheet provided with the layer of the printed pattern and the culture medium mixture prepared as described above, the outer frame layer formed of the hydrophobic ink thereon, and the cover sheet provided with the layer of the water-absorbent resin were 10 It was cut to a size of × 10 cm square.
The base sheet is cut so that the outer frame layer is located in the center. Each of the above base sheet and cover sheet,
That is, 5 sets were prepared and sterilized by γ-ray irradiation to obtain the culture device of Example 2.

(Cultivation test) On the layer of the medium mixture surrounded by the outer frame layer of the hydrophobic ink of each substrate sheet of the 5 sets of the culture apparatus of the above Example 2, the bacterial solution prepared in the above Example 1 was applied. Inoculate 1 ml of each with a sterilized pipette, overlay the cover sheet with the surface with the layer of water-absorbent resin on top, and press lightly from the top of the cover sheet to apply the test liquid to the entire inner surface of the outer frame layer. The mixture was spread and left to stand for about 1 minute to form a gel, which was then placed in an incubator and cultured at 37 ° C. for 48 hours.

[Embodiment 3] In the culture apparatus of Embodiment 3, the means for making the spread of the inoculated test liquid constant consisted of an outer frame layer made of a hydrophobic ink and a porous film filter provided inside the outer frame layer. First, the second embodiment is a combination type.
In the same manner as above, the base sheet was provided with a layer of the grid printed pattern and the medium mixture, and an outer frame layer of hydrophobic ink was provided thereon.
At this time, printing was repeated using a plate having the same pattern so that the outer frame layer had a thickness of about 40 μm.
A sheet of the main body portion was created by cutting into cm square dimensions. Also, the same cover sheet as in Example 2 was prepared. And as a porous film filter,
A hydrophilic PTFE type membrane filter (manufactured by ADVANTEC) having a pore size of 0.
2 μm and a thickness of 35 μm were punched out into a circle having a diameter of 48 mm. As described above, three sets were set as one set, five sets were prepared, and each set was sterilized by γ-ray irradiation to prepare the culture device of Example 3.

(Culture test) A hydrophilic PTFE type membrane filter was placed in each of the outer frame layers of the five sheets of the main body of Example 3 above, and the bacterial solution prepared in Example 1 was placed thereon. Inoculate 1 ml each with a sterile pipette,
Cover it with a cover sheet and press it lightly, spread the bacterial solution over the entire inner surface of the outer frame layer, and let it stand for about 1 minute to form a gel, put it in an incubator at 37 ° C for 48 hours. Cultured.

[Preparation of culture sample for comparison and culture test] Separately from the above-mentioned example, for comparison, 20 ml of standard agar medium prepared in a Petri dish was prepared by a conventional pour method using 1 ml of the above-mentioned bacterial solution.
A culture sample for comparison was prepared by pouring. Created 5 samples. This culture sample was placed in an incubator and cultured at 37 ° C. for 48 hours as in the example.

The number of colonies generated by the culture test of the above-mentioned Examples 1, 2 and 3 and the culture sample for comparison was counted, and each data and average value are shown in Table 1. (Below margin)

[0043]

[Table 1] Bacteria count measurement result (E. coli) [unit: pieces]

As is clear from the results of the culture tests shown in Table 1, the results of the culture tests using the culture devices of Examples 1, 2 and 3 of the present invention were carried out using the standard agar medium prepared by the conventional pour method. The results are almost the same as the results of the culture test, indicating that the culture device is easy to operate and is practical enough. When a membrane filter was used as in Example 3 and a bacterial solution was inoculated on the membrane filter, the bacteria could be stopped on the filter for culturing, and thus grown colonies could be clearly observed.

[0045]

As described in detail above, the culturing apparatus of the present invention has a waterproof base sheet and a cover sheet covering the waterproof base sheet, and the base sheet or the medium mixture layer provided on the cover sheet. As a good adhesion to the substrate sheet and the cover sheet, and absorb the water of the inoculated test liquid, to form a gel suitable for culture,
With a water-absorbent resin that can supply the water necessary for the growth of microorganisms,
A dry coating film of a mixture of a nutrient component for microorganisms, and, if necessary, a cold water-soluble substance that enhances water retention and gel formation and a dye capable of being metabolized by microorganisms, is further inoculated with the test liquid. As a means for making the spread area of the cells uniform, an outer frame layer made of hydrophobic ink, or a porous film filter, or a combination of both is provided, and the entire culture device is sterilized in advance. There is. Therefore, it has storability, and does not require an autoclave or a special instrument for spreading the test liquid to a certain area even when it is used, and it is possible to efficiently and accurately measure the number of bacteria and the like by extremely simple operation. An effect that a culture device can be provided is exhibited. Further, in the production of the culture device of the present invention, when a layer of the medium mixture or a layer of the water absorbent resin is provided on the base material sheet or the cover sheet, it is necessary to provide an adhesive (adhesive) layer as an intermediate layer. Resource saving,
It can be rationalized and has excellent productivity. In addition, it is possible to easily incinerate in terms of disposability after use, and it is possible to inexpensively provide a culture device excellent in usability and performance.

[Brief description of drawings]

FIG. 1 is a schematic perspective view showing the configuration of an embodiment of a culture device of the present invention.

FIG. 2 is a schematic cross-sectional view taken along line AA showing the configuration of the main body of the culture device shown in FIG.

FIG. 3 is a schematic perspective view showing the configuration of another embodiment of the culture apparatus of the present invention.

FIG. 4 is a schematic cross-sectional view taken along line BB showing the configuration of the main body of the culture device shown in FIG.

FIG. 5 is a schematic perspective view illustrating the configuration of still another embodiment of the culture device of the present invention.

FIG. 6 is a schematic cross-sectional view taken along line C-C showing the configuration of the main body of the culture device shown in FIG.

[Explanation of symbols]

 1 Base Material Sheet 2 Cover Sheet 3 Cell Print Pattern 4 Layer of Medium Mixture 5 Outer Frame Layer with Hydrophobic Ink 6 Porous Film Filter

Claims (3)

[Claims] 1. A cover for a microorganism culturing apparatus comprising a waterproof base sheet and a cover sheet which covers the base sheet, wherein a layer of a medium mixture is provided on at least the base sheet. An outer frame layer made of a hydrophobic ink is provided on the inner surface of the base material sheet that overlaps with the sheet, or on the inner surface of both the base material sheet and the cover sheet to make the spread of the test liquid to be inoculated constant. Culture device. 2. A microbial culture device comprising a waterproof base material sheet and a cover sheet covering the base material sheet, and a layer of a medium mixture provided on at least the base material sheet. A culture device, characterized in that a porous film filter is provided on the inner surface of a material sheet or a cover sheet to make the spread of the test liquid to be inoculated constant. 3. A microbial culturing apparatus comprising a waterproof base sheet and a cover sheet covering the base sheet, and a layer of a medium mixture provided on at least the base sheet. A material sheet or an inner surface of the cover sheet, an outer frame layer made of a hydrophobic ink for making the spread of the test liquid to be inoculated constant, and a region surrounded by the outer frame layer inside the outer frame layer. A culture device comprising a porous film filter having a corresponding shape.