JPH11113562A - Detection of microorganism, device for detecting microorganism and plate for detecting microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a rapid method for detecting microorganisms, by which a detecting examination of the microorganisms is carried out cheaply, rapidly and with high accuracy, and further to provide a detecting device. SOLUTION: This detecting method comprises gathering microorganisms in a culturing layer 5 formed on a light-transmissive and water-nonpermissive supporter 1, reacting the gathered microorganisms with a reagent for labeling the microorganisms in the culturing layer 5, irradiating the light on the culturing layer 5, converting the transmitted light or the reflected right of the irradiated light into an electric signal, and detecting the label based on the converted electric signal.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to water, raw materials, semi-finished products, and microorganisms used in the production and quality control processes of foods, cosmetics, pharmaceuticals, medical tools, industrial products, and the like. The present invention relates to a method and an apparatus for quick and accurate detection.

[0002]

2. Description of the Related Art Heretofore, in a microorganism test, a microorganism has been collected from a subject to be examined, cultured on an agar (agarose gel) and a plate medium containing components necessary for culture, and grown to a measurable size. It has been performed by a method of evaluating colonies with the naked eye (hereinafter, this method is referred to as a plate method). This plate method is the most widely used method at present because it is easy to operate and anyone can use it if there is a simple culture device.

However, according to this method, even if a microorganism having a relatively high growth rate, such as Escherichia coli or Bacillus subtilis, is colonized by a microorganism of interest on a plate medium until it becomes observable with the naked eye, It takes more than 48 hours, and more than 10 days for slower growing bacteria. In this way, the plate method takes a considerable amount of time to detect bacteria, so it is currently impossible to meet the need to know what bacteria are present and how much are present in the survey target as soon as possible. It is.

[0004] As a method of detecting microorganisms other than the plate method, 1) the microorganisms are filtered through a membrane filter, a fluorescent substance is incorporated into the body of the microorganism, and then the laser is irradiated to the microorganism to emit fluorescence emitted from the microorganism. A method of detecting the presence of a microorganism by detecting. 2) From a microorganism, adenosine triphosphate (AT
A method for detecting the presence of microorganisms by extracting P) and causing it to emit light chemically. However, it is not common at present and the penetration rate is low for 1) and 2) because of the large size of the apparatus and the high unit price per inspection.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a microorganism which has conventionally required a long time to obtain a result, or obtained a result in a short time, but has a low practicality due to a high unit price. Is to perform a low-cost, quick and accurate detection test.

[0006]

The above objects of the present invention have been attained by the following constitutions.

(1) A microorganism is collected in a culture layer provided on a light-permeable, water-impermeable support, and the collected microorganism is reacted with a reagent for labeling the microorganism in the culture layer. A method for detecting microorganisms, comprising irradiating the culture layer with light to convert transmitted light or reflected light into an electric signal using a solid-state imaging device, and detecting the label based on the electric signal.

(2) The method for detecting a microorganism according to the above (1), wherein after collecting the microorganism in a culture layer, a reagent for labeling the microorganism is added.

(3) The method for detecting a microorganism according to the above (1), wherein a reagent for labeling the microorganism is contained in the culture layer in advance.

(4) The method for detecting a microorganism according to the above (1), wherein the microorganism is collected in a culture layer containing a reagent for labeling the microorganism in advance, and then a reagent for labeling another microorganism is added. Method.

(5) The method as described in any one of (1), (2), (3) and (4) above, wherein the electric signal is an electric signal obtained by scanning a solid-state imaging device with respect to a culture layer. A method for detecting microorganisms.

(6) The culture layer before collecting the microorganisms is irradiated with light, the transmitted light or the reflected light is converted into an electric signal by a solid-state imaging device, and then the light is irradiated on the culture layer from which the microorganisms are collected. The method according to any one of claims 1 to 3, wherein the color tone or the gradation of the culture medium is corrected by performing a process of reducing an electric signal obtained from the culture layer before the microorganism collection from the signal.
The method for detecting a microorganism according to any one of 3, 4, or 5.

(7) A light source for irradiating a microorganism detection plate having a culture layer on a light-transmitting and insoluble support for collecting microorganisms, a transmitted light or a reflected light of the irradiated light. An organism detecting device, comprising: a solid-state imaging device that converts light into an electric signal.

(8) The microorganism detecting apparatus according to the above (7), wherein the light source is a white light or an ultraviolet light.

(9) The microorganism detection according to the above (7) or (8), further comprising a filter for passing only a light beam having a specific wavelength, and irradiating the light from the light source to the microorganism detection plate via the filter. apparatus.

(10) A plate for detecting microorganisms, comprising a culture layer on a light-permeable, water-insoluble support.

(11) The microorganism detecting plate as described in (10) above, wherein the culture layer has a thickness of 1 mm or less.

(12) The microorganism detection plate as described in (10) or (11) above, further comprising a film covering the entire surface of the culture layer.

The present inventor has studied the plate method and other alternative methods for detecting microorganisms which have been carried out so far in detail. (1) In order to perform the detection in a short time, the cultivation time is reduced. Must be shorter. (2) When the culture time is shortened, it is almost impossible to observe colonies with the naked eye. (3) It is difficult to detect a colony as a substance by short-term culture. (4) Specific problems such as the fact that the laser device itself is large and impractical are extracted.

In order to solve the above problems, as a result of intensive studies, the present invention aims to develop a method and an apparatus which can obtain a microorganism detection result with high accuracy in a short time and have a high price-performance ratio. Successful. Hereinafter, the present invention will be described in detail.

The method for detecting microorganisms according to claim 1 of the present invention is not particularly limited in the type of microorganism to be adapted, and is applicable to all microorganisms that grow on a medium and give rise to colonies. Can be done.

In the method for detecting a microorganism according to the second aspect of the present invention, first, a sample in which a target microorganism is considered to exist is used for detecting a microorganism provided with a culture layer containing nutrients suitable for the growth of the microorganism. Grow on plate (eg, FIG. 1). At that time, a reagent for labeling the microorganism is added to the microorganism detection plate. Depending on the type of the reagent or the microorganism, the labeling reagent may be added to the culture layer in advance, or may be immediately after the microorganism is sprayed on the microorganism detection plate or after the colony is formed by the microorganism. After the culture is completed, the microorganism detection plate is irradiated with light, and the transmitted or reflected light is converted into an electric signal by a solid-state imaging device. The obtained electric signal is transferred to an electronic computer as an image information file, and is recorded in a main storage device of the electronic computer or an auxiliary storage device such as a fixed disk device, a magneto-optical disk device, or a semiconductor storage device. Colonies are counted on an electronic computer. Thus, the microorganisms are counted.

The microorganism detecting plate according to the tenth aspect of the present invention has the structure shown in the sectional view of FIG. In this cross-sectional view, it is necessary to use a water-impermeable and highly transparent resin as a material constituting the water-impermeable support 1, especially polyethylene terephthalate (PE).
Resins such as T) and polyvinyl chloride are preferred. The culture layer 5 includes a water-absorbent layer 2 and a medium mixture layer 3. For the water-absorbing layer 2, it is effective to use a substance having an effect of adhering the respective layers so as to be sandwiched between the water-impermeable support 1 and the following medium mixture layer 3. Specifically, polyethylene oxide, cross-linked polyacrylamide and the like are preferable. In the medium mixture layer 3, it is necessary to use nutrients for culturing microorganisms and components for absorbing the moisture of the sample and forming a gel. Examples of microbial nutrients include peptone, dipotassium hydrogen phosphate, glucose, lactose, sodium chloride, potassium chloride, yeast extract, meat extract and the like. Carrageenan, guar gum, xanthan gum, or a mixture thereof can be used as a component for absorbing the moisture of the sample and forming a gel. Some of these nutrients can be selected according to the target microorganism, and can be used in combination with the gel-forming component. The water-absorbing layer 2 may or may not be provided depending on the measurement conditions, the measurement target, and the conditions of the medium mixture layer. In this case, the culture layer 5 is constituted only by the medium mixture layer 3, and has a form in which the medium mixture layer 3 is provided directly on the water-impermeable support 1.

The cover sheet 4 is a film that covers the entire culture layer of the microorganism detection plate,
It has a function of preventing dust from being mixed in and evaporating water in the culture layer. This cover sheet is also water-impermeable,
The material must be light-transmitting, and the constituent materials are preferably polyvinyl chloride, polyethylene, and polyvinylidene chloride.

[0025] In the method for detecting microorganisms according to claim 3 of the present invention, a sample in which the microorganism of interest is present is prepared by culturing a sample containing a nutrient suitable for the growth of the microorganism and a reagent for labeling the microorganism. On the plate for detecting microorganisms of the present invention provided with. After the culture is completed, the microorganism detection plate is irradiated with light, and the transmitted light is converted into an electric signal by a solid-state imaging device. The obtained electric signal is transferred to an electronic computer as an image information file, and is recorded in a main storage device of the electronic computer or an auxiliary storage device such as a fixed disk device, a magneto-optical disk device, or a semiconductor storage device. Colonies are counted on an electronic computer. Thus, the microorganisms are counted.

[0026] In the method for detecting microorganisms according to claim 4 of the present invention, a sample in which a microorganism of interest is present is prepared by culturing a sample containing a nutrient suitable for the growth of the microorganism and a reagent for labeling the microorganism. On the plate for detecting microorganisms of the present invention provided with. At that time, a reagent for labeling the microorganism is added to the microorganism detection plate. Depending on the type of the reagent and the microorganism, the timing of adding the labeling reagent may be immediately after the microorganism is sprayed on the microorganism detection plate or after the colony is formed by the microorganism. After the culture is completed, the microorganism detection plate is irradiated with light, and the transmitted light is converted into an electric signal by a solid-state imaging device. The obtained electrical signal is
The data is transferred to the computer as an image information file, and is recorded in the main storage device of the computer or in an auxiliary storage device such as a fixed disk device, a magneto-optical disk device, or a semiconductor storage device. Colonies are counted on an electronic computer. Thus, the microorganisms are counted.

The method for detecting microorganisms of the present invention will be described with reference to the schematic diagram of FIG. An installation table (not shown) for installing the microorganism detection plate 8 on the imaging device 10 that is sealed so that light does not enter during the detection operation, and irradiates the microorganism detection plate 8 installed on the installation table with light. And a solid-state imaging device 9 for converting transmitted light of the irradiated light into an electric signal. In addition, it is preferable that a filter 7 is provided between the light source 6 and the microorganism detection plate 8 for comparison. The electric signal obtained by the imaging operation is transmitted to the electronic computer 1
Sent to 1. According to a command from the input device 12, the obtained electric signal is converted and counted as the number of colonies. The obtained result is displayed on the display device 13.

In the method for detecting microorganisms according to claim 5 of the present invention, it is desirable that the imaging operation by the solid-state imaging device 9 be performed before and after culturing the microorganism. This is because a more accurate result can be obtained by comparing both pieces of image information. However, if it is difficult to perform an imaging operation before culturing, it may be omitted. The electric signal obtained by the imaging operation can be recorded as an image information file including the surface information of the culture layer using a semiconductor storage device, a magnetic recording device, a magneto-optical recording device, or the like.

In the method for detecting microorganisms according to claim 6 of the present invention, the image information after culturing and before culturing are compared,
By subtracting the information before culturing from the information after culturing, it is possible to remove noise that is not derived from microorganisms and occurs during culturing. The noise means an image on the culture layer that is not derived from microorganisms, such as a scratch or a stain unintentionally attached during the operation before and after the culture operation.

In the microorganism detecting apparatus according to claim 7 of the present invention, about 5 to 10 microorganism detecting plates can be installed on the mounting table of the microorganism detecting plate in the imaging device 10 of FIG. A reading operation is performed for each of the installed plates, and when one of the plates is read, the next plate for detecting microorganisms is automatically conveyed, and the image of the plate for detecting microorganisms can be taken.

Further, in the microorganism detecting apparatus according to the eighth aspect of the present invention, it is possible to use a light source that generates light in the ultraviolet region in addition to white light as the light source of the microorganism detecting apparatus. For example, when a reagent that emits fluorescence by ultraviolet light is used for labeling a microorganism, a light source of ultraviolet light is particularly effective. In the case of performing a color reaction due to the biological activity of microorganisms on the microorganism detection plate 8, the color is formed by installing a filter 7 that transmits only light of a specific wavelength between the light source 6 and the microorganism detection plate 8. The reaction can be detected more effectively. The change in hue by the filter 7 can be returned to the original hue in the image processing stage, and a hue close to an actual image can be obtained.

In practicing the method of the present invention, when the sample containing microorganisms is a liquid, it can be cultured by adding the sample directly onto a plate for detecting microorganisms. When a microorganism present on the solid surface is used as a sample, the microorganism is peeled off from the solid surface, or wiped off with a damp swab or the like,
It can be suspended in a liquid and cultured like a liquid sample. In addition, when testing microorganisms in the air, forcibly collect them using an air sampler, or moisten the culture layer with water, etc., and then capture the falling bacteria by leaving them to stand. It is also possible to culture as it is.

As a reagent for characteristically labeling a microorganism which can be used in the present invention, 2,3,5-triphenyl-2H-tetrazolium chloride (TT
C), (2- (2'-benzothiazolyl) -5-styryl-3- (4'-phthalhydridyl) tetrazolium chloride, 3, (4,5-dimethylthiazol-2-
Yl) -2,5-diphenyl-2H-tetrazolium bromide (MTT), 2,2 ', 5,5'-tetraphenyl-3,3'-(p-biphenylene) ditetrazolium chloride, p-nitroblue tetrazolium chloride , P-tolyltetrazolium red, o-tolyltetrazolium red, tetrazolium blue, tetrazolium red, tetrazolium violet, piperonyltetrazolium blue, etc. Reagents that stain colonies. A reagent capable of generating a fluorescent substance by the action of an enzyme possessed by a microorganism, such as 4-methylumbelliferyl-β-D-galacto, which is a sugar substrate to which 4-methylumbelliferone is bound. Reagents such as pyranosides; Trypan blue, which directly stains the surface or body of microorganisms,
4 ', 6-diamidino-2-phenylindole (DA
PI), acridine orange, rhodamine and the like. The reagents that can be used are not limited to those listed here.Reagents that can label only specific microorganisms, or reagents that specifically label microorganisms that meet certain conditions, depending on the type of microorganism to be detected Further, it is possible to select from reagents suitable for the purpose, such as a reagent capable of labeling microorganisms in general. According to a fourth aspect of the present invention, a reagent to be added to the culture layer and a reagent to be added later are added to a substance or substance resulting from the reaction of the microorganism with the reagent contained in the culture layer by a combination of the reagent to be added later. And detecting those reactions, for example, a method using an antigen-antibody reaction is also possible.

The cultivation can be performed by setting conditions such as temperature and humidity which are convenient for culturing the target microorganism. The apparatus used for culturing can realize conditions for the growth of the target microorganism, such as a commonly used closed thermostat, a thermostat with a program function, and a thermostat with a carbon dioxide gas concentration adjustment function. Any type of device can be used.

In the method of the present invention, the size of a colony formed by the growth of microorganisms is 0.01 mm to 0.2 mm, which is smaller than the size that can be distinguished by the naked eye.
Since the determination can be made at the time of reaching 1 mm, the culturing time is set at 5,000 to 10,000 for one target microorganism.
This is the time to multiply to about an individual. The actual cultivation time varies depending on the microorganism species and culture conditions (environment).
It is desirable to make appropriate settings according to the target microorganism species. For example, in the case of Escherichia coli having a high growth rate, the conventional plate method required about 48 hours for the colony to be confirmed by the naked eye, whereas the colony was detected within 6 hours by using the method of the present invention. Becomes possible.

A known method is used as a method for irradiating the reacted microorganism with light, converting the reflected light or transmitted light into an electric signal, and detecting the label. For example, among the obtained electric signals, a color of a predetermined specific wavelength for detecting a label of a bacterial colony is identified, and binarization is performed for each pixel with a predetermined threshold. Then, if necessary, the image is displayed with the marker portion highlighted. The color of the specific wavelength and the predetermined threshold may be determined based on the color and the degree of coloring determined by the reagent that labels the microorganism. In addition, a labeling process may be performed to count the number of bacterial colonies from the obtained binary image. For example, the image is scanned, and a pixel having a value of 1 (1-pixel) having no number assigned thereto is found, and a new number is assigned. Next, the same numbers are assigned to 1-pixels in the vicinity of 8 of this image. In addition, the ones near eight of them
-Assign the same number to the pixels. Repeat this process,
If there are no more pixels to be newly numbered in the neighborhood of 8, 1
All the connected components, that is, one colony, are given the same number. Scanning is continued again, and when an unnumbered 1-pixel is found, a new number is assigned and the same processing as above is performed. Thus, the number is detected by sequentially numbering all the colonies.

In the conventional method of observing with the naked eye and using a colony counter using a CCD camera, it was difficult to count the colonies unless the colonies were cultured up to about 0.5 mm to 1 mm. By using a sensor, it has become possible to dramatically improve the resolution of the obtained image information. CC
As for the D-line sensor, a high-resolution product can be obtained at a low price even at the present time. In addition, it is expected that it will be possible to further increase the resolution in the future, because it is technically advanced every day. In addition, the use of a dye that stains microorganisms characteristically makes it possible to detect a colony of a target microorganism even with a culture time in which colonies cannot be confirmed with the naked eye.

[0038]

The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 A polyethylene film having a thickness of 0.1 mm (hereinafter abbreviated as PET film) was used as a water-impermeable support. 10% polyacrylamide on PET, N, N'-methylene-b
is-acrylamide, N, N, N ', N'-tetramethylethylenediamine, 2,3,5-triphenyl-2
Applying a mixed aqueous solution of H-tetrazolium chloride,
A water-absorbing layer of a crosslinked polyacrylamide was formed.
Further, a mixed aqueous solution of peptone, glucose, meat extract, and guar gum was applied thereon, and dried at 100 ° C. to form a medium mixture layer. These water absorbing layers,
Two layers of the medium mixture layer were used as a culture layer, and this was used as a plate for detecting microorganisms.

Escherichia coli was used as the strain. The sample solution was diluted so that the number of Escherichia coli in the sample solution became 100, 500, and 1,000 / ml, thereby preparing a dilution series of Escherichia coli. Spray 0.5 ml of this sample solution onto the microorganism detection plate prepared above, cover with a cover sheet,
The cells were cultured in an incubator at 6 ° C. for 6 hours.

After the cultivation, the plate for detecting microorganisms was taken out and set in a microorganism detecting device, and the plate for detecting microorganisms was scanned by a solid-state imaging device. At this time, a white light was used as a light source. Based on the obtained images, the number of colonies was counted on an electronic computer. The results are shown in Table 1 together with the results of Example 2 and Comparative Example 1.

Example 2 A polyethylene film (hereinafter abbreviated as PET film) having a thickness of 0.1 mm was used as a water-impermeable support. 10% polyacrylamide on PET, N, N'-methylene-b
A mixed aqueous solution of is-acrylamide and N, N, N ', N'-tetramethylethylenediamine was applied to form a water-absorbing layer of cross-linked polyacrylamide. further,
A mixed aqueous solution of peptone, glucose, yeast extract, and xanthan gum was applied thereon and dried at 100 ° C. to form a medium mixture layer. Two layers, a water-absorbing layer and a medium mixture layer, were used as a culture layer, and this was used as a plate for detecting microorganisms.

0.5 ml of the sample solution prepared in Example 1 was sprayed on the plate for detecting microorganisms prepared above, covered with a cover sheet, and cultured in a 37 ° C. incubator for 6 hours.

After the completion of the culture, the cover sheet was peeled off, and a 0.1 M citrate buffer (pH 4.5) containing 1 mM 4-methylumbelliferyl-β-D-galactopyranoside was placed on the plate for detecting microorganisms. After the addition, the cover sheet was reattached, left in a thermostat at 25 ° C. for 15 minutes while shielding light. Thereafter, the plate for detecting microorganisms was taken out and set in a microorganism detecting device, and the plate for detecting microorganisms was scanned by a solid-state imaging device. At this time, the light source is 340 nm.
UV lamp was used. Based on the obtained images, the number of colonies was counted on an electronic computer. The results are shown in Table 1 together with the results of Example 1 and Comparative Example 1.

Comparative Example 1 A plate method which is a conventional method for detecting microbial colonies was used.
The number of bacteria was verified using the dilution series of Escherichia coli prepared in Example 1.

On a standard agar medium prepared in a Petri dish, 0.5 ml of a dilution series of E. coli was diluted to prepare a sample for comparison. This sample was cultured in an incubator at 37 ° C. for 48 hours, and the number of generated colonies was counted. The results are shown in Table 1 together with Examples 1 and 2.

[0047]

[Table 1]

From Table 1, it can be seen that the method of the present invention can reduce the culturing time to about 1/5 to 1/10 of the conventional plate method and can accurately count the number of colonies as compared with the conventional plate method.

Example 3 A polyethylene film having a thickness of 0.1 mm (hereinafter abbreviated as PET film) was used as a water-impermeable support. 10% polyacrylamide on PET, N, N'-methylene-b
A mixed aqueous solution of is-acrylamide and N, N, N ', N'-tetramethylethylenediamine was applied to form a water-absorbing layer of cross-linked polyacrylamide. further,
A mixed aqueous solution of peptone, glucose, yeast extract, and xanthan gum was applied thereon and dried at 100 ° C. to form a medium mixture layer. Two layers, a water-absorbing layer and a medium mixture layer, were used as a culture layer, and this was used as a plate for detecting microorganisms.

Legionella was used as the strain. The number of Legionella bacteria in the sample solution is 100, 500, 1.00
After dilution to 0 cells / ml, a dilution series of Legionella was prepared. A 0.5-fold dilution of the anti-Legionella mouse monoclonal antibody (IC
After adding 0.1 ml of N, 699551) and stirring,
The mixture was sprayed on the plate for detecting microorganisms prepared as described above, covered with a cover sheet, and cultured in an incubator at 37 ° C. for 18 hours.

After the completion of the culture, the cover sheet was peeled off, and the sample solution on the microorganism detection plate was removed. The plate for detecting microorganisms was lightly washed twice with PSB, and then the anti-mouse IgG-labeled FITC was labeled on the plate for detecting microorganisms.
The sheep antibody was added and left in a thermostat at 37 ° C. for 30 minutes. Then, the sample solution on the microorganism detection plate was removed, and the plate was lightly washed twice with PBS, then set on the microorganism detection device, and the microorganism detection plate was scanned by the solid-state imaging device. At this time, a white light was used as a light source. Based on the obtained images, the number of colonies was counted on an electronic computer.
The results are shown in Table 2 together with the results of Example 4 and Comparative Example 2.

Example 4 A polyethylene film having a thickness of 0.1 mm (hereinafter abbreviated as PET film) was used as a water-impermeable support. By applying a mixed aqueous solution of peptone, glucose, yeast extract and xanthan gum on PET and drying at 100 ° C,
A medium mixture layer was formed. Only one layer of the culture mixture layer was formed as a culture layer, and this was used as a plate for detecting microorganisms.

In 0.5 ml of the sample solution prepared in Example 3,
0.1 ml of a 100-fold diluted anti-Legionella mouse monoclonal antibody (699551, manufactured by ICN) was added, and the mixture was stirred. The mixture was sprayed on the microorganism detection plate prepared above, covered with a cover sheet, and covered with a cover sheet at 37 ° C. For 18 hours.

After completion of the culture, the cover sheet was peeled off, and the sample solution on the microorganism detection plate was removed. The plate for detecting microorganisms was lightly washed twice with PSB, and then the anti-mouse IgG-labeled FITC was labeled on the plate for detecting microorganisms.
The sheep antibody was added and left in a thermostat at 37 ° C. for 30 minutes. Then, the sample solution on the microorganism detection plate was removed, and the plate was lightly washed twice with PBS, then set on the microorganism detection device, and the microorganism detection plate was scanned by the solid-state imaging device. At this time, a white light was used as a light source. Based on the obtained images, the number of colonies was counted on an electronic computer.
The results are shown in Table 2 together with the results of Example 3 and Comparative Example 2.

Comparative Example 2 Using a plate method which is a conventional method for detecting microbial colonies,
The number of bacteria was verified using the dilution series of Legionella bacteria prepared in Example 3.

On a standard agar medium prepared in a Petri dish, 0.5 ml of a dilution series of Legionella was diluted and a sample for comparison was prepared. This sample was cultured in a 37 ° C. incubator for 7 days, and the number of generated colonies was counted. The results are shown in Table 2 together with the other Examples 3 and 4.

[0057]

[Table 2]

As is evident from Table 2, the method of the present invention can reduce the culturing time by 1/5 to 1/1 compared to the conventional plate method.
It can be seen that the number of colonies can be reduced to about 10 and the number of colonies can be counted accurately.

[0059]

According to the conventional plate method, it takes about 48 hours to visually confirm colonies of microorganisms, and 5 to 10 days for long ones. For example, Escherichia coli and Salmonella used for the Ames test require 48 hours for cultivation to confirm colonies,
Legionella requires a culture period of 7 days.

By using the method and apparatus of the present invention, the culturing time can be reduced to about 1/5 to 1/10 of the conventional one, and the number of colonies can be counted accurately. The number was almost the same level as the number of colonies visually counted by the conventional plate method. According to the present invention, it has become possible to greatly reduce the time required for counting the colonies of the target microorganism.

[Brief description of the drawings]

FIG. 1 is a cross-sectional view of a plate for detecting microorganisms of the present invention.

FIG. 2 is a schematic diagram illustrating a configuration of a detection device according to the present invention.

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 water-impermeable support 2 water-absorbent layer 3 medium mixture layer 4 cover sheet 5 culture layer 6 light source 7 filter 8 microorganism detection plate 9 solid-state image sensor 11 electronic computer 12 input device 13 display device

Claims (12)

[Claims] A microorganism is collected in a culture layer provided on a light-permeable, water-impermeable support, and the collected microorganism is reacted with a reagent for labeling the microorganism in the culture layer.
A method for detecting microorganisms, comprising irradiating the culture layer with light to convert transmitted light or reflected light into an electric signal using a solid-state imaging device, and detecting the label based on the electric signal. 2. The method according to claim 1, wherein after collecting the microorganism in the culture layer, a reagent for labeling the microorganism is added.
The method for detecting microorganisms according to the above. 3. The method for detecting a microorganism according to claim 1, wherein a reagent for labeling the microorganism is contained in the culture layer in advance. 4. The method for detecting microorganisms according to claim 1, wherein the microorganisms are collected in a culture layer containing a reagent for labeling microorganisms in advance, and then a reagent for labeling another microorganism is added. Method. 5. The electric signal according to claim 1, wherein the electric signal is an electric signal obtained by scanning a solid-state imaging device with respect to a culture layer. A method for detecting microorganisms. 6. A method for irradiating a culture layer before collecting microorganisms with light, converting transmitted light or reflected light into an electric signal by a solid-state imaging device,
Thereafter, from the electric signal obtained by irradiating the culture layer from which the microorganisms were collected with light, the color tone or gradation of the culture medium is corrected by performing a process of subtracting the electric signal obtained from the culture layer before the microorganism collection. Claims 1, 2, 3, and 4
Or the method for detecting a microorganism according to any one of 5. 7. A light source for irradiating light, a transmitted light or a reflected light of the irradiated light, to a microorganism detection plate having a culture layer on a light-permeable and insoluble support for collecting microorganisms. An organism detection device having a solid-state imaging device that converts the signal into an electric signal. 8. The microorganism detecting device according to claim 7, wherein the light source is a white light or an ultraviolet light. 9. The microorganism detection method according to claim 7, further comprising a filter that passes only a light beam having a specific wavelength, and irradiating the microorganism detection plate with light from a light source through the filter. apparatus. 10. A plate for detecting microorganisms, comprising a culture layer on a light-permeable, water-insoluble support. 11. The plate for detecting microorganisms according to claim 10, wherein the thickness of the culture layer is 1 mm or less. 12. The plate for detecting microorganisms according to claim 10, further comprising a film covering the entire surface of the culture layer.