JPH07147997A - Inspection of microorganisms and inspection device therefor - Google Patents

Abstract

PURPOSE:To provide an inspection process for microorganisms which can provide colony counts of high exactness in a shortened culture time. CONSTITUTION:Agar plate culture medium inoculated with a specimen is cultured for a specific time, then a non-fluorescent substance 16 which produces a fluorescent substance by reaction with an enzyme born by the tested microorganism is allowed to disperse on the surface of the agar plate medium 12. Then, the surface is irradiated with light to count the spots emitting the fluorescence.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an inspection method for speeding up a microorganism inspection for colony counting using a plate medium such as agar and an inspection apparatus used in the inspection method.

[0002]

2. Description of the Related Art From the viewpoint of hygiene control, it is important to carry out a microbiological test for checking whether harmful microbes are mixed in foods and cosmetics. Microbial inspection method by the conventional colony counting method, the sample prepared with sterile water is spread on the agar plate medium, this is incubated at a predetermined temperature for a predetermined time, and the number of bacterial groups that have proliferated to be visible is counted. Therefore, the presence and number of harmful microorganisms mixed in the sample are inspected.

In such an inspection method, it takes a culture time for the bacterial groups to grow and grow before the inspection results can be obtained. Even fast growing coliforms,
Cultivation for about 24 hours is required for the growth to be visible, and for fungi, it is required for 5 days or more.

In food manufacturers and the like, strict hygiene control of shipped products is necessary, and microbiological inspection is indispensable. However, for products in which freshness is important, they must be shipped before the inspection results are obtained. The situation is unavoidable.

Therefore, there has been proposed a method for conducting a microbiological test more quickly using a fluorescent substrate. As an example, diffuse the sample into a selective medium containing desoxycholic acid,
This is grown under a culture condition of 37 ° C. and the bacteria to be inspected are cultured while suppressing the growth of bacteria other than the inspected substance, and a specific enzyme (β-galactosidase (β-Gal in the case of E. coli).
to produce an actosidase)). Non-fluorescent substance (4-methylumbelliferyl galactoside) for this enzyme
Fluorescent substance (4 methyl umbelliferone)
Occurs. The amount of the fluorescent substance converted is proportional to the amount of the enzyme possessed by the bacterium, that is, the amount of the bacterium. Therefore, if the fluorescence intensity and the result of the conventional microorganism inspection method based on the colony count are associated in advance, the number of bacterial groups can be estimated from the fluorescence intensity, and the presence or absence and the number of harmful microorganisms can be inspected.

According to such a method, it is sufficient to culture the bacteria to such an extent that the fluorescence intensity can be detected, and the culture time can be greatly shortened as compared with the colony counting method by visual observation. Approximately 1/3), quick inspection is possible.

[0007]

In the inspection method for measuring the fluorescence intensity and estimating the number of bacterial groups, the inspection can be performed quickly, but the inspection accuracy is unstable. This is because the amount of enzyme produced varies depending on the culture conditions of the bacterium, and there is a high possibility that the test results will fluctuate. In addition, it is an indirect test that estimates the number of bacterial groups from the fluorescence intensity, and is less accurate than the colony counting method that directly counts the number of bacterial groups.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a microorganism inspection method and a microorganism inspection apparatus which can perform an inspection rapidly and have the accuracy of a colony counting method.

[0009]

In order to achieve such an object, the method for microbial examination of the present invention is to spread a sample prepared with sterile water on a plate medium and culture the medium containing the sample for a predetermined time. Then, in this cultured sample-containing medium, a non-fluorescent substance that produces a fluorescent substance in response to an enzyme of the microorganism to be tested is diffused, and the surface of the plate medium is irradiated with light to determine the number of places that emit fluorescence. The number of test microorganisms contained in the sample is measured.

Then, the surface of the plate culture medium is virtually divided into a large number of matrix-like compartments, and fluorescence detecting means for detecting the presence or absence of fluorescence in each of these compartments, and a detection signal of the fluorescence detecting means are provided in the compartments. And a display means for displaying the detection signal, the storage means for storing the detection signal and the display means for storing the detection signal are provided, and the test included in the specimen is determined from the number of the sections or section groups in which fluorescence is displayed on the display means. The number of target microorganisms may be measured.

Further, the microorganism inspection apparatus used in the above-mentioned microorganism inspection method comprises an irradiation means for irradiating the surface of the plate medium with light, a fluorescence detection means for detecting the presence or absence of fluorescence emitted according to the irradiation of the light, and the fluorescence. Moving means for relatively moving the detecting means and the plate medium to the surface of the plate medium while keeping the fluorescence detecting means along, and a large number of sections virtually dividing the surface of the plate medium into a matrix by the relative movement. Storage means for storing the detection signal of the fluorescence detection means corresponding to the address of the section, and display means for displaying the detection signal stored in the storage means.

Further, the irradiation means irradiates the strip-shaped light with a narrow width so as to straddle the surface of the plate medium, and the light receiving part of the fluorescence detecting means is opposed to the irradiation position of the strip-shaped light so that the plate is irradiated. A large number are arranged so as to straddle the culture medium, and the moving means relatively moves the flat plate culture medium in a direction orthogonal to the length direction of the band-shaped light with respect to the irradiation means and the arranged light receiving parts, and this relative The fluorescence detecting means may be configured to periodically sample the presence or absence of fluorescence according to the movement.

[0013]

[Operation] The non-fluorescent substance is diffused into the microorganism to be inspected that has been cultivated in a plate medium, and the fluorescent substance is generated by the reaction with the enzyme produced by the microorganism. The place where fluorescence is emitted is the place where there are bacterial groups, and the inspection accuracy similar to the conventional colony counting method can be obtained. Moreover, it is sufficient that the bacterial group is grown and grown to such an extent that fluorescence can be detected, the culture time can be shortened as compared with the conventional colony counting method, and rapid inspection is possible.

If the surface of the plate culture medium is virtually divided into a large number of matrix-like sections and the presence or absence of fluorescence is detected by the fluorescence detecting means, and these detection signals are stored and displayed on the display means, A weak fluorescence emission that can be detected by the fluorescence detection means is sufficient, and the culture time can be shortened accordingly. Also, if the display means is displayed in an enlarged manner than the plate medium, the number of bacterial groups can be easily counted. Furthermore, it is possible to reproduce the past detection signal by reading the detection signal stored in the storage means at a later date and displaying it.

Further, the fluorescence detection means for detecting the presence or absence of fluorescence and the plate medium in which the microorganism to be inspected is cultivated are relatively moved to sample the detection signals in a large number of sections in which the surface of the plate medium is divided into a matrix. If these detection signals are stored in the storage means in correspondence with the addresses of the compartments, the number of compartments for which the presence or absence of fluorescence should be detected by the fluorescence detecting means at one time may be less than all the compartments of the plate medium. The detection device can be manufactured inexpensively.

Further, the irradiation means irradiates the strip-shaped light with a narrow width so as to straddle the surface of the plate culture medium, and the light receiving part of the fluorescence detection means is arranged so as to face the strip-shaped light irradiation position and straddle the plate culture medium. If a large number are arranged and relatively moved in the direction orthogonal to the length direction of the band-shaped light, the detection signal is sampled according to this relative movement, so that the rows by the arrangement of the light receiving units and the sampling by the relative movement of once are performed. All the detection signals of each section obtained by dividing the surface of the plate medium in a matrix by rows are obtained.

[0017]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS First, the basic principle of the microorganism testing method of the present invention will be described with reference to FIG. FIG. 1 is a schematic process diagram of the inspection method of the present invention.

An example of detection of coliforms will be described with reference to FIG. An agar plate medium 12 containing desoxycholic acid is provided on a Petri dish 10, and a predetermined amount of a specimen 14 prepared with sterile water is dropped and diffused on the surface of the selective medium. Then, this is grown under the culture condition of 37 ° C. to grow the bacterial group up to a predetermined amount. The selection medium is used to grow Escherichia coli while suppressing the growth of bacteria other than E. coli.
Furthermore, on the surface of the agar plate medium 12, the non-fluorescent substance 16
(4 Methyl umbelliferyl galactoside) is dropped and diffused to react with the enzyme (β-galactosidase) possessed by the grown bacterial group 18 to generate a fluorescent substance (4 methyl umbelliferone). Further, light having a wavelength of, for example, 360 nm, at which the fluorescent substance is most excited, is emitted from a light source such as the xenon lamp 20 through the lens 22 and the first filter 24. Further, the fluorescence emitted at the wavelength of, for example, 460 nm generated by the excitation of the fluorescent substance is selectively transmitted by the second filter 26, and the number of places where fluorescence is generated is visually counted.

In the method for inspecting microorganisms according to the present invention, the conventional colony counting method was used to grow bacteria until they could be visually confirmed. The number of bacterial groups consisting of the above bacteria can be counted. Moreover, it is only necessary to be able to determine the presence or absence of the site where fluorescence is generated, and since the inspection accuracy is not affected by the fluorescence intensity, there is no change in the inspection results due to slight differences in culture conditions. Therefore, in the microorganism inspection method of the present invention, only the conventional colony counting method and the means for confirming the presence or absence of bacterial groups are different,
The inspection accuracy is the same and the reliability is high.
And it is not necessary to grow until the bacteria can be seen,
The higher the sensitivity of the non-fluorescent substance 16, the shorter the culture time can be. Non-fluorescent substances for testing coliforms 16
Is currently marketed by Ajinomoto Co., Inc. under the trade name of "Biorapid E". When this is used, the culture time is about one-third of that of the conventional one and the accuracy is sufficiently high. Good test results have been obtained, and rapid microbiological tests are possible. As the non-fluorescent substance 16 for this microbe test, in addition to the above-mentioned products for coliform bacteria, fungi,
Commercially available bacteria, Escherichia coli in foods containing lactic acid bacteria, and the like are commercially available from Ajinomoto Co., Inc. as "Biorapid" series.

Next, one embodiment of the microorganism inspection apparatus used for carrying out the above-described microorganism inspection method of the present invention will be described with reference to FIG.
And it demonstrates with reference to FIG. FIG. 2 is a schematic diagram of the structure of the microbe inspection apparatus, FIG. 3 is a diagram for explaining the operation of the microbe inspection apparatus of FIG. 2, and (a) is a virtual matrix of the surface of the agar plate medium. FIG. 2 is a simplified illustration of an array of divided sections and a light receiving section of the fluorescence detecting means,
(B) shows an example of the screen displayed on the display means according to the detection signal.

In FIG. 2, a Petri dish 10 in which the prepared sample is diffused on the surface of the agar plate medium 12 and further cultured on a moving means 30 such as a belt conveyor, and the non-fluorescent substance 16 is dropped and diffused on the petri dish 10. It is installed and moved.

Toward the moving petri dish 10,
A narrow band-shaped light having a width orthogonal to the moving direction of the moving means 30 is emitted from the emitting means 32. The irradiation means 32 applies light from a light source such as the xenon lamp 20 to the first filter 24 via the lens 22. For example, the wavelength of 360 nm at which the fluorescent substance produced by the reaction with the enzyme is most excited is selected and the glass is selected. The light enters the light guide tube 34 such as a fiber. The other end of the light guide tube 34 is arranged as an example so as to be orthogonal to the moving direction of the moving means 30, and the light emitted from the light guide tube 34 is agar of the petri dish 10 via the first lens array 36. Irradiation is performed so that the surface of the plate medium 12 is the focal point and straddles the surface.

Further, the moving means 30 is moved toward the focal point of the surface of the agar plate medium 12 irradiated with the light from the irradiation means 32.
The fluorescence detecting means 38 is arranged orthogonal to the moving direction of the.
The fluorescence detection means 38 is provided with a second lens array 40 directed toward the focal point of the irradiation means 32, and the other end of the second lens array 40 is of, for example, 460 nm through the second filters 26, 26, ... C with high sensitivity to light
Connected to a light receiving portion 42 such as a CD one-dimensional image sensor,
The fluorescence received by the second lens array 40 is given to the light receiving section 42. In this light receiving unit, for example, about 4100 pixels are arranged in a length of 9 cm that straddles the Petri dish 10.

The detection signal of the light receiving portion 42 of the fluorescence detecting means 38 is given to the binary conversion circuit group 46 via the signal output line 44, and converted into a binary signal by comparison with a predetermined threshold value. Then, these binary signals are transferred to the latch circuit group 4
8 and is latched according to an appropriate sampling signal. The sampling period and the moving speed of the moving means 30 are related to each other, and are set so that the Petri dish 10 having a diameter of 9 cm can perform sampling, for example, 4100 times. Therefore, by the rows of the pixel array of the light receiving unit 42 and the columns of the sampling by the movement of the Petri dish 10,
The surface of the agar plate medium 12 is virtually divided into a large number of matrix-shaped sections, and a detection signal is obtained for each section.

Then, the binary signal latched by the latch circuit group 48 is appropriately stored in the storage means 50 by using the pixel array number s _n of the light receiving section 42 and the sampling period number t _n as addresses. Further, when the petri dish 10 completes the passage under the fluorescence detecting means 38, the stored data is read from the storage means 50 and given to the display means 52 to be appropriately enlarged and displayed. As shown in FIG. 3 (a),
Bacteria group 18,18 on agar plate medium 12 of Petri dish 10
.. is present, the display means 52 is shown in FIG.
It is displayed as shown in (b). This display means 52
By counting the number of bacterial groups displayed in, the same colony count as in the past can be easily obtained.

In the apparatus used in the above-mentioned microorganism inspection method, the petri dish 10 is moved relative to the fluorescence detection means 38 by the moving means 30, but the invention is not limited to this, and the petri dish 10 is kept fixed. The fluorescence detecting means 38 may be moved. Further, the fluorescence detecting means 38 may be provided so as to cover the entire Petri dish 10 so that the detection signals of all the sections can be obtained at one time. Further, the fluorescence detecting means 38 is configured with a small number of pixels in the light receiving portion. Then, the Petri dish 10 and the fluorescence detecting means 38 may be relatively two-dimensionally moved so that the detection signals of all the sections can be obtained.

Although the agar plate medium 12 is used for the description, the present invention is not limited to this, and needless to say, it is not limited to agar if the bacteria to be inspected are selectively cultured. The non-fluorescent substance used in the present invention is not limited to the above-mentioned products, and may be any substance as long as it reacts with the enzyme contained in the bacterium to produce a fluorescent substance.

[0028]

As described above, in the method for testing a microorganism of the present invention, a non-fluorescent substance is reacted with an enzyme of a bacterium to be tested to produce a fluorescent substance, and the fluorescent substance is irradiated with light. The fluorescent light that is emitted makes it possible to recognize the bacterial group, if any, and the inspection accuracy similar to that of the conventional colony counting method can be obtained. Moreover, unlike the conventional colony counting method, it is not necessary to grow the bacterial group until the bacterial group can be directly visually recognized, and the examination can be speeded up because the culture time is short.

If the presence or absence of fluorescence is discriminated by the fluorescence detecting means and the detection signal is displayed on the display, even weak fluorescence can be detected with high accuracy, and the culture time can be shortened accordingly.

Further, in the microorganism inspection apparatus of the present invention, the fluorescence detecting means is moved relative to the plate medium in which the microorganism to be inspected is cultured to detect the detection signals of a number of virtually matrix-shaped compartments. Since this is stored and displayed by the display means, the number of sections for detecting the detection signal at once by the fluorescence detection means may be smaller than the number of the entire sections, and the device can be manufactured at such a low cost.

If the irradiating means and the light receiving portion of the fluorescence detecting means are arranged so as to straddle the plate medium, a row of pixels of the light receiving portion and a column by sampling by relative movement of the Petri dish once, All detection signals of each section obtained by virtually dividing the surface of the plate medium into a matrix are obtained, and the operation for detecting fluorescence is easy.

[Brief description of drawings]

FIG. 1 is a schematic process chart for explaining the basic principle of a microorganism inspection method of the present invention.

FIG. 2 is a schematic configuration diagram of a microorganism inspection device of the present invention.

FIG. 3 is a diagram for explaining the operation of the microbe inspection apparatus of FIG. 2, in which (a) is a simplified arrangement of the compartments that virtually divide the surface of the agar plate medium into a matrix and the light receiving portions of the fluorescence detection means. And (b) shows an example of a screen displayed on the display means according to the detection signal.

[Explanation of symbols]

 12 agar plate medium 14 prepared sample 16 non-fluorescent substance 18 bacteria group 20 xenon lamp 22 lens 24 first filter 26 second filter 30 moving means 32 irradiating means 38 fluorescence detecting means 42 light receiving section 48 latch circuit group 50 storage means 52 Display means

Claims (4)

[Claims] 1. A sample prepared with sterile water is spread on a plate medium, the sample-containing medium is incubated for a predetermined time, and the cultured sample-containing medium reacts with an enzyme of a microorganism to be tested. Diffuse non-fluorescent material that produces fluorescent material,
A method for inspecting microorganisms, characterized in that the number of microorganisms to be inspected contained in the specimen is measured from the number of places where the surface of the plate medium is irradiated with light to emit fluorescence. 2. The method for detecting microorganisms according to claim 1, wherein the surface of the plate medium is virtually divided into a large number of matrix-like compartments, and fluorescence detection means for detecting the presence or absence of fluorescence in each of these compartments. Storage means for storing the detection signal of the fluorescence detection means in association with the address of the section and display means for displaying the detection signal are provided, and the section or section group in which the fluorescence displayed on the display means is detected The number of microbes to be inspected contained in the sample is measured from the number of the microbes. 3. An irradiation means for irradiating the surface of a plate culture medium with light, a fluorescence detection means for detecting the presence or absence of fluorescence emitted in response to the irradiation of the light, the fluorescence detection means and the plate culture medium, and the plate medium. A moving means for relatively moving the fluorescence detecting means along the surface of the plate, and a detection signal of the fluorescence detecting means for a number of sections virtually dividing the surface of the plate medium into a matrix by the relative movement. And a display means for displaying the detection signal stored in the storage means. 4. The microorganism testing apparatus according to claim 3, wherein the irradiation means irradiates light in a narrow width band so as to straddle the surface of the plate culture medium, and the light receiving part of the fluorescence detection means is made into the band-shaped light. A plurality of them are arranged so as to straddle the plate culture medium so as to face the irradiation position, and the moving means crosses the plate culture medium with respect to the irradiation means and the arranged light receiving portions at right angles to the length direction of the band-shaped light. And a fluorescence detecting means for periodically sampling the presence or absence of fluorescence according to the relative movement.