JP3049961B2 - Bacterial test equipment - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a bacteria testing apparatus for measuring the number of microorganisms and somatic cells in a liquid or a liquid.

[0002]

2. Description of the Related Art Here, bacteria are treated as a generic term including microorganisms, somatic cells, and the like. Counting the number of these bacteria is required in various fields such as food, brewing, clinical, water and sewage, and semiconductors. It is extremely important for quality control and environmental management. For example, a colony counting method is generally used for measuring the number of microorganisms or the activity of microorganisms. The colony counting method is a method in which a certain amount of a sample solution is sprayed or mixed on an agar medium and cultured, and the resulting colonies are counted. However, the culturing time requires several tens of hours, and the operation is complicated.

[0003] In addition, there are a method of measuring ATP, in which ATP is caused to emit light by a luciferin-luciferase substrate-enzyme mixture, and the number of bacteria is estimated from the amount of the emitted light, and a method in which bacteria are stained with a fluorescent dye and observed. The operation is complicated, lacks quickness, and the accuracy is poor. Overcoming these shortcomings of conventional methods, rapid and accurate methods have been developed in recent years. For example, JP-A-2-51063, magazine J.A. C
lin. Chem. Clin. Biochem. , Vo
l. 26, 1988, pp. 147-148, a luminescent enzyme is labeled on a two-dimensionally dispersed bacterium using an antigen-antibody reaction, and the luminescent base point when a substrate is added is determined. This is a method of capturing and counting as an image. FIG. 6 is a schematic diagram showing a configuration of a main part of a measuring unit of an apparatus for performing the method. FIG. 6 shows a luminescence image of adherent bacteria 2 (a luminescence base point) filtered by a membrane filter (hereinafter sometimes simply referred to as a membrane) 1. The camera lens 3 forms an image on the input surface 5 of the high-sensitivity imaging means 4, and an image signal is taken out by a cable 6. This method has excellent characteristics in terms of quickness and accuracy of measurement by using high-sensitivity imaging means such as an image intensifier.

[0004]

However, the method disclosed in the above document has the following problems.
This method has a photon level sensitivity as an imaging means, but there is a loss of light amount in the preceding optical system, and as a result, for example, a reaction system having a low luminescence intensity such as emitting ATP contained in bacteria is: Can not be used.

Further, as shown in FIG. 6, since the light-emitting surface is imaged from above, it should be originally disposed above the bacteria-adhering surface, such as an automatic injection mechanism for synchronizing the reagent with the imaging means. However, it is difficult to install the camera because it may hinder imaging. Therefore, after the luminescence reaction is started in advance, the film must be installed and imaging must be performed. This is difficult to apply to, for example, most ATP luminescence reactions in which the luminescence lifetime is short and the light amount peaks several seconds after the start of the reaction. This means that automation of this type of measurement is difficult.

The present invention solves the above-mentioned problems, and enables high-sensitivity and rapid measurement even at a low light emission amount and a low concentration.
It is an object of the present invention to provide a bacterium testing apparatus that can measure a luminescent base point immediately after the start of a luminescent reaction.

[0007]

In order to solve the above-mentioned problems, a bacterium testing apparatus according to the present invention comprises a bacterium obtained by filtering a predetermined amount of a sample through a membrane filter in advance, and a predetermined reagent at a capturing position. The membrane filter is configured to capture a luminescent image by directly contacting the membrane filter with the input surface of an optical fiber plate connected to the imaging surface of the high-sensitivity imaging means.

[0008]

With the above configuration, the bacteria test apparatus of the present invention can receive light from the bacteria capturing position with the maximum light receiving efficiency determined by the numerical aperture of the optical fiber plate, and can perform high-sensitivity imaging of the membrane filter. Since the luminous reaction reagent was added and penetrated from the back surface of the membrane filter to the image-forming surface of the means, that is, the input optical fiber plate in close contact with the bacterium-adhering surface, the luminescence reaction was started on the bacterium-adhered surface, and the luminescence was started. Immediately after the start of the reaction, the luminescence image can be taken. Further, by using a tapered optical fiber plate capable of expanding the size of an input image to a predetermined magnification as the input optical fiber plate of the high-sensitivity imaging means, the effective visual field region of the film surface can be expanded.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below based on embodiments. FIG. 1 is a schematic cross-sectional view showing the configuration of the measuring section of the bacteria test apparatus of the present invention. In FIG. 1, a membrane filter 7 in which bacteria are captured by a filtration device (not shown) is placed on an input optical fiber plate 8a which is a part of a high-sensitivity imaging means 8 of an image intensifier incorporating a multi-channel plate. At this time, the surface of the main membrane filter 7 on which the bacteria are attached is in contact with the input optical fiber plate 8a. Then, the image multiplied inside the high-sensitivity imaging means 8 of the image intensifier incorporating the multi-channel plate is
The image is projected on the output optical fiber plate 8b as a fluorescent image, imaged by a camera lens 9 installed below the image, and an image sensor, for example, a CCD camera 10, and output as an image signal. Further, the lid 11 of the measuring unit
Is provided with an injection nozzle 12 for a luminescence reaction substrate reagent, and a reagent can be injected from the back side of the bacteria-adhered surface of the membrane filter 7 after the pretreatment, and the luminescence reaction is started from the time when it permeates and diffuses on the surface. Is started. A high voltage is applied to the voltage lead wire 13 of the high-sensitivity imaging means 8 so that the photocathode 8c is at the ground potential. The ground potential is
This is because, when a high voltage is applied to the photocathode 8c, there is a danger that the wet film 7 or the reagent may leak, or may generate noise on the photocathode 8c.

Here, the optical superiority of the bacteria test apparatus of the present invention will be described in comparison with the above-described imaging method using a camera lens (FIG. 6). First, FIG. 2 schematically shows a state in which light from a bacterial cell position (base point) in the conventional method is imaged by a lens on an input optical fiber plate of an image intensifier with a built-in multi-channel plate.
FIG. 2A is a ray tracing diagram showing light rays from the point light sources a and b on the film to the image plane traced, and FIG.
Is the numerical aperture of the optical fiber plate (NA = 0.1)
FIG. 4 is a ray tracing diagram in which light rays having a light receiving angle θ limited by the above are traced back to respective base points. As can be seen from FIGS. 2A and 2B, the brightness of an image usually depends on the size of the lens and the focal length, that is, the f-number. The brightness is determined by the numerical aperture (NA). Moreover, since the size of the film is usually larger than that of the optical fiber plate, a reduced projection (magnification <1) is obtained, and the light receiving solid angle ω from the light emitting base point is obtained.
Is smaller than the light receiving angle θ of the fiber. That is, the light receiving angle θ is not sufficiently used.

The magnitudes of θ and ω will be compared with a specific calculation example. The size of the membrane (line segment bc) is 35 m in diameter
m, effective field of view of optical fiber plate (line segment b ₁ c
₁₎ The diameter of 15 mm, when the distance f ₁ to the film surface and 100mm from the lens, the focal length (image-side focal length) f of the lens is 42.9 mm. Here, assuming that the magnification of the lens is m and the amount of displacement from the optical axis on the image plane is x, the light receiving solid angle ω at the object point is expressed by the following equation.

Ω = tan ⁻¹ {(1 + 1 / m) x / f + 1 / m (tan θ / 2)} −tan ⁻¹ {(1 + 1 / m) x / f−1 / m (tan θ / 2)} (1 ) Also, θ is N. A = n · sin (θ / 2) = 0.1 (2) Calculated from n = 1.00 (refractive index of air) and set to 11.5 °. Assuming that the magnification m (line segment bc / line segment b ₁ c ₁ ) is 2.333, the value of ω was calculated. As a result, the film was 4.94 degrees at the center (optical axis) and 4.6 at the periphery.
5 degrees.

On the other hand, FIG. 3 is a schematic diagram for explaining the light receiving angle at the base point in the apparatus of the present invention. FIG.
FIG. 3A shows the case where a normal optical fiber plate is used, and FIG. 3B shows the case where a tapered optical fiber plate is used. As shown in FIG. 3, the device of the present invention has no image forming process by the lens, and the light emitting base point 14 is in close contact with the input surface 15 a of the optical fiber plate 15.
The solid angle of light reception from the light emitting base point 14 is equal to the light reception angle 16 of the optical fiber, and the loss due to light absorption of the lens itself (about 1).
0%). 80% light transmittance when the optical fiber plate 15 is replaced by a tapered optical fiber plate 15 having an incident diameter / outgoing diameter ratio of 40:15, and the refractive index of the liquid film layer 17 between the membrane filter 7 and the optical fiber plate 15 (Approximately 1.3), the light collection efficiency is 2.8 compared to the imaging method using a lens.
It will be improved twice.

FIGS. 4 (a) to 4 (c) are schematic diagrams showing a process when a viable cell count is performed by applying the ATP luminescence method using luciferin-luciferase to the apparatus of the present invention. 4 (a) to 4 (c), E (symbol) represents an enzyme, ie, luciferase here, and S (（) represents a substrate, here, luciferin. Since the luciferin-luciferase mixture is infiltrated in advance on the membrane surface 7a on which the bacteria C are captured, a liquid membrane layer 17a containing an enzyme-substrate is provided between the input optical fiber surface 15a and the membrane surface 7a. Exists. On the other hand, since ATP18 (marked by A) is confined inside the cell by the cell membrane 19, no luminescence reaction occurs [FIG. 4 (a)].

Here, from the film back surface 7b, the AT indicated by an arrow
When a P extract (usually a reagent for dissolving cell membranes such as a surfactant) 20 is injected, the P extract is diffused on the membrane surface 7a, the attached bacterial cell membrane 19 is dissolved, and the internal ATP 18 is released to the outside. [FIG. 4 (b)]. ATP1 released
8 reacts with the substrate luciferin S by the luminescent enzyme luciferase E present in the liquid film layer 17a to emit light (thick arrow 21) [FIG. 4 (c)].

FIG. 5 shows the response characteristics of the luminescence intensity of this reaction. The response curve reaches a light intensity peak a few seconds after the start of the reaction, and then the light intensity rapidly decreases. As described above, by using the apparatus of the present invention, the reagent can be injected from the back surface of the film, and the measurement can be easily performed from the early stage of the luminescence reaction. The present invention particularly relates to a measuring unit of a measuring device that counts emission base points distributed two-dimensionally, and the subsequent processing of an image signal output during measurement is the same as that of the related art,
After a predetermined number of frames are added to the image signal and subjected to appropriate image processing, the image signal can be binarized and counted.

In the embodiment of the present invention, an image intensifier having a built-in multi-channel plate is used as the high-sensitivity imaging means, but a normal image intensifier may be used. Further, an optical fiber plate can be directly coupled to a photocathode or an image sensor of a SIT camera or a cooled CCD camera.

Furthermore, as the luminescence reaction using the apparatus of the present invention, ATP luminescence by luciferin-luciferase has been described as an example, but DNA inside bacteria is immobilized on a membrane,
It is also applicable to a method in which an antibody or a probe labeled with a luminescent enzyme is reacted with the DNA, and finally, a substrate of the luminescent enzyme is added to emit light.

[0019]

As described in the embodiment, the bacteria test apparatus of the present invention uses an optical fiber plate instead of an image forming method using a lens as a measuring unit, and makes the bacteria adhering surface of a membrane filter adhere to an input surface of the optical fiber plate. In addition to improving the light collection efficiency of the light from the luminescence base point, measurement can be performed immediately after the start of the luminescence reaction, and complete measurement is possible. Automation can be achieved.

[Brief description of the drawings]

FIG. 1 is a schematic cross-sectional view showing a configuration of a measuring section of a bacteria test apparatus of the present invention.

FIGS. 2A and 2B show a conventional image forming method using a lens, wherein FIG. 2A is a ray tracing diagram from a point light source to an image plane, and FIG.

FIG. 3 shows a light receiving angle at a base point in the apparatus of the present invention;
(A) is a case where a normal optical fiber plate is used,
(B) is a schematic diagram when a tapered optical fiber plate is used.

FIG. 4 shows a process of viable cell count by the ATP luminescence method of the apparatus of the present invention, wherein (a) is a state before a luminescence reaction, (b) is a state in which a reagent is injected, and (c) is a luminescence state. Pattern diagram

FIG. 5 is a response characteristic diagram of luminescence intensity of an ATP luminescence reaction by the apparatus of the present invention.

FIG. 6 is a schematic diagram showing a configuration of a main part of a measuring unit of the conventional device.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Membrane filter 2 Adhering bacteria 3 Camera lens 4 High sensitivity imaging means 5 Input surface 6 Cable 7 Membrane filter 7a Membrane surface 7b Backside of membrane 8 High sensitivity imaging means 8a Input optical fiber plate 8b Output optical fiber plate 8c Photoelectric surface 9 Camera lens 10 CCD camera DESCRIPTION OF SYMBOLS 11 Cover 12 Injection nozzle 13 Voltage lead wire 14 Emission base point 15 Optical fiber plate 15a Input surface 16 Receiving angle 17 Liquid film layer 17a Liquid film layer 18 ATP 19 Cell membrane 20 ATP extract 21 Light emission

Claims (4)

(57) [Claims] A bacterium testing apparatus for filtering a fixed amount of a sample through a membrane filter in advance and causing a luminescent reaction of the captured bacterium using a predetermined reagent, and taking an image of the luminescent image by a high-sensitivity imaging means to measure the number of bacteria. A bacterium testing apparatus, wherein a bacterium-adhering surface of a membrane filter and an input surface of a high-sensitivity imaging means having an optical fiber plate on an image forming surface are in close contact with each other. 2. An apparatus according to claim 1, wherein said high-sensitivity imaging means has an output surface of an optical fiber plate in contact with said imaging device. 3. The apparatus according to claim 1, wherein a reagent is added and permeated from the back surface of the bacteria-adhered surface to start a luminescence reaction. 4. An apparatus according to claim 1, wherein said optical fiber plate is a tapered optical fiber plate capable of expanding the size of an input image to a predetermined magnification.