JPH06237791A - Detection of fine body - Google Patents

Abstract

PURPOSE:To directly count fine bodies such as bacteria without culturing the fine bodies. CONSTITUTION:The method for detecting fine bodies comprises enzymatically labeling a sheet catching the fine bodies, bringing the sheet into contact with a luminous substrate for a definite time, removing the luminous substrate, again giving the luminous substrate to the sheet, and subsequently imaging the luminous image with a highly sensitive imaging device in a state reduced in the back ground light.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting a minute object which uses a luminescent reaction as a detecting means to quickly and easily measure the number of minute objects such as bacteria present without using a culturing means.

[0002]

2. Description of the Related Art Counting and quantitatively analyzing the number of microorganisms such as bacteria, somatic cells, and minute objects such as inorganic or organic fine particles is an environmental management and hygiene management in each field such as semiconductor industry, food, water and sewage, and clinical. It is extremely important for quality control. In the following, bacteria will be described as a specific example. Conventionally, a colony counting method has been generally used to control the number of bacteria. The colony counting method is a method in which a fixed amount of a sample solution is sprayed or mixed on an agar medium and cultured until the colonies have a size that can be observed with the naked eye or a microscope, and the number of generated colonies is counted. This quantification method requires the growth of bacterial colonies to the extent that it can be measured with the naked eye or with a microscope, so it takes a long time to measure the number of bacteria, and the operation is complicated and accurate. bad.

Recently, a method has been developed which overcomes the drawbacks of the conventional method and enables rapid and highly accurate measurement. For example, Japanese Unexamined Patent Publication No. 4-104799 discloses a method in which two-dimensionally dispersed bacteria are labeled with an enzyme that catalyzes a luminescence reaction, and a luminescent base point when a luminescent substrate is added is imaged and counted. ing. As a concrete practical example of this method, a luminescence image of bacteria (light-emission base point) captured by a sheet is formed on the image input surface of the high-sensitivity image pickup means by a camera lens, and an image signal is taken out to detect the light-emission base point. Show how to know the bacterial count.

Further, by improving this, an optical fiber plate is provided on the image input surface of the high-sensitivity image pickup means, the bacteria adhering surface of the sheet is placed on this optical fiber plate surface to cause a luminescent reaction, and the luminescent image of the bacterial cells is counted. A method is also conceivable. However, even with this means having the most optically advantageous structure, it was not possible to detect the luminescence emitted from one bacterium corresponding to the luminescence as a luminescent base point that can be discriminated against the background light.

In the above-mentioned rapid measuring method of the number of bacteria, it is necessary to clearly distinguish the luminescent base point on the secondary plane from the background light in order to accurately detect the bacterium as the luminescent base point.

[0006]

In order to solve the above problems, it is conceivable to reduce the background light or to enhance the luminescence amount corresponding to one bacterium. Luminescence emitted from bacteria is caused by an enzymatic reaction, and the amount of luminescence is proportional to the amount of enzyme. Here, there is a limit to the amount of enzyme that can label one bacterium, and therefore there is a limit to enhancing the luminescence amount equivalent to one bacterium.

Further, considering that the optical signal entering the image input surface of the high-sensitivity image pickup means is accumulated and finally imaged, if the background light is strong, an effective signal is buried in the background light. It becomes difficult to detect the light emission origin.

Therefore, the present inventor considered that the reduction of background light would be an effective means for solving the above problems.

In the present invention, when a luminol-based luminescent substrate is used, it has been observed that luminol is an unstable substance and emits light by being significantly catalyzed by an extremely small amount of substance. That is, in order to measure the background light due to the luminescent substrate itself containing luminol, when a background substrate was measured by adding a luminescent substrate to a film to which no enzyme was attached, strong luminescence of luminol due to some catalytic action was observed. Background light was observed to the extent that it hindered detection of the luminescent origin originating from the enzyme to be measured. Therefore, when making the luminescence-labeled bacteria emit light, first, the aqueous solution contained in the sheet is removed, and then the luminescence substrate is applied to the bacteria-adhering surface to diffuse the luminescence substrate into the sheet, after which the substrate is removed and the luminescence substrate is added again. The present invention is completed by knowing that when a sheet is put on and then the sheet and the luminescent image input surface are brought into close contact with each other to capture a luminescent image, background light is reduced and the luminescent base point of bacteria can be clearly distinguished. The object of the present invention is to provide a method for rapidly detecting the number of microscopic objects such as bacteria by clearly distinguishing the light emission corresponding to one microscopic object such as bacteria and the background light. Especially.

[0010]

In order to achieve the above object, the present invention makes a micro object captured on a sheet emit light by using a predetermined luminescent substrate, and captures the luminescent image by a high-sensitivity imaging means. In the method for detecting a micro object for detecting the micro object, a sheet pre-capturing the micro object is impregnated with a luminescent substrate and left for a predetermined time, the impregnated luminescent substrate is removed, and then the luminescent substrate is again sheeted. A method for detecting a minute object is configured such that the sheet is impregnated with the enzyme-labeled micro substance to emit light, and then the sheet and the luminescence image input of the high-sensitivity imaging device are brought into close contact with each other to capture the luminescence image of the sheet. The micro object is a microorganism or a somatic cell, the luminescent substrate is mainly composed of luminol, and a sheet is pressure-bonded to the luminescent image input surface during imaging.

The present invention will be described in detail below with reference to the drawings.

[0012] As very small objects to be measured in the present invention, microorganisms such as bacteria, somatic cells, inorganic, there are organic fine particles, E is a specific example. coli , Pseudom
onas . sp . Bacteria such as, eukaryotes such as yeast,
Somatic cells such as cancerous cells and interstitial cells, iron particles, silica particles, low molecular weight organic compounds such as pesticides attached to fine particles such as fine activated carbon, and the like can be mentioned.

When the minute object is dispersed in liquid or gas, the minute object can be captured on the sheet by filtering the liquid or gas with a sheet.

The material of the sheet is nitrocellulose,
A mixture of nitrocellulose and acetate, nylon, polysulfone, and the like are preferable, and those formed of these materials as a filtration membrane are preferable. The filtration pore size of the sheet is determined by the particle size of the fine particles to be captured,
When measuring bacteria or the like, the pore size is 0.1 to 0.
About 5 μm is preferable, and as commercially available products, nitrocellulose acetate filter (manufactured by Japan Millipore Industry), nitrocellulose filter (manufactured by Toyo Filter Paper), nylon filter (MICRON SEPARATIO)
NS INC. Manufactured by Fuji Film Co., Ltd.) and the like.

The sheet that has captured the minute objects is first impregnated with a luminescent substrate. When the luminescent substrate emits light by an enzyme, the micro-object captured on the sheet is bound with the labeling enzyme in advance. As the labeling enzyme, known ones such as peroxidase enzyme can be used, and the binding method is also known.

When the micro object is a bacterium, a labeling enzyme may be bound as follows. That is, a certain amount of a sample is filtered in advance and the bacteria are two-dimensionally dispersed and captured on a sheet, and the captured bacteria are dissolved by a bacterial solution to expose the nucleic acid in the bacteria to the outside of the cells. A nucleic acid binding substance labeled with a peroxidase enzyme is applied onto this sheet to bind the nucleic acid derived from the bacterial cell with the nucleic acid binding substance. It is preferable to wash and remove the nucleic acid-binding substance that has not bound to the nucleic acid from the sheet.

After binding the labeling enzyme as described above,
The sheet is impregnated with a luminescent substrate corresponding to the labeling enzyme.

In this case, in order to efficiently impregnate the sheet, it is preferable to remove the aqueous solution contained in the sheet by a method such as suction.

The luminescent substrate is preferably a luminescent substrate that emits light by radicalization of the luminescent substrate. Illustrative examples of such a luminescent substrate include luminol and luminol isomers,
There are lucigenin, luciferin, isoluminol derivative, bis (2,4,6-trichlorophenyl) oxalate and the like.

A mixture of luminol, H ₂ O ₂ and an enhancer is particularly preferable as a luminescent substrate.

Then, the sheet impregnated with the luminescent substrate is left for a certain period of time. The standing time is preferably about 1 to 10 minutes, and the standing temperature is preferably 5 to 40 ° C.

After standing, the luminescent substrate impregnated in the sheet is removed, and the luminescent substrate is again impregnated in the sheet.

As a method for removing the luminescent substrate, for example, a suction operation such as placing a sheet impregnated with the luminescent substrate on the upper part of the filter plate and reducing the pressure from the lower part with an aspirator, or washing is preferable.

The sheet thus re-emitted is
Then, the luminescent image is picked up by being brought into close contact with the luminescent image input surface of the high-sensitivity imaging device. As the image pickup device, a known device can be used, but a device having the following configuration is particularly preferable.

FIG. 1 is a block diagram showing an example of the image pickup apparatus.

In the figure, reference numeral 12 is a two-dimensional photon counter, and an image input surface (optical fiber surface) 14 is formed on the upper surface thereof, and a luminescent image is input through this input surface 14. Reference numeral 16 denotes a light-shielding cover which covers the upper portion of the counter tube 12 so that light does not enter the input surface 14 from the outside. In addition, the light shielding lid 16
A metal block 18 is fixed to the inner wall of the upper part of the sheet. The sheet 20 is pressed by the load of the block 18, and the minute object capturing surface 22 and the image input surface 14 of the sheet 20 are pressed.
And are crimped. The input image is converted into an image signal by the imaging camera 24, sequentially sent to the image integration device 26 and the image processing device 28, and finally displayed on the monitor 30. In addition, 32 is an arithmetic processing unit and 34 is a video copy.

When a load is applied by the block 18 at the time of inputting an image using the high sensitivity image pickup device,
The measurement results for the case with no load and the case with no load are as shown in FIG. That is, when a load is applied, the background light is low and the background light is uniformly pressed, and one bacterium is detected as an independent light emission base point. On the other hand, when no load is applied, the background light is non-uniform and high, and the luminescence derived from one bacterium is buried in the background light and is not observed as an independent luminescent origin. Furthermore, it can be seen that the amount of light between the background light and the light emission base point is also reduced by applying a load as shown in FIG.

The load pressure for crimping is 0.00045N /
mm ² or more, preferably 0.001 N / mm ² or more, and the higher the pressure, the clearer the distinction between the light emission base point and the background light is, which is preferable. Should.

Although bacteria are used in the above description, the present invention is not limited to this, and the same applies to detection of somatic cells, inorganic and organic microscopic objects.

[0030]

【Example】

(Example 1) First, Pseudomonas cultivated in advance
fluorescens diluted appropriately to the 50Cell
/ Ml Pseudomonas fluores
Prepare sample water containing cens, and sample water 1
ml is a polysulfone sheet with a pore size of 0.2 μm (25 mm
φ), and then Pseudomonas flu
orescens were captured on a sheet.

The sheet is treated with 0.2N caustic soda solution and 1% S.
The solution is lysed by contacting with 5 ml of a mixed solution of DS to expose the nucleic acid inside the cell to the outside of the cell, and then a buffer solution of pH 7.2 (1.5 M NaCl, 0.5 M Tris-HC
1, 0.01 M NA ₂ EDTA mixture)
Further, the sheet was treated with a blocking agent containing albumin to prevent non-specific binding of anti-nucleic acid antibody that reacts with nucleic acid to the sheet without reacting with nucleic acid.

Next, the anti-nucleic acid antibody obtained by immunizing a mouse was labeled with peroxidase, and the obtained enzyme-labeled anti-nucleic acid antibody was diffused on a sheet and reacted with the nucleic acid immobilized on the sheet. After the reaction, the unreacted and non-specifically adsorbed enzyme-labeled anti-nucleic acid antibody on the sheet was removed by washing.

Next, the sheet was brought into contact with a luminescent substrate (a solution containing luminol and hydrogen peroxide as a main component) to cause an enzymatic reaction to emit light. This luminescence was detected by the high-sensitivity imaging means shown in FIG.

In the above-mentioned luminescence measurement, first, the luminescent substrate was applied to the bacteria-adhering surface to diffuse the luminescent substrate into the sheet, the substrate was removed, the luminescent substrate was placed on the sheet again, and the luminescent base points were counted. The luminescent base score was obtained. (Comparative Example 1) When the luminescent substrate was applied only once, that is, when the measurement was performed in the same manner as in Example 1 without previously washing the sheet with the luminescent substrate, the luminescent base point was buried in the background light. Pseudomonas fluoresc
It was impossible to count the location of ens as the luminescent origin . (Control Example 1) Pseud in sample water by culture method
When the number of Omonas fluorescens was counted, the number of colonies of 47 ± 6 was counted, which was in good agreement with the number of luminescent groups when the sheet was washed with a luminescent substrate in advance and measured. (Example 2) After microfloc filtration of industrial water,
Pure water is prepared by treating with a two-bed, three-bed type pure water production device and a mixed bed polisher, and the pure water is processed by microfiltration and a reverse osmosis membrane device, followed by UV oxidation, a mixed bed cartridge polisher, ultra 20 L of ultrapure water obtained by filtration was added to 0.45
μm nitrocellulose acetate sheet (37mm
φ) and 0.2% caustic soda solution and 1% to destroy the bacterial cell membrane trapped on this sheet.
It was brought into contact with 5 ml of a mixed solution of SDS. When the subsequent operation was performed in the same manner as in Example 1, 19 ± 1 luminescent base points were obtained. (Comparative Example 2) When the measurement was performed without washing the sheet, the luminescent base point was buried in the background light, and it was impossible to count the location of bacteria as the luminescent base point. (Comparative Example 2) On the other hand, in accordance with "Low temperature, long-time culture method" of JIS K-0550 "Test method of bacterial count in ultrapure water", 2
When the cells were cultured at 5 ° C. for 5 days, 21 ± 3 colonies were detected and it was confirmed that the measurement accuracy of this example was high. (Example 3) The effect of the load of the high-sensitivity imaging device and the effect of the substrate cleaning of the present invention were examined by the S / N ratio. (1) Examination of Effect of Load on High-Sensitivity Imaging Device The sheet used was one impregnated with a luminescent substrate again (sheet of Example 1). The image pickup apparatus shown in FIG. 1 was used, and the load was adjusted by attaching and detaching the block.

[0035]

[Table 1] (2) The effect of removing background light by causing the sheet to emit light in advance, washing and removing the luminescent substrate, and then re-emitting light. The high-sensitivity imaging device is that of FIG.
It was set to m ² .

[0036]

[Table 2] Considering the above effects (1) and (2) as independent effects, the effects of the two methods (loading and cleaning) are 1.27 × 1.
There was an improvement in S / N ratio of 71 = 2.17 times.

[0037]

INDUSTRIAL APPLICABILITY According to the present invention, the background light is reduced to the level of the luminescence of the non-catalytic luminescent reagent, a low and uniform background light is obtained, and the luminescence from one bacterium can be clearly distinguished from the background light. , A single bacterium can be detected rapidly and accurately.

[Brief description of drawings]

FIG. 1 is a block diagram showing an example of a high-sensitivity imaging device used for implementing the present invention.

FIG. 2 is a graph showing a difference in image signal with respect to pressing force between a sheet and an image input surface.

[Explanation of Codes] 12 two-dimensional photon counter 14 image input surface 16 light-shielding lid 18 block 20 sheet 22 microscopic object capturing surface 24 imaging camera 26 image accumulator 28 image processor 30 monitor

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI Technical indication location G06F 15/62 380 9287-5L 390 9287-5L (72) Inventor Yasushi Zaitsu Kawasaki-ku, Kawasaki-shi, Kanagawa No. 1 Nitta Tanabe, Fuji Electric Co., Ltd. (72) Inventor Masanori Morimoto No. 1 Nitta Tanabe, Kawasaki-ku, Kawasaki-shi, Kanagawa Fuji Electric Co., Ltd.

Claims (4)

[Claims] 1. A method for detecting a micro object in which a micro object captured on a sheet is caused to emit light by using a predetermined luminescent substrate, and the luminescence image is picked up by a high-sensitivity imaging means to detect the micro object. After impregnating the sheet that has captured the object with the luminescent substrate and leaving it for a predetermined time, the impregnated luminescent substrate is removed, and then the sheet is again impregnated with the luminescent substrate to emit light from the micro substance, and then the sheet. A method for detecting a minute object, which comprises closely contacting a luminescent image input surface of a high-sensitivity imaging device with a luminescent image of a sheet. 2. The detection method according to claim 1, wherein the minute object is a microorganism or somatic cell. 3. The detection method according to claim 1, wherein the luminescent substrate is mainly composed of luminol. 4. The detection method according to claim 1, wherein the sheet is pressure-bonded to the light-emission image input surface during image pickup.