JPH10215859A - Filter device for trapping microbe, microbial numbermeasuring kit and method for measuring microbial number - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microbe-trapping filter device capable of easily and quickly trapping only microbes even in the case where a sample is contaminated with animal and plant cells, etc., and applicable to a kit and a method for measuring a microbial number, and to provide the kit and the method for measuring a microbial number in a sample easily and quickly through a single stage filtration without needing special equipment or expertise. SOLUTION: This filter device consists of the first filter having pore sizes of 30-70μm and the second filter having pore sizes of 0.45-4μm. This microbial number-measuring kit consists of a microbe-trapping and filtering device for detection, which are composed of the first filter having pore sizes of 30-70μm and the second filter having pore sizes of 0.45-4μm made of a hydrophobic material, a dye solution composition and a colorimetric device. This method for measuring a microbial number comprises the introduction of a colored sample to trapping microbes in such a manner that the sample comes to contact at first with the first filter in the microbe-trapping and filtering device for detection, which are composed of the first filter having pore sizes of 30-70μm and the second filter having pore sizes of 0.45-4μm made of a hydrophobic material, and the detection of the microbial number in the sample based on the degree of coloration of the second filter.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a filter for collecting bacteria which can simultaneously remove animal and plant cells and the like present in a sample and collect bacteria, a kit for measuring the number of bacteria using such a filter as an element, Also, the present invention relates to a measurement method for rapidly measuring the number of cells using such a kit for measuring the number of bacteria.

[0002] The filter for collecting bacteria of the present invention can easily and quickly collect only bacteria, and thus can be used in the kit for testing the number of bacteria and the method for measuring the number of bacteria of the present invention. The bacterial count measuring kit and the bacterial count measuring method of the present invention can rapidly and simply determine the bacterial count in a sample by a simple one-step filtration operation without requiring advanced technology and specialized knowledge. Can be measured. Therefore, the present invention can be effectively used in a wide range of fields such as the food field, including the diagnostic field in which bacteria in urine is a problem.

[0003]

2. Description of the Related Art Conventionally, in the field of urinalysis, the number of bacteria in a sample such as urine has been measured in order to discover inflammation and to estimate a disease state. For example, if bacteria in the urine multiply, it is diagnosed as bacteriuria, suggesting a urinary tract infection. Therefore, the number of bacteria is measured for early treatment and drug administration.

In the field of foods, the number of bacteria in foods is measured in order to determine whether or not the foods are spoiled. In the case of fermented milk and lactic acid bacteria drinks, the number of lactic acid bacteria in foods is measured. The number of lactic acid bacteria in food is measured for control. The measurement of the number of bacteria in such a case is mainly performed by an agar plate culture method.

[0005] However, this culturing method requires specialized techniques and equipment such as a thermostat, and it takes 24-48 hours to obtain results. For example, in urinalysis, outpatient urine samples are subjected to a culture test in a bacterial laboratory or a testing center, so that the results are only available to the doctor a few days later. In order to perform highly accurate diagnosis, a rapid bacterial measurement method is required.

[0006] On the other hand, in the case of food, there are an E. coli test and a general bacterial test. Although the E. coli test gives a result after culturing for 18 hours, the general bacterial test requires culturing for 24 hours or more. Usually, results are obtained after product shipment. In particular, in the case of measuring the number of lactic acid bacteria in fermented milk, etc., it takes about 72 hours in a normal culture method, so it is after shipping the product, and in many cases, after the consumer has already finished eating and drinking the product, At last, the number of lactic acid bacteria can be determined, and improvement has been demanded from the viewpoint of the control of the number of bacteria.

[0007] The present applicant has solved the conventional drawbacks by measuring the number of bacteria in a sample.
A method has already been proposed in which bacteria are stained, collected on a hydrophobic filter, and the number of bacteria in a sample is measured from the degree of coloration of the bacteria (Japanese Patent Application Laid-Open No. Hei 8-140698). According to this method, the number of bacteria in a sample can be measured quickly and easily without requiring a skilled technique, specialized knowledge, or special equipment.

However, in the case of this method, when animal and plant cells and the like are present in the sample, they are also stained, and there is a problem that it is difficult to accurately measure the number of bacteria in the sample. This publication also discloses a technique for collecting animal cells using a prefilter, but the disclosed prefilter has a filter pore size slightly larger than a filter for collecting bacteria, so that a sample is used. When the number of animal cells inside is large, there is a problem that clogging occurs and filtration becomes difficult.

[0009]

DISCLOSURE OF THE INVENTION The present invention solves such conventional drawbacks, and enables simple and quick collection of bacteria only, even if animal and plant cells are mixed in the sample, and the determination of the number of bacteria. It is an object of the present invention to provide a filter for collecting bacteria that can be used in a kit or a measuring method. The present invention also provides a quick, simple, and accurate one-stage filtration operation without the need for special equipment or specialized knowledge, and even when animal and plant cells are present in the sample. It is an object of the present invention to provide a method for measuring the number of bacteria in a sample and a kit for measuring the same.

[0010]

Means for Solving the Problems The present inventors paid attention to the size and difference in size of bacteria and animal and plant cells, and efficiently combined animal and plant cells with a filter having a specific filtration pore size. The inventors focused on the fact that the bacteria were removed and only the bacteria were collected, and completed the present invention.

That is, the gist of the present invention is as follows. (1). A filter for collecting bacteria having a first filter having a pore size of 30 to 70 μm and a second filter having a pore size of 0.45 to 4 μm. (2). The filter for collecting bacteria according to the above (1), wherein the thickness of the first filter is 1.5 to 7 mm. (3). The filter for collecting bacteria according to the above (1) or (2), wherein the first filter is a polyvinyl alcohol filter. (4). The filter for collecting bacteria according to any one of (1) to (3), wherein the first filter is a filter for removing animal and plant cells, and the second filter is a filter for collecting bacteria. (5). The filter for collecting bacteria according to any one of (1) to (4), wherein the second filter is a filter made of a hydrophobic material. (6). Bacterial collection and detection comprising a first filter having a pore size of 30 to 70 μm and a second filter having a pore size of 0.45 to 4 μm made of a hydrophobic material, a filter for detection, a dye solution composition, and a bacterial count comprising a colorimetric means kit. (7). A filter for collecting and detecting bacteria having a first filter having a pore size of 30 to 70 μm and a second filter having a pore size of 0.45 to 4 μm made of a hydrophobic material was colored so as to come in contact with the first filter first. A method for measuring the number of bacteria, which comprises introducing a sample, collecting bacteria, and detecting the number of bacteria in the sample from the degree of coloring of the second filter.

[0012]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS First, a filter for collecting bacteria according to the present invention will be described. The filter for collecting bacteria of the present invention has two types of filters (first filter and second filter) having specific pore sizes. In the present invention, the pore size of the filter is defined by the bubble point method (JIS K38).
32) or a pore diameter obtained by a particle retention measuring method such as JIS Z8901, which is measured using a powder having a constant particle size. In addition, the filter of the present invention,
The first filter having a large pore size is used in such a manner that the first filter comes in contact with the sample containing bacteria.

The first filter has a function of collecting and removing animal and plant cells other than bacteria and the like. Here, animal and plant cells and the like refer to animal cells such as leukocytes and epithelial cells, plant cells such as algae, cells having a larger form than bacteria, and the like. The size of animal and plant cells differs depending on the type, but is about 8 to 15 μm for leukocytes, about 20 to 40 μm for epithelial cells, and about 3 to 10 μm for green alga chlorella corresponding to plant cells.

In order to exhibit the function of collecting and removing animal and plant cells other than bacteria, the pore diameter must be 30 to 70 μm. Further, the thickness is preferably 30 to 60 μm in terms of the filtration speed and the ability to collect animal and plant cells. If the pore size is less than 30 μm, clogging due to animal and plant cells is likely to occur, and if it exceeds 70 μm, collection of leukocytes and the like becomes incomplete. In this case, since the pore size of the second filter described later of the present invention is small, there is a possibility that the filterability may decrease due to clogging of the second filter. In addition, when used in the components of the kit for measuring the number of bacteria of the present invention, the accuracy of measuring the number of bacteria may be reduced.

[0015] The thickness of the filter is preferably 1.5 to 7 mm, more preferably 3 to 6 mm. If it is less than 1.5 mm, collection of animal and plant cells may be incomplete, and if it exceeds 7 mm, the passage of bacteria may be incomplete and the collection rate of bacteria by the first filter may be high. There is. In this case, the performance of collecting bacteria does not decrease, but when used in the components of the kit for measuring the number of bacteria of the present invention, the accuracy of measuring the number of bacteria may decrease.

The material of the filter is not particularly limited, but is preferably a material made of polyolefin such as polypropylene or a material made of polyvinyl alcohol in that bacteria are hardly adsorbed. A polyvinyl alcohol filter is particularly preferable because it is porous and has a high collecting ability. Among them,
Formal filter-treated polyvinyl alcohol filters are preferred.

The second filter has a function of collecting bacteria, and its pore size needs to be 0.45 to 4 μm.
On the other hand, the size of bacteria is generally 1-2 μm. That is,
The second filter of the present invention can capture even a pore size larger than that of bacteria. However, 4μ
m, it becomes difficult to collect bacteria, and 0.45 μm
If it is smaller, filtration becomes difficult.

The material of the filter is not particularly limited, and may be a filter made of a hydrophilic material such as cellulose acetate. However, in the case of employing a method in which bacteria are stained with a dye and the number is determined, if a filter made of a hydrophilic material is used, the filter itself is also stained, and it is difficult to remove excess dye. Therefore, when this method is employed, it is preferable to employ a filter made of a hydrophobic material (hereinafter abbreviated as a hydrophobic filter).

As such a hydrophobic filter, for example, a hydrophobic filter such as a nylon filter, a fluorine filter such as polytetrafluoroethylene (tetrafluoroethylene resin), a polyolefin filter such as polypropylene, a polycarbonate filter, or a glass filter is used. No. Among these filters, a hydrophobic filter made of polytetrafluoroethylene or polypropylene is particularly preferable because an excessive dye can be easily removed.

If a hydrophilic material, for example, a nitrocellulose filter is made hydrophobic by surface treatment, it can be used as a hydrophobic filter. Here, the surface treatment is coating or the like, and as the material used for the coating, the above-mentioned nylon-based, fluorine-based, or polyolefin-based material can be used. The filter for collecting bacteria of the present invention has the two types of filters as described above. When collecting bacteria, the sample containing bacteria passes through the first filter first as described above. Need to be used in In addition, if a second filter made of a hydrophobic material is used, the filter for collecting bacteria of the present invention can be suitably used as a filter for collecting and detecting bacteria in the kit for measuring the number of bacteria of the present invention. It can be suitably used for the method for measuring the number of bacteria of the present invention.

The filtering device of the present invention may be any device that uses the first filter and the second filter described above. However, if a filtering container for storing the filter is used, it is advantageous because there is no bacterial contamination. The filtration container has a tubular shape, that is, a tubular shape or a tubular shape, and can be made of either plastic such as vinyl chloride or the like or glass. Further, the capacity may be appropriately selected according to the amount of the sample or the like to be used.

The filter container may be composed of two connectable elements. In this case, one filter container stores the second filter and the other filter container stores the first filter. An embodiment is employed. Then, the filtration container storing the first filter may have a tapered shape suitable for collecting a sample, and a scale for measuring the sample may be provided on a portion ahead of the storage portion of the first filter.

In the above embodiment, the filtration container storing the second filter may be one in which a support for the second filter is arranged in advance. The material and shape of the filter support are not limited as long as the filter support can support the second filter. Usually, it is made of silicone resin or the like. As a filtration device for collecting bacteria using such a filtration container, the one described as reference symbol A in FIG. 1 can be suitably used.

Next, the kit for measuring the number of bacteria according to the present invention will be described. The kit for measuring the number of bacteria of the present invention comprises a filter for collecting and detecting bacteria, a dye solution composition and a colorimetric means. Bacterial collection and detection filter has a pore size of 30 to 70 μm
And a pore size 0.45 made of a hydrophobic material
The filter has a second filter of about 4 μm, and has basically the same configuration as the above-mentioned filter for collecting bacteria. However, since the kit of the present invention uses the dye solution composition, it is necessary to use a second filter made of a hydrophobic material for the above-described reason. In addition, the second filter in the present kit has a function as a filter for detecting bacteria as well as a function as a filter for collecting bacteria. Specifically, it has a detection function that shows a correlation between the number of collected bacteria and the degree of coloring.

The description of the first filter and the second filter is the same as described above. As the first filter, a filter having a thickness of 1.5 to 7 mm or a filter made of polyvinyl alcohol can be suitably used. The description of the filtration container and the like is the same as above. In the bacteria count kit of the present invention, an embodiment in which the filtration container storing the first filter and the filtration container storing the second filter are separate bodies is preferable in terms of simplicity of detection of the collected bacteria. is there.

As the dye solution composition, a composition capable of performing both staining and washing is used. Specifically, a dye which can be used for staining bacteria, water or various buffers, and if necessary, And a surfactant to be added. Examples of dyes that can be used for staining bacteria include safranin, fuchsin, methylene blue, methyl green, crystal violet, gentian violet, and Victoria blue B. Safranin, fuchsin, and methylene blue are particularly preferred from the viewpoint of ease of determination. And methyl green are preferred.

The usable pH of the buffer is from 3 to 9, preferably from 4 to 7 from the viewpoint of the stability of the dye. If necessary, a preservative such as ethanol, a surfactant or the like can be added. Available dye concentrations are
In the case of fuchsin, usually 0.00005 to 0.0006
% (W / v), preferably 0.0002 to 0.0004
5% (w / v). Here, the concentration of the dye is 0.00
If it is less than 005%, coloring will be insufficient, while if the concentration of the dye solution exceeds 0.0006%, it will be difficult to remove excess dye.

If necessary, a preservative such as ethanol may be added in an amount of 0.1 to 15% (v / v). When a surfactant is added, the surfactant may be added at a ratio of 0.001 to 1.0% (w / v).

The colorimetric means in the kit for measuring the number of bacteria of the present invention is a means for quantifying the number of bacteria based on the degree of staining of the filter with bacteria. Measurement of bacterial count
(1) It is easiest to visually compare using a color comparison table, but (2) it can also be performed by a colorimetric method by measuring optical density (OD). In the case of visual judgment, it can be measured not only from the surface on the front side of the second filter (the surface facing the first filter) but also from the degree of coloring on the back side. In these cases, a color control table for each side or a calibration curve of absorbance and bacterial count may be prepared.

Next, the method for measuring the number of bacteria according to the present invention will be described. In the measurement method of the present invention, the pore diameter is 30 to 70.
μm first filter and a pore size of 0.
Bacteria are collected by introducing a colored sample from the first filter into the filter for bacteria collection and detection having a second filter of 45 to 4 μm so as to come in contact with the first filter first, and the degree of coloration of the second filter From the number of bacteria in the sample. As the filter for collecting and detecting bacteria used in the measurement method of the present invention, the filter for collecting and detecting bacteria in the above-mentioned kit for measuring the number of bacteria can be suitably used.

As a measuring method, the following embodiment can be specifically adopted. 70 to 140 microliters of the sample is added to a container containing 200 to 800 microliters of the dye liquid composition for coloring the sample (the dye liquid composition may be added to the container containing the sample). Color the sample. Next, a syringe is attached to the second filter side of the filter for collecting and detecting bacteria, and the sample is suction-filtered by pulling the piston. If suction is difficult, attaching a locking device to the piston can prevent the piston from moving backward. When the sample has passed, the filter is removed from the syringe, the surface of the second filter is dried, and the color of the second filter is compared with a color control table to determine the bacterial count.

Hereinafter, the kit for measuring the number of bacteria and the method for measuring the number of bacteria according to the present invention will be described in more detail with reference to the drawings. FIG. 1 is an explanatory view showing one embodiment of the kit for measuring the number of bacteria of the present invention. FIG. 2 is an explanatory diagram showing a state in which the kit for measuring the number of bacteria is prepared by combining the kit for measuring the number of bacteria of the present invention with a commercially available syringe.

In FIG. 1, a filtration container A has a second filter 1, a first filter 3, and a filter support 2 therein. This filtration container A is composed of two components, a cylindrical filtration container A1 containing the second filter 1 and the filter support 2 and a filtration container A2 containing the first filter 3 and having a narrow end. Consists of

In the method of the present invention, a second filter (hydrophobic filter) 1 is used as a filter for collecting and detecting bacteria.
Is used. A hydrophobic filter is weak or non-polar and easily removes excess dye, so that it is suitable as a filter for collecting bacteria. The pore size of the second filter 1 needs to be in the range of 0.45 to 4 μm as described above.

The size of the suction filtration area (colored area) of the second filter 1 is not particularly limited as long as it is easy to see. The shape of the colored region is not particularly limited, but a circular shape is desirable from the point of viewability, and the diameter is preferably about 1 to 3 mm.
The size of the whole second filter is not particularly limited, but at least the size of the colored area is required at least. As a color of the second filter 1, a color that can be easily determined may be determined in consideration of a dye to be used. In order to easily determine the degree of coloring, white is preferable. Further, a transparent or translucent hydrophobic filter can also be used. When the filter support 2 is made white, the determination becomes easy.

On the other hand, the other end of the filtration container A1 can be attached to the tip of a syringe B, thereby enabling suction by the syringe B. In addition, if necessary, the syringe B itself may be included in the kit for measuring the number of bacteria of the present invention. Further, a piston locking device (not shown) may be included. And
As shown in FIG. 1, a first filter 3 is mounted inside the filter container A2 (that is, the first filter 3 is provided inside) at the tip (the side provided with the second filter 1) of the filter container A1. (The element for sampling) is detachably combined. Usually, as shown in FIGS. 1 and 2,
The first filter 3 attached to the inside of the filtration container A2 is prepared so as to be fitted into the inside of the filtration container A2 so that the tip of the filtration container A1 partially enters the inside of the filtration container A2. And the second filter 1 attached to one end of the filtration container A1.

The filter container A2 is a tube having one end tapered, that is, a tube or a tube. A sample collection port 4 is provided in the filter container A2 so that a sample can be collected from one end of the tapered shape. Have been. Further, the filtration container A2 may be provided with a measurement scale. in this case,
It is advisable to scale the sample in the range of 50 to 200 microliters, which is the amount of the sample normally used.

The first filter 3 mounted inside the filtration container A2 is used for removing animal and plant cells and the like, and is used when animal cells larger than bacteria, such as leukocytes, coexist in a bacterial sample. For the purpose of allowing bacteria to pass through but not passing through such large animal cells and collecting them.

In a preferred embodiment of the method for measuring the number of bacteria according to the present invention, when measuring the number of bacteria in a sample, the bacterial sample is first introduced into the filtration vessel A by a suction operation. Will be described. That is, first, a filtration container A2 in which the first filter 3 is mounted is fitted (fitted) to the tip of the filtration container A1 (the side provided with the second filter 1). At this time, the first filter 3 and the second filter 1 are in contact with each other. Next, the other end of the filtration container A1 (the second filter 1)
Attach the syringe B to the side opposite to the side provided with to prepare for the suction operation. However, the above operations may be performed in the reverse order. FIG. 2 shows the state at the stage of preparing for the suction operation in this way.

As shown in FIG. 1, the dye solution composition D is placed in a suitable container (eg, an eye drop bottle 5 or the like) to form a kit, and is used in about one suction filtration operation. A suitable container E capable of containing an amount of the dye solution composition D, for example, a microtube having a volume of about 0.5 to 1.5 milliliters is combined, and the dye solution composition D is put therein and the number of bacteria is counted. Will be used for measurement.

A container E for holding such a dye solution composition
(A microtube or the like) in combination, into which a small amount of the dye solution D is added in advance, and then a bacterial sample is collected using a dropper (not shown) and the container E
And mix well. As a result, the bacterial sample is stained with the dye solution composition D. The amount of the dye solution composition per sample is suitably 2 to 5 times the amount of the sample. On the other hand, the amount of the sample is usually 50 to 200 microliter.
Thus, for example, if the sample volume is 100 microliters, the dye solution composition requires 200-500 microliters. However, an amount exceeding 5 times the amount of the sample is not required. The amount of the dye solution composition used is preferably the same as the amount used for preparing a color control table or a calibration curve, and the amount used for a sample with an unknown number of bacteria, from the viewpoint of suppressing errors.

Immediately after mixing as described above, a filtration container (sample collection element) A2 attached to the tip of the syringe B
The mixture of the dye solution composition and the bacterial sample contained in the container E is pulled from the sampling port 4 provided at the tip of the syringe B by pulling the piston 6 of the syringe B and suction-filtered.

By the suction filtration operation, the stained bacteria are collected on the second filter 1. At the same time, excess dye is removed. That is, when the piston 6 of the syringe B is pulled and sucked, a liquid containing the stained bacterial sample (a mixture of the bacterial sample solution and the dye solution composition D) is collected by the second filter, and at the same time, excess dye is removed. Removed.

As another mode of sampling, the following method can be adopted. In this case, a measurement scale is attached to the filtration container A2. After preparing for the suction operation as shown in FIG. 2, a predetermined amount of the bacterial sample is introduced from the sampling port 4 provided at the tip of the filtration container A2 by the suction operation of the syringe B. The amount of the bacterial sample to be introduced may be based on the measurement scale provided in the filtration container A2. Next, after the bacterial sample was introduced by the suction operation of the syringe B as described above,
It is once extruded and injected into the dye solution composition D contained in the container E. By this operation, the bacterial sample and the dye solution composition are mixed, and the bacteria are stained. Thereafter, the piston 6 of the syringe B is pulled and suction filtered in the same manner as described above, and the bacteria are collected by the second filter.

According to the method of the present invention, the bacteria thus stained can be simultaneously collected on the second filter and the excess dye can be removed, and the operation steps can be simplified. In the measurement method of the present invention, bacteria collection,
The collection of stained bacteria in the second filter 1 for detection and the removal of excess dye are performed simultaneously, and the number of bacteria in the sample is measured from the degree of coloring of the second filter 1.

That is, after the stained bacteria are collected on the second filter 1 in this manner, when the filtration vessel A2 attached to the tip of the syringe B is removed, the tip of the filtration vessel A1 is exposed. It will be a shape. This filtration container A1
The degree of coloration of the bacteria present on the second filter 1 provided at the tip of the sample, that is, the color intensity, is compared with, for example, a color control table F prepared in advance using a sample having a known amount of bacteria. By doing so, the number of bacteria in the sample is measured. The color control table F shown in FIGS. 1 and 2 is an example of the color control table, and the symbols (-, +, ++) described in the color control tables are the bacterial numbers. Indicates the degree of presence.
1 and 2, the color control table F is divided into three parts to compare the degree of coloring (that is, the number of bacteria), but it is also possible to make more detailed divisions for comparison.

In the measuring method of the present invention, the collection of bacteria and the collection of stained bacteria in the second filter for detection and the removal of excess dye are simultaneously performed, and the hydrophobicity for the detection of bacteria is simultaneously measured. The number of bacteria in the sample is detected from the degree of color of the filter. Specifically, the measurement of the number of bacteria by the color control table is performed by measuring the degree of coloration of the bacteria present on the second filter, that is, the color intensity, by using a sample of a known amount of the bacteria in advance. The comparison may be performed by comparing with the comparison table of the above.

The color control table is obtained by taking a color photograph of the second filter stained and washed by the measuring method of the present invention using a sample having a known number of bacteria, or the same as the dyed second filter. It can be created by coloring filter paper or the like to a certain degree, or printing a similar color on paper. For example, when measuring the number of bacteria in urine, a color comparison table is created as follows. That is, although the urinary bacteria is present in the urine of healthy people at a low concentration (about 10,000 per 1 ml (milliliter) at most), a total of 1 ml
If the number is 100,000 or more per liter, it is determined that bacteriuria is suspected. If the number is 1,000,000 or more per ml, bacterial infection (bacteriuria) is definite. Therefore, a color comparison table that can be detected before and after this value may be created. Normally, it is sufficient to prepare the following standard color contrast table, but it is needless to say that the present invention is not limited to this. -: Negative (number of bacteria less than 10 ³ / ml) +: Positive (number of bacteria 10 ⁴ to 10 ⁵ / ml) ++: Strongly positive (number of bacteria 10 ⁶ to 10 ⁷ / ml)

In the colorimetric determination by measuring the optical density (OD), a dye coloring bacteria present on a second filter for collecting and detecting bacteria is eluted with an organic solvent, and the elution is carried out. The degree of coloration of the liquid may be measured by absorbance, and may be quantified by comparing it with a previously prepared calibration curve of optical density (OD) and the number of bacteria. The wavelength at the time of measuring the absorbance may be appropriately determined depending on the dye used. Here, various alcohols can be used as the organic solvent, but ethanol is particularly preferable.

The calibration curve may be created, for example, as follows. That is, when fuchsin is used as a dye, the degree of coloring of a sample diluted to various concentrations is measured by an optical density (OD) of 492 nm using a microplate reader. On the other hand, the number of bacteria in the sample solution of the same sample as the sample diluted to various concentrations was calculated by counting the number of colonies in the agar plate culture method, and a calibration curve of the number of bacteria and the optical density was created from both results. do it.

Here, the sample to which the present invention can be applied is not particularly limited. For example, urine, liquid seasonings (soy sauce, sauce, etc.), liquid foods and drinks (eg, fermented milk
Liquid foods such as lactic acid bacteria beverages), alcoholic beverages (eg, wine, sake, etc.), washed samples in the case of powders and solid foods (eg, vegetables, fish meat, etc.), water-soluble paints, etc., and river water, pond water, hot springs It can be widely applied to water, aquarium water, domestic wastewater, cooling tower water and the like.
The sample may be appropriately diluted as necessary, or, for example, in the case of a solid food, may be crushed and then diluted to obtain a measurement sample. The measurement method of the present invention relates to the method for determining the presence of bacteria such as Escherichia Coli, Staphylococcus aureus (Staphyl) in such a sample.
ococcus aureus), Pseudomonas aeruginosa
) Etc. are to be measured quickly.

[0052]

Next, the present invention will be described in detail with reference to examples. [Example 1] Staphylococcus aureum
, IFO 3183 strain) and Escherichia coli (E. coli, ATCC 113O3 strain) were cultured in broth broth liquid medium at 30 ° C. for 48 hours to prepare a culture solution. Then, it diluted with sterile water and produced the sample. The number of bacteria in the sample was determined from the number of colonies cultured on an agar plate medium (CLED medium). Filtration is diameter 1
The bacteria collecting material cut into a 3 mm circle was mounted on a filter housing, and 1 ml of the sample was filtered to calculate a collection rate. Filters made of polytetrafluoroethylene (PTFE) and those made of cellulose acetate were used. Regarding the number of bacteria present in the filtrate,
The cells were cultured on an agar plate medium (CLED medium) and determined from the number of colonies. The results are shown in Table 1.

[0053]

[Table 1] From Table 1, it was confirmed that a high collection rate was obtained by using the bacteria collection material having a pore size of 0.45 to 4 μm.

Example 2 A sample was prepared by culturing and diluting Escherichia coli of the same strain as that used in Example 1 in the same manner as in Example 1. Further, the number of bacteria in the sample and the number of bacteria in the filtrate were determined in the same manner as in Example 1.

As animal cells, hybridoma SHM
Using -D33 (ATCC 1668) strain, the cells were cultured in a GIT medium (manufactured by Daigo Kunitachi Chemical Co., Ltd.) for 3 days, and diluted with a PBS buffer (pH 7) to obtain a sample. The number of animal cells was counted under a microscope. Each collection material was attached to a straw having an inner diameter of 3 mm, and 1 ml of the sample was suction-filtered with a syringe to determine a collection rate. A filter made of formal-treated polyvinyl alcohol and having a thickness of 3 mm was used as the filter. The collection rate was determined by a method similar to the number of animal cells / bacteria in the sample before filtration and compared with the value after filtration. In addition, the filterability was evaluated, and the case where the filtration was smooth was evaluated as ○, the time required for the filtration was long, but the case where the entire amount could be filtered was evaluated as △, and the case where the entire amount could not be filtered even over time was evaluated as ×.
And The results are shown in Table 2.

[0056]

[Table 2] From Table 2, it was confirmed that when the pore size was 30 to 70 µm, a high collection rate of animal cells and a high passage rate of bacteria were obtained, and also good filterability was obtained.

Example 3 In Example 2, the effect of thickness was examined on a filter having a pore size of 60 μm, which was confirmed to have a high collection rate of animal cells, a high passage rate of bacteria, and good filterability. The sample and the test method were performed in the same manner as in Example 2. The results are shown in Table 3.

[0058]

[Table 3] From Table 3, although the influence of the thickness is smaller than that of the pore diameter, it is suggested that when the diameter is less than 1.5 mm, the collection rate of the hybridoma is decreased, and when the diameter is 9.0 mm, the collection rate of Escherichia coli is increased. .

Example 4 (1) Preparation of a Color Control Table for Measuring the Number of Bacteria in Urine Escherichia coli (ATCC 11) was used as a bacterium.
303 strain) and Staphylococcus au
reus ) (IFO 3183 strain), the following experiment was performed.

The broth broth medium was inoculated with the bacterial species, and
For 24 hours, and the culture solution was used as a standard sample bacterial suspension. On the other hand, collecting urine released from healthy males,
The bacteria dilution was used. A predetermined amount of this bacterial dilution and the hybridoma SHM-D are added to the standard sample bacterial suspension.
33 (ATCC 1668) strain in PBS buffer (pH
7) Add the suspension and count the number of each bacteria about 1,000 / ml, about 10,000 / ml, about 100,000 / ml,
Five standard samples were prepared so as to have a concentration of about 100,000 cells / ml and about 10 million cells / ml, and to have about 100,000 cells / ml for the hybridoma SHM-D33 strain. For comparison, the hybridoma SH was also used.
A sample of strain MD-33 alone was also prepared. The number of bacteria by the culturing method was counted based on the number of colonies cultured at 37 ° C. for 24 hours on a CLED agar plate medium. The number of hybridoma SHM-D33 strains was counted using a microscope.

The kit for measuring the number of bacteria shown in FIGS. 1 and 2 was used for the experiment. The dimensions and the dye solution composition were as follows. -Dye liquid composition D: 0.0004% of fuchsin (w /
v), pH containing salt at 0.85% (w / v) and Tween 20 (Kanto Chemical) at 0.15% (w / v).
Is 4.9 ・ Filtration container A1: 4.6 mm in outer diameter, 4 mm in inner diameter, and 2 in length
0 mm, polypropylene tube • Filtration vessel A2: Wide-mouth side inner diameter 5 mm (manufactured by Quality Corporation, USA) chip • Microtube (vessel E containing dye solution composition):
1.5 ml volume, made of polypropylene (manufactured by U.S.A. Quality Co., Ltd.)-Hydrophobic filter for bacteria detection (second filter) 1:
Polytetrafluoroethylene membrane filter (pore diameter 3 μm, colored area diameter 2.0 mm) Filter support 2: silicone tube (outer diameter 4 m)
m, inner diameter 2.0 mm) cut into 7.5 mm in length ・ Eye drop bottle 5: 10 ml volume, made of polypropylene ・ First filter 3: diameter 4 mm, thickness 4 mm, made of formal-treated polyvinyl alcohol, pore size = 60μ
m, locking device (not shown): length 55 mm, made of polypropylene

As shown in FIG. 2, a syringe B having a capacity of 3 ml was attached to one end of the filtration container A1.
At the other end (on the side provided with the second filter 1), a filtration container (sample collection element) A in which the first filter 3 is mounted.
2 was detachably fitted. Next, a bacterial sample was collected using a dropper (not shown) and 350 μl (7 to 8).
2) (80 to 100 μl) were added dropwise to a container E (microtube) in which the dye solution composition D of (1) was previously placed and mixed. Then, immediately, the mixture of the dye solution composition and the bacterial sample contained in the container E is transferred from the sampling port 4 provided at the tip of the filtration container A2 attached to the tip of the syringe B to the piston of the syringe. 6 was suction-filtered. The suction was performed by using a lock device to prevent the piston from moving backward.

By this suction filtration operation, the stained bacteria were collected on the second filter 1, and at the same time, excess dye was removed. After the stained bacteria are collected on the second filter 1 in this manner, the filter container A2 is removed, the tip of the filter container A1 is exposed, and the second filter provided at the tip of the filter container A1 is exposed. Filter 1
As a result of visually observing the degree of coloration of the bacteria present on the
The results are shown in Table 4 below.

[0064]

[Table 4]

From Table 4, it can be seen that, in the absence of the first filter, the hybridoma was trapped by the second filter, and strong coloring was observed regardless of the number of bacteria. On the other hand, when the first filter was provided, the coloring became stronger with the increase in the number of bacteria, and the effect of the present invention was confirmed. With reference to the coloring degree of the second filter, a three-stage color control table was prepared. For the production, UTI of Japan
Reference (bacteria count 10 less than ^four urinary tract infection negative per 1 ml, criteria for positive 10 ⁴ or more) according to the less than 10 ⁴ - (negative), 10 ⁴ or more to + (positive ) And 10 ⁶ or more were determined as ++ (strong positive).

(2) Measurement of the number of bacteria in urine Using the urine released from a healthy person and the urine released from various patients as samples, the same operation as shown in the above (1) for preparing the color control table for urinary bacteria measurement was performed. Then, the number of urinary bacteria was measured by visual comparison with the color control table prepared in the above (1). The results are shown in Table 5. For comparison, CLE
The number of bacteria measured on a D-agar plate medium at 37 ° C. for 24 hours is also shown. In Table 5, "filtration impossible" means a state in which filtration could not be performed at all due to clogging.

[0067]

[Table 5]

From Table 5, it was found that when the first filter was not used, a defect that filtration was impossible or that a healthy person was judged to be positive was found. On the other hand, when the first filter was installed, it was confirmed that accurate judgment was made.

[0069]

According to the filter for collecting bacteria of the present invention, even if animal and plant cells are mixed in the sample, only the bacteria can be collected easily and quickly, and the bacteria counting kit and the measuring method can be used. Available. In this case, the number of bacteria can be detected simply and accurately. Further, the filter for collecting bacteria of the present invention can be suitably used for the kit or the method for measuring the number of bacteria of the present invention.

According to the kit for measuring the number of bacteria of the present invention, one-stage filtration by a syringe is sufficient, so that the operation is extremely simple. In addition, since no skill is required and no equipment is required, any place can be selected for measurement. Further, the bacteria count kit of the present invention is extremely simple and inexpensive,
Since no special equipment is required, there is an advantage that it can be used at any site. In particular, since ordinary laymen can use it, they can easily use river water,
Inspection of the water quality around us such as pond water, sea water, hot spring water, etc. is possible, which not only contributes to environmental preservation, but also has the characteristic of being able to inspect the number of bacteria in foods such as liquid foods and solid foods. .

According to the kit for measuring the number of bacteria of the present invention, after completion of the measurement, a sample requiring identification of the bacteria can be collected by suction, and if the tubule is sealed by heat treatment or the like, it can be transported to the laboratory. It can be a container. Therefore, the present invention
It can be widely used for various tests including the urine test field (diagnosis area).

According to the measuring method of the present invention, the collection of stained bacteria on the hydrophobic filter and the removal of excess dye can be performed simultaneously by a one-stage suction filtration operation using a syringe, and the number of bacteria in the sample can be reduced. Can be measured quickly and easily. According to this method, determination can usually be made within 5 minutes. Further, according to this method, the number of bacteria in a sample can be measured without requiring specialized skills or knowledge.

In addition, according to the method of the present invention, the presence or absence of bacteria can be determined without culturing the bacteria, so that the method can be used for specimen screening for bacterial tests in hospitals, which contributes to reducing costs for patients and hospitals. it can. In addition, the method of the present invention does not require special equipment, so that it can be measured by small hospitals and clinics, or even by ordinary people.

Further, the method of the present invention can be applied to, for example, yeast other than bacteria by selecting the filtration pore size of the second filter, so that the application range is extremely wide.
According to the method of the present invention, the number of bacteria can be determined before the food (eg, yogurt) is shipped, which can contribute to the quality control of the food.

[Brief description of the drawings]

FIG. 1 is an explanatory view showing one embodiment of a kit for measuring the number of bacteria of the present invention.

FIG. 2 is an explanatory diagram showing a state in which the kit for measuring the number of bacteria is prepared by combining the kit for measuring the number of bacteria of the present invention with a commercially available syringe.

[Description of sign]

A Filtration vessel (consisting of filtration vessel A1 and filtration vessel A2) A1 Filtration vessel (filtration vessel containing second filter) A2 Filtration vessel (sample collection element) B Syringe D Dye liquid composition E Dye liquid composition Container F to be collected F Color control table 1 Second filter 2 Filter support 3 First filter 4 Sampling port 5 Eye drop bottle 6 Piston

Claims (7)

[Claims] 1. A filter for collecting bacteria having a first filter having a pore size of 30 to 70 μm and a second filter having a pore size of 0.45 to 4 μm. 2. The thickness of the first filter is 1.5 to 7 mm.
The filter for collecting bacteria according to claim 1, wherein 3. The filter for collecting bacteria according to claim 1, wherein the first filter is a filter made of polyvinyl alcohol. 4. The filter for collecting bacteria according to claim 1, wherein the first filter is a filter for removing animal and plant cells, and the second filter is a filter for collecting bacteria. 5. The filter for collecting bacteria according to claim 1, wherein the second filter is a filter made of a hydrophobic material. 6. A bacteria collection and detection filter, a dye solution composition, and a colorimetric means having a first filter having a pore size of 30 to 70 μm and a second filter made of a hydrophobic material having a pore size of 0.45 to 4 μm. A bacteria count kit comprising: 7. The filter for bacteria collection and detection having a first filter having a pore size of 30 to 70 μm and a second filter made of a hydrophobic material having a pore size of 0.45 to 4 μm is contacted first from the first filter. A method for measuring the number of bacteria, which comprises introducing a sample colored as described above, collecting bacteria, and detecting the number of bacteria in the sample from the degree of coloring of the second filter.