JPH0965893A - Rapid bacterium counting method and bacterium counter kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a convenient method and apparatus for counting rapidly the number of bacteria at one step that samples a specimen only by filtration by suction without using a sampling tool such as a syringe and without special equipment and technical knowledge. SOLUTION: The objective method for counting the number of bacteria in a sample comprises introducing a bacterial sample into a filter vessel which incorporates a hydrophobia filter for detecting bacteria, injecting the sample into a liquid pigment composition by extrusion, filtering the contents in the vessel by suction, collecting stained bacteria on to the hydrophobic filter, and determining the number of bacteria in the sample from the degree of coloring of the filter; and the other objective kit comprises a filter vessel A that contains a hydrophobic filter 1 for detecting bacteria and a filter support 2, a sampling member C in which a prefilter 3 is fitted if necessary and which can be mounted on the tip of the filter vessel, a liquid pigment composition D, a container E which contains the liquid pigment composition, and a color reference table F.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention does not require tools such as a dropper for collecting a sample, and only requires a suction filtration operation with a syringe, so that it does not require advanced technology or specialized knowledge and is simple. The present invention relates to a method and a measurement kit capable of quickly and simply measuring the number of bacteria in a sample by a one-step filtration operation. INDUSTRIAL APPLICABILITY The present invention includes the field of metalworking oils, the field of dyes where rot is a problem, as well as the field of diagnosis where bacteria in urine are a problem.
It can be effectively used in a wide range of fields such as food fields and environmental fields such as hot spring water.

[0002]

2. Description of the Related Art Conventionally, in the field of urinalysis and the like, the number of bacteria in a sample such as urine has been measured in order to detect inflammation and estimate the condition. 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 addition, in the food field, etc., the number of bacteria in the food is measured to determine whether the food is not spoiled,
Further, in fermented milk and lactic acid bacterium beverages, the number of lactic acid bacteria in the food is measured to control the number of lactic acid bacteria in the food.

The number of bacteria in such cases is usually measured by the agar plate culture method. However, this culturing method requires specialized techniques and equipment such as a constant temperature bath, and it takes 24 to 48 hours for results to be obtained. 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. On the other hand, in the case of food, there are Escherichia coli test and general bacteria test.
Cultivation for general bacteria requires culturing for 24 hours or more, and in the case of many food products, the result is usually obtained after the product is shipped. Especially when measuring the number of lactic acid bacteria in fermented milk,
Since it takes about 72 hours with a normal culture method, after the product has been shipped, and in many cases, after the consumer has already finished eating and drinking the product, the lactic acid bacterium count is finally known. Improvement was required from the aspect of management.

Further, in the field of metalworking oils such as water-soluble cutting oils, bacteria sometimes proliferate and the oil agent is decomposed. Currently, a simple culture kit (eg, trade name = Easy Cult) is used to check for the presence of bacteria, but the results are 4
It takes 8 hours. Therefore, anti-corruption measures will be delayed and
Occurrence of bad odor due to decay may not be prevented. Furthermore, although not currently in practical use, a method for measuring the enzyme activity of bacteria (JP-A-57-74095), a fluorescent dye method (JP-A-62-138185), a filter dyeing method (JP-A-1) However, there are drawbacks such as instability of reagents for measuring enzyme activity, necessity of special equipment (fluorimeter), and complexity of dyeing method.

To solve the above-mentioned conventional drawbacks, the applicant of the present invention measures the number of viable microorganisms in a sample by staining the microorganisms and then collecting them on a hydrophobic filter or a hydrophobic filter. A method has already been proposed in which the microorganisms are dyed after being collected in the sample, then the excess dye is removed by washing, and the number of the microorganisms in the sample is measured from the coloring degree of the microorganisms (Japanese Patent Laid-Open No. 4-218392). . According to this method, it is possible to quickly and easily measure the number of microbial cells in a sample without requiring skilled techniques, specialized knowledge, special equipment, and the like. However, in the case of this method, it is not only necessary to use a device such as a dropper for collecting a sample, but also a two-step filtration operation is required, so there is a problem that the operation is complicated when actually used. It was

The inventors of the present invention have solved the above-mentioned conventional drawbacks, do not use a tool such as a dropper for collecting a sample, and can perform suction filtration operation with a syringe, so special equipment and specialized knowledge are required. It is an object of the present invention to provide a method for measuring the number of bacteria in a sample quickly and easily (within 1 minute) by a simple one-step filtration operation and a measurement kit therefor. Is.

[0007]

The present invention according to claim 1 is a method for measuring the number of bacteria in a sample, wherein a bacterial sample is introduced into a filtration container having a hydrophobic filter for detecting bacteria by suction operation. After that, it is extruded and injected into the dye liquid composition, and then suction-filtered to collect the stained bacteria on the bacteria-detecting hydrophobic filter, and the sample is obtained from the coloring degree of the bacteria-detecting hydrophobic filter. The present invention provides a rapid method for measuring the number of bacteria, which is characterized by measuring the number of bacteria therein.

Next, the present invention according to claim 8 is for collecting a sample which is equipped with a filtration container having a hydrophobic filter for detecting bacteria and a filter support therein, and a prefilter, which can be mounted on the tip of the filtration container. The present invention provides a kit for measuring the number of bacteria, which comprises a member, a dye solution composition, a container containing the dye solution composition, and a color control table.

Furthermore, the present invention according to claim 9 is a filtration container having a hydrophobic filter for detecting bacteria and a filter support therein, a sample-collecting member that can be attached to the tip of the filtration container, a dye solution composition, The present invention provides a kit for measuring the number of bacteria, which comprises a container for containing the dye liquid composition and a color control table. The invention according to claim 9 is different from the invention according to claim 8, in that the part for sampling is not equipped with a prefilter.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The method of the present invention described in claim 1 above can be suitably carried out by the kit for measuring the number of bacteria of the present invention described in claim 8 or claim 9. Therefore, hereinafter, the measuring method of the present invention according to claim 1 will be described with reference to the bacterial count measuring kit of the present invention according to claim 8 or 9.

The present invention according to claim 1 will be described.
In this measuring method, when measuring the number of bacteria in a sample, first, a bacterial sample (for example, fermented milk or fermented milk drink) is introduced by suction into a filtration container having a hydrophobic filter for detecting bacteria therein.

The filtration container has a tubular shape, that is, a tubular shape or a tubular shape, and either a plastic such as vinyl chloride or a glass can be used. Further, the capacity may be appropriately selected according to the amount of the liquid such as the dye liquid composition used. This filtration container has a hydrophobic filter for bacteria detection inside, and a support for the filter is arranged in advance. As such a filtration container, the one described in claim 8 can be preferably used.

The present invention will be described below with reference to the drawings. FIG.
FIG. 9 is an explanatory view showing one embodiment of the bacterial count measuring kit of the present invention described in claim 8. Further, FIG. 2 is an explanatory view showing a state in which the bacterial count measuring kit of the present invention described in claim 8 is combined with a commercially available syringe to prepare for bacterial count measurement. In the figure, reference numeral A is a filtration container.

The filtration container A in the kit for measuring the number of bacteria according to claim 8 has a hydrophobic filter for bacteria detection 1 and a filter support 2 inside. The filtration container A has a tubular shape, that is, a tubular shape or a tubular shape as described above, and is provided with the hydrophobic filter 1 for detecting bacteria at one end thereof. As the filtration container A,
As described above, either plastic such as vinyl chloride or glass can be used, but plastic is preferable from the viewpoint of handling.

In the method of the present invention, the hydrophobic filter 1 is used as a filter for detecting bacteria. A hydrophobic filter is weak or non-polar and easily removes excess dye, so that it is suitable as a filter for collecting bacteria. On the other hand, a polar filter is not preferable because it is not easy to remove excess dye.

As such a hydrophobic filter 1,
For example, a hydrophobic filter such as a nylon type, a fluorine type such as polytetrafluoroethylene (tetrafluoroethylene resin), a polyolefin type such as polypropylene, a polycarbonate type, or a glass type is used. Among these filters, a hydrophobic filter made of polytetrafluoroethylene or polypropylene is particularly preferable because an excessive dye can be easily removed. In addition, a hydrophilic material such as a nitrocellulose filter can be used as a hydrophobic filter by rendering it hydrophobic by surface treatment. Here, the surface treatment is coating or the like, and as the material used for coating, the above-mentioned nylon-based, fluorine-based, or polyolefin-based materials can be used.

The filtration pore size of the hydrophobic filter 1 for detecting bacteria is 0.2 to 3.0 which is usually used for collecting bacteria.
A range of about μm is sufficient. The size of the suction filtration area (area of the colored region) of the hydrophobic filter 1 for detecting bacteria is not particularly limited as long as it is easy to see. The shape of this colored area is not particularly limited, but a circular shape is desirable in terms of visibility, and its diameter is 1
It is preferably about 3 mm. The size of the entire hydrophobic filter 1 for detecting bacteria is not particularly limited, but at least the size of the colored area is required.

As the color of the hydrophobic filter 1 for detecting bacteria, a color that can be easily determined may be set in consideration of the dye used. In order to easily determine the degree of coloring, a white hydrophobic filter 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.

The filter support 2 may be made of any material, shape, etc., as long as it can support the bacteria detection hydrophobic filter 1. Usually, it is made of silicone resin or the like.

On the other hand, the other end of the filtration container A can be attached to the tip of a syringe B so that the syringe B can aspirate. In addition, if necessary, the syringe B itself may be included in the kit for measuring the number of bacteria of the present invention.

Then, as shown in FIG. 1, a pre-filter 3 is mounted inside the tip of the filtration container A (the side provided with the bacteria-detecting hydrophobic filter 1) (that is, the pre-filter 3 is attached). (Internal) Sampling member C
However, they can be freely combined. Usually, as shown in FIGS. 1 and 2, the sample collecting member C is prepared so that the tip of the filtration container A partially fits inside the member C for sampling, and when it is fitted. First, the pre-filter 3 mounted inside the member C for sampling and the hydrophobic filter 1 for bacteria detection mounted at one end of the filtration container A are in contact with each other. The sample-collecting member C has a tube shape with one end tapered, that is, a tubular shape or a tubular shape.
A sample collection port 4 is provided there so that a sample can be collected from one end of the tapered shape. Further, it is preferable that the sample collecting member C is provided with a measuring scale. In FIG. 1, one with three measuring scales is shown. It is advisable to calibrate the range of 50 to 100 μl, which is the volume of the sample that is usually used.

The prefilter 3 mounted inside the sample-collecting member C is for removing animal cells and is used when animal cells larger than bacteria, such as white blood cells, coexist in the bacterial sample. , Bacteria are allowed to pass, but such large-sized animal cells are not passed, but to collect. Therefore, in the case of a bacterial sample in which such large-sized animal cells are not present, such as fermented milk or fermented milk drink, the prefilter 3 may be omitted as shown in claim 9. When the pre-filter 3 is used, it may be pre-treated with a filter having a pore size of about 6 to 60 μm. As such a pre-filter, a polypropylene-based filter (having a filtration pore size of 7
urine collection filter (material: PVC type) for pregnancy diagnostic agents such as "Gonavi slide / Shin Gona slide" (manufactured by Mochida Pharmaceutical Co., Ltd.) in the examples described later, commercially available oil-based pens, etc. It is possible to use felt, non-woven fabric, etc.

As described above, in the present invention according to claim 1, in measuring the number of bacteria in a sample, first, a bacterial sample (for example, a fermentation sample) is placed in a filtration container A having a hydrophobic filter 1 for detecting bacteria therein. Milk or fermented milk beverage) is introduced by a suction operation, and the procedure for performing this suction operation will be described.

That is, first, the sample collecting member C having the prefilter 3 mounted therein is attached (fitted) to the tip of the filtration container A (the side provided with the bacteria detecting hydrophobic filter 1). At this time, the pre-filter 3 and the hydrophobic filter 1 for detecting bacteria are brought into contact with each other.
However, as shown in claim 9, a sample collecting member C to which the pre-filter 3 is not attached may be combined and attached. Next, the syringe B is attached to the other end of the filtration container A (the side opposite to the side provided with the bacteria detection hydrophobic filter 1) 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 preparation for the suction operation in this way. In this state, a predetermined amount of the bacterial sample is introduced by the suction operation of the syringe B from the sample collection port 4 provided at the tip of the sample collection member C. The introduction amount of the bacterial sample may be determined by using the measurement scale provided on the sample collecting member C as a guide.

The sample to which the present invention can be applied is not particularly limited, and examples thereof include urine and cutting oil,
Water-soluble metal processing oils such as rolling oil and heat treatment oil, liquid seasonings (soy sauce, sauces, etc.), liquid foods and drinks (eg fermented milk, lactic acid beverages, etc.), liquors (eg wine, sake, etc.), liquid foods, powders And solid foods (eg vegetables, fish)
In the case of, it can be widely applied to washed samples, water-soluble paints, etc., as well as river water, pond water, hot spring water, aquarium water, domestic waste water, etc. 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 measuring method according to claim 1 is a bacterium such as Escherichia coli (Escherichia coli) present in such a sample.
richia Coli), Staphylococcus au
reus), Pseudomonas aeruginosa, etc.
In particular, the measurement kit of the present invention according to claim 9 can be effectively used for rapid measurement of the number of bacteria in fermented milk and fermented milk beverages.

Next, after the bacterial sample is introduced by the suction operation of the syringe B as described above, it is once extruded and injected into the dye liquid composition D. Here, the pigment liquid composition D is put in a suitable container (for example, an eye drop bottle 5 or the like) as a kit as shown in FIG. 1, and the amount of the dye liquid composition D to be used for about one suction filtration operation is used. An appropriate container E capable of containing the dye liquid composition D, for example, a microtube having a volume of about 0.5 to 1.5 ml is combined, and the dye liquid composition D is put in this container and used for measurement of the number of bacteria. Will be forced. A container E (microtube or the like) containing such a dye liquid composition is used in combination, and a small amount of the dye liquid composition D is previously added thereto from the eye drop bottle 5 or the like, and then introduced into the syringe B. Once the bacterial sample has been extruded, it is injected into it and mixed well. As a result, the bacterial sample is stained with the dye solution composition D.

As the dye liquid composition D, a composition which can be used for both dyeing and washing is used. Specifically, a dye that can be used for dyeing bacteria and water or various buffer solutions,
It further comprises a surfactant added as necessary.

Examples of pigments that can be used for staining bacteria include safranine, fuchsin, methylene blue, methyl green, crystal violet, gentian violet, and Victoria blue B. Safranine, Fuchsin, methylene blue and methyl green are preferred. The usable pH of the buffer solution is 4 to 9, and preferably 5 to 7 from the viewpoint of dye stability. If necessary, a preservative such as ethanol or a surfactant may be added. In the case of fuchsin, the usable dye concentration is usually 0.00005 to 0.
0004% (w / v), preferably 0.0001 to 0.0003%
(W / v). If the concentration of the dye is less than 0.00005%, coloring will be insufficient, while the concentration of the dye solution will be
If it exceeds 0.0004%, it becomes difficult to remove an excessive amount of dye, which is not preferable.

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

It is appropriate that the amount of the dye liquid composition per sample is 2 to 5 times the amount of the sample. On the other hand, the amount of the sample is usually 50 to 100 μl. So, for example, if the sample volume is 1
For 00 μl, 200-500 μl of dye solution composition is required. 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 the injection as described above, a container E (such as a microtube) for containing the dye solution composition is introduced from the sample collection port 4 provided at the tip of the sample collection member C attached to the tip of the syringe B. ), The mixture of the pigment liquid composition and the bacterial sample is suction filtered by pulling the piston 6 of the syringe B. By this suction filtration operation, the stained bacteria are collected by the bacteria-detecting hydrophobic filter 1. At the same time, excess dye is removed. That is, when the piston 6 of the syringe B is pulled and sucked, the liquid containing the stained bacterial sample (mixed liquid of the bacterial sample liquid and the dye liquid composition D) is collected by the hydrophobic filter for bacteria detection 1. At the same time, excess dye is removed. According to the method of the present invention, the bacteria thus stained can be collected on the hydrophobic filter and the excess dye can be removed at the same time, which simplifies the operation process.

In the measuring method of the present invention as set forth in claim 1, the thus-collected bacteria on the hydrophobic filter 1 for detecting bacteria and the removal of excess dye are simultaneously carried out to detect bacteria. The number of bacteria in the sample is measured from the degree of coloration of the hydrophobic filter 1 of 1. That is, after the dyed bacteria are collected by the bacteria-detecting hydrophobic filter 1 in this manner, the sample-collecting part C attached to the tip of the syringe B is removed, and the tip of the filtration container A is removed. Is exposed. The degree of coloring of the bacteria present on the bacteria-detecting hydrophobic filter 1 provided at the tip of the filtration container A,
That is, the number of bacteria in the sample is measured by comparing the intensity of the color with, for example, a control table F of the color prepared in advance using a sample having a known amount of bacteria. The color comparison table F shown in FIGS. 1 and 2 is an example of the color comparison table, and the numbers (from) described in the color comparison table are shown.
Indicates "lognumber per ml".

In the measuring method of the present invention as set forth in claim 1, the thus-collected bacteria on the hydrophobic filter for bacteria detection and the removal of excess dye are simultaneously carried out to detect bacteria. The number of bacteria in the sample is measured from the degree of coloring of the hydrophobic filter. The number of bacteria in the sample is measured from the degree of coloration of the bacteria in the sample from which the excess dye has been removed in this way. The easiest way to measure the number of bacteria is to perform a visual comparison using (1) a color control table, but (2) use a colorimetric quantitative method based on optical density (OD) measurement. You can also In the case of visual judgment, it can be measured not only from the front surface of the hydrophobic filter for detecting bacteria but also from the degree of coloring on the back surface. In these cases, a color control table for each side or a calibration curve of absorbance and bacterial count may be prepared.

The above-mentioned (1) visual determination of the number of bacteria is
Specifically, the degree of coloration of the bacteria present on the hydrophobic filter for collecting bacteria, that is, the color intensity, is compared with a color control table prepared in advance using a sample having a known number of bacteria. It should be done by doing. The color control table uses a sample having a known number of bacteria, and takes a color photograph of a hydrophobic filter for detecting bacteria, which is stained and washed by the measuring method according to claim 1, or detects the stained bacteria. It can be prepared by coloring a filter paper or the like to the same extent as the hydrophobic filter for printing, or printing the same color on the paper.

For example, when measuring the number of bacteria in urine, a color control table is prepared as follows. That is,
Low concentration of urinary bacteria in healthy human urine [1ml
(Approximately 10,000 per milliliter)], but if the total number is more than 100,000 per 1 ml, it is judged that bacteriuria is suspected. Urine) is definitive. Therefore, a color comparison table that can be detected before and after this value may be created. Normally 1000 / ml, 10,000 / ml, 100,000 / m
It is sufficient to prepare a standard color contrast table for five points of 1, 1 million pieces / ml and 10 million pieces / ml, but of course, the present invention is not limited to this.

Further, the optical density (OD) of the above (2).
The colorimetric quantification by measurement is based on eluting a dye that colors bacteria present on a hydrophobic filter for detecting bacteria with an organic solvent, measuring the degree of coloration of the eluate by absorbance, and preparing a previously prepared optical density ( OD) and the calibration curve of the number of bacteria. The wavelength at the time of measuring the absorbance may be appropriately determined depending on the dye used. As the organic solvent, various alcohols can be used, 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.

[0039]

Next, the present invention will be described in detail with reference to examples. Example 1 (1) Preparation of a color control table for measuring the number of bacteria in urine As bacteria, Escherichia coli (ATCC 11
303) and Staphylococcus aure
us ) (IFO 3183) was used to perform the following experiments.
The broth broth medium was inoculated with the bacterial species and allowed to stand at 37 ° C. for 24 hours, and the culture solution was used as a standard sample bacterial suspension. On the other hand, the discharged urine of a healthy man was collected and filtered through a polytetrafluoroethylene membrane filter (manufactured by Toyo Roshi Kaisha, Ltd.) having a pore size of 0.5 μm to prepare a bacterial dilution solution. A predetermined amount of this solution for diluting bacteria was added to the above-mentioned suspension for standard samples so that the number of each bacteria was about 1,000 / ml,
10,000 pieces / ml, 100,000 pieces / ml, 100
Five kinds of standard samples were prepared so as to have a concentration of about 10 million / ml and about 10 million / ml. 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 bacterial count measuring kit 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: 0.002% of fuchsin and 0.8% of salt
Liquid containing 5% and Tween 20 at 0.05% ・ Filtration container A: outer diameter 4.6 mm, inner diameter 4 mm, length 20
mm, polypropylene tube-Sample-collecting member C: 10, 50, 100 µl tip with measuring scale, wide-mouth side inner diameter 5 mm (manufactured by Quality Corporation of the United States) -Microtube (container E for containing dye solution composition):
1.5 ml volume, polypropylene (manufactured by American Quality Co., Ltd.)-Bacterial detection hydrophobic filter 1: Polytetrafluoroethylene membrane filter (pore size 3 μm, colored area diameter 1.5 mm) -Filter support 2: Silicone tube (outside) Diameter 4m
m, inner diameter 1.5 mm) cut into a length of 7 mm ・ Eye drop bottle 5:10 ml volume, made of polypropylene ・ Prefilter 3: diameter 4 mm, length 4 mm, made of polyvinyl alcohol, filtration hole diameter = 60 μm

As shown in FIG. 2, a 3 ml syringe B was attached to one end of the filtration container A, while the other end of the filtration container A (the side provided with the hydrophobic filter 1 for detecting bacteria) was used.
The sample collecting member C having the pre-filter 3 mounted therein is detachably fitted therein, and the bacterial sample 100 μm is taken through a sample collecting port 4 provided at the tip of the sample collecting member C.
1 was collected by the suction operation of the syringe B. Next, after introducing the bacterial sample by the suction operation of the syringe B as described above, it was once extruded and injected into a container E (microtube) containing 350 μl (7 to 8 drops) of the dye liquid composition D.

Immediately after the injection, the pigment liquid composition contained in the container E (the above-mentioned microtube) is supplied from the sample collection port 4 provided at the tip of the sample collection member C attached to the tip of the syringe B. The mixture of bacterium and bacterial sample was suction filtered by pulling on the piston 6 of the syringe. By this suction filtration operation, the stained bacteria were collected on the bacteria-detecting hydrophobic filter 1, and at the same time, the excess dye was removed. After collecting the stained bacteria on the hydrophobic filter for bacteria detection 1 in this manner, the syringe B
Remove the sampling member C attached to the tip of
The tip of the filtration container A was exposed, and the degree of coloration of bacteria existing on the bacteria-detecting hydrophobic filter 1 provided at the tip of the filtration container A was visually observed. there were.

[0043]

[Table 1]

These colored filters were color photographed as standard samples and used as a color control table. In addition, there was no significant difference in the degree of coloring between Escherichia coli and Staphylococcus aureus.

(2) Measurement of number of bacteria in urine Using the urine discharged from healthy subjects and urine discharged from various patients as samples, the same operation as shown in the above (1) Preparation of color control table for measuring urinary bacteria was performed. Then, the number of bacteria in urine was measured by visual judgment in comparison with the color control table prepared in (1) above. The results are shown in Table 2. For comparison, CLE
The number of bacteria measured on a D-agar plate medium at 37 ° C. for 24 hours is also shown.

[0046]

[Table 2]

Example 2 (1) Preparation of a color control table for measuring the number of bacteria in fermented milk beverages Commercially available yogurt (manufactured directly by Agricultural Co., Ltd., trade name: grated apple) was treated with water to obtain 10 ⁵ bacteria / About 1 ml
Three kinds of standard samples were prepared by diluting to about 0 ⁶ pieces / ml and about 10 ⁷ pieces / ml. The number of bacteria was measured by the culturing method by counting the number of colonies after culturing at 37 ° C. for 72 hours on a plate-count agar medium containing BCP (manufactured by Eiken Chemical Co., Ltd.).

The bacterial count measuring kit used was the measuring kit used in Example 1 from which the prefilter was removed, and the reagents, instruments, and operating method were the same as in Example 1.

100 μl of the standard sample was stained in the same manner as in Example 1, suction filtered, and then visually observed for the degree of coloring of bacteria present on the hydrophobic filter 1 for detecting bacteria. The results are shown in Table 3.

[0050]

[Table 3]

These colored filters were color photographed as standard samples and used as a color control table.

(2) Measurement of the number of bacteria in fermented milk beverages Three kinds of yogurt (manufactured by direct sales of Agricultural Products, brand name: grated apple, plain type, grated carrot) stored in a refrigerator for a predetermined period were multiplied by 1000 times with water. The sample was diluted. As in the case of the standard sample in (1) above, 100 μl of each was subjected to a staining operation, and suction filtration was performed. By this operation, the dyed bacteria were collected on the bacteria-detecting hydrophobic filter 1 and the excess dye was removed at the same time. The degree of coloring of the bacteria present on the hydrophobic filter for detecting bacteria 1 was visually observed, and compared with the color control table prepared in (1) above to make a visual judgment. To determine the number of bacteria in yogurt, a dilution factor of 10
The number of bacteria was measured by multiplying by 00 times (10 ³ ). The measurement time was 60 seconds. The results are shown in Table 4. In addition,
For comparison, the number of bacteria measured by the culture method (BCP-added plate count agar medium, Eiken Chemical Co., Ltd., 37 ° C., 72-hour culture) is also shown.

[0053]

[Table 4]

Comparative Example 1 According to the method described in JP-A-4-218392,
When the number of bacteria in urine was measured, the measurement time was 3 minutes.

[0055]

According to the method of the present invention described in claim 1,
Without using a dropper or other device to collect the sample, it is possible to collect the stained bacteria on the hydrophobic filter and remove excess dye at the same time by a one-step suction filtration operation using a syringe. Can be measured quickly and easily. According to the method of the present invention as set forth in claim 1, it is possible to make the determination within 1 minute (about 30 seconds if used). Further, according to the method of the present invention described in claim 1, it is possible to measure the number of bacteria in a sample without requiring specialized technology or knowledge. Moreover, according to the method of the present invention as set forth in claim 1, since the presence or absence of bacteria can be detected without culturing the bacteria, it can be utilized for sample screening of bacterial tests in hospitals, which contributes to cost reduction of patients and hospitals. be able to. Further, in the method of the present invention according to claim 1,
Since no special equipment is required, it can be measured by small hospitals, clinics, or even ordinary people. Furthermore, the method of the present invention according to claim 1 can be applied to many bacteria by selecting the filtration pore size of the hydrophobic filter, and therefore the application range is extremely wide. According to the method of the present invention as set forth in claim 1, the number of bacteria can be known before the shipment of food (yogurt or the like), which can contribute to the quality control of food.

According to the bacteria count measuring kit of the present invention as set forth in claims 8 and 9, there is no need to perform operations such as collecting a sample with a dropper and dropping a reagent with a reagent bottle, and performing a one-step filtration operation with a syringe. Therefore, the operation is extremely simple. Also, since no skill is required and no equipment is required,
Any place for measurement. Further, the bacterial count measuring kit of the present invention according to claims 8 and 9 is extremely simple and inexpensive, and does not require any special equipment, so that it can be used in any field. There is an advantage. In particular, since it can be used by ordinary amateurs, it is possible to easily inspect the water quality around us, such as river water, pond water, seawater, and hot spring water, which not only contributes to environmental conservation, but also contributes to the preservation of liquid foods and solids. It has the characteristic that it is possible to inspect the number of bacteria in foods such as foods. Furthermore, according to the bacterial count measuring kit of the present invention according to claims 8 and 9,
After the measurement is completed, a sample that requires identification of bacteria can be collected by suction, and if the thin tube is sealed by heat treatment or the like, it can be used as a transport container to an examination room. Therefore, the present invention includes the field of urinalysis (diagnosis area) and the field of metal processing,
It can be widely used for various inspections.

[Brief description of drawings]

FIG. 1 is an explanatory view showing one embodiment of the kit for measuring the number of bacteria of the present invention according to claim 8.

FIG. 2 is an explanatory view showing a state in which the bacterial count measuring kit of the present invention according to claim 8 is combined with a commercially available syringe to prepare for bacterial count measurement.

[Description of sign]

 Reference Signs List A Filtering container B Syringe C Sampling member D Dye solution composition E Container for containing the dye solution F Color control table 1 Hydrophobic filter for bacteria detection 2 Filter support 3 Prefilter 4 Sampling port 5 Eye drop bottle 6 Piston

Claims (9)

[Claims] 1. A method for measuring the number of bacteria in a sample, which comprises introducing a bacterial sample into a filtration container having a hydrophobic filter for detecting bacteria by a suction operation, and then extruding and injecting the composition into a dye liquid composition, Then, suction filtration is performed to collect the stained bacteria on the bacteria detection hydrophobic filter, and the number of bacteria in the sample is measured from the coloring degree of the bacteria detection hydrophobic filter. How to quickly measure numbers. 2. The method according to claim 1, wherein the dye liquid composition comprises a dye, a buffer solution and a surfactant. 3. The dye concentration in the dye liquid composition is 0.00005.
The method according to claim 1, which is about 0.0004% (w / v). 4. The concentration of the surfactant in the dye liquid composition is 0.
The method according to claim 1, which is 001 to 1.0% (w / v). 5. The method according to claim 1, wherein the filtration container has a sample-collecting part having a pre-filter mounted therein at the tip thereof. 6. The method according to claim 1, wherein the sample is urine. 7. The method according to claim 1, wherein the sample is fermented milk or a fermented milk drink. 8. A sampling container having a filtration container having a hydrophobic filter for detecting bacteria and a filter support therein, and a prefilter, which can be attached to the tip of the filtration container, a dye solution composition, and the dye solution. A kit for measuring the number of bacteria, comprising a container for containing the composition and a color control table. 9. A filtration container having therein a hydrophobic filter for detecting bacteria and a filter support, a sample-collecting member that can be attached to the tip of the filtration container, a dye solution composition, a container for containing the dye solution composition, and Bacteria count kit consisting of a color control table.