JPH08322594A - Bacteria count measuring method and bacteria count measuring tool - Google Patents

Abstract

(57) [Abstract] (Correction) [PROBLEMS] To provide a method and a measuring tool for rapidly (within 1 minute) and simply measuring the number of bacteria in a sample by a one-step filtration operation. To do. Kind Code: A1 A hydrophobic filter for detecting bacteria is provided inside, and a dye solution composition is pre-disposed on the side from which a bacterial sample is collected as seen from the filter, and the filter is provided on the opposite side to the filter. In a tube-shaped filtration container in which the support of the above is preliminarily placed, the bacterial sample is collected from the side from which the bacterial sample is collected, and then suction-filtered from the side of the support to collect and collect the stained bacteria on the filter. A method for rapid measurement of the number of bacteria, which comprises simultaneously performing removal of a dye and measuring the number of bacteria in a sample from the degree of coloring of the filter.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method and a method for rapidly and simply measuring the number of bacteria in a sample by a one-step filtration operation without requiring advanced technology or specialized knowledge. Regarding ingredients. INDUSTRIAL APPLICABILITY The present invention can be effectively used in a wide range of fields such as a diagnostic field in which bacteria in urine are a problem, a metal processing oil field, a dye field in which decay is a problem, a food field, and an environmental field such as hot spring water. You can

[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, bacteriuria is diagnosed, 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 a urinalysis, outpatient urine samples are tested for culture in a bacterial laboratory or laboratory, and the results are only available to the doctor a few days later. In order to make a highly accurate diagnosis, a rapid bacterial assay 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. But,
Since a two-step filtration operation is required, there is a problem that the operation is complicated when actually used.

The present inventors have solved such conventional drawbacks, do not require special equipment or specialized knowledge, and can quickly (within 1 minute) and simply by one filtration operation. An object of the present invention is to provide a method for measuring the number of bacteria in a sample and a measuring tool therefor.

[0007]

The present invention according to claim 1 provides a method for measuring the number of bacteria in a sample, which has a hydrophobic filter for detecting bacteria therein and which is seen from the filter. A dye solution composition is pre-disposed on the side from which a bacterial sample is collected, and a tube-shaped filtration container in which the support of the filter is pre-disposed on the side opposite to the filter, and the bacterial sample is collected from the side from which the bacterial sample is collected. After putting in and dyeing, suction filtration from the support side, collecting the stained bacteria on the filter and removing excess dye at the same time, and measuring the number of bacteria in the sample from the degree of coloring of the filter The present invention provides a rapid measurement method for the number of bacteria.

Next, the present invention as set forth in claim 2 is a method for measuring the number of bacteria in a sample, which has a hydrophobic filter for detecting bacteria therein and which is a bacterial sample as seen from the filter. Dispose the dye composition in advance on the collecting side,
A bacterial sample is placed in a tubular filtration container in which the support of the filter, a piston, and an aqueous solution are arranged on the opposite side of the filter as seen from the filter, and after staining, filtration is performed by moving the piston in the aqueous solution in the filtration container. The present invention provides a rapid method for measuring the number of bacteria, wherein the number of bacteria in a sample is measured from the degree of coloration of the filter by simultaneously collecting the stained bacteria on the filter and removing excess dye. To do. The invention according to claim 2 is characterized in that, in the invention according to claim 1, a piston and an aqueous solution are further arranged in advance in the filtration container.

Further, according to the present invention as set forth in claim 8, a tube-shaped filtration container having a sample collection port at one end and capable of suction by a syringe at the other end is sequentially arranged from the sample collection port side. And a dye solution composition, a hydrophobic filter for detecting bacteria, and a filter support, which provide a bacteria count measuring tool.

Further, the present invention according to claim 9 is such that a thin tube for sampling is attached to one end, and the other end is placed in a tubular filtration container capable of being sucked by a syringe in order from the thin tube side. Provided is a bacteria count measuring device comprising a prefilter, a dye liquid composition, a hydrophobic filter for detecting bacteria, a filter support, a piston movable in the filtration container, an aqueous solution, and a stopper for preventing the leakage of the aqueous solution. To do. In the invention according to claim 9, in the invention according to claim 8, a prefilter, a piston movable in the filtration container, an aqueous solution, and a stopper for preventing leakage of the aqueous solution are further added to the inside of the filtration container. It has a characteristic.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The method of the present invention described in claim 1 can be suitably carried out by the bacteria count measuring tool of the present invention described in claim 8. Further, the above-mentioned claim 2
The described method of the present invention can be suitably carried out by the bacterial count measuring instrument of the present invention described in claim 9.
Therefore, hereinafter, the measuring method of the present invention described in claim 1 will be described with reference to the bacterial count measuring tool of the present invention described in claim 8, and then the book described in claim 9. The measuring method of the present invention described in claim 2 will be described with reference to the bacterial count measuring device of the invention.

[0012] First, the present invention as set forth in claim 1 will be described. In this measuring method, in measuring the number of bacteria in a sample, a bacteria-detecting hydrophobic filter is provided inside and the bacteria are seen from the filter. The dye solution composition is pre-disposed on the side from which the sample is collected (on the left side of the filter in FIG. 1, which will be described later), and on the opposite side from the filter (on the right side of the filter in FIG. 1, which will be described later). A bacterial sample (for example, fermented milk or a lactic acid bacterium beverage) is placed and dyed from the side from which the bacterial sample is collected, in a tubular filter container in which the support of the filter is previously arranged. 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 inside, and a dye solution composition is previously arranged on the side from which a bacterial sample is collected as seen from the filter, and the filter support is provided on the opposite side as seen from the filter. The body is arranged in advance. As such a filtration container, the one described in claim 8 can be preferably used.

Next, the present invention as set forth in claim 2 will be described. In this measuring method, when the number of bacteria in a sample is measured, a hydrophobic filter for detecting bacteria is provided inside, and the bacteria are not seen from the filter. The dye solution composition on the side from which the bacterial sample is collected (on the left side of the filter in FIG. 2, which will be described later), and on the opposite side of the filter (on the right side of the filter in FIG. 2, which will be described later). 1) A bacterial sample is placed in a tubular filtration container in which a support for the filter, a piston, and an aqueous solution are previously arranged and stained. The filtration container according to the present invention described in claim 2 has a tubular shape, that is, a tubular shape or a tubular shape, as in the invention described in claim 1, and both a plastic material such as vinyl chloride and a glass material are used. can do. Further, the volume may be appropriately selected depending on the amount of the dyeing composition to be used. This filtration container has a hydrophobic filter for bacteria detection inside, and a dye solution composition is pre-disposed on the side from which a bacterial sample is collected as seen from the filter, and the dye solution composition is placed on the opposite side as seen from the filter. A filter support, a piston, and an aqueous solution are previously arranged. As such a filtration container, the one described in claim 9 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 bacteria count measuring device of the present invention described in claim 8. In the figure, reference numeral A is a filtration container.

That is, the filtration container A in the device for measuring the number of bacteria according to claim 8 is a tubular container having a sample collection port 15 at one end and capable of suction by the syringe 13 at the other end. Then, inside the filtration container A, the dye liquid composition 5 is sequentially provided from the side of the sampling port 15 side.
Hydrophobic filter 6 for detecting bacteria and filter support 7
Are arranged. In addition, if necessary, the syringe 13 can be included in the bacteria count measuring device of the present invention.

The tip of the sample collection port 15 provided at one end of the filtration container A may be open, or a suitable stopper 16 may be arranged to prevent the dye liquid composition 5 from leaking. It may be. An internal insertion tube or the like may be used instead of the plug 16.

On the other hand, the other end of the filtration container A can be fitted with the tip of a syringe 13 to enable suction by the syringe 13. However, as described above, the syringe 13 itself may be included in the bacteria count measuring instrument of the present invention, if necessary.

As the pigment liquid composition 5 disposed inside the filtration container A, a composition capable of both staining and washing is used, and specifically, a pigment that can be used for staining bacteria, It is composed of water or various buffer solutions.

Examples of pigments that can be used for staining bacteria include safranin, fuchsin, methylene blue, methyl green, crystal violet, gentian violet, and Victoria blue B. Safranin, especially from the viewpoint of easy determination, 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 0.0001 to 0.00.
045% (w / v), preferably 0.00015 to 0.0003%
(W / v). Here, if the concentration of the dye is less than 0.0001%, the coloring is insufficient, while the concentration of the dye solution is 0.
If the content exceeds 0%, it becomes difficult to remove the excess dye, so that both are not preferable.

If necessary, 0.1 to 15% (v / v) may be added when a preservative such as ethanol is added, and a surfactant may be added when a surfactant is added. May be added at a rate 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, it is not necessary to use more than 5 times the amount of the sample. The amount of the dye liquid composition used is preferably the same as the amount used when creating a color control table or a calibration curve and the amount used for a sample of unknown bacterial count in order to suppress errors.

Further, inside the filtration container A, when viewed from the side of the sample collection port 15, a hydrophobic filter 6 for bacteria detection is arranged next to the dye liquid composition 5. As the filter for detecting bacteria, the hydrophobic filter 6 is used in the method of the present invention. The hydrophobic filter has weak polarity or no polarity and is easy to remove excess dye, and is therefore 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 6,
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 it is easy to remove excess dye. 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 6 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 (colored area) of the hydrophobic filter 6 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 6 for detecting bacteria is not particularly limited, but at least the size of the colored area is necessary.

As the color of the hydrophobic filter 6 for detecting bacteria, a color that can be easily determined may be set in consideration of the dye used. A white hydrophobic filter is preferable for easily determining the degree of coloring. A transparent or semi-transparent hydrophobic filter can also be used, and the determination becomes easy when the hydrophobic filter is placed on a white sheet for determination.

Further, when viewed from the side of the sampling port 15 inside the filtration container A, next to the pigment liquid composition 5 and the hydrophobic filter 6 for detecting bacteria, a filter support 7 is further provided in order. It is arranged. The filter support 7 may be made of any material and may have any shape as long as it can support the bacteria detection hydrophobic filter 6. Usually, it is made of silicone resin or the like.

As described above, the filtration container A in the device for measuring the number of bacteria according to claim 8 has a sampling port 15 at one end as described above.
And the other end of which is a tubular shape capable of being sucked by a syringe 13, and inside the filtration container A, the dye liquid composition 5 is sequentially provided from the side of the sampling port 15 side. A hydrophobic filter 6 for detecting bacteria and a filter support 7 are arranged.

The bacterial count measuring instrument of the present invention according to claim 8 is:
It has a structure as described above. In the measuring method according to the first aspect, first, an appropriate bacterial sample, preferably fermented milk or a lactic acid bacterium beverage, is put into such a filter container A and dyed.

The sample to which the present invention can be applied is not particularly limited, and examples thereof include urine, 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. It should be noted that the sample may be appropriately diluted as needed, or, for example, in the case of a solid food or the like, it may be diluted after crushing and then used as the 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 invention according to claim 1 and the invention according to claim 8 can be effectively used for rapid measurement of the number of lactic acid bacteria in fermented milk or a lactic acid bacterium beverage.

In the invention according to claim 1, the filtration container A
The procedure for adding and staining the bacterial sample as described above will be described below. First, the syringe 13 is attached to a predetermined site on the opposite side of the sampling port 15 side of the filtration container A (note that this operation is not necessary when the syringe is set in advance), and then sampling is performed. A bacterial sample is added through the mouth 15 and stained. Specifically, for example, a bacterial sample is dropped from the sample collection port 15 into the dye liquid composition 5 using a dropper or the like, and is thoroughly mixed with the dye liquid composition 5 to stain the bacterial sample. When a plug 16 is provided near the sample collection port 15, the plug 16 may be removed and then a bacterial sample may be dropped into the dye liquid composition 5 using a dropper or the like. When an internal insertion tube is provided in the vicinity of the sampling port instead of the stopper 16, a dropper can be passed through the center of the tube to drop the bacterial sample into the dye liquid composition 5. , Convenient.

Next, the syringe 14 of the syringe 13 is pulled and sucked, and filtered (suction-filtered from the filter support 7 side) to collect the stained bacteria on the bacteria-detecting hydrophobic filter 6 and to collect excess bacteria. Dye removal is performed at the same time. That is, when the syringe 14 of the syringe 13 is pulled and sucked, the liquid containing the stained bacterial sample (mixed liquid of the bacterial sample liquid and the dye liquid composition 5) is transferred to the hydrophobic filter 6 for detecting bacteria.
At the same time, the 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 for detecting bacteria and the removal of the 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 determined 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 determination, it can be measured not only by the front surface of the hydrophobic filter for detecting bacteria but also by the coloring degree of the back surface. In these cases, a color control table for each side and a calibration curve for absorbance and bacterial count may be created.

The above-mentioned (1) visual determination of the number of bacteria is
Specifically, compare the degree of coloring of bacteria present on the hydrophobic filter for collecting bacteria, that is, the intensity of color, with a color comparison table prepared in advance using a sample with a known number of bacteria. It can 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,
Bacteria in urine are low in urine of healthy people [1 ml even when high)
(Around 10,000 per milliliter)], but a total of 100,000 or more per ml is judged to be suspected to be bacteriuria, and if 1 million or more per ml, bacterial infection (bacteria Urinary) is definite. Therefore, it suffices to create a color contrast table that can be detected before and after this value. Normally 1,000 pieces / ml, 10,000 pieces / ml, 100,000 pieces / ml, 1
It is sufficient to create a standard color contrast table for 5 points of, 000,000 pieces / ml and 10,000,000 pieces / ml, but of course, the present invention is not limited to this.

Further, the optical density (OD) of the above (2).
Colorimetric quantification by measurement is a dye coloring bacteria present on a hydrophobic filter for bacteria detection, eluting with an organic solvent, the coloring degree of the eluate is measured by absorbance, and an optical density created in advance ( OD) and the number of bacteria can be collated and quantified. 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 the dye, the degree of coloring of the sample diluted to various concentrations is measured by a microplate reader by the optical density (OD) of 492 nm, On the other hand, the number of bacteria in the sample solution of the same sample diluted to various concentrations was calculated by counting the number of colonies in the agar plate culture method, and a calibration curve for the number of bacteria and optical density was created from the results of both. do it.

Next, the invention according to claim 2 will be described with reference to the invention according to claim 9 with reference to the drawings. Figure 2
FIG. 11 is an explanatory view showing one embodiment of the bacteria count measuring tool of the present invention described in claim 9. In addition, FIG. 3 is an explanatory view showing a manner in which a syringe is attached to the bacteria count measuring tool shown in FIG. 2 to sample a sample. Furthermore, FIG. 4 ((A) to FIG. 4)
(F) shows the procedure of the method of using this bacteria count measuring tool. In the figure, reference numeral A is a filtration container.

That is, the filtration container A in the device for measuring the number of bacteria according to claim 9 is a tubular container in which the sample collecting thin tube 1 is attached to one end, and the other end allows suction by the syringe 13. The inside of the filtration container A is sequentially moved from the side of the thin tube 1 through the pre-filter 4, the dye liquid composition 5, the hydrophobic filter 6 for detecting bacteria, the filter support 7, and the inside of the filtration container. A possible piston 8, an aqueous solution 9 and a plug 12 preventing the leakage of said aqueous solution are arranged. In addition, if necessary, the syringe 13 can be included in the bacteria count measuring device of the present invention.

The sampling thin tube 1 attached to one end of the filtration container A is made of a flexible material, for example, a resin such as polyethylene.
A sampling scale 2 is provided, which indicates a standard for collecting a bacterial sample. 50-100 μl, which is the volume of the sample that is normally used
It is good to add a scale to the range. Furthermore, the tip of the sample-collecting thin tube 1 may be open, but it is usually sealed to avoid contamination, and it is advisable to add a cutting mark 3 as shown in the drawing.

On the other hand, the other end of the filtration container A can be fitted with the tip of the syringe 13 so that the syringe 13 can suck the tip. However, as described above, the syringe 13 itself may be included in the bacteria count measuring instrument of the present invention, if necessary.

Inside the filtration container A, the thin tube 1
When viewed from the side, first, the pre-filter 4 is arranged. This pre-filter 4 is for removing animal cells, and when larger animal cells than bacteria, such as white blood cells, coexist in a bacterial sample, bacteria are allowed to pass through, but such large animal cells are It is for collecting without passing. As the pre-filter 4, for example, one having a filtration pore size of about 6 to 8 μm may be used for pre-treatment.
As such a pre-filter, a polypropylene-based filter (having a filter pore size of about 7 μm), a urine collection filter for a pregnancy diagnostic drug such as “Gonavi Slide / New Gonavi Slide” (manufactured by Mochida Pharmaceutical Co., Ltd.) in Examples described later (Material: PVC type), felt, non-woven fabric, etc. used in commercially available oil-based pens can be used.

Next, inside the filtration container A, the thin tube 1
When viewed from the side, the dye solution composition 5 is disposed next to the prefilter 4. However, usually, in order to prevent the contact between the prefilter 4 and the dye liquid composition 5, the internal insertion tube 10 is arranged between them. The inner insertion tube 10 is made of a synthetic resin such as a silicone resin, and can draw a bacterial sample into the dye solution composition 5 through the prefilter 4 when aspirated by a syringe, but before use. The purpose is to prevent contact between the prefilter 4 and the dye liquid composition 5.

Further, inside the filtration container A, when viewed from the side of the thin tube 1, the pre-filter 4 and the dye liquid composition 5 are disposed next to the bacteria-detecting hydrophobic filter 6. Here, as a filter for detecting bacteria,
The same hydrophobic filter 6 as described in the description of the invention of claim 1 and the invention of claim 8 is used.

Further, inside the filtration container A, when viewed from the side of the thin tube 1, next to the prefilter 4, the dye liquid composition 5, and the hydrophobic filter 6 for detecting bacteria, a filter support 7 is further provided. A piston 8, which is movable in the filtration container, an aqueous solution 9, and a stopper 12 which prevents the aqueous solution from leaking are sequentially arranged. The filter support 7 may be made of any material and may have any shape as long as it can support the bacteria detection hydrophobic filter 6. Usually, it is made of silicone resin or the like.

Next, the piston 8 movable in the filtration container has a slightly smaller outer diameter than the filtration container A, and is slid in the filtration container A by suction or pressurization of the syringe 13. And can move. This piston 8
The material is not particularly limited, but polyacrylamide gel, konjac, etc. are preferable from the viewpoints of light weight and low cost.

In addition, the aqueous solution 9 is combined with the piston 8 to prevent a bacterial sample from entering the syringe 13 and to protect the syringe 13 from bacterial contamination.
This is to isolate the 3rd. Therefore, usually, water or an alcohol-containing aqueous solution can be used. The amount of the aqueous solution varies depending on the shape and size of the filtration container, the amount of the dyeing solution, and the like, but is usually about 500 to 1000 μl.

Furthermore, a stopper 12 for preventing the leakage of the aqueous solution 9
Is disposed at the end of the filtration container A and the end on the side to which the syringe 13 is attached. Usually, the stopper 12 is removed, and the syringe 13 is attached and used as shown in FIG. The plug 12 is not particularly limited in its material and shape as long as it can play a role as a plug for preventing the leakage of the aqueous solution 9. In addition, normally, this stopper 1
In addition to No. 2, in order to effectively prevent the aqueous solution 9 from leaking, an internal insertion tube 11 may be arranged inside (inside) the stopper 12. This internal insertion tube 11 is made of a synthetic resin such as a silicone resin, like the internal insertion tube 10 described above, and effectively prevents the aqueous solution 9 from leaking when the plug 12 is removed. belongs to.

The filtration container A in the device for measuring the number of bacteria according to claim 9 has the structure as described above. In the measuring method according to claim 2, first, such a filter container A is used.
Bacterial sample is put in and stained.

The sample to which the present invention described in claim 2 can be applied is not particularly limited, and examples thereof include those described in the description of the present invention described in claim 1. However, for the measurement of the number of lactic acid bacteria in the fermented milk, the method according to claim 1 and the measuring tool according to claim 8 are simple and more suitable.

The procedure of placing the above-mentioned bacterial sample in the filtration container A and staining it will be described with reference to FIG. First, the syringe 13 is attached to a predetermined portion of the filtration container A on the side opposite to the sample collection thin tube 1 side (note that this operation is not necessary if the syringe is set in advance), and then cut. After cutting off the tip of the sampling thin tube 1 by the mark 3,
As shown in FIG. 4A, the side of the filtration container A on which the syringe 13 is mounted is placed below the horizontal position to collect a bacterial sample. Next, as shown in FIG. 4 (a), after separating the tip of the sample collecting thin tube 1 from the sample solution, the syringe 14 of the syringe 13 is pulled and sucked to extract the bacterial sample in the filtration container A, specifically Is introduced into the upper portion of the dye liquid composition 5 (on the side of the sample collecting thin tube 1 from the dye liquid composition). Thereafter, as shown in FIG. 4C, the syringe 14 of the syringe 13 is pushed (pressurized) to push the dye liquid composition 5 back to its original position. At this time, the air that has entered at the time of introducing the bacterial sample is expelled. Then, FIG.
As shown in (d), the air bubbles are moved with the syringe 13 mounting side above the horizontal position, and subsequently, as shown in FIG. To the original position. Due to the movement of the bubbles, the bacterial sample liquid and the pigment liquid composition 5 are mixed, and the bacterial sample is stained. Usually, this operation is performed at least once.

After staining the bacterial sample in this manner, the bacterial sample is filtered by the movement of the piston 8 in the aqueous solution 9 of the filtration container A, and the hydrophobic filter 6 for bacterial detection collects the stained bacteria and the excess dye. And simultaneously remove. 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.

The procedure for filtering by moving the piston 8 in the aqueous solution 9 of the filtration container A is as shown in FIG. 4F, and the syringe of the syringe 13 is kept in the state of FIG. 4E. When 14 is pulled and sucked, the piston 8 moves in the aqueous solution 9 of the filtration container A, and along with this, a liquid containing a stained bacterial sample (mixed liquid of the bacterial sample liquid and the dye liquid composition 5) is generated. The excess dye is removed at the same time as being collected by the bacteria detection hydrophobic filter 6.

In this way, after collecting the stained bacteria on the hydrophobic filter for bacteria detection and removing excess dye at the same time, the bacteria detection is carried out in the same manner as in the present invention according to claim 1. The number of bacteria in the sample may be measured from the degree of coloring of the hydrophobic filter for use.

[0054]

EXAMPLES 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 lactic acid bacteria Commercially available yogurt (manufactured directly by Agricultural Farm, brand name: grated apple) was mixed with water to give a lactic acid bacteria count of about 10 ⁵ cells / ml.
Three types of standard samples were prepared by diluting to about 10 ⁶ cells / ml and about 10 ⁷ cells / ml. In addition,
The number of lactic acid bacteria by the culturing method was counted by the number of lactic acid bacteria after culturing at 37 ° C. for 72 hours in a BCP-added plate count agar medium (manufactured by Eiken Chemical Co., Ltd.).

The bacteria count measuring tool shown in FIG. 1 was used for the experiment. The dimensions and reagents were as follows.・ Filtration container A: outer diameter 5 mm, inner diameter 4 mm, length 135 m
m, made of vinyl chloride-Dye liquid composition 5: pH 7 phosphate buffer containing 0.0002% of fuchsin and 0.05% of Tween 20 300 μl-Hydrophobic filter for detecting bacteria 6: Polytetrafluoroethylene Made membrane filter (pore diameter 3 μm, colored area diameter 1.5 mm) ・ Filter support 7: Silicone resin perforated plug (outer diameter 4 mm, hole diameter 1.5 mm, length 7 mm)

100 μl of each of the standard samples was added to the dye liquid composition from the sampling port, and the syringe 14 of the syringe 13 was pulled and suction filtered. By this operation, the dyed lactic acid bacteria were collected on the bacteria detection hydrophobic filter 6 and the excess dye was removed at the same time. The coloring degree of lactic acid bacteria (coloring degree of circle having a diameter of 1.5 mm) existing on the hydrophobic filter 6 for detecting bacteria was visually observed and the results are shown in Table 1 below.

[0057]

[Table 1]

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

(2) Measurement of Lactic Acid Bacteria Count Using the same bacteria count measuring device as shown in (1) above and shown in FIG. 1, yogurt stored in a refrigerator for 2 weeks, 3 weeks, and 4 weeks, respectively, has the following lactic acid bacteria count. It measured like this. The size and reagent of the bacteria count measuring tool shown in FIG. 1 were the same as those in (1) above.

That is, three kinds of yogurt (manufactured directly by Agricultural Farm, brand name: grated apple, plain type, grated carrot) stored in a refrigerator for a predetermined period in a bacteria count measuring device shown in FIG. 100 μl of each diluted product was added and suction filtration was performed. By this operation, the dyed bacteria were collected on the bacteria detection hydrophobic filter 6 and the excess dye was removed at the same time. The degree of coloring of the lactic acid bacteria present on the hydrophobic filter 6 for detecting bacteria (the degree of coloring of a circle having a diameter of 1.5 mm) was visually observed, and compared with the color control table prepared in (1) above, and visually observed. Was determined by. In order to measure the number of lactic acid bacteria in yogurt, the number of lactic acid bacteria was measured by multiplying the dilution ratio by 1000 times (10 ³ ). The measurement time was 60 seconds. The results are shown in Table 2. For comparison, the culture method (BCP-added plate count agar medium, manufactured by Eiken Chemical Co., Ltd., 3
The number of lactic acid bacteria measured at 7 ° C for 72 hours was also displayed.

[0061]

[Table 2]

Example 2 (1) Preparation of color control table for measuring the number of bacteria in urine Escherichia coli (ATCC 11303) was used for the experiment. First, the urine discharged from a healthy male was collected, 0.1% of glucose was added thereto, and sterile filtered with a polytetrafluoroethylene (PTFE) membrane filter (made by Toyo Roshi Kaisha, Ltd.) having a pore size of 0.5 μm. The medium was used. This medium was inoculated with the above-mentioned Escherichia coli, and static culture was carried out at 37 ° C. for 24 hours to prepare a bacterial culture solution as a bacterial suspension for standard samples.
On the other hand, the same discharged urine of a healthy male was obtained with a filtration pore size of 0.5μ
m was filtered through a polytetrafluoroethylene membrane filter (manufactured by Toyo Roshi Kaisha, Ltd.) to prepare a solution for bacterial dilution. By adding a predetermined amount of this bacterial dilution solution to the standard sample bacterial suspension, the number of bacteria will be about 1,000 cells / ml, 10,000 cells / ml, 100,000 cells / ml, 1 million cells / ml. m
Five kinds of standard samples were prepared so that the concentration was about 1 and about 10 million / ml. The number of bacteria by the culturing method was counted by the number of colonies after culturing on a CLED agar plate medium at 37 ° C. for 24 hours.

The bacteria count measuring device shown in FIG. 2 was used for the experiment. The dimensions and reagents were as follows.・ Filtration container A: outer diameter 5 mm, inner diameter 4 mm, length 135 m
m, made of vinyl chloride ・ Sampling thin tube 1: outer diameter 2 mm, inner diameter 1 mm, polyethylene ・ Pre-filter 4: outer diameter 4 mm, inner diameter 4 mm, polyvinyl alcohol, filtration pore size = 60 μm ・ Staining liquid composition 5: pH 7 Phosphate buffer solution containing 0.0002% of fuchsin and 0.05% of Tween 20 300 μl ・ Bacterial detection hydrophobic filter 6: Polytetrafluoroethylene membrane filter (pore size 3 μm, colored area diameter 1.5 mm)・ Filter support 7: Perforated stopper made of silicone resin (outer diameter 4 mm, hole diameter 1.5 mm, length 7 mm) ・ Piston 8: Material; Konjac, length 5 mm ・ Aqueous solution aspirated by syringe 9: Water 800 μl

In the bacteria count measuring instrument as described above, when the syringe 13 sucks the aqueous solution 9, the aqueous solution 9 is sucked into the syringe 13, the piston 8 moves accordingly, and the dyeing liquid composition 5 is filtered to remove the stained bacteria. It had a structure of being collected by the hydrophobic filter 6 for detecting bacteria. Aspirate the standard sample (bacterial sample) to the scale at the position shown in FIG.
l), the thin tube 1 for sampling was separated from the bacterial sample, further sucked, and the bacterial sample was introduced into the filtration container A. Then, the syringe 14 of the syringe 13 is pushed to give the dye solution composition 5
Was pushed back to its original position. At that time, the air that entered when the bacterial sample was introduced was expelled. Next, the bubbles were moved with the syringe 13 mounting side above the horizontal position, and subsequently, the bubbles were moved to the original position with the syringe 13 mounting side below the horizontal position. Due to the movement of the bubbles, the staining liquid composition 5 and the bacterial sample liquid were mixed, and the bacteria were stained. Finally,
It was sucked with a syringe 13 and filtered by moving the piston 8 in the aqueous solution 9 of the filtration container A. By this operation, the dyed bacteria were collected on the bacteria detection hydrophobic filter 6 and the excess dye was removed at the same time. Coloring degree of bacteria present on the hydrophobic filter 6 for detecting bacteria (diameter: 1.5 mm
As a result of visually observing the degree of coloring of the circle),
It was as in the table.

[0065]

[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 urine The urine discharged from healthy people and the urine discharged from various patients were collected using the same bacterial count measuring tool shown in FIG. 2 as described in (1) above,
The number of bacteria in urine was measured. The size and reagent of the bacteria count measuring tool shown in FIG. 2 were the same as those in (1) above.

That is, the urine discharged from a healthy person and the urine discharged from various patients are respectively sucked up to the scale (100 μl) at the positions shown in FIG. 2, the sampling capillary 1 is separated from the bacterial sample, and further sucked. Then, the bacterial sample was introduced into the filtration container A. Then, the syringe 14 of the syringe 13 was pushed to push the staining liquid composition 5 back to the original position. At that time, the air that entered when the bacterial sample was introduced was expelled. Next, syringe 1
Bubbles were moved with the 3 mounting side above the horizontal position, and subsequently the bubbles were moved to the original position with the syringe 13 mounting side below the horizontal position. Due to the movement of the bubbles, the staining liquid composition 5 and the bacterial sample liquid were mixed, and the bacteria were stained. Finally, it was sucked by the syringe 13 and filtered by moving the piston 8 in the aqueous solution 9 of the filtration container A. By this operation, the dyed bacteria were collected on the bacteria detection hydrophobic filter 6 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 (coloring degree of a circle having a diameter of 1.5 mm) is visually observed,
A visual judgment was made in comparison with the color control table prepared in (1) above. The measurement time was 60 seconds. The results are shown in Table 4. For comparison, the number of bacteria measured on a CLED agar plate medium (cultured at 37 ° C. for 24 hours) is also shown.

[0069]

[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.

[0071]

According to the method of the present invention as set forth in claims 1 and 8, it is possible to simultaneously collect the stained bacteria on the hydrophobic filter and remove the excess pigment by one-step filtration operation. The number of bacteria in a sample can be measured quickly and easily. According to the method of the present invention as set forth in claims 1 and 8,
Usually, the determination can be made within 1 minute (about 30 seconds if used), and particularly in the case of the method of the present invention according to claim 1, more rapid measurement is possible. In addition, according to the method of the present invention as set forth in claims 1 and 8, without requiring specialized technology or knowledge,
The number of bacteria in the sample can be measured. Moreover, according to the method of the present invention as set forth in claims 1 and 8, since the presence or absence of bacteria can be detected without culturing the bacteria, the method can be utilized for sample screening for bacterial tests in hospitals, and can reduce costs for patients and hospitals. You can contribute. In addition, since the method of the present invention according to claims 1 and 8 does not require any special equipment, it can be measured by a small-scale hospital, clinic, or general person. Furthermore, the method of the present invention according to claims 1 and 8 can be applied to many bacteria by selecting the filtration pore size of the hydrophobic filter, so that the application range is extremely wide.

According to the bacterial count measuring instrument of the present invention as set forth in claims 2 and 9, operations such as sampling of a sample with a dropper and dropping of a reagent with a reagent bottle are not necessary, and filtration with a syringe is sufficient. Is extremely simple. Further, since no skill is required and no equipment is required, any place for measurement can be selected. Furthermore, although the bacteria count measuring instrument of the present invention according to claim 9 is a disposable type (disposable type) having a simple structure, there is no risk of the sample entering the syringe and there is concern about contamination such as bacterial contamination. The syringe can be reused because it does not exist. On the other hand, since the bacterial count measuring instrument of the present invention according to claim 8 is mainly used for measuring the bacterial count of lactic acid bacteria, there is no fear of causing a serious problem of bacterial contamination, and the syringe can be reused. . Further, according to the bacterial count measuring instrument of the present invention as set forth in claims 2 and 9, it is extremely simple and inexpensive, and no special equipment is required, so that it can be used in any field. The advantage is that 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 bacteria count measuring instrument of the present invention as set forth in claims 2 and 9, after the measurement, a sample requiring identification of bacteria can be collected by suction, and the thin tube is sealed by heat treatment or the like. For example, it also serves as a transport container to the laboratory. Therefore, the present invention can be widely used for various tests including the field of urinalysis (diagnosis area) and the field of metal processing.

The invention described in claim 2 and the invention described in claim 9 are the same as the invention described in claim 1 and the invention described in claim 9, which are very simple. In view of the above, it can be said that the syringe is slightly complicated so that the syringe can be reused without causing such a problem.

[Brief description of drawings]

FIG. 1 is an explanatory view showing one embodiment of the bacteria count measuring instrument of the present invention described in claim 8.

FIG. 2 is an explanatory view showing one embodiment of the bacteria count measuring device of the present invention described in claim 9.

FIG. 3 is an explanatory diagram showing a manner in which a syringe is attached to the bacteria count measuring tool shown in FIG. 2 to collect a sample.

FIG. 4 ((a) to (f)) shows a procedure of a method of using the bacterial count measuring instrument of the present invention.

[Description of sign]

 A Filtration vessel 1 Sampling tube 2 Sampling scale 3 Cutting mark 4 Prefilter 5 Dye liquid composition 6 Hydrophobic filter for bacteria detection 7 Filter support 8 Piston 9 Aqueous solution 10 Internal insertion tube 11 Internal insertion tube 12 Plug 13 Syringe 14 Syringe 15 Sampling port 16 Stopper

Claims (9)

[Claims] 1. A method for measuring the number of bacteria in a sample, which has a hydrophobic filter for detecting bacteria therein, and
Collect the bacterial sample in a tubular filtration container in which the dye solution composition is pre-disposed on the side from which the bacterial sample is taken when viewed from the filter, and the support of the filter is pre-disposed on the opposite side when viewed from the filter. After putting a bacterial sample from the side to stain, suction filtration from the side of the support, simultaneously collecting the stained bacteria to the filter and removing excess dye, the bacteria in the sample from the coloring degree of the filter. A rapid method for measuring the number of bacteria, which comprises measuring the number of bacteria. 2. A method for measuring the number of bacteria in a sample, which has a hydrophobic filter for detecting bacteria therein, and
A dye solution composition is placed in advance on the side from which a bacterial sample is collected as seen from the filter, and a support tube of the filter, a piston, and an aqueous solution are placed on the opposite side of the filter in a tubular filtration container. , After the bacterial sample is put into the stain and then filtered by the movement of the piston in the aqueous solution of the filtration container, the stain of the stained bacteria to the filter and the removal of the excess dye are carried out at the same time. A method for rapidly measuring the number of bacteria, which comprises measuring the number of bacteria in a sample. 3. The method according to claim 1, wherein the sample is fermented milk or a lactic acid bacterium beverage. 4. The method according to claim 2, wherein the sample is urine. 5. The method according to claim 1, wherein the dye liquid composition comprises a dye, a buffer solution and a surfactant. 6. The dye concentration in the dye liquid composition is from 0.0001 to
The method according to claim 1 or 2, which is 0.00045% (w / v). 7. The surfactant concentration in the dye liquid composition is 0.
The method according to claim 1 or 2, wherein the amount is 001 to 1.0% (w / v). 8. A dye solution composition and a bacterium for detecting bacteria in a tubular filtration container having a sample collection port at one end and a suction port capable of being sucked by a syringe at the other end in order from the sample collection port side. A bacteria count measuring tool comprising a hydrophobic filter and a filter support. 9. A thin tube for sampling is attached to one end, and
The other end can be sequentially moved from the capillary side into a prefilter, dye solution composition, hydrophobic filter for bacteria detection, filter support, and inside the filtration container into a tube-shaped filtration container that allows suction with a syringe. Piston, aqueous solution,
And a bacteria count measuring tool comprising a stopper for preventing the leakage of the aqueous solution.