JP2000116396A - Stain of microorganism, and staining composition and its preservation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microorganism-staining composition by which number of microorganisms can quickly, simply and easily be measured and high detectability can be retained even in a specimen is which the concentration of microorganisms is low, and also to provide a method for preserving the composition and excellent in preservation stability. SOLUTION: This microorganism-staining composition includes coloring matter, a surfactant and, optionally, a salt. The composition is regulated so as to show pH 4-6 and has the coloring matter concentration of >0.0005% (W/V) and <=0.0007% (W/V), the surfactant concentration of 0.1-0.3% (W/V) and the salt concentration of 0.5-2.0% (W/V). The preservation method is as follows: (1) preserving an aqueous solution of coloring matter and a salt and an aqueous solution of a surfactant separately; (2) mixing both solutions immediately before use.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a composition for staining microorganisms. More specifically, animal urine, metalworking oils,
The present invention relates to a microorganism staining composition that can be used for measuring the number of bacteria contained in dyes, foodstuffs, water, and the like.

[0002]

2. Description of the Related Art Conventionally, when performing urine tests on humans, livestock, or various animals bred in homes, zoos, and the like, the types of bacteria in the urine are determined in order to discover inflammation and to estimate the disease state. A measurement of that number has been made. 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.

[0003] In this case, the measurement of the number of bacteria is usually carried out by a plate culture method, and the test result becomes clear on the day after urine is collected. For this reason, there has been a problem that prompt and appropriate emergency treatment may not be performed. In the food field, the types of bacteria in the food and the number of inhabitants are measured in order to determine whether the food is spoiled. Furthermore, at the production and storage sites of fermented milk and lactic acid bacteria drinks, the number of lactic acid bacteria is measured to control the number of lactic acid bacteria in these drinks.

[0004] Also in this case, the measurement of the number of bacteria is mainly performed by the agar plate culture method, and it takes 24 to 48 hours for the result to become clear. in this way,
As a method for quickly measuring the number of bacteria inhabiting various things, a technique of staining the bacteria has been developed. For example, Japanese Patent Publication No. 56-38196 proposes a method for staining bacteria by using a composition comprising a chelating agent and a dye and adjusting the pH value to 7.0 or more. Also,
In JP-A-1-124767, the pH value is 8
There has been proposed a method using a dye which is soluble in 緩衝 12 buffers and functions in this pH range. In each of these methods, an acidic solution is added to remove excess dye after staining. Further, it does not show a method for maintaining the degree of coloring after dyeing.

Further, in JP-A-6-209790, a microorganism is stained using monoethanolamine together with the dye fuchsin, then collected by a hydrophobic filter, and then excess fuchsin is removed to color the microorganism. A method for measuring the number of microorganisms in a sample from the degree has been proposed. According to this method, the number of microorganisms can be measured quickly and easily, but there is a drawback that the degree of coloring may change with time. In JP-A-10-136996, a microorganism-staining composition containing a dye and a surfactant or a dye, a surfactant and a salt is adjusted to a pH value in the range of 4 to 6, and the microorganisms are stained. A method for measuring the number of microbial bacteria in a sample by using it for staining is proposed. This method is an excellent method because the degree of coloration of microorganisms does not change with the passage of time, but is still not sufficiently satisfactory in detection sensitivity.

[0006] Further, if the microorganism staining composition fades during storage, accurate results cannot be obtained when the number of microorganisms in a sample is measured from the degree of coloring of microorganisms.
After the preparation, a dye having the ability to maintain the same dyeability even after a long storage period of more than two months is desired.

[0007]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned problems of the prior art, and enables the presence or the number of microorganisms in a sample to be measured quickly and easily. Does not change over time,
Moreover, it is an object of the present invention to provide a microorganism staining composition capable of staining microorganisms with high detection sensitivity, a microorganism staining method using the same, and a method for storing the staining composition.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, the dye and the surfactant, or the dye, the surfactant and the salt are contained at specific concentrations, respectively. And, by using the composition for microbial staining adjusted to a specific pH value range,
They have found that the above problems can be solved, and have completed the present invention based on these findings.

That is, the gist of the present invention is as follows. [1] It contains a dye and a surfactant, and has a pH value of 4 to 6.
Wherein the dye concentration exceeds 0.0005% (W / V) and is 0.0007%.
(W / V) A composition for staining microorganisms which is not more than (W / V). [2] The dye concentration is 0.00052 to 0.00068%
(W / V) The composition for staining microorganisms according to the above [1], which is (W / V). [3] The composition for microorganism staining according to [1] or [2], wherein the surfactant concentration is 0.1 to 0.3% (W / V). [4] a microbial staining composition containing a dye, a surfactant and a salt, and having a pH value adjusted to a range of 4 to 6, wherein the dye concentration exceeds 0.0005% (W / V); 0.
0007% (W / V) or less, a surfactant concentration of 0.1 to 0.3% (W / V), and a salt concentration of 0.5 to 2.0.
% (W / V). [5] A microbial staining composition containing a dye, a surfactant, and a salt and having a pH value adjusted to a range of 4 to 6, and having a dye concentration of 0.00052 to 0.00068% (W
/ V), the surfactant concentration is 0.1 to 0.3% (W /
V), a composition for staining microorganisms having a salt concentration of 0.7 to 1.0% (W / V). [6] A method for staining microorganisms, wherein the composition for staining microorganisms according to any one of [1] to [5] is brought into contact with microorganisms in a pH range of 4 to 6. [7] The dye concentration exceeds 0.0005% (W / V),
0.0007% (W / V) or less and a salt concentration of 0.5
A method for preserving a microorganism staining composition, in which a dye solution composition component having a concentration of 2.0% (W / V) and a surfactant component are separated and stored separately. [8] The dye concentration is 0.00052 to 0.00068%
(W / V) and the salt concentration is 0.7 to 1.0% (W / V).
A method for preserving a composition for microbial staining, in which the dye liquid composition component V) and the surfactant component are separated and stored separately.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism staining composition of the present invention is a microorganism staining composition containing a dye and a surfactant and having a pH value adjusted to a range of 4 to 6, wherein the concentration of the dye is Over 0.0005% (W / V) and 0.0007%
(W / V) or less, preferably 0.00052 to 0.00
068% (W / V).

The above-mentioned pigment may be any pigment that can stain microorganisms. Usually, the microbial staining composition is used as an aqueous solution. Therefore, the dye used in the microorganism staining composition is preferably water-soluble. Suitable examples of such a dye include fuchsin, safranin-O, methylene blue, methyl green, crystal violet, gentian violet, and Victoria blue B. Among these dyes,
Fuchsin and safranin-O are particularly suitable because there is a large difference in the degree of coloring depending on the concentration of microorganisms, and it is easy to determine the presence or absence of microorganisms and the number of microorganisms.

It is important that the concentration of the pigment is sufficient to color the microorganism and that the excess pigment not used for coloring the microorganism can be removed. In particular, by staining microorganisms at a dye concentration at which the microorganisms can be sufficiently colored, the detection sensitivity of microorganisms can be maintained at a high level. Even if the microorganism concentration is, for example, about 10 ⁵ CFU / milliliter, it is desirable that the pigment concentration be such that it can be colored so that the number of the microorganisms can be sufficiently determined.

In the present invention, the detection density of microorganisms can be maintained at a high level, and the dye concentration at which excess dye can be removed is 0.0005%.
(W / v) and 0.0007% (w / v) or less. This is because the dye concentration is 0.0005% (w /
If the value is less than v), the coloring of the microorganisms may be insufficient, which may hinder the determination of the number of microorganisms, particularly for a sample having a relatively low concentration of the microorganisms. If the ratio exceeds (w / v), the operation of removing excess dye that has not been used for coloring the microorganisms becomes complicated, and the time required for the removal is prolonged. Then, the dye concentration is adjusted to 0.00052 to
When the content is in the range of 0.00068% (W / V), the detection sensitivity of microorganisms can be maintained even higher, which is more preferable.

The composition for staining microorganisms contains a surfactant together with the above-mentioned dye, and the surfactant is used for washing and removing excess dye used for staining microorganisms. I have. As this surfactant,
Although various types can be used, it is preferable that the composition has a property that does not affect the pH value of the composition for microbial staining or the sample at the time of staining. As such a surfactant, a nonionic surfactant is suitably used.
The content of the surfactant in the composition for staining microorganisms is preferably from 0.1 to 0.3% (w / v). The reason is that the surfactant content is 0.1%.
% (W / v), it may be difficult to remove excess dye, and if it exceeds 0.3% (w / v), even dyes attached for staining microorganisms may be removed. Because there is.

If necessary, a preservative such as ethanol may be added to the composition for staining microorganisms.
When these preservatives such as ethanol are added, the use ratio is preferably 1 to 15% (v / v). Further, the composition for staining microorganisms of the present invention is adjusted so that the pH of the composition becomes 4 to 6. When the pH value is less than 4, the degree of coloring for microorganisms is too weak, and when the pH value exceeds 6, the degree of coloring for microorganisms is too strong. Further, when the pH value is outside the range of 4 to 6, the degree of coloring with the passage of time after coloring, particularly the fading after coloring, may proceed. When the pH value is out of the range of 4 to 6, the storage stability of the composition for staining microorganisms decreases, and the staining property for microorganisms changes even after storage for about 2 to 3 weeks. There is.

As a reagent for adjusting the pH value of the composition for staining microorganisms, any reagent which does not easily decompose by microorganisms may be used. As such a reagent, an inorganic acid or an inorganic alkali is suitable, and it is preferable to use hydrochloric acid, sodium hydroxide, or the like which is easily available, inexpensive and easy to handle. In the composition for staining microorganisms of the present invention, those containing salts in addition to the above-mentioned dyes and surfactants are preferable. The concentration of the salt contained in the composition for staining microorganisms is 0.5 to 2.0% (w /
v), preferably 0.7 to 1.0% (w / v).
As the salt used here, a water-soluble salt is preferable, and an inorganic salt of an alkali metal is preferable in view of solubility in water. Among them, sodium chloride and potassium chloride are particularly preferable because they are easily available and easy to handle.

If the salt concentration in the microorganism staining composition is out of the above range, the microorganisms colored by the pigment are collected by filtration, and if the concentration of the colored microorganisms is high, the collection by filtration becomes difficult. This is because it may be. Next, when preparing the composition for staining microorganisms, water may be used as a diluent, and the content of the dye or the surfactant may be adjusted to a predetermined amount. In this case, in addition to a dye and a surfactant, a diluent and, if necessary, a pH adjusting reagent or a salt are used. In general, a composition for staining microorganisms can be prepared by simply mixing these components, and the order of mixing these components is not particularly limited. Preferably,
A dye, a surfactant and, if desired, a salt are added to the diluent, and if the pH is out of range, the pH may be adjusted by adding a pH adjusting reagent.

With respect to the method of staining microorganisms using the composition for staining microorganisms thus prepared, the composition for staining microorganisms is stained by contacting the composition with microorganisms at a pH value in the range of 4 to 6. . In this case, the amount of the microorganism staining composition used for staining is suitably 2 to 5 times the amount of the sample. The amount of the sample used for this staining is usually 50 to 1
It is about 00 microliter. Therefore, when the measurement sample is 100 microliters, the amount of the microorganism staining composition used for one measurement is 200 to 50.
It is about 0 microliter. Furthermore, when measuring the number of microorganisms in the sample, the amount of the microorganism staining composition used, the amount used to create a color control table and calibration curve,
It is preferable that the amount used for a sample with an unknown number of bacteria is made equal to suppress the error.

There are no particular restrictions on the samples to which the method for staining microorganisms using the microorganism staining composition of the present invention can be applied, and humans, livestock, animals raised at home, zoos and the like are not particularly limited. Water-soluble metalworking oils such as cutting oils, rolling oils, and heat-treated oils; water-soluble paints; liquid seasonings such as soy sauce and sauces; liquid foods and beverages such as fermented milk and lactic acid bacteria beverages; and wine and sake Alcoholic beverages; solid foods such as vegetables and fish meat; seawater, river water, pond water, hot spring water, aquarium water,
Water such as household wastewater is included. These samples may be used as they are, or may be used after diluting them at an appropriate magnification.For solid foods, etc., samples that have been subjected to treatment such as crushing and dilution are measured. It may be a sample for use.

The concentration of microorganisms contained in the sample is preferably about 1 × 10 ⁴ to 1 × 10 ⁷ cells / milliliter. If it is less than 1 × 10 ⁴ / ml, it may be difficult to color, and 1 × 1
0 exceeds ⁷ / ml, is because the filtration in the case of employing a measurement method which requires a filtration step may be difficult. Therefore, when the concentration of microorganisms is particularly high, the sample may be appropriately diluted and used so that the concentration of microorganisms contained in the sample falls within the above range.

Next, after preparing the composition for staining microorganisms, it may be necessary to store the composition until it is used. In such a case, even if the prepared microbial staining composition is stored as it is, the liquidity of the microbial staining composition of the present invention is adjusted to have a pH value in a weakly acidic region of 4 to 6. Therefore, although the storage stability is better than that of a conventional composition for staining microorganisms which has been adjusted to neutral or basic, if the storage period is prolonged, the dyeability and the retention of the degree of coloring may be reduced.

Therefore, in the present invention, among the constituents of the composition for microbial staining, a dye and a salt are contained, and the concentration of the dye exceeds 0.0005% (W / V) and
007% (W / V) or less, preferably 0.00052 to
0.00068% (W / V), and the salt concentration is set to 0.1.
5 to 2.0% (W / V), preferably 0.7 to 1.0%
(W / V), and the pH component of which is adjusted to be in the range of 4 to 6, and the other component, the surfactant component, are separated and stored separately. As described above, the composition for microbial staining is separated into two components and stored, and these components are mixed at the time of use to be used for dyeing, thereby improving the stainability of the microorganism and the retention of the degree of coloring. Storage for a long period, for example, six months or more.

As described above, when the dye liquid composition component and the surfactant component are stored separately, the surfactant component is 1 component.
It is stored as a concentrated solution of 0% by weight or more, preferably 10 to 50% by weight, for example, at 1 to 20 ° C. If within 5 months, 5 ℃
When used in a degree, it is better to use it within three months.

Next, when the number of microorganisms is measured using the composition for staining microorganisms of the present invention, it is desirable to remove excess staining composition. As means for removal, centrifugation or filtration can be applied, but separation by filtration is preferred in terms of simplicity and reliability, and suction filtration and pressure filtration are particularly preferred in terms of filtration speed. As a filter used for this filtration, a hydrophobic filter is preferable.
This is because hydrophobic filters are weak or non-polar, and it is easy to remove excess dye. As the material of the hydrophobic filter, for example, polytetrafluoroethylene or polypropylene is particularly preferable because it is easy to remove excess dye.

The filtration pore size of this hydrophobic filter is 0.2 to 3.0 μm which is usually used for collecting microorganisms.
Those in the range of about are preferably used. The size of the filtration area (area of the colored area) of the hydrophobic filter is
There is no particular limitation as long as it is easy to see. Regarding the shape of the colored area, a circle is desirable from the viewpoint of easy viewing, and its diameter is 1 to 3.
mm is preferable. There is also no particular limitation on the size of the entire hydrophobic filter, but at least the size of the colored area is at least necessary. Further, as the color of the hydrophobic filter, a color that can be easily determined may be appropriately used in consideration of the dye to be used. Usually, in order to easily determine the degree of coloring, it is preferable to use a white hydrophobic filter.

When the number of microorganisms is measured using the staining composition or the staining method of the present invention, a method of removing other components that can be stained by using a prefilter can also be adopted. For example, by employing a prefilter, when animal cells larger than bacteria, such as leukocytes, coexist in a sample containing bacteria, the bacteria are allowed to pass through but are not passed through such large animal cells. Can be gathered.

When a prefilter is used, for example, the pretreatment may be performed using a filter having a filtration pore size of about 6 to 60 μm. As a material of such a pre-filter, a polypropylene-based filter or the like is suitable.
Next, by suction filtration of the mixture of the staining composition and the sample containing bacteria, the collection of stained bacteria in the hydrophobic filter for bacteria detection and the removal of excess dye were performed at the same time. The number of bacteria in the sample is determined from the degree of coloration of the hydrophobic filter for detection.

The number of bacteria in the sample is determined from the degree of coloring of the bacteria in the sample from which the excess dye has been removed. The measurement of the bacterial count is most conveniently performed by visual comparison using (1) a color control table, but (2) the optical density (O.D.
D. ) Can be performed by a colorimetric method. 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.

In the case of measuring the number of bacteria by visual observation in the above (1), the degree of coloration of bacteria existing on the hydrophobic filter for collecting bacteria, that is, the color intensity, is measured by using a sample having a known amount of bacteria. The comparison may be performed by using a color comparison table prepared in advance by using the color comparison table. This color control table is obtained by taking a color photograph of a hydrophobic filter for detecting bacteria which has been stained and washed by the above-mentioned measuring method using a sample having a known number of bacteria, and also for detecting the stained bacteria. It can be prepared by coloring filter paper or the like to the same extent as a hydrophobic filter, or by printing a similar color on paper.

For example, when measuring the number of bacteria in urine, a color comparison table is prepared as follows. That is, the urinary bacteria are present in the urine of healthy people at a low concentration, and are present at about 10,000 per 1 ml (milliliter) at most, but when the total number is 100,000 or more per 1 ml, It is determined that urine is suspected, and bacterial infection (bacteriuria) is definitive if the number is 1,000,000 or more per ml. Therefore, a color comparison table that can be detected before and after this value may be created. Normally, it is sufficient to prepare the following standard color contrast table, but it is needless to say that the present invention is not limited to this.

-: Negative (number of bacteria 10 ³ / ml or less) +: Positive (number of bacteria 10 ⁴ to 10 ⁵ / ml) ++: Strongly positive (number of bacteria 10 ⁶ to 10 ⁷ / ml) Since the standard color control table is used to judge whether bacteria are positive or negative, not only make a color control table that prints each color of + and ++ that are positive for bacteria, but also print a color that is negative for bacteria, If the color is darker than this color, a color control table for judging bacteria positive may be created.

Further, the optical density (OD) of the above (2)
In the case of colorimetric quantification by measurement, a dye that colored bacteria present on a hydrophobic filter for bacteria detection was eluted using an organic solvent, and the degree of coloration of the eluate was measured by absorbance to prepare in advance. The quantification may be performed by collating with a calibration curve between the optical density (OD) and the number of bacteria. The wavelength at the time of measuring the absorbance may be appropriately determined depending on the dye used. As the organic solvent, various alcohols can be used, and ethanol is particularly preferable.

The calibration curve may be created, for example, in the following manner. That is, when fuchsin is used as a dye, the degree of coloration of a sample diluted to various concentrations is measured using a microplate reader at an optical density (OD) of 492 nm. 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 obtained from both results. Just create it.

[0034]

Next, the present invention will be described in more detail with reference to examples. [Reference Example 1] Degree of coloration of standard bacteria by staining composition [D1] As a bacterium, Escherichia coli (ATCC 11) was used.
303) and Staphylococcus aure
us; IFO 3183), the following experiment was performed.

The broth broth medium was inoculated with the bacterial species, and
For 24 hours, and the culture solution was used as a standard sample bacterial suspension. On the other hand, collecting urine released from healthy men,
Filtration was carried out with a polytetrafluoroethylene membrane filter (Advantech Toyo) having a filtration pore size of 0.5 μm to prepare a bacteria diluting solution.

A predetermined amount of a bacteria diluting solution is added to the bacterial suspension for the standard sample, and the number of bacteria is about 1,000 / ml.
About 10,000 pieces / ml, about 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 according to the culture method was determined at 37 ° C. for 2 days on a CLED agar plate medium.
Counting was performed based on the number of colonies after culturing for 4 hours.

In the experiment for preparing the color control table for measuring the number of bacteria in urine, the kit for measuring the number of bacteria shown in FIGS. 1 and 2 was used. The bacteria count kit is shown in FIG. 1 as A; filtration container, B; syringe, C;
D: Dyeing composition, E: Dyeing composition container, F: A color control table. The filtration container indicated by A is
It is a polypropylene tube with an outer diameter of 4.6 mm, an inner diameter of 4 mm, and a length of 20 mm.
The plastic sampling syringe shown in FIG. 2 is a plastic sampling syringe, and the sampling member indicated by C is a 10, 50, or 100 μL measuring tip (manufactured by Quality Corporation, USA) having a wide-bore side inner diameter of 5 mm. And these A, B,
C are adapted to be interconnected as shown in FIG.

The filter container A is provided with a filter support 2 and a hydrophobic filter 1 for detecting bacteria at the end of the sample collecting member C side. The filter support 2 has a shape obtained by cutting a silicone tube having an outer diameter of 4 mm and an inner diameter of 1.5 mm into a length of 7 mm. The hydrophobic filter 1 for detecting bacteria is made of a polytetrafluoroethylene membrane having a pore diameter of 3 μm. It consists of a filter and has a colored area diameter of 1.5 mm. In addition, the syringe B has a built-in piston 6 used for inhaling and discharging liquid, and the sampling member C has the pre-filter 3 attached to the filtration container A side, and the distal end portion thereof. Is provided with a sampling port 4. The prefilter 3 is a filter made of polyvinyl alcohol having a filtration hole diameter of 60 μm, and has a diameter of 4 m.
m, length 4 mm.

Next, D is a dyeing composition, and E is a container for containing the dyeing composition.
This is a 5 ml microtube made of polypropylene (manufactured by Quality Corporation, USA). As the dyeing composition [D1] used here, fuchsin was used as a dye at a concentration of 0.1%.
00005% (w / v) and 0.85% salt (w / v)
v) an aqueous solution containing 0.17% (w / v) of a surfactant (Kanto Kagaku; Tween 20),
A liquid whose pH was adjusted to 4.9 was used.

As shown in FIG. 2, a sample having a 3 ml syringe B attached to one end of a filtration container A and a pre-filter 3 attached to the other end of the filtration container A as shown in FIG. The sampling member C was detachably fitted and fitted, and 100 μl of the bacterial sample was collected from the sampling port 4 at the distal end of the sampling member C by the suction operation of the syringe B.

Next, after the bacterial sample was introduced by the suction operation of the syringe B as described above, the bacterial sample was once placed in the container E containing 350 μl (7 to 8 drops) of the staining composition [D1]. Extruded and injected and mixed gently. In the present embodiment, the sample is collected by the suction operation of the syringe B, but a syringe may be used instead of the syringe B. A method of sucking a sample with a dropper and dropping two drops (about 100 μl) into the container E may be adopted.

Immediately after the injection of the bacterial sample, the mixed solution of the staining composition and the bacterial sample contained in the container E is pulled from the sampling port 4 at the tip of the sample collecting member C to the piston 6 of the syringe B. Thereby, suction filtration was performed. By this suction filtration operation, the mixed solution is converted into the hydrophobic filter 1 for detecting bacteria.
, Whereby the stained bacteria are collected on the hydrophobic filter 1 for detecting bacteria, and at the same time, excess dye is removed.

After the stained bacteria are collected by the hydrophobic filter 1 for detecting bacteria in this manner, the sampling member C at the tip of the syringe B is removed to expose the tip of the filtration container A. Filter 1 for detecting bacteria
The coloration of the upper stained bacteria was visually observed. Next,
In order to determine the degree of coloring of the filter 1, using dye magenta, the dot% of the dye was 5%, 10%, 20%.
%, 30%, 40%, 50%, 60%, 70%, 80%
A color chart was prepared by printing a 90% and 90% color sheet on white paper, and the degree of coloring was indicated by the numerical value of the dot% of this dye. Table 1 shows the observation results of the coloring degree.

Example 1 The following five types of bacteria were cultured to prepare a culture solution. (1) Staphylococcus aureus: IFO
(3183 strain) (2) Escherichia coli (E. coli HV 1184 strain) (3) Klebsiella pneumoniae (clinical isolate) (4) Pseudomonas aerogenes (clinical isolate) (5) Enterococcus faecalis (clinical isolate) As a medium, broth bouillon Using a liquid medium, each bacterial species was inoculated into the liquid medium, and static culture was performed at 30 ° C. for 48 hours, and the culture solution was used as a standard sample bacterial suspension.

On the other hand, urine released from healthy males was collected and filtered through a polytetrafluoroethylene membrane filter (Advantech Toyo Co., Ltd.) having a filtration pore size of 0.5 μm to prepare a bacterial dilution. A sample was prepared by adding a predetermined amount of a bacterial dilution to the bacterial suspension. The number of bacteria in the sample was measured by counting the number of colonies of the above bacteria (1) to (4) when cultured on a normal agar plate medium at 30 ° C. for 48 hours. In addition, the above (5) Enterococcus faecalis was purified on a bird soy blood agar medium (manufactured by Kyokuto Pharmaceutical).
Counting was performed based on the number of colonies cultured at 48 ° C. for 48 hours.

Next, fuchsin was added as a pigment at 0.000.
52% (w / v), salt 0.85% (w / v), surfactant (Kanto Chemical Co .; Tween 20) 0.17%
An aqueous solution containing (w / v) was prepared, and the pH value was adjusted to 4.9 with a dilute hydrochloric acid aqueous solution to prepare a dyeing composition. Next, this staining composition was added to the sample, and the bacteria in the sample were stained using the kit for measuring the number of bacteria, and the degree of coloring of the filter was observed. The degree of coloring was compared with the color chart prepared in Reference Example 1, and the degree of coloring was indicated by dot%. The results are shown in Table 2. In the column of coloring degree in Table 2, ○
Those marked with a symbol indicate that the sample was clearly distinguished from the sample without bacteria, and those marked with a Δ indicate that the sample was light in color and difficult to distinguish from the sample without bacteria.

[Reference Example 2] Degree of coloring of standard bacteria by dyeing composition [D2] Instead of dyeing composition [D1] used in Reference Example 1, the composition was changed to a fuchsin concentration of 0.0006% (w /
v), 0.19% (w / v) concentration of a surfactant (Kanto Chemical; Tween 20), 0.85% (w / v) of salt
The coloring degree of the standard bacterium was indicated in dot% in the same manner as in Reference Example 1 except that a certain dyeing composition [D2] was used as v). Table 1 shows the results of observation of the degree of coloring of the standard bacteria.

Example 2 Instead of the dyeing composition [D1] used in Example 1, the composition was changed to a fuchsin concentration of 0.0006% (w / v) and a surfactant (manufactured by Kanto Chemical Co .; Tween 20) was used in the same manner as in Example 1 except that a dyeing composition [D2] having a concentration of 0.19% (w / v) and a salt concentration of 0.85% (w / v) was used. The degree of coloration of the filter was observed. The results are shown in Table 2.

Reference Example 3 Degree of Coloring of Standard Bacteria by Dyeing Composition [D3] Instead of the dyeing composition [D1] used in Reference Example 1, the composition was replaced with a fuchsin concentration of 0.00068% (w /
v), the concentration of a surfactant (Kanto Chemical; Tween 20) 0.20% (w / v), the concentration of salt 0.85% (w / v)
Except for using a certain dyeing composition [D3] as v), the coloring degree of the standard bacterium was indicated by dot% in the same manner as in Reference Example 1. Table 1 shows the results of observation of the degree of coloring of the standard bacteria.

Example 3 Instead of the dyeing composition [D1] used in Example 1, the composition was changed to a fuchsin concentration of 0.00068% (w / v) and a surfactant (manufactured by Kanto Chemical Co .; Tween 20) was used in the same manner as in Example 1 except that a dyeing composition [D3] having a concentration of 0.20% (w / v) and a salt concentration of 0.85% (w / v) was used. The degree of coloration of the filter was observed. The results are shown in Table 2.

Reference Example 4 Degree of Coloring of Standard Bacteria by Dyeing Composition [D4] In place of the dyeing composition [D1] used in Reference Example 1, the composition was changed to a fuchsin concentration of 0.00045% (w /
v), 0.16% (w / v) concentration of a surfactant (Kanto Chemical; Tween 20), 0.85% (w / v) of salt
The coloring degree of the standard bacterium was indicated in dot% in the same manner as in Reference Example 1 except that a certain dyeing composition [D4] was used as v). Table 1 shows the results of observation of the degree of coloring of the standard bacteria.

[Comparative Example 1] Instead of the dyeing composition [D1] used in Example 1, the composition was changed to a fuchsin concentration of 0.00045% (w / v) and a surfactant (manufactured by Kanto Chemical Co .; Tween 20) was used in the same manner as in Example 1 except that a dyeing composition [D4] having a concentration of 0.16% (w / v) and a salt concentration of 0.85% (w / v) was used. The degree of coloration of the filter was observed. The results are shown in Table 2.

[Reference Example 5] Degree of coloration of standard bacteria by dyeing composition [D5] Instead of dyeing composition [D1] used in Reference Example 1, the composition was changed to a fuchsin concentration of 0.0008% (w /
v), a surfactant (Kanto Chemical Co .; Tween 20) concentration of 0.22% (w / v), and a salt concentration of 0.85% (w / v)
The coloring degree of the standard bacterium was represented by dot% in the same manner as in Reference Example 1, except that a certain dyeing composition [D5] was used as v). Table 1 shows the results of observation of the degree of coloring of the standard bacteria.

[Comparative Example 2] Instead of the dyeing composition [D1] used in Example 1, the composition was changed to a fuchsin concentration of 0.0008% (w / v) and a surfactant (Kanto Chemical; Tween 20) was used in the same manner as in Example 1 except that a dyeing composition [D5] having a concentration of 0.22% (w / v) and a salt concentration of 0.85% (w / v) was used. The degree of coloration of the filter was observed.

The results are shown in Table 2.

[0056]

[Table 1]

[0057]

[Table 2]

[0058]

[Table 3]

Reference Example 6 A urine sample from a healthy person (10
As a result of measuring the degree of coloring of ³ samples / ml or less in 30 samples, the dot% was all around 5%. In addition, urine samples of patients with urinary tract infection (10 ⁵ to 1 bacteria)
0 ⁷ / ml) results of degree of coloration was measured of 1 dot%
It was 0% or more, and the dot count was 20 to 40% for the specimens having a bacterial count of 10 ⁶ / ml or more.

From the above results, taking the coloring degree of the urine sample as a reference,-: negative (coloring degree of about 5) +: positive (coloring degree of about 10) ++: strong positive (coloring degree of about 30) A comparison table was created.

Example 4 As urine samples, urine samples A to C of healthy subjects and urine sample A of patients with urinary tract infection shown in Table 3
The bacteria were stained with the staining composition [D3] used in Example 3 for each of the 10 specimens (G) to (G), and judged by the color control table of Reference Example 6. The results of these determinations are shown in Table 3 together with the results of measuring the number of bacteria in the sample.

[0062]

[Table 4]

Example 5 A staphylococcus aureus (IFO 3183 strain) was used as a bacterium, and a broth broth liquid medium was used as a medium, and a culture solution obtained by culturing at 30 ° C. for 48 hours was used. Dilute with water to reduce bacterial count to 10
A sample of about ⁵ / ml was obtained. And this sample 10
Using 0 μL, the concentration of fuchsin was 0.0006% (w / v), the concentration of surfactant was 0.19% (w / v) of surfactant (Tween 20; manufactured by Kanto Kagaku Co., Ltd., Tween 20), and Bacteria were stained by the same operation as in Example 1 using the staining composition immediately after being adjusted to a concentration of 0.85% (w / v), and the degree of coloration of the filter was observed.

Next, in order to evaluate the storage stability of the composition for staining microorganisms, the concentration of fuchsin was set to 0.000.
6% (w / v) and a salt concentration of 0.85% (w / v)
v) a dye solution composition component comprising an aqueous solution and a surfactant (Kanto Chemical Co .; Tween 20) at a concentration of 19% (w
/ V) Two components of a surfactant component consisting of an aqueous solution were prepared separately.

Then, these two components were stored at room temperature (15 to 25 ° C.) for 2 weeks, 4 weeks, 6 weeks, 8 weeks, and 10 weeks, respectively. The number of samples of both components for storage was 10 for each storage period.

Next, after the respective storage periods, a surfactant component was added to and mixed with the dye solution composition component to prepare a dyeing composition, and the same operation as in Example 1 was carried out. The bacteria were stained by the method described above, and the degree of coloring of the filter was observed. Table 4 shows the measurement results.

[0067]

[Table 5]

[Comparative Example 3] Fuchsin concentration 0.0006
% (W / v), surfactant (manufactured by Kanto Chemical Co .; Tween 2)
0) concentration 0.19% (w / v), salt concentration 0.85
% (W / v) of the dyeing composition was prepared.

Next, in the state of an aqueous solution containing these three components, each of
Stored at room temperature (15-25 ° C.) for weeks, 4, 6, 8 and 10 weeks. The number of samples was 10 for each storage period. Then, for each of these storage periods, it was confirmed in the same manner as in Example 4 whether or not there was a change in the degree of coloring upon storage.

Table 5 shows the results of these measurements.

[0071]

[Table 6]

[0072]

According to the composition for staining microorganisms of the present invention,
The presence or absence and the number of microorganisms in a sample can be measured quickly and easily, and the detection sensitivity can be maintained high even in a sample having a low concentration of microorganisms. The effect of having excellent storage stability is obtained.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram of a kit for measuring the number of bacteria used in Reference Examples and Examples.

FIG. 2 is an explanatory view showing a state in which a kit for measuring the number of bacteria is prepared by combining the kit for measuring the number of bacteria used in Reference Examples and Examples with a commercially available syringe.

[Description of sign]

 Reference Signs List A Filtering container B Syringe C Sampling member D Staining composition E Staining container for staining composition 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 (8)

[Claims] 1. A microorganism staining composition containing a pigment and a surfactant and having a pH value adjusted to a range of 4 to 6, wherein the pigment concentration exceeds 0.0005% (W / V), 0.
A composition for staining microorganisms, which is not more than 0007% (W / V). 2. A dye concentration of 0.00052 to 0.000.
The composition for staining microorganisms according to claim 1, wherein the composition is 68% (W / V). 3. A surfactant concentration of 0.1 to 0.3% (W
/ V), the composition for staining microorganisms according to claim 1 or 2. 4. A microorganism staining composition containing a dye, a surfactant and a salt and having a pH value adjusted to a range of 4 to 6, wherein the dye concentration is 0.0005% (W / V). Exceeding 0.0007% (W / V), a surfactant concentration of 0.1 to 0.3% (W / V) and a salt concentration of 0.5 to
A composition for staining microorganisms which is 2.0% (W / V). 5. A composition for dyeing microorganisms containing a dye, a surfactant and a salt and having a pH value adjusted to a range of 4 to 6, wherein the dye concentration is 0.00052 to 0.00068.
% (W / V), the surfactant concentration is 0.1 to 0.3% (W
/ V), a microorganism staining composition having a salt concentration of 0.7 to 1.0% (W / V). 6. A method for staining a microorganism, wherein the composition for staining a microorganism according to any one of claims 1 to 5 is brought into contact with a microorganism in a pH range of 4 to 6. 7. When the dye concentration exceeds 0.0005% (W / V) and is 0.0007% (W / V) or less, and the salt concentration is 0.5 to 2.0% (W / V). Yes, its pH value is 4 ~
A method for preserving a composition for staining microorganisms, in which a dye solution composition component and a surfactant component adjusted to the range of 6 are separated and stored separately. 8. A dye concentration of 0.00052 to 0.000
68% (W / V) and a salt concentration of 0.7 to 1.0% (W / V
V), a method for preserving a microorganism staining composition, in which a pH component is adjusted to a range of 4 to 6 and a dye component composition and a surfactant component are separated and stored separately.