JP2002202302A - Bacteria dyeing method, method for classifying and counting bacteria and diluent liquid for dyeing bacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a dyeing method capable of detecting bacteria rapidly and efficiently even if nitrite ions are present in a sample in high concentration. SOLUTION: Polymethine type coloring matter is allowed to act on bacteria in the presence of a substance capable of reducing nitrite ions to dye bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for staining bacteria,
More specifically, the present invention relates to a method for classifying and counting bacteria and a diluting solution for bacterial staining, and more particularly to a method for staining bacteria in clinical samples, particularly preferably bacteria in urine samples, a method for classifying and counting bacteria, and a method for staining bacteria. For diluent.

[0002]

2. Description of the Related Art The urinary bacterial count is an important parameter in clinical diagnosis for determining the presence or absence of infection. Generally, as a criterion of urinary tract infections (Urinary Tract Infection), a case where urine bacteria count appeared 10 ⁵ cells / ml or more and positive. Further, when the concentration is 10 ³ cells / ml or less, the urine is regarded as contaminated urine (resident bacteria), which is regarded as negative. In the case of about 10 ⁴ cells / ml, it is a determination suspension area, but is often a retest area.

[0003] Conventional methods for confirming urinary bacteria include methods such as confirmation under a microscope after gram staining, confirmation under a microscope without staining, and confirmation under a microscope after fluorescent staining. No. In the urine, non-clinically useful mucus threads, crystals, amorphous salts, and cell fragments, often referred to as contaminants, often interfere with important measurement particles (especially bacteria), which can lead to an accurate bacterial count. Was difficult to count. Practically, there is no method for accurately counting the number of bacteria of about 10 ⁴ cells / ml.

[0004] For example, in Gram staining, bacteria and contaminants are simultaneously stained, so that a small number of bacteria are often overlooked under a microscope. In addition, there were a plurality of dyeing steps, and time was required for dyeing (about 15 minutes), so that the working efficiency was poor. Staining-free microscopy is a rapid method, but it is impossible to discriminate bacteria from bacteria, especially when cocciform contaminants appear. Regarding fluorescence staining microscopy, bacteria detection performance is higher than the above two methods.However, when contaminants other than bacteria are mixed, a method for effectively removing them and rapidly staining bacteria is disclosed. It has not been. In the case of the agar medium method, which is a standard method, it takes 16 hours or more to measure the number of bacteria, which is hardly quick.

Also, US Pat. No. 4,622,298,
JP-A-9-119926 and JP-A-9-32959
No. 6 proposes a method of detecting bacteria by measuring a fluorescently stained urine sample with a flow cytometer, and using a polymethine dye for fluorescent staining.

[0006]

However, when a polymethine dye is used to stain bacteria, the bacteria may not be sufficiently stained. For a sample in which bacteria capable of reducing nitrate are growing and producing a large amount of nitrite,
Nitrite ion decomposes the polymethine dye and does not work effectively for staining bacteria. In particular, acidic pH
In, the effect can be reduced when the bacteria are well stained and the mucus filaments coexist as in a urine sample, but the effect of nitrite ions is remarkable at acidic pH.

[0007] The present invention provides a staining method, a method for classifying and counting bacteria, and a diluent for bacterial staining that enable bacteria to be detected quickly and efficiently even when nitrite ions are present at a high concentration in a sample. The purpose is to:

[0008]

SUMMARY OF THE INVENTION The present invention is a method for staining a bacterium, which comprises reacting a polymethine dye in the presence of a substance capable of reducing nitrite ions. Further, according to the present invention, (1) bacteria in a sample are stained with a polymethine dye in the presence of a substance capable of reducing nitrite ions, and (2) the obtained sample is subjected to flow cytometry. Introduced into the detection part of the meter, irradiate each of the stained bacterial cells with light, measure the scattered light and fluorescence emitted from these cells, and (3) scattered light intensity and fluorescence intensity or particle A method for classifying and counting bacteria is provided by the pulse width reflecting the length. Further, according to the present invention, there is provided a diluent for bacterial staining, comprising an effective amount of a nitrite ion-reducible substance and a buffer for maintaining acidity.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In practicing the present invention, the sample is not particularly limited as long as it is used for counting the presence or absence of bacteria or, if bacteria are contained, the number thereof. The present invention can be applied to all clinical samples such as urine samples, blood, and cerebrospinal fluid. Among them, urine samples are effective. Here, the bacterium is a bacterium having a nitrate reducing power and producing nitrite, for example, an intestinal bacterium such as Staphyrococcus aureus,
Gram-negative facultative bacilli such as E. coli, Klebsiella sp. Or Proteus sp., Staphyrococcus sp., Pseudomonas sp., S
erratia sp., Enterobacter sp., Enterococcus sp., S
treptpococcus sp. and Citrobacter sp. The sample may be diluted 4 to 15 times, preferably about 4 to 10 times with water or the like.

[0010] Substances capable of reducing nitrite ions include:
For example, ascorbic acid, isoascorbic acid, sulfamic acid, sulfanilic acid, aminomethanesulfonic acid, aminoethanesulfonic acid, glycine, glutamic acid, glutamine, aspartic acid, asparagine, methionine, glutathione, cysteine, mercaptoacetic acid,
3-Mercaptopropionic acid, sulfurous acid, pyrosulfite, hydroxylamine, phosphinic acid and their salts and a combination of one or more substances selected from the group consisting of sulfanilamide, aminomethane, mercaptoethanol, thiophenol, and urea Can be.
Examples of the salt include an alkali metal salt, and in the case of hydroxylamine, a hydrochloride, a sulfate, a phosphate and the like. As a guide of the concentration, it can be used at a concentration of 10 mM or more in a diluent for diluting the sample and can be used at a concentration of 5000 mM.
The following are appropriate: Specifically, for ascorbic acid, 85 to 115 mM, and for sulfamic acid, 40 to 2
A suitable concentration is 10 mM to 50 mM for cysteine, glutathione and sodium sulfite, and 0.5 M or more for urea, and more preferably 5 M or less for urea. If the concentration of urea is too high, cell denaturation may occur. It is generally said that in the presence of 10 ⁵ nitrate-reducing bacteria / ml, nitrite ions are produced at 0.06 mg / ml. In general, the growth of bacteria is 10 ⁸ to 10 ⁹ cells / m 2.
It is said that the upper limit is 1 and the growth does not (cannot) occur any more. Therefore, preferably the amount of 10 ⁵ to 10 ⁸ cells / ml of bacteria capable of reducing nitrite ions produced.

In the bacterial staining method of the present invention, the pH at the time of staining is not particularly limited as long as it is a pH at which bacteria can be stained. Advantageously, 1) bacteria are stained better than neutral or alkaline, and (2) non-specific staining of mucus threads can be suppressed and mucus threads can be dissolved to some extent.

In order to maintain acidity, an acid or pKa1 ~
Five buffers can be used. Preferably pH 2.0-
As long as it can maintain 3.0, the buffer is not particularly limited, but preferably, citrate, phosphate, phthalate, glycine, succinic acid, lactic acid, β-alanine, ε
-Aminocaproic acid and fumaric acid and their alkali and alkaline earth metal salts and the like can be used. The amount used can be an amount capable of maintaining the pH range, and is 10 to 5
It can be suitably used in the range of 00 mM.

In order to stain bacteria efficiently, it is preferable to damage the cell membrane (cell wall) of the bacteria so that the dye can penetrate efficiently. For example, the purpose can be achieved by adding a cationic surfactant, an anionic surfactant, an amphoteric surfactant, a nonionic surfactant, and the like. Similar results can be obtained by using an antibacterial preservative (antibacterial agent), an organic solvent such as alcohol, or the like in addition to the surfactant. Furthermore, sufficient dyeability can be obtained, for example, even with the effect of heat or damage due to microwaves (such as a microwave oven).
Among them, cationic surfactants are not only capable of staining bacteria, but also have a large effect of reducing the influence on bacterial detection by dissolving and shrinking mucus threads, red blood cells, cell debris, etc. present in the sample. It can be suitably used.

The cationic surfactant is not particularly restricted but includes, for example, quaternary ammonium salts represented by the following formula.

Embedded image(Where R^TenIs an alkyl group having 6 to 18 carbon atoms or (C
₆H_Five) -CH_Two-; R¹¹, R¹²And R¹³Are the same or different
An alkyl group having 1 to 3 carbon atoms or a benzyl group; ^-
Is a halogen ion).

Examples of the alkyl group having 1 to 3 carbon atoms include methyl, ethyl, propyl and the like. 6-18 carbon atoms
Examples of the alkyl group include hexyl, heptyl, octyl, decyl, dodecyl, tetradecyl and the like.
Examples of the halogen include fluorine, bromine, iodine, and chlorine. Specifically, hexyl trimethyl ammonium salt, octyl trimethyl ammonium salt, decyl trimethyl ammonium salt, dodecyl trimethyl ammonium salt, tetradecyl trimethyl ammonium salt, hexadecyl trimethyl ammonium salt, octadecyl trimethyl ammonium salt, benzyl trimethyl ammonium salt are preferably used. Can be

Another example of the cationic surfactant is a pyridinium salt. _{[(C 5 H 5) N} + - (CH 2) n-CH 3] Y - ( wherein, n 7 to 17, Y ^- is a halogen ion). Specifically, octylpyridinium salts, decylpyridinium salts, dodecyltrimethylpyridinium salts, tetradecyltrimethylpyridinium salts, hexadecyltrimethylpyridinium salts, and the like are preferably used. The concentration of the cationic surfactant is 10-50,000 mg / ml, preferably 100-3000 mg
/ ml is preferred.

Although the anionic surfactant is not particularly limited, for example, lauroyl sarcosinate, cocoyl sarcosinate, myristoyl sarcosinate, oleyl sarcosinate, etc. are preferably used as N-acylaminoacetates. The concentration of anionic surfactant is 0.1mg / ml ~ 10mg
/ ml, preferably from 0.5 mg / ml to 5 mg / ml.

The amphoteric surfactant is not particularly limited,
Preferable examples include the following betaine acetates.

Embedded image (Wherein, R ¹⁴ is an alkyl group having 8 to 20 carbon atoms; R ¹⁵ and R ¹⁶ are the same or different and are an alkyl group having 1 to 3 carbon atoms;
An alkenyl group or an alkynyl group having 2 to 3 carbon atoms).

As the alkyl group having 1 to 3 carbon atoms, those similar to the above can be mentioned. Examples of the alkenyl group having 2 to 3 carbon atoms include vinyl and allyl. Examples of the alkynyl group having 2 to 3 carbon atoms include acetylenyl and propynyl. Examples of the alkyl group having 8 to 20 carbon atoms include octyl, decyl, dodecyl, and tetradecyl. Specific examples include betaine dodecyldimethylammonium acetate, betaine hexadecyldimethylammonium acetate, betaine decyldimethylammonium acetate, and the like. The concentration of the amphoteric surfactant is 1 mg / ml to 100 mg / ml, preferably 5 mg / ml to 20 mg / ml.

The nonionic surfactant is not particularly restricted but includes, for example, polyoxyethylene (n) alkyl ethers having an alkyl group having 10 to 20 carbon atoms and n: 10 to 20 carbon atoms. In polyoxyethylene (n) alkylphenyl ether, the number of carbon atoms of the alkyl group is C8 to C10, n: 2 to 2
A thing of 0, for example, POE (10) octyl phenyl ether and the like are suitable.

In addition, Triton X is known to have the property of solubilizing membrane proteins with other surfactants.
−100 (polyethylene-glycol-mono [p- (1,1,3,3-tetrame
thylbutyl) phenyl] ether), CHAPS (3-[(3-cholamidopr
opyl) dimethylammonio] propanesulfonic acid), CHAP
SO (3-[(3-cholamidopropyl) dimethylammonio] -2-hydr
oxypropanesulfonic acid), BIGCHAP (N, N-bis (3-D-gl
uconamidopropyl) cholamide), deoxy-BIGCHAP (N, N-bi
s (3-D-gluconamidopropyl) deoxycholamide), sucrose monomerca plate (sucrose monocaprate), sucrose monocholate (sucrose monocholate), n-
Octyl-α-D-glucopyranoside (n-octy-α-D-glucop
yranoside), n-heptyl-α-D-thioglucopyranoside, n-octyl-
α-D-thioglucopyranoside (n-octyl-α-D-thioglucop
yranoside), n-dodecyl-α-D-maltopyranoside (nd
odecyl-α-D-maltopyranoside), n-nonyl-α-D-thiomaltopyranoside
r) and the like. These surfactant concentrations range from 0.5 to
50 mg / ml, preferably 1.0 to 10 mg / ml is suitable.

Furthermore, examples other than the surfactant include a bactericidal preservative (antibacterial agent) and the like. As the antibacterial agent, for example, an isothiazoline antibacterial agent and a biguanide antiseptic are suitably used. The concentration of the antibacterial agent is 1.0-30m
g / ml is preferred. As an organic solvent such as alcohol,
For example, methanol, ethanol, isobutyl alcohol, phenoxyethanol, methoxyethanol, ethoxyethanol, butoxyethanol and the like are suitable.
The concentration of these alcohols is preferably 1.0 to 100 mg / ml.

The dye is not particularly limited as long as it can stain bacteria. However, when the sample is a urine sample, a dye capable of staining bacteria in an acidic region is preferable for the above-mentioned reason. Regarding the concentration, a suitable concentration differs for each dye, but for example, it can be used in a range of 0.1 to 100 ppm (final concentration). From the viewpoint of the ability to detect bacteria, it is advantageous to use a fluorescent dye that emits fluorescence by binding to at least one of the components constituting bacteria. In this regard,
Polymethine dyes are preferred, for example, the following (1) to
The dye of (11) can be used.

(1) Thiazole orange

(2)

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(3)

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(4)

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(5)

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(6)

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(7)

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(8)

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(9)

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(10) A compound represented by the following general formula:

Embedded image (Wherein, R ₁ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ₂ and R ₃ are a hydrogen atom, an alkoxy group having 1 to 3 alkyl groups or carbon atoms having 1 to 3 carbon atoms; R ₄ is hydrogen An atom, an acyl group or an alkyl group having 1 to 3 carbon atoms; R ₅ is a hydrogen atom, an alkyl group having 1 to 3 carbon atoms which may be substituted;
Z is a sulfur atom, an oxygen atom or a carbon atom substituted by an alkyl group having 1 to 3 carbon atoms; n is 1 or 2; X ⁻ is an anion) (11) Compound represented by the following general formula:

Embedded image (Wherein, R ₆ is an alkyl group having 1 to 18 carbon hydrogen atom or a carbon; R ₇ Beauty R ₈ is a hydrogen atom, an alkyl group or an alkoxy group having 1 to 3 carbon atoms having 1 to 3 carbon atoms; R ₉ is hydrogen atom, an acyl group, or an alkyl group having 1 to 18 carbon atoms; Z is a carbon having sulfur, oxygen or an alkyl group having 1 to 3 carbon atoms; n is 0, 1 or 2; X ^- is an anion is there).

Of these dyes, (1) is commercially available. (2) and (3) are Japan Photographic Dye Laboratories, Inc.
More available. (5) to (9) are Molecular Probe
s, Inc. (10) is disclosed in
No. 104683, (11) is disclosed in JP-A-10-3190.
No. 10 describes a production method.

Among the dyes represented by the general formula (10), particularly, the following dyes:

Embedded image

In addition, among the dyes represented by the general formula (11), particularly the following dyes:

Embedded image Is preferred.

Furthermore, when the sample is a urine sample, by performing staining in the co-presence of either sulfate or nitrate, the fluorescent stainability of bacteria is increased and nonspecific staining of foreign substances is suppressed. Is preferred. The amount used can be in the concentration range of 10 to 500 mM, preferably 50 to 200 mM.

The staining method of the present invention can be carried out by mixing a sample, an aqueous solution containing a substance capable of reducing nitrite ions, and a solution containing a dye. The dye may be contained in an aqueous solution containing a substance capable of reducing nitrite ions, but if the dye to be used is unstable in the aqueous solution, the dye may be dissolved in a water-soluble material such as methanol, ethanol or ethylene glycol. If dissolved in an organic solvent and mixed with an aqueous solution containing a substance capable of reducing nitrite ions at the time of use, the storage stability of the dye can be improved.

The reaction temperature and time are not particularly limited, but the reaction can be carried out at a temperature of 15 to 50 ° C. and for a time of 15 minutes immediately after mixing.

The sample stained by the staining method of the present invention
Although bacteria can be detected by observation with a microscope or an image recognition device, bacteria can be detected and counted with high accuracy by flow cytometry. That is, a sample containing bacteria is diluted with an aqueous solution containing a substance capable of reducing nitrite ions, the sample is subjected to a staining reaction with a polymethine fluorescent dye for a certain period of time, and the sample treated in the above step is subjected to a flow cytometer. Irradiates each of the stained bacterial cells with light, measures the scattered light and fluorescence emitted from the cells, and measures the measured signal intensity of the scattered light and fluorescence or the length of the particles. Bacteria can be detected by separating and counting bacteria and other components based on the pulse width that reflects this.

In particular, when the sample is a urine sample, various components other than bacteria coexist. In such a case, to separate and count bacteria and other components, the signals obtained by the measurement are combined. be able to. Examples of the signal combination include forward scattered light intensity and forward scattered light pulse width, forward scattered light intensity and fluorescence intensity, and forward scattered light pulse width and fluorescence intensity. Preferably, for example, first, a two-dimensional distribution map (scattergram) is created using a combination of the forward scattered light intensity and the forward scattered light pulse width, and a population including bacteria is specified on the distribution map to perform gating. The mucus thread was separated, and a two-dimensional distribution map was created for the gated population using a combination of the forward scattered light intensity and the fluorescence intensity, and bacteria and other components (crystals and Cell debris). A conceptual diagram is shown in FIG. If only bacteria are contained in the sample, a two-dimensional distribution map can be created by combining the forward scattered light intensity and the fluorescence intensity, and the bacteria can be counted.

[0042]

EXAMPLES Preferred examples are shown below, but the present invention is not limited thereto. Example 1 Reagent composition (diluent) Citric acid 92.3 mM Sodium hydroxide 0.75 g / l (amount to become pH 2.5) Tetradecyltrimethylammonium bromide 0.1% (w / v) Sodium sulfate 90 mM Ascorbic acid 85 mM (Staining solution) Dye A (the following structural formula) 40 ppm (ethylene glycol solution)

Embedded image

A sample containing a large amount of nitrite ions (bacteria concentration)
5.0 × 10 6 cells / ml; hospital urine) To 140 μm, 952 μl of a diluent of the above composition and a staining solution were added so that the final concentration of the dye A was 1 ppm, and the mixture was reacted at 40 ° C. for 20 seconds, and a red semiconductor laser was used as a light source. The scattered light and fluorescence were measured with a flow cytometer (8.0 μl of analyzed urine), and a two-dimensional distribution diagram (scattergram) was created by taking the fluorescence intensity on the horizontal axis and the forward scattered light intensity on the vertical axis. . The results are shown in FIG. As a control,
The measurement was performed using a reagent containing no ascorbic acid (FIG. 2). When a reagent containing no ascorbic acid was used, bacteria were not stained and the fluorescence intensity was 0, whereas when ascorbic acid was added, the bacteria were stained and could be detected. .

Example 2 The same measurement was carried out using 100 mM of sulfamic acid instead of ascorbic acid in the diluent of the above example. The results are shown in FIG. It was confirmed that the bacteria were stained and detected in the same manner as in Example 1.

[0045]

According to the method for staining bacteria of the present invention, a substance capable of reducing nitrite ions is added.
Even if bacteria having nitrate reducing power produce nitrite ions, the bacteria can be detected without being affected by the nitrite ions.

[Brief description of the drawings]

FIG. 1 is a scattergram of fluorescence intensity-forward scattered light intensity when ascorbic acid is used as a reducing agent in Example 1 of the present invention.

FIG. 2 is a scattergram of fluorescence intensity-forward scattered light intensity when no reducing agent is contained in Example 1 of the present invention.

FIG. 3 is a scattergram of fluorescence intensity-forward scattered light intensity when sulfamic acid is used as a reducing agent in Example 2 of the present invention.

FIG. 4 is a conceptual diagram showing a method for separating and counting bacteria after performing the bacterial staining method of the present invention.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Junya Inoue 1-5-1, Wakihama Kaigandori, Chuo-ku, Kobe City Inside Sysmex Corporation (72) Inventor Yoshiro Ikeuchi 1-5-1, Wakihama-kaigandori, Chuo-ku, Kobe City No. Sysmex Corporation F-term (reference) 2G045 BB25 BB41 CB21 FB12 GC11 GC15 2G054 AA10 BB08 CE02 EA03 EA05 GA05

Claims (12)

[Claims] 1. A method for staining bacteria, comprising reacting a polymethine dye in the presence of a substance capable of reducing nitrite ions. 2. The substance capable of reducing nitrite ion is ascorbic acid, isoascorbic acid, sulfamic acid, sulfanilic acid, aminomethanesulfonic acid, aminoethanesulfonic acid, glycine, glutamic acid, glutamine, aspartic acid, asparagine, methionine. , Glutathione, cysteine, mercaptoacetic acid, 3-mercaptopropionic acid, sulfite, pyrosulfite, hydroxylamine, phosphinic acid and salts thereof, and sulfanilamide, aminomethane, mercaptoethanol, thiophenol, urea 2. The method according to claim 1, which is one or more compounds. 3. A polymethine dye comprising: (1) thiazole orange (2) (3) (4) (5) (6) (7) (8) (9) (10) A compound represented by the following general formula: (Wherein, R ₁ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ₂ and R ₃ are a hydrogen atom, an alkoxy group having 1 to 3 alkyl groups or carbon atoms having 1 to 3 carbon atoms; R ₄ is hydrogen An atom, an acyl group or an alkyl group having 1 to 3 carbon atoms; R ₅ is a hydrogen atom, an alkyl group having 1 to 3 carbon atoms which may be substituted;
Z is a sulfur atom, an oxygen atom or a carbon atom substituted by an alkyl group having 1 to 3 carbon atoms; n is 1 or 2; X ⁻ is an anion) (11) A compound represented by the following general formula: Formula 10 (Wherein, R ₆ is an alkyl group having 1 to 18 carbon hydrogen atom or a carbon; R ₇ Beauty R ₈ is a hydrogen atom, an alkyl group or an alkoxy group having 1 to 3 carbon atoms having 1 to 3 carbon atoms; R ₉ is hydrogen atom, an acyl group, or an alkyl group having 1 to 18 carbon atoms; Z is a carbon having sulfur, oxygen or an alkyl group having 1 to 3 carbon atoms; n is 0, 1 or 2; X ^- is an anion is there).
The method according to claim 1, wherein the method is at least one selected from the group consisting of: 4. The method according to claim 1, wherein the polymethine dye is further allowed to act in the presence of a surfactant. 5. The method according to claim 1, wherein the polymethine dye is allowed to act under acidic conditions. 6. The method according to claim 1, wherein the polymethine dye is further allowed to act in the presence of an inorganic salt of either sulfate or nitrate. 7. Bacteria in a sample are stained by the action of a polymethine dye in the presence of a substance capable of reducing nitrite ions, and the obtained sample is detected by a flow cytometer. And irradiate each of the stained bacterial cells with light, and measure the scattered light and fluorescence emitted from these cells. (3) Measure the scattered light intensity and fluorescence intensity or particle length. A method to classify and count bacteria by the reflected pulse width. 8. The method according to claim 1, wherein the step (1) comprises diluting the sample containing bacteria with an aqueous solution containing a substance capable of reducing nitrite ions to promote the permeability of the dye to the bacteria. The method according to claim 7, further comprising a step of staining with a fluorescent dye for a certain period of time. 9. In step (3), at least one of (i) forward scattered light intensity and forward scattered light pulse width, (ii) forward scattered light intensity and fluorescent intensity, and (iii) forward scattered light pulse width and fluorescent intensity. 9. The method according to claim 7, wherein the bacteria are classified and counted by one. 10. In an effective amount of step (3), (i)
9. The method according to claim 7, wherein bacteria are classified and counted based on at least one of forward scattered light intensity and forward scattered light pulse width, (ii) forward scattered light intensity and fluorescence intensity, and (iii) forward scattered light pulse width and fluorescence intensity. The described method. 11. A diluent for bacterial staining, comprising an effective amount of a nitrite-reducing substance and a buffer for maintaining acidity. 12. The substance capable of reducing nitrite ion is ascorbic acid, isoascorbic acid, sulfamic acid, sulfanilic acid, aminomethanesulfonic acid, aminoethanesulfonic acid, glycine, glutamic acid, glutamine,
Aspartic acid, asparagine, methionine, glutathione, cysteine, mercaptoacetic acid, 3-mercaptopropionic acid, sulfite, pyrosulfite, hydroxylamine,
The diluent for cell staining according to claim 11, which is one or more compounds selected from the group consisting of phosphinic acid and salts thereof, and sulfanilamide, aminomethane, mercaptoethanol, thiophenol, and urea.