JP2000232897A - Prompt discrimination of fungi - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for promptly and accurately discriminating the number and kind of both living and dead fungi by effectively making a fluorescent dye penetrate and stain the inside of cells of various living and dead fungi and by irradiating liquid containing the fluorescent dye and the fungi in which fluorescent dye particles not penetrating the cells are prevented from firefly luminescence by intracellular enzyme with pulsed excitation light having high light energy and proper excitation wavelength. SOLUTION: This method for promptly discriminating the number and kind of both lining and dead fungi is to irradiate liquid 4 containing a fluorescent dye and fungi with pulsed excitation light 6 having high light energy and proper excitation wavelength to cause high-intensity firefly luminescence. In the liquid 4 the inside of living fungus cells is effectively penetrated and stained with a fluorescent dye for staining living fungus cells such as acetoxymethyl esterified calcein or acetoxymethyl esterified BCECF, or CFSE, carboxylfluorescein diacetate or fluorescein diacetate together with a dyeing accelerator comprising salts, chitinase and cellulase, and the inside of dead fungus cells is penetrated and stained with a fluorescent dye for staining dead fungus cells such as propidium iodide, ethidium bromide or ethidium homodimer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a viable cell and a killed cell by effectively penetrating and dyeing fluorescent dyes having different fluorescence wavelengths into viable and dead cells of a fungus and irradiating them with excitation light. The present invention relates to a method for instantly discriminating a fungus for immediately discriminating the number and the bacterial species.

[0002]

2. Description of the Related Art Although hygiene management and safety management of foods have been well managed conventionally, cooked foods have been increased due to the recent trend toward healthier eating out and eating out as well as eating out. Etc. are distributed in various ways, and the speed from production to consumption of these foods has been remarkably increased. In conjunction with the legislation on product liability, foods are more quickly and strictly sanitary. Management and safety management have become indispensable.

[0003] The hygiene control of foods is generally judged based on the degree of bacterial contamination, that is, the number of coliforms.
Since the number of coliforms is determined by inoculating the fungi of the specimen on an agar medium or the like and culturing for about 24 to 48 hours, and then forming a colony by reading with an optical microscope, the current distribution speed is high. Food hygiene control cannot be dealt with at all, and the accuracy of visual discrimination by cultured colonies is extremely poor, and furthermore, infectious Salmonella, Vibrio parahaemolyticus, and toxin-type staphylococci, which cause food poisoning, It has been pointed out that it is unfit for the actual situation to carry out strict hygiene management, such as the use of a specific medium for discrimination of special strains such as Clostridium botulinum.

In view of such circumstances, the present inventors have developed fluorescein or fluorescein which can selectively penetrate into viable and dead bacterial cells of fungi appropriately collected from a specimen and emit light at different fluorescence wavelengths upon irradiation with excitation light. A fluorescent dye composed of the derivative and propidium iodide was mixed with physiological saline at a required concentration, and a dye, which promoted the penetration into cells, a dye accelerator composed of chitines or cellulase was mixed at a required concentration. It is mixed into a dyeing solution, heated to a required temperature and subjected to fluorescent dyeing, and a runoff inhibitor comprising diethylstilbestrol or N, N'-dicyclohexylcarbodiimide to prevent backflow of the fluorescent dye. Is mixed at a required concentration to form a fluorescent stained bacterial solution, and the fluorescent stained bacterial solution is dropped on a light-transmitting plate and irradiated with excitation light having a center wavelength of 488 nm to obtain different fluorescent stains. Expanded viewing the emission shape and emission number of wavelengths, and investigate that following Te live bacteria and dead and the number of bacteria, may determine the species immediately, Japanese Patent Application No. 8-1252 already as prior application
No. 18 discloses the contents thereof. Further, the stained bacterium solution is injected into the cell, and the center wavelength of the cell is 488 nm, and the cell is irradiated with excitation light composed of pulsed light. After converting the number of luminescence into an electric signal, a fungus instantaneous discrimination device capable of discriminating and displaying the number of viable bacteria and dead bacteria and the type of bacteria by image processing and numerical processing was also developed, which was disclosed in Japanese Patent Application No. Hei 9-370157. The contents are disclosed.

[0005] However, fungi adhered and collected from a specimen for discrimination include a large number of bacteria consisting of prokaryotic cells, as well as eukaryotic fungi, yeasts and microorganisms. In addition, it has been clarified by gram staining or the like that bacteria, which are the main target of discrimination, differ greatly in cell structure and components depending on the bacterial species. That is, staphylococci, pneumococci, streptococci, tuberculosis, etc. are listed as typical examples of Gram-positive bacteria stained by Gram staining, and Escherichia coli, Shigella, spirochetes, and the like are typical examples that are not Gram stained. No. One of the stainings in this Gram staining is considered to be due to the cell structure.Prokaryotic cells have a cell membrane around the cytoplasm, and have a relatively thick membrane called a cell wall outside the cell membrane. Cell wall is about 15-60n for Gram-positive bacteria
m, and has a single-layer structure. Gram-negative bacteria have a two-layer cell wall consisting of a thin inner wall of about 2 to 3 nm and a slightly thick outer layer of about 8 to 10 nm. The cell wall epidermis contains ribonucleic acid (RNA) and a special protein complex, which is thought to react with a dye and be stained. Therefore, a fluorescent dye consisting of fluorescein or a derivative thereof to penetrate into living cells and a fluorescent dye consisting of propidium iodide to penetrate into dead cells were mixed in physiological saline at a required concentration. When fluorescent dyeing is performed by mixing fungi in the staining solution, large variations occur in the osmotic staining properties due to the cell structure and constituents of the bacterium, and bacteria, which are difficult to perform osmotic staining, are treated with salts, chitines or cellulase. If the penetration enhancer is blended at the required concentration,
Fluorescent dyes dispersed and mixed in the staining solution are hydrolyzed by the pH and temperature of the staining solution or slight component fluctuations in physiological saline, etc., and become fluorescent, and furthermore, live bacterial cells, especially decay In the transformed viable cells, the intracellular enzymes are easily washed away from the cell membrane and the cell wall, whereby the fluorescent dye particles dispersed and mixed in the fluorescent stained bacterial solution are hydrolyzed, and the viable cells and the dead cells are irradiated by the excitation light. This results in a mixture of the fluorescent light emitted by the fluorescent dye penetrated into the bacterial cells and the fluorescent light emitted by the unpermeated fluorescent dye particles.

According to the invention of the prior application, fluorescent dyes having different fluorescence wavelengths are selectively penetrated into live and dead cells of fungi, and the cells are irradiated with excitation light at different fluorescent wavelengths. Fluorescent light rays corresponding to the light emission shape and the number of light emissions are detected by a high-sensitivity photodiode or electron multiplier, converted to electrical signals, digitized and image-processed, and the number of live and dead bacteria and The purpose is to immediately and accurately determine the bacterial species. Therefore, the intensity of the fluorescent light emitted from the fluorescent dye penetratingly stained into viable cells and dead cells is accompanied by the fluorescent emission of the fluorescent dye particles that have not penetrated into the fluorescent dye solution by irradiation with the excitation light. If they are detected relatively small, and they are detected by a photodiode or electron multiplier with high detection capability, these are mixedly detected, and the problem that the type of bacteria, such as the accurate number of live and dead bacteria, cannot be determined, is newly added. appear.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made to solve such a problem, and the present invention is effective in live and dead fungal cells of fungi comprising various cell structures and forming substances. Fluorescent dye is then permeated with a fluorescent dye, and the pulsed excitation light having a high light energy and an appropriate excitation wavelength is applied to a fluorescent stained bacterium solution in which the fluorescence of unpermeated fluorescent dye particles due to the elimination of intracellular enzymes is suppressed. Irradiate and produce fluorescent light with high luminescence intensity from viable bacterial cells and dead bacterial cells stained by permeation staining,
Accordingly, it is an object of the present invention to provide a method for instantaneously discriminating a fungus capable of immediately and accurately discriminating the number of live bacteria and the number of dead bacteria and the type of bacteria.

[0008]

Means for Solving the Problems According to the present invention for solving the above-mentioned problems, the technical means adopted by the present invention for solving the above-mentioned problems is particularly problematic in the fluorescent dye, particularly when the permeability of the fluorescent dye varies. Acetoxymethyl esterified acetoxymethyl ester, which has excellent permeability into cells for viable cells consisting of poorly permeable cell structures and constituents in order to effectively penetrate into viable bacterial cells that cause Viable bacterial cells composed of calcein bromide, acetoxymethyl esterified BCECF, and a cell structure and components that have relatively good intracellular permeability include CFSE, carboxylfluorescein diacetate, fluorescein diacetate, and live bacterial cells. In order to increase the permeability, a fluorescent dye selected from acridine orange etc. is used for further penetration dyeing into dead cells. A fluorescent dye selected from propidium iodide, etidue bromide and etidue homodimer is mixed with physiological saline at a concentration of 3 to 15 μmol / ml, respectively, and the temperature is 24 to 37. The fungus adhered and collected from the specimen by a suitable means is mixed into the staining solution kept at ° C., and the viable cells and the dead cells are permeated and stained to form a fluorescent stained bacterial solution.

Further, when carboxylfluorescein diacetate or fluorescein diacetate is used for the permeation staining of live bacterial cells, the dyeing solution containing salts, chitines or cellulase at a concentration of 1 to 10 μmol / ml in the staining solution. The fluorescent stained bacterial solution mixed with bacteria and adhered and collected from the specimen and subjected to fluorescent staining was mixed with an anti-runoff agent comprising diethylstilbestrol or N, N'-dicyclohexylcarbodiimide. 5 to 20 μmol /
Then, the fluorescent dye solution is injected into a cell made of a translucent material, and the white pulse light from the white pulse light source is converted into an appropriate wavelength having a transmission wavelength in the range of 480 to 550 nm. Irradiating a pulse excitation light of a required wavelength through a band pass filter, and detecting the light emission shape and the number of light emission related to the fluorescence emission having high fluorescence intensity from the viable cell and the dead cell, and performing an electrical treatment. Thus, the present invention resides in a configuration in which live bacteria, dead bacteria, and bacterial species are immediately and accurately distinguished.

[0010]

The present invention having the above construction has the following functions. In other words, when the fluorescent dye has poor penetrating ability due to the cell structure such as the cell membrane and cell wall of the viable cell of the fungus to be discriminated and the forming components, acetoxymethyl esterified calcein or acetoxymethyl esterified BCEC is used.
By using F as a fluorescent dye for staining live cells,
The acetylation of the hydroxyl group significantly increases the lipophilicity, so that not only the permeability into the cell membrane is increased and sufficient permeation staining is realized, but also the fluorescent dye itself does not emit fluorescent light, and Hydrolysis by intracellular enzymes such as esterase during permeation creates fluorescence,
Fluorescence of the fluorescent dye particles dispersed in the unpermeated fluorescent dye solution is also prevented.

[0011] Further, if the penetration dyeability is relatively good,
CFSE, carboxyl fluorescein diacetate,
Alternatively, since fluorescein diacetate is used and a staining accelerator consisting of salts, chitines or cellulase is mixed and used in physiological saline at a concentration of 1 to 10 μmol / ml, the cell structure, especially the cell membrane, is softened and the fluorescent dye is used. After the osmotic staining, an anti-runoff agent consisting of diethylstilbestrol or N, N'-dicyclohexylcarbodiimide is added to the fluorescent stained bacterial solution in an amount of 5%.
Since the mixture is mixed at a concentration of about 20 μmol / ml, the elimination of enzymes and the like from the cells, particularly from the attenuated viable cells, is prevented. Fluorescence of the dye particles will also be blocked. Fluoride dyes consisting of propidium iodide or ethidium bromide and ethidium homodimer for infiltration into dead cells can penetrate freely without being affected by cell membranes and cell walls. Can be used in combination with a fluorescent dye for staining any viable cell.

There is a Stokes law between the excitation light wavelength and the fluorescence wavelength of the fluorescent dye, and each of the fluorescent dyes must be retained in order to emit fluorescent light with high fluorescence intensity by effective excitation. In this case, the excitation light beam is irradiated with a specific excitation wavelength that matches the specific excitation wavelength, and the allowable excitation light wavelength is set to 10 nm before and after the specific excitation wavelength, preferably 3 to 5 nm or less. The use of various fluorescent dyes to enhance permeation staining in accordance with the components results in a unique excitation wavelength for each fluorescent dye, and the fluorescent dye for dead cell staining also has a unique excitation wavelength. Since the wavelength is maintained, there is a danger that fluorescence emission with high fluorescence intensity cannot be obtained by irradiating excitation light with a single excitation wavelength. Because of this, white pulse light from a white pulse light source with strong light energy,
A band-pass filter is provided for transmitting and converting the transmission wavelength to an appropriate wavelength in the range of 480 to 550 nm, and the fluorescence injected into the cell as pulse excitation light having a high light energy which is adapted to the excitation wavelength inherent to the fluorescent dye and has a high light energy. Because the stained bacterial solution is irradiated, the fluorescent dye penetratingly stained into live cells and dead cells emits fluorescent light with high fluorescence intensity, and the light emission shape and the number of light emission are accurately detected and electrical Processing will be performed.

[0013]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below in detail with reference to the accompanying drawings. FIG. 1 is an explanatory view of a procedure for preparing a fluorescent stained bacterial solution 4 which emits fluorescent light by irradiating pulse excitation light in the present invention. In this fluorescently stained bacterial solution 4, physiological saline 1 is used as a main component because it is necessary to mix and disperse the fungi adhered and collected from the specimen by appropriate means and to survive. Fluorescent dye 1A for staining live cells and fluorescent dye 1 for staining dead cells to selectively penetrate into viable cells and dead cells of the fungus dispersed and dispersed.
B is mixed to a required concentration, and if the mixing ratio of the fluorescent dye 1A for staining viable cells and the fluorescent dye 1B for staining dead cells becomes too low, the fluorescence emission energy after permeation staining also decreases. A concentration of at least 3 μmol / ml or more with respect to physiological saline 1 is desired because it becomes small and there is a risk that sufficient discrimination cannot be made. However, if the concentration is too high, there is a risk that live bacteria may be weakened or killed. Should be kept at most within 15 μmol / ml of saline 1.

By the way, fungi to be distinguished include those of prokaryotic cells and those of eukaryotic cells, as well as cell structures such as cell membranes and cell walls and their forming components as classified into Gram-positive bacteria and Gram-negative bacteria in Gram staining. In particular, the cell membrane greatly contributes to the penetration into cells, and the permeability to living cells is greatly influenced by these factors. For this reason, calcein or BCECF (biscarboxyethylcarboxylfluorescein) is acetoxymethyl ester as a fluorescent dye 1A for viable cell staining of viable cells having poor permeation staining as seen in eukaryotic cells and gram negative bacteria in gram staining. It is desired to enhance the lipid solubility and significantly increase the permeability into the cell membrane by the conversion. Furthermore, CFS is used for fungi that are relatively easy to penetrate into the cell membrane, such as prokaryotic cells and Gram-positive bacteria in Gram staining.
E. Carboxyfluorescein diacetate or fluorescein diacetate is convenient, but in such a case, in order to ensure more reliable intracellular permeability, a dye accelerator 1C consisting of salts (eg, magnesium chloride), chitines or cellulase is required. It is desired that the dye is mixed with the dye at a desired concentration. Since the dyeing accelerator 1C is intended to soften the cell membrane and particularly enhance the permeability, which affects the permeability of the fluorescent dye 1A for dyeing viable cells, it enhances the permeability. Since the mixed use of the concentration may affect the viable cells as well as re-bleach the fluorescent dye 1A for the viable cell stain which has been permeated and stained, the staining solution 2 is at least 1 μmol / ml to at most 10 μmol. / Ml range.
In addition, it should be particularly noted that as a fluorescent dye for staining viable bacterial cells of eukaryotic cells, acridine orange is preferred from many staining processes.

On the other hand, the fluorescent dye 1B for staining dead cells easily penetrates into cells and intercalates with nucleic acids, and emits at a fluorescent wavelength different from that of the fluorescent dye 1A for live cells upon irradiation with excitation light. Since fluorescent light with high brightness is desired,
Specific examples of the fluorescent dye 1B for staining dead cells include the specific dye of the fluorescent dye 1A for live cell staining, which is used in combination with propidium iodide and ethidium bromide and ethidium homodimer. An excitation wavelength that is similar to the excitation wavelength of the above is used. By the way, as an example of the combination of the fluorescent dye 1A for staining viable cells and the fluorescent dye 1B for staining dead cells, when carboxylfluorescein diacetate or fluorescein diacetate is used as the fluorescent dye 1A for staining viable cells, When propidium iodide is used as the fluorescent dye 1B for staining dead cells and acetoxymethyl esterified calcein or acetoxymethyl esterified BCECF is used as the fluorescent dye 1A for staining live cells, it is used for staining dead cells. If the fluorescent dye 1B is ethidium homodimer, and if CFSE is used as the fluorescent dye 1A for live cell staining, etidue bromide or the like is preferred as the fluorescent dye 1B for dead cell staining. . However, the fluorescent dye 1A for staining viable cells and the fluorescent dye 1B for staining dead cells have significant differences in their excitation wavelengths depending on the production method and the manufacturer. It is.

Thus, the required concentration of the fluorescent dye 1A for staining viable cells and the fluorescent dye 1B for staining dead cells are mixed with the physiological saline solution 1, and further the staining accelerator 1C is mixed with the required concentration. The staining solution 2 is mixed with a fungus 3 for discrimination, which is appropriately adhered and collected from the specimen, and dispersed, and the fluorescent dye 1A for live cell staining and the dead cell stain When performing penetration dyeing with fluorescent dye 1B,
In particular, in order to promote the activity of viable bacteria and enhance permeation dyeability, it is desired to perform permeation dyeing by keeping the dyeing solution 2 heated at 24 to 37 ° C., and heating conditions vary depending on the type of bacteria. The temperature is preferably about 24 to 27 ° C for fungi composed of eukaryotic cells, and about 34 to 36 ° C for bacteria of prokaryotic cells.

As described above, the fluorescent dye-stained bacterial solution 4 is prepared by mixing and dispersing the fungi 3 into the staining solution 2 and heating 4 B to a required temperature to perform permeation staining. The more easily the fluorescent dyes such as fungi and decayed fungi can penetrate, the higher the effluent is. Therefore, in the case of the penetration dyeing of these fungi, diethylstilbestrol or N, N'-dicyclohexylcarbodiimide is used after the penetration staining. 5A to the fluorescent stained bacterial solution 4
It is desired to mix them by 20 to 20 μmol / ml.

In order to determine the number of viable and dead bacteria and the type of fungi adhered and collected from a specimen using the fluorescent stained bacterial solution 4, as shown in FIG. A fluorescent dye bacterium solution 4 is injected into a cell 5 formed of a translucent material, which is required to be converted into an electric signal after the injection of an amount of light and irradiation with the excitation light. A pulse excitation light 6 is used as an excitation light in order to irradiate an excitation light having a high light energy intensity and a required excitation wavelength to emit fluorescent light having a high fluorescence emission intensity. The pulse excitation light 6 is provided with a white pulse light source 6A having a very high light energy intensity on one side capable of irradiating the cell 5, and the white pulse light from the white pulse light source 6A is supplied to the fluorescent dye bacterium solution in the cell 5. Fluorescent dye 1A for viable cell staining used in No. 4
In addition, the transmission wavelength is 480 to 550 in order to convert and transmit to an excitation wavelength that matches or approximates a specific excitation wavelength held by each of the fluorescent dyes 1B for staining dead cells.
A band-pass filter 6B capable of freely converting to a specific wavelength in the range of nm is provided and transmitted, so that the pulse excitation light 6 having a high light energy intensity can be easily obtained.

In such a case, the white pulse light source 6A
The reason for this is that a xenon lamp or the like can be used normally, and the reason that the pulse excitation light 6 is used is that the maximum fluorescence emission intensity when the fluorescent dye is irradiated with excitation light of general continuous light is approximately 200.
The fluorescent dye 1A for staining viable cells and the fluorescent dye 1B for staining dead cells, which were permeated into live cells and dead cells, exhibiting the behavior of attenuating after peaking at about 250 μsec. In order to immediately determine the number of viable and dead cells and the type of the bacterium based on the fluorescence emitted from, it is desirable to capture the maximum fluorescence intensity. This requires pulsed excitation light 6
And averaging the number of fluorescent light beams having the maximum fluorescent light emission intensity for each pulse leads to extremely reliable discrimination. The pulse interval of the pulse excitation light 6 is such that the peak of the maximum fluorescence emission intensity accompanying the irradiation of the pulse excitation light 6 is attenuated in about 200 to 250 μsec and thereafter,
A pulse interval of at least 750 μs is preferred.

The selection of the transmission wavelength of the band-pass filter 6B for converting the white pulse light to the pulse excitation light 6 depends on the combination with the fluorescent dye 1B for staining dead cells used in combination with the fluorescent dye 1A for staining viable cells. Therefore, it is naturally determined that the mutual fluorescent dyes to be used together are preferably excited and emitted with the same excitation wavelength because they are determined by the relationship with the unique excitation wavelength held by each. However, when there is a large divergence between the specific excitation wavelengths, the pulsed excitation light 6 converted by the band-pass filter 6B adapted to one of the excitation wavelengths is irradiated to capture the fluorescent light emission, and then the other excitation light is emitted. It is also possible to irradiate the pulse excitation light 6 converted by the band-pass filter 6B so as to match the wavelength, capture the fluorescence emission, and electrically process each of them.
Even when the fluorescent dye 1A for staining viable cells and the fluorescent dye 1B for staining dead cells are used in various ways, the excitation wavelength can be dealt with in the range of 480 to 550 nm. Is an excitation wavelength range of 480 to 550 nm and an arbitrary number of sheets so as to transmit and convert to an excitation wavelength that can cope with the combined use of the fluorescent dye 1A for staining live cells and the fluorescent dye 1B for staining dead cells. It is convenient to provide the band pass filters 6B in a switchable manner. Fluorescent light that has been fluorescently illuminated to a high fluorescence intensity by irradiation with the pulsed excitation light 6 emits fluorescent light from live bacterial cells in two directions orthogonal to the irradiation optical axis of the pulsed excitation light 6. On the other side, fluorescent light from the dead cells is detected by a detector tube 6D such as a photodiode or an electron multiplier through a transmission filter 6C that transmits a predetermined wavelength in accordance with Stokes law, and is then subjected to electrical processing. Is performed to make a determination.

[0021]

As described above, the present invention provides acetoxymethyl esterified calcein and acetoxymethyl esterified BCECF even when the fluorescent dye penetrates into viable bacterial cells of a fungus collected from a specimen for discrimination as described above. Can be used, the lipophilicity is strong, and the permeability is significantly increased in combination with the warming of the fluorescently stained bacterial solution, and the fluorescent staining of the viable bacterial cells can be carried out well, and the intracellular cells are produced with the acetoxymethyl esterification. Is hydrolyzed by the above enzyme to impart fluorescence, and thus the fluorescence of the fluorescent dye particles for staining viable cells dispersed in the fluorescence stained bacterial solution is also suppressed. Furthermore, if the fluorescent dye has relatively good permeation dyeing ability into the viable cells, CFSE
Using carboxylfluorescein diacetate or fluorescein diacetate with a fluorescent dye for viable cell staining and using a dye, a salt, chitines or cellulase as a dye accelerator, the penetration staining into viable bacterial cells is not successfully performed. It will be. In particular, in fungi having good permeation staining properties, bleeding after permeation staining is apt to occur, and the bleeding also leads to the loss of intracellular enzymes. Therefore, diethylstilbestrol or N, N'-
By preventing the runoff with the runoff inhibitor comprising dicyclohexylcarbodiimide, the fluorescent dye of the fluorescent dye particles for viable cell staining, which is sufficiently dispersed in the fluorescent dye-stained bacterial solution, is also suppressed. Thus, according to the present invention, a wide range of fluorescent dyes for staining viable cells can be used in accordance with the characteristics of fungi, and as a fluorescent dye for staining dead cells, propidium iodide, etidue bromide or etidueme is used. Since a homodimer or the like can be appropriately used in combination, a fluorescently stained bacterial solution effectively penetrated into live cells and dead cells is prepared, and light energy is applied to the cells into which the fluorescently stained bacterial solution is injected. High intensity pulsed light with a transmission wavelength of 480
Since the pulse excitation light is irradiated through a band-pass filter that can be converted to an appropriate transmission wavelength in the range of 550 nm to 550 nm, viable cells and dead cells emit fluorescent light with high fluorescent emission intensity and are detected. Since the electrical treatment is carried out, the method can be said to be an extremely excellent method for immediately distinguishing fungi such that the number of dead bacteria and the number of dead bacteria can be immediately determined with high accuracy.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram of a procedure for preparing a fluorescently stained bacterial solution.

FIG. 2 is an explanatory diagram of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Physiological saline 1A Fluorescent dye for live cell staining 1B Fluorescent dye for dead cell staining 1C Staining accelerator 2 Staining solution 3 Fungi 4 Fluorescent stained bacterial solution 4A Anti-spill agent 4B Warming 5 Cell 6 Pulse excitation Light 6A White pulse light source 6B Bandpass filter 6C Transmission filter 6D Detector tube

Claims (2)

[Claims] 1. Acetoxymethyl esterified calcein, acetoxymethyl esterified BCECF, CFS
E, fluorescent dye for live cell staining selected from carboxylfluorescein diacetate, fluorescein diacetate or acridine orange, and dead cell staining selected from propidum iodide, etidue bromide or etidueme homodimer Fluorescent dye is added to physiological saline in 3 to 15 μmol each
/ Ml at a concentration of 24 to 37 ° C
A fluorescently stained bacterial solution obtained by mixing the appropriately collected fungi into the staining solution held in the above and performing fluorescent staining is injected into a cell made of a translucent material, and a white pulse light is transmitted through the cell. The cell is irradiated as a pulse excitation light of a required wavelength through a band-pass filter capable of converting the wavelength to an appropriate wavelength in the range of 480 to 550 nm, and the fluorescent light emission shape and the number of fluorescent light emission at different fluorescent wavelengths are detected and electrically processed. Thus, a method for immediately distinguishing fungi in which the number of live and dead bacteria and the type of bacteria are immediately distinguished. 2. A dyeing solution in which the fluorescent dye for viable cell staining is carboxyl fluorescein diacetate or fluorescein diacetate, and a dyeing accelerator comprising salts, chitines or cellulase is 1 to 10 μm.
ol / ml and added to diethylstilbestrol or N, N.
An anti-runoff agent selected from N'-dicyclohexylcarbodiimide is added to the fluorescent stained bacterial solution in an amount of 5 to 20 μmol / ml.
2. The method of claim 1, wherein the fungi are mixed.