JP2008054509A - Method for determining ratio of viable bacteria, dead bacteria and pseudo-viable bacteria - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily and quickly determining the life or death of bacteria in high accuracy. <P>SOLUTION: The method for effectively and easily determining the life or death of bacterial cell comprises semi-drying treatment of a specimen containing bacterial cells and the treatment with physiological salt solution and/or a surfactant solution before or after the staining of the specimen with two kinds of dyes, i.e. a dye specifically staining viable bacteria and a dye specifically staining dead bacteria. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for efficiently and accurately determining the viability of bacteria contained in a sample. More specifically, the present invention relates to a bacterium that has stopped substantial life activity such as growth and metabolism, such as some enzyme systems. The present invention relates to a method for distinguishing between live and dead bacteria that can be identified as killed even when the activity of the microorganism is retained.

  The determination of the life or death of bacteria contained in a sample is important for verifying the effect of sterilization treatment in foods, for example. In particular, it is necessary to quickly and accurately determine whether a bacterium is alive or not, such as when many samples are assayed. Conventionally, as a method of discriminating the life and death of bacteria contained in a sample, a method of detecting cell viability from the wavelength difference and color development amount of colored light by contacting cells with a plurality of chemical substances that develop color, A method for measuring the number of viable bacteria by measuring the amount of ATP, a method for culturing a part of the sample, and calibrating the proliferating bacteria are disclosed (Patent Documents 1-3). However, the conventional method has a problem that the accuracy of the test is insufficient or the processing process takes too much time.

Also, especially when the sample is sterilized, even though substantial life activity itself such as growth and metabolism has stopped, the “pseudo-viable bacteria” (defined later) ”)” Was also recognized as a living bacterium in the conventional bacterial viability determination method.
Patent application title: Microorganism weighing method and microorganism weighing apparatus Method for measuring the number of viable bacteria in plant beverages Method for estimating viable cell count

  In view of the above-mentioned present situation, the present invention uses a combination of a plurality of types of dyes, that is, a dye that selectively stains viable bacteria and a dye that selectively stains dead bacteria, thereby accurately determining the viability ratio of bacteria. An object of the present invention is to provide a method for simple and quick discrimination.

  Another object of the present invention is to provide a method of correctly recognizing “pseudo-viable bacteria”, which may be misidentified as viable bacteria in the conventional method, in determining whether bacteria are alive or dead.

In the present specification and claims, the following terms shall each be used according to the following definitions: A “viable dye” refers to a dye that is taken into cells by viable bacteria (living bacteria) and colors viable bacteria, but does not have the ability to color dead bacteria (dead bacteria).
“Dead fungus pigment” means that it cannot pass through the cell membrane pores of viable bacteria, or once discharged into live cells, it cannot be colored, but Refers to a dye that has the ability to enter cells, stay in the cells by binding to DNA, etc., and color dead bacteria.
“Whole fungal pigment” refers to a pigment capable of staining bacteria regardless of whether they are viable or dead.
A “pseudo-viable bacterium” refers to a dead bacterium that has lost its functions of growth and metabolism and has stopped substantial vital activity, but retains activity in some enzyme systems such as cell membranes.

  The present inventors examined various methods to obtain a method for accurately and quickly knowing the existence state of bacteria in a sample, such as verification of the bactericidal effect, and as a result, reliably measured pseudo-viable bacteria as dead bacteria. The method that can be obtained was found and the present invention was completed.

  That is, the first aspect of the present invention is a method for determining the abundance of each bacterium in a mixed sample containing live bacteria, dead bacteria, and pseudo-viable bacteria that retains enzyme activity by stopping life activity, Each state characterized by comprising a step of semi-drying the mixed sample and then treating with a salt solution and / or a surfactant solution, a bacterial staining step with a viable dye, and a bacterial staining step with a dead fungus dye A method for determining the abundance of bacteria present in

  According to a second aspect of the present invention, in the determination method according to the first aspect, the mixed sample is subjected to a semi-drying treatment, and then a step of treating with a salt solution and / or a surfactant solution, and thereafter using a viable dye. Provided is a method for discriminating the existence ratio of live bacteria, dead bacteria, and pseudo-bacteria, which comprises performing a bacterial staining process and a bacterial staining process with a dead bacteria pigment simultaneously or sequentially.

  According to a third aspect of the present invention, there is provided the method for determining the abundance ratio of viable, dead, and pseudobacteria according to the first or second aspect, wherein the viable dye is a fluorescein fluorescent dye.

  According to a fourth aspect of the present invention, there is provided the method for determining the abundance ratio of viable, dead, and pseudobacteria according to the third aspect, wherein the viable dye is 6-carboxyfluorescein diacetate.

  According to a fifth aspect of the present invention, there is provided the method for determining a presence ratio of viable bacteria, dead bacteria, and pseudo-bacteria according to any one of the first to fourth aspects, wherein the dead bacteria pigment is propidium iodide. To do.

  According to a sixth aspect of the present invention, there is provided a viable cell, dead cell or pseudo-bacteria according to the first or second mode, wherein the viable cell dye is 6-carboxyfluorescein diacetate and the dead cell dye is propidium iodide. Provide a method for determining the existence ratio of.

  According to a seventh aspect of the present invention, in any one of the first to sixth aspects, the viable bacteria, dead bacteria and Provided is a method for determining the existence ratio of pseudo-bacteria.

  The eighth aspect of the present invention is the method for determining the abundance ratio of viable, dead, and pseudobacteria according to the seventh aspect, wherein the total microbial pigment is 4 ′, 6-diamidino-2-phenylindole dihydrochloride I will provide a.

  According to a ninth aspect of the present invention, there is provided the method for determining the abundance ratio of viable, dead, and pseudobacteria according to any one of the first to eighth aspects, wherein the salt solution is an aqueous sodium chloride solution. .

  According to a tenth aspect of the present invention, the concentration ratio of the aqueous sodium chloride solution is within a range of 0.75% to 0.95%, and the existence ratio determination of the viable bacteria, dead bacteria and pseudobacteria according to the ninth aspect is performed. Provide a method.

  The eleventh aspect of the present invention is the presence of viable, dead, and pseudobacteria according to any one of the first to tenth aspects, wherein the surfactant is polyoxyethylene sorbitan monolaurate. Provide a percentage discrimination method.

  The twelfth aspect of the present invention is the live bacteria, killed bacteria and pseudo-bacteria according to the eleventh aspect, wherein the concentration of polyoxyethylene sorbitan monolaurate is in the range of 10-5% to 10-9% Provide a method for determining the existence ratio of.

  In a thirteenth aspect of the present invention, each of the steps described in any one of the first to twelfth steps is used to fluorescently stain a mixed sample containing live bacteria, dead bacteria, and pseudobacteria, and a spectrophotometer is used. The present invention provides a method for measuring the existence ratio of viable / dead bacteria, which comprises measuring the fluorescence of the mixed sample.

  By using the method for determining the viability of bacteria provided by the present invention, it becomes possible to discriminate pseudo-viable bacteria that could not be distinguished from viable bacteria as dead bacteria, and a more accurate method for verifying the bactericidal effect Can provide. In addition, the present invention does not include the “bacterial culture process” that is the standard in the bacterial life / death discrimination method, and can greatly reduce time and human labor. Conventionally, especially in food-related sterilization processing, 3-4 days were required for the determination of availability, but this can be performed in several hours by using the present invention.

The present invention is a mixed sample containing live bacteria, dead bacteria and pseudo-viable bacteria (mixed sample refers to a sample that may contain bacteria in these three states, and actually This is also a case where only one or two types are present), a semi-drying process followed by a salt solution and / or a surfactant solution process, a bacterial dyeing process with viable pigment In addition, by performing the bacterial staining step with the dead bacteria pigment in an appropriate order or simultaneously, it is possible to discriminate the proportion of bacteria and the number of existing individuals. Of course, the above-described steps can be performed on one sample, or the sample can be divided and processed at one or two of the steps. Table 1 shows the relationship between each step and the corresponding bacterial state.

  In Table 1, (1) and (3) are colored by the A treatment, and the presence thereof (the total amount when all the fungal dyes are used) is confirmed. It is also possible to calculate the abundance from the colored state by creating a calibration curve or the like in advance. Depending on the B treatment, (2) is colored and the presence of dead bacteria is confirmed. As in the case of process A, the abundance can be calculated using a calibration curve or the like. Next, by performing the A process after performing the C process, it becomes possible to confirm the presence of (1) and at the same time not to color (3), which may be identified as viable bacteria, and further perform the C process. Then, if the B process is performed, the presence of the total amount of (2) and (3) is confirmed, and the amount can also be calculated. If both the A process and the B process are performed after the C process, the color development by the A process (including fluorescence) and the color development by the B process (including fluorescence) are changed to different colors (1), ( It is also possible to determine each of 2) and (3), and of course, it is possible to know their abundance ratio.

  Furthermore, a mixed sample of bacteria is divided into a plurality of samples, the state of each bacteria is grasped for a sample that has been subjected to A treatment and / or B treatment in advance, and C If processing is performed, then processing A and / or processing B is performed, and the data obtained for each processing is compared, it is possible to determine the quantity, ratio, etc., of each bacterial state in the mixed sample. Depending on the purpose of knowing the state of the bacteria in the sample, either the viable dye or the dead fungus dye can be replaced with the whole fungus dye. Is included.

  Any combination of viable and dead dyes may be used as long as they can be distinguished. For example, fluorescent dyes having different autofluorescence wavelengths, preferably red fluorescence and green If a dye having fluorescence is combined, it is possible to easily determine the viability of the bacteria by observation using a normal fluorescence microscope, and it is possible to measure the viable / dead ratio within the same field of view.

In the present invention, as an important means for assisting discrimination between viable and dead bacteria, it is effective to perform a semi-drying treatment and a treatment with a salt solution and / or a surfactant on a bacterial sample before staining. When measuring substances related to the physiological activity of bacteria, such as quantifying the amount of ATP, if the bacteria are already dead but membrane enzymes are active, the activity of these enzymes It is often recognized as “live bacteria”. In order to solve this problem, a solution containing a bacterial sample is dropped in advance on an agar plate or the like, and a semi-drying process is performed by absorbing moisture into the agar, and further, a salt solution and / or a surfactant is used. It is effective to reduce the metabolic activity of the enzyme system of dead bacteria. Even with these treatments, viable bacteria are not affected by their activity. The method for the semi-drying treatment is not particularly limited. For example, as described above, the solution may be dropped on the plate for about 5 to 30 minutes, preferably 10 minutes. The kind of the salt solution is not particularly limited, but an alkali metal salt or an alkaline earth metal salt, particularly a sodium salt or a potassium salt is preferable. These salt solutions are generally aqueous solutions, and the concentration thereof is, for example, 0.75% -0. It is effective to treat with 95% NaCl, preferably 0.85% NaCl for about 1-3 hours. The surfactant may be appropriately selected according to the sample, and the type of the surfactant is not limited to the present invention. However, the neutral surfactant is preferable from the viewpoint of not affecting the living bacteria. In the case of polyoxyethylene sorbitan monolaurate (trade name Tween 20, manufactured by ICI Americas) generally used in the experiment, it is within a range of 10 ⁻⁵ to 10 ⁻⁹ %, preferably at a concentration of 10 ⁻⁷ % for 1 to 3 hours. By processing, it is possible to reduce the metabolic activity of dead bacteria to such an extent that it can be distinguished from live bacteria. The concentration of Tween 20 is preferably 10 ⁻⁷ to 10 ⁻⁹ % in the case of Gram negative bacteria such as E. coli, and 10 ⁻⁶ to 10 ⁻⁷ % in Gram positive cocci. The treatment with the salt solution and the treatment with the surfactant may be performed separately, or may be performed simultaneously from the viewpoint of shortening the reaction time.

  In more detail, the viable dye used in the present invention is preferably a fluorescein fluorescent dye. Fluorescent dyes with fluorescein as the basic skeleton, such as calcein-AM, fluorescein diacetate, succinimidyl ester, etc., are rich in lipid solubility because the phenolic hydroxyl group of the fluorescein skeleton is protected by acetyl groups, etc., and can easily pass through cell membranes have. Outside the cell, the fluorescence is suppressed due to the ester protecting group, but when entering the cell, it is hydrolyzed by the esterase present in the cell and becomes fluorescent. In addition, these dyes are less lipophilic due to hydrolysis and cannot be eluted out of the cell, resulting in staying in living cells and staining them. A series of reactions does not occur in dead cells without esterase activity, and the cells are not stained with fluorescence. The viable dye is preferably a fluorescent dye having the above properties, more preferably 6-carboxyfluorescein diacetate.

  The dead dye in the present invention will be described in more detail. It is a DNA-binding fluorescent dye that has a molecular size that cannot pass through the cell membrane of a living cell. These dyes can enter dead cells and bind to nuclear DNA within the cell. Propidium iodide (molecular weight 668.39) is an example of a killed dye suitable for the above purpose.

  When using a pigment | dye in this invention, what is important is the combination of a living microbe pigment | dye and a dead microbe pigment | dye. That is, by staining bacterial samples with fluorescent dyes having different wavelengths, it is possible to distinguish between live bacteria and bacteria in which membrane-based enzymes and the like remain active in addition to dead bacteria. As described in the following examples, viable and dead bacteria can be distinguished effectively by using 6-carboxyfluorescein acetate (or SYT9 dye) as a viable dye and propidium iodide as a killed dye as a combination of the above dyes. It becomes.

  In the present invention, it is also possible to replace either the viable dye or the dead fungus dye with a whole fungus dye (a dye that can stain bacteria regardless of whether it is live or dead). Even in this case, any substance that emits fluorescence different from that of dead bacteria when replaced with viable dyes, or any substance that emits fluorescence different from that of viable bacteria when replaced with dead bacteria dyes may be used. And the like may be appropriately selected from those conventionally used for bacterial staining. Among them, 4 ', 6-diamidino-2-phenylindole dihydrochloride (DAPI) is particularly suitable as a control for DNA fluorescence staining in many cells.

  As shown in the examples below, in the present invention, in order to perform viable and dead bacterial staining on the bacteria contained in the sample, the intensity of the detected fluorescence is detected by detecting a specific wavelength using a spectrophotometer. Can be converted into the abundance ratio of live and dead bacteria. Measure the fluorescence intensity of a solution containing no bacteria in advance and the fluorescence of a solution containing a specific amount of viable bacteria, and measure the fluorescence intensity for each of the wavelengths of viable and dead dyes in the sample. By taking this ratio, it is possible to easily calculate how many live bacteria and how many dead bacteria exist in the solution. Standard solutions, spectrophotometers, and the like may be used as necessary, and are not intended to limit the present invention.

  By applying the method for discriminating between live bacteria and dead bacteria in the present invention to a sample for sterilization, it can be used as a method for verifying the sterilization effect. For example, a sample of drinking water that needs to be sterilized, and after sterilization, this sample is stained with live and dead dyes, and the salt solution and / or surfactant solution treatment described above is performed. It is possible to calculate the presence of live bacteria and dead bacteria contained therein and verify how much the effect of sterilization was compared with the sample before sterilization. Examples of the present invention will be described below, but the present invention is not limited to the examples.

  (Preparation of E. coli) In the examples, Escherichia coli was used as a bacterium in all operations. E. coli was inoculated into 5 ml of LB medium (1% peptone, 0.5% yeast extract, 0.5% NaCl) and cultured at 37 ° C. for about 14 hours. After 14 hours, the absorbance (670 nm) of the culture solution was measured, and the solution was adjusted to a value of 2.0. 1 ml of a culture solution (suspension) of Escherichia coli was centrifuged at 10,000 × g for 10 minutes at 4 ° C. to precipitate the bacteria and resuspended in 1 ml of 0.85% NaCl aqueous solution.

  (Preparation of control for E. coli standard of life and death) 50 μl of the above E. coli resuspension was added to 1 ml of 70% isopropyl alcohol, treated at room temperature for 1 hour and completely sterilized to obtain a dead cell sample. In addition, 50 μl of E. coli resuspension was added to 1 ml of 0.85% NaCl aqueous solution, and this was used as a viable cell sample. The dead bacteria and live bacteria sample solution was centrifuged at 10,000 × g for 10 minutes at 4 ° C., and the bacterial precipitate was collected. The precipitate was washed twice with 1 ml of 0.85% NaCl aqueous solution and then suspended in 0.1 ml of 0.85% NaCl aqueous solution.

(Sterilization by UV irradiation) Several 50 μl of live Escherichia coli suspension was placed on a plastic plate, and the UV non-irradiated part (control of live bacteria) was covered with aluminum foil. Under room temperature conditions, ultraviolet rays of 254 nm were irradiated at an intensity of ² J / cm ² for 1 minute. After irradiation, the suspension was collected in a tube, centrifuged at 10000 × g for 10 minutes at 4 ° C. to precipitate bacteria, and resuspended in 0.1 ml of 0.85% NaCl aqueous solution.

(Sample treatment after UV irradiation) Each 0.1 ml E. coli suspension subjected to UV irradiation (non-irradiation as a control) was dropped on an LB agar medium and left for about 10 minutes to temporarily suspend the suspension. The cells were absorbed in an agar medium and the cells were placed in a semi-dry state. Thereafter, E. coli was recovered from the surface of the agar medium using 1 ml of 0.85% NaCl solution, centrifuged at 10000 × g for 10 minutes at 4 ° C. to precipitate the bacteria, and 0.1 ml of 0.85% NaCl solution ( ^10-7 % Tween 20). This resuspension was treated for 60 minutes at 37 ° C. As a control for the semi-drying treatment, a 0.1 ml E. coli suspension subjected to UV irradiation was placed in a tube and allowed to stand on ice for 10 minutes.

  (Dyeing of live and dead bacteria) 50 μl is taken from the re-suspension of Escherichia coli after semi-drying treatment, and 6-carboxyfluorescein diacetate (or SYT9 dye) is used as a live fungus dye and propidium iodide as a dead fungus dye Was added respectively. The tube with the solution was shaken for 15 minutes, the stained E. coli suspension was diluted 30-50 times with 0.85% NaCl aqueous solution, and fluorescence was measured using a fluorescence spectrometer. For observation using a fluorescence microscope, the suspension was collected on a slide glass without dilution and observed. The excitation wavelength of the fluorescence spectrometer was 488 nm, green fluorescence was measured at 515-530 nm, and red fluorescence was measured at 590-620 nm.

  (Results of dye staining of live and dead bacteria) As shown in FIG. 1, it is observed that living E. coli is not stained with propidium iodide but is stained only with 6-carboxyfluorescein diacetate and emits green fluorescence. (Left figure). On the other hand, the bacteria (dead bacteria) completely sterilized with isopropyl alcohol were stained with propidium iodide and red fluorescence was observed (right figure). Thereby, it became clear that it is possible to distinguish between live bacteria and dead bacteria by the staining method of the present invention. In addition, using the prepared live bacteria / dead bacteria sample for E. coli standard curve, the ratio of live bacteria to dead bacteria is as shown on the horizontal axis in FIG. 2 (survival rate = live cell ratio). A live cell sample and a dead cell sample were mixed and prepared. Using the mixed bacterial solution, green fluorescence and red fluorescence were measured, and the ratio was calculated. The horizontal axis of the graph represents the ratio (%) of the living cells in the whole cells, and the vertical axis represents the ratio of green (515 nm) / red (590 nm). It becomes clear that the ratio of viable bacteria in the whole cell and the ratio of green / red fluorescence show a positive correlation as shown in the graph, and the ratio of viable bacteria in the solution is obtained using this green / red ratio. It became possible.

(Results of UV disinfection and dye staining) By irradiation with UV for 1 minute, bacteria became non-growth bacteria (dead bacteria) (FIG. 3). In FIG. 3, the left figure shows the schematic diagram of the spot on a plate, and a number shows the dilution series of bacteria. The right picture shows a picture of the actual UV irradiated / unirradiated plate. In the UV-irradiated plate (left), Escherichia coli grows at each spot and colonies are observed. On the UV-irradiated plate (right), no colonies are formed even in a solution containing high-density bacteria. This shows that the bacteria are surely dead by UV irradiation. However, by staining with 6-carboxyfluorescein diacetate and propidium iodide, bacteria that could not grow by UV irradiation (dead bacteria) showed the same stained image as live bacteria. The dead bacteria were treated with the semi-dry treatment shown in the above method and a 0.85% NaCl aqueous solution (containing 10 ⁻⁷ % Tween 20) for 1 hour or longer, and the 6-carboxyfluorescein acetate and propidium iodide were used in the above staining method. Staining was performed and the green / red ratio of the fluorescence was calculated (FIG. 4). Since the measurement was performed using a device different from the device used in FIG. 2, the fluorescence intensity ratio of the completely dead bacteria is a value near 3.3 in FIG. 4. Bacteria that were not treated with sodium chloride / surfactant solution after UV irradiation showed a green / red ratio similar to that of the living bacteria treated with sodium chloride / surfactant. However, the number of green / red ratios for bacteria that were treated with sodium chloride / surfactant solution after UV irradiation was similar to that of bacteria that were completely sterilized using isopropyl alcohol. It was shown that the bacteria after UV irradiation could be distinguished as dead bacteria. In addition, regarding the salt / surfactant solution treatment, there was no clear difference between the one-hour treatment and the 2-3-hour treatment, and it was revealed that the treatment for one hour can provide a sufficient effect.

The dyeing | staining image of the bactericidal control for the life-and-death standard curve of produced E. coli is shown. Both live and dead samples are stained with 6-carboxyfluorescein diacetate (trade name: SYT9), which is a viable dye, and propidium iodide, which is a killed dye, using an excitation wavelength of 480 nm and green fluorescence (left figure). 520 nm, and red fluorescence (dead bacteria in the right figure) was observed at 600 nm. In the left figure (live bacteria), dead dyes are excreted from the cells by metabolism, and almost no red staining is seen, whereas in the right figure (dead bacteria), the cells are stained with dead dyes and vivid Red fluorescence was observed. The relationship between the ratio of live bacteria and dead bacteria and green fluorescence / red fluorescence is shown. In the graph, the horizontal axis represents the proportion (%) of the living cells in the whole cells, and the vertical axis represents the green / red ratio. Using the prepared sample for the standard curve for life and death of Escherichia coli, the ratio of live cells to dead cells was adjusted as shown in the graph. Using the mixed bacterial solution, green fluorescence and red fluorescence were measured, and the ratio was calculated. It shows a state in which the growth ability has been lost by ultraviolet irradiation. The left figure shows a schematic diagram of spots on the plate, and the numbers indicate the bacterial dilution series. The right picture shows a picture of the actual UV irradiated / unirradiated plate. In the UV-irradiated plate (left), Escherichia coli grows at each spot and colonies are observed. On the UV-irradiated plate (right), no colonies are formed even in a solution containing high-density bacteria. This shows that the ability of bacteria to grow is lost by UV irradiation. The ratio of green fluorescence to red fluorescence by treatment with bacteria (NaCl, Tween 20) after UV irradiation is shown in comparison with untreated ones. Treatment of NaCl-unirradiated bacteria with NaCl or Tween 20 has almost no effect on the green / red fluorescence ratio (= viable / dead bacteria ratio), and samples that are not treated with NaCl or Tween 20 after UV irradiation (left on ice) ), The ratio of viable bacteria / dead bacteria was not different from that of unirradiated UV, but when the bacteria after UV irradiation were treated with NaCl and Tween 20, the proportion measured as viable bacteria decreased, and treatment for 1 hour was sufficient It became clear that this effect was seen.

Claims (13)

  In a mixed sample containing live bacteria, dead bacteria, and pseudo-viable bacteria that have retained enzyme activity by stopping life activity, a method for determining the abundance ratio of each bacterium, the mixed sample being semi-dried, A method for determining the abundance of bacteria in each state, comprising a step of treating with a salt solution and / or a surfactant solution, a step of staining with a bacterium dye, and a step of staining with a dead bacterium dye .   2. The discrimination method according to claim 1, wherein the mixed sample is subjected to a semi-drying treatment, and then a step of treating with a salt solution and / or a surfactant solution, followed by a step of staining the bacteria with viable pigment and a bacterium with dead pigment. A method for discriminating the presence ratio of viable bacteria, dead bacteria, and pseudo-bacteria, characterized in that the staining step is simultaneously or sequentially performed.   The method for determining the abundance ratio of viable, dead, and pseudobacteria according to claim 1 or 2, wherein the viable dye is a fluorescein fluorescent dye.   The method for determining the abundance ratio of viable, dead, and pseudobacteria according to claim 3, wherein the viable dye is 6-carboxyfluorescein diacetate.   The method for determining the presence ratio of live bacteria, dead bacteria, and pseudo-bacteria according to any one of claims 1 to 4, wherein the dead bacteria pigment is propidium iodide.   The method for determining the presence ratio of viable, dead, and pseudobacteria according to claim 1 or 2, wherein the viable dye is 6-carboxyfluorescein diacetate and the dead fungus is propidium iodide.   The method for determining the presence ratio of viable bacteria, dead bacteria, and pseudo-bacteria according to any one of claims 1 to 6, wherein either one of the viable dye or the dead fungus dye is replaced with a whole fungus dye.   The method for determining the presence ratio of viable, dead, and pseudobacteria according to claim 7, wherein the total fungal pigment is 4 ', 6-diamidino-2-phenylindole dihydrochloride.   The method for determining the presence ratio of viable, dead, and pseudobacteria according to any one of claims 1 to 8, wherein the salt solution is an aqueous sodium chloride solution.   The method for determining the presence ratio of viable, dead, and pseudobacteria according to claim 9, wherein the concentration of the aqueous sodium chloride solution is in the range of 0.75% to 0.95%.   The method for determining the presence ratio of viable, dead, and pseudobacteria according to any one of claims 1 to 10, wherein the surfactant is polyoxyethylene sorbitan monolaurate. The method according to claim 11, wherein the concentration of polyoxyethylene sorbitan monolaurate is in the range of ^10-5 % to ^10-9 %.   A mixed sample containing live bacteria, dead bacteria, and pseudo bacteria is fluorescently stained by each step according to any one of claims 1 to 12, and fluorescence measurement of the mixed sample is performed using a spectrophotometer. A method for measuring the existence ratio of viable / dead bacteria, characterized in that

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