JPH11146798A - Quick discrimination of microorganism cell and discrimination kit for microorganism cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for quickly discriminating a microorganism cell capable of more rapidly and surely discriminating the life and death of a microorganisms in a specimen than a conventional method and to obtain a discrimination kit for a microorganism cell therefor. SOLUTION: This method for quickly discriminating the life and death of a microorganism cell comprises treating a microorganism containing specimen with a first fluorescent coloring matter to emit fluorescence by an enzyme reaction in a living cell and a second fluorescent coloring matter to emit fluorescent having a wave length different from that of the first fluorescent coloring matter and discriminating the life and death of the microorganism by difference in wavelength of the fluorescence produced. A discriminating kit for a microorganism cell is provided with the first fluorescent coloring matter to emit fluorescence of an enzyme reaction in a living cell and the second fluorescent coloring matter to emit fluorescence having a wavelength different from that of the first fluorescent coloring matter, treats a microorganism-containing specimen with the first and the second fluorescent coloring matters and discriminating the life and death of the microorganism by difference in wavelength of the fluorescence produced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for rapidly identifying microbial cells in a microbial application field such as the medical field and the food field, which is capable of quickly identifying the viability of microorganisms in a specimen, and a microbial cell identification kit therefor. The present invention is, for example, to confirm the effect of sterilization treatment of foods and the like, to quickly confirm the sterilization effect of pharmaceuticals such as antibiotics, and to quickly measure the viability ratio for the selection of optimal culture conditions in the production process of useful substances by fermentation. Or, it can be applied to a wide range of fields such as textbooks for experiments and research on microorganisms.

[0002]

2. Description of the Related Art As a method for discriminating the viability of living cells, a method is known in which a plurality of fluorescent dyes are used to detect differences in staining of nucleic acids by flow cytometry. However, the conventional identification method stains nucleic acid with a fluorescent dye and detects a slight difference in staining between live cells and dead cells, so that it is unclear whether microorganism cells are alive or dead, especially. Further, in the field where the identification by the conventional method requires a long time for staining, and in which it is necessary to identify the life and death of microorganisms in a short time, it has been desired to provide a method which can obtain an identification result more simply and quickly.

[0003]

SUMMARY OF THE INVENTION The present invention provides a method for rapidly identifying microbial cells, which can more quickly and reliably identify the viability of microorganisms in a specimen than conventional methods, and an identification kit for microorganisms therefor. That is the task.

[0004]

According to the present invention, there is provided a method for rapidly identifying microbial cells, comprising the steps of:
And a second fluorescent dye, which is taken into dead cells and emits fluorescence of a different wavelength from the first fluorescent dye,
The method is characterized in that it is applied to a specimen containing a microorganism, and the difference between the wavelengths of the generated fluorescence is used to discriminate the life and death of the microorganism. In the method for rapidly identifying microbial cells of the present invention, the viability of microorganisms may be identified by flow cytometry. It is preferable that a calcein derivative is used as the first fluorescent dye. Further, as the second fluorescent dye,
Propidium iodide may be used.

[0005] The identification kit for microbial cells of the present invention comprises a first fluorescent dye that emits fluorescence by an enzymatic reaction in living cells;
A first fluorescent dye which is taken into dead cells and a second fluorescent dye which emits fluorescence having a different wavelength; and reacts the first and second fluorescent dyes on a specimen containing microorganisms to produce a fluorescent light. The difference between the wavelengths is used to determine whether the microorganism is alive or dead. In the identification kit for microbial cells of the present invention,
It is desirable that a calcein derivative be used as the first fluorescent dye. Further, propidium iodide may be used as the second fluorescent dye.

[0006]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, a first fluorescent dye which emits fluorescence by an enzymatic reaction in a living cell and a second fluorescent dye which is taken in dead cells and emits a fluorescence having a wavelength different from that of the first fluorescent dye. And two fluorescent dyes. As the first fluorescent dye, a calcein derivative, which is efficiently absorbed into microbial cells and emits strong fluorescence upon undergoing an enzymatic reaction (hydrolysis) in the cells, preferably various esters of calcein, can be used. For example, a commercially available product is Calce
in-AM (manufactured by Dojin Chemical Laboratory Co., Ltd.). It exhibits little fluorescence per se, is easily taken into microbial cells, is hydrolyzed by various esterases in microbial cells, and emits strong yellow-green fluorescence (excitation wavelength: 490 nm, fluorescence wavelength: around 515 nm).

As the second fluorescent dye which is taken into dead cells and emits fluorescence having a wavelength different from that of the first fluorescent dye, a fluorescent dye such as propidium iodide or ethidium homodimer can be used, and in particular, propidium Iodide is preferably used.

The microbial cell identification kit of the present invention only needs to include the first fluorescent dye and the second fluorescent dye, and can be used for analyzing a buffer solution, a physiological saline for dilution, or a test tube. Other necessary reagents and equipment may also be included. It is also possible to apply a form in which the first fluorescent dye and the second fluorescent dye are packed for each required amount of one analysis, or are enclosed in a graduated container for dilution.

In the method for rapidly identifying microbial cells of the present invention, the first fluorescent dye and the second fluorescent dye are allowed to act on a specimen containing a microorganism, and the viability of the microorganism is identified based on the wavelength difference of the generated fluorescence. This identification can be performed using a method of observing the staining of microorganisms using a fluorescence microscope or a flow cytometry method.

[0010] In order to discriminate the viability of microbial cells after staining using flow cytometry, the light receiving wavelength of the light receiving section of the discriminating apparatus is determined by comparing the light receiving wavelength of the first fluorescent dye (after hydrolysis) with the light receiving wavelength of the second fluorescent dye. To the fluorescence wavelength of the fluorescent dye. In this identification method by flow cytometry, a first fluorescent dye and a second fluorescent dye are allowed to act on a microorganism culture solution, and the stained test solution is simply passed through an identification device, and the microorganisms in the microorganism culture solution are removed. It is particularly preferable because viability can be measured, many samples can be measured continuously, and automation can be performed.

The present invention can be applied to the identification of bacteria such as Escherichia coli, Staphylococcus aureus, Streptococcus, Pneumococcus, Pseudomonas sp., Bacillus sp., Clostridium sp. It is. The present invention is, for example, to confirm the effect of sterilization treatment of foods and the like, to quickly confirm the sterilization effect of pharmaceuticals such as antibiotics, and to quickly measure the viability ratio for the selection of optimal culture conditions in the production process of useful substances by fermentation. Or, it can be applied to a wide range of fields such as textbooks for experiments and research on microorganisms.

[0012]

EXAMPLES Example 1 Discrimination of Viable and Dead Microorganisms by Flow Cytometry Escherichia coli (Escherichia)
E. coli) and heat-killed death sterilization were used to stain the cells according to the method of the present invention, and life and death were identified by flow cytometry.

(Reagent) Solution A: Fluorescent dye solution for live cells prepared by dissolving Calcein-AM (manufactured by Dojindo Laboratories Co., Ltd.) in DMSO at 1 mM / ml. Solution B: Dissolve propidium iodide in ethanol,
Fluorescent dye solution for dead cells at 1 mg / ml.

(Operation) 10 ml of the culture of the microorganism is collected in a 35 ml centrifuge tube, and centrifuged at 4000 rpm for 10 minutes (4 ° C.). The supernatant was discarded, 5 ml of ice-cold 10 mM HEPES (buffer) was added, and the suspension was slowly suspended on ice.
Centrifuge at 0 rpm for 10 minutes (4 ° C.). Discard the supernatant, and add 5 ml of ice-cold PBS (physiological saline).
Add and suspend slowly on ice. 500 μl of the suspension obtained in a 1.5 ml centrifuge tube
Solution A (fluorescent dye solution for living cells) 500 μl (P
500 μl of BS plus 5 μl of Calcein-AM) (on ice) and then incubate at 30 ° C. for 30 minutes. Further, 5 μl of solution B (fluorescent dye solution for dead cells) is added to the centrifuge tube, and left at room temperature for 5 minutes. After the reaction, measurement is performed by flow cytometry.

In the case of dead cells, the culture of the microorganism was heated at 100 ° C. for 10 minutes, and then the above-mentioned steps were performed as in the case of living cells.

The measuring device is FACS manufactured by BECTON DICKINSON.
Using a Calibur, the measurement conditions were an excitation wavelength of 488 nm, a fluorescence wavelength of 515 nm (green side), and 635 nm (red side). FIG. 1 shows the results of measuring the fluorescence wavelength by flow cytometry using live cells of Escherichia coli, and FIG. 2 shows the results of using the dead cells. As is clear from the comparison between FIG. 1 and FIG. 2, the plot of FIG. 1 where live cells were measured and the plot of FIG. 2 where dead cells were measured clearly distinguished the fluorescence wavelength. Thus, it was found that by implementing the present invention, the viability of microorganisms in a specimen can be measured quickly and reliably.

Example 2 Growth Curve and Time-Dependent Changes of Living and Dead Cells Escherichia coli was cultured using a jar fermenter.
The culture solution was taken out at predetermined time intervals, the absorbance (OD) was measured, and the ratio of living cells to dead cells (and dying cells) was examined in the same manner as in the above-described Example 1. FIG. 3 shows the time course of the absorbance, that is, the growth curve, and FIG. 4 shows the results of the time course of the living and dead cells. 3 and 4, the viable cell rate is low and the dead cell rate is high at the start of culture, but the viable cell rate is high and the dead cell rate is low in the logarithmic growth phase.
In the stable period, the viable cell rate gradually decreased and the dead cell rate tended to gradually increase. This measurement method revealed that the change over time in the viable cell rate can be measured.

[0018]

As described above, according to the present invention,
In the field of microbial applications such as medical and food fields, it is possible to provide a microbial cell rapid identification method and a microbial cell identification kit that can more rapidly and reliably identify the viability of microorganisms in a sample than conventional methods. it can.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of flow cytometry using living cells.

FIG. 2 is a graph showing the results of flow cytometry using dead cells.

FIG. 3 is a graph showing a growth curve of bacteria used.

FIG. 4 is a graph showing the change over time of living and dead cells.

────────────────────────────────────────────────── (5) Continuation of the front page (51) Int.Cl. ⁶ Identification code FI C12R 1: 185) (72) Inventor Yoshinori Hiraoka 4-12-9 Agananami, Kure City, Hiroshima Prefecture

Claims (7)

[Claims] 1. A first fluorescent dye which emits fluorescence by an enzymatic reaction in a living cell, and a second fluorescent dye which is taken into dead cells and emits fluorescence having a wavelength different from that of the first fluorescent dye, A method for rapidly identifying microbial cells, wherein the method is applied to a specimen containing a microorganism, and the viability of the microorganism is identified based on the wavelength difference of the generated fluorescence. 2. The method for rapidly identifying microbial cells according to claim 1, wherein viability of the microorganism is identified by flow cytometry. 3. The method for rapidly identifying microbial cells according to claim 1, wherein a calcein derivative is used as the first fluorescent dye. 4. The method according to claim 3, wherein propidium iodide is used as the second fluorescent dye. 5. A method according to claim 1, wherein the first fluorescent dye emits fluorescence by an enzymatic reaction in living cells, and the second fluorescent dye emits fluorescence having a wavelength different from that of the first fluorescent dye when taken into dead cells. An identification kit for microbial cells, wherein the first and second fluorescent dyes act on a specimen containing a microorganism, and the difference between the wavelengths of the generated fluorescence is used to determine the viability of the microorganism. 6. The identification kit for microbial cells according to claim 5, wherein a calcein derivative is used as the first fluorescent dye. 7. The microbial cell identification kit according to claim 6, wherein propidium iodide is used as the second fluorescent dye.