JP2003284592A - Method for measurement of physiological activity of microorganism and kit for measurement of physiological activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for measurement of physiological activity of microorganisms which have physiological activity, etc., such as refining an environmental pollutant and to provide a kit to carry out the method. <P>SOLUTION: The method subjects a sample containing the microorganisms to a dying treatment using a first fluorescent dye, a second fluorescent dye with a different wave length from the first fluorescent dye and a third fluorescent dye with a different wave length from the first and the second fluorescent dyes and then measures the fluorescence in flow cytometry or laser scanning cytometer. The method reveals, from the result of the measurement, the number and refining activity to the environmental pollutant and state of living of cells of the microorganisms refining the environmental pollutant by decomposition, dechlorination, or the like. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring physiological activity of microorganisms and a kit for measuring physiological activity. The present invention is, for example, a measurement for grasping the total cell number, purification activity and survival state of microorganisms that can purify environmental pollutants such as polychlorinated biphenyls contained in soil etc. by metabolism, decomposition, dechlorination, etc. Can be applied to.

[0002]

2. Description of the Related Art In recent years, environmental pollution due to environmental pollutant compounds has become a problem, and among them, polychlorinated biphenyl (hereinafter, PCB) is a chlorinated aromatic compound which is highly toxic to the human body, and is particularly attracting attention. . It is known that these environmental pollutant compounds are detoxified by being decomposed by a predetermined microorganism, etc., and attempts to improve environmental pollution by various methods have been studied. Aerobic PC, for example, for cleaning soils extensively contaminated with PCB
Environmental restoration (bioremediation) using microorganisms such as B-decomposing bacteria and anaerobic PCB dechlorination consortia (hereinafter, decomposing bacteria (group)) is used as an effective method.

By the way, PCB in such a method
In order to evaluate the decomposing efficiency of, the accurate total cell number of the decomposing bacteria (group) contained in the contaminated soil, the survival state, and the physiological activity such as the metabolic activity of PCB are important. To measure the physiological activity, for example, as a method for specifically detecting a predetermined microorganism in soil, there is a FISH (fluorescence in situ hybridization) method using 16S rRNA as a probe.

[0004]

However, in the FISH method, since the microorganisms are fixed and detected, the physiological activity of the microorganisms cannot be measured simultaneously with the detection. Also,
The colony counting method (CFU) is widely used to evaluate the viability of microorganisms in soil. However, the colony counting method is time-consuming to measure.

The present invention has been made on the basis of such a technical problem, and specifically detects microorganisms having physiological activity, in particular, the activity of purifying environmental pollutant compounds, and at the same time with the detection, the survival state of the microorganisms. It is an object of the present invention to provide a method for measuring physiological activity, a kit for measuring physiological activity, and the like, which can measure physiological activity.

[0006]

In order to solve the above-mentioned problems, the method for measuring the physiological activity of a microorganism of the present invention comprises a first fluorescent dye which binds to a predetermined microorganism and emits fluorescence of a predetermined wavelength, Incorporated in a gene involved in physiological activity in the microorganism, expressed by the gene, a second fluorescent dye that emits fluorescence of a wavelength different from that of the first fluorescent dye, and is incorporated in dead cells of the microorganism, A first fluorescent dye and a third fluorescent dye that emits fluorescence of a wavelength different from those of the second fluorescent dye are allowed to act on a sample containing the microorganism, and the sample is detected based on a wavelength difference of fluorescence generated in the sample. It is characterized by measuring the total cell number, physiological activity and survival state of the microorganism contained in

The physiological activity measuring method of the present invention is characterized in that the total cell number is determined by the wavelength based on the first fluorescent dye,
The physiological activity can be measured by the wavelength based on the fluorescent dye, and the survival state can be measured by the wavelength based on the third fluorescent dye. Further, the physiological activity is preferably a purification activity of environmental pollutant compounds. Further, by using GFP (green fluorescent protein) or luciferase as the second fluorescent dye, and introducing the expression gene of GFP or luciferase into the promoter downstream of polychlorinated biphenyl metabolism and / or dechlorination enzyme gene,
It is desirable to measure the purification activity of polychlorinated biphenyl as the purification activity. Further, the wavelength of fluorescence generated by the sample can be detected by flow cytometry or a laser scanning cytometer.

The kit for measuring physiological activity of a microorganism of the present invention is based on the wavelength of fluorescence generated by acting on a specimen containing a predetermined microorganism, based on the total number of cells, physiological activity and survival state of the microorganism contained in the specimen. A kit for measuring physiological activity for measuring, wherein a first fluorescent dye that binds to the microorganism and emits fluorescence of a predetermined wavelength, and is incorporated into a gene involved in the physiological activity in the microorganism, And a second fluorescent dye that emits fluorescence of a wavelength different from that of the first fluorescent dye and that is incorporated into living cells or dead cells of the microorganism to express the first fluorescent dye and the second fluorescent dye. And a third fluorescent dye that emits fluorescence of a wavelength different from the above. Further, the physiological activity is preferably a purification activity of environmental pollutant compounds. In addition, the physiological activity measuring kit of the present invention measures the polychlorinated biphenyl purification activity as the environmental pollutant, and thus the G as the second fluorescent dye is used.
It can be equipped with FP (green fluorescent protein) or luciferase.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION There is no particular limitation on the physiological activity of the microorganism to which the measuring method of the present invention is applied, but the physiological activity of the microorganism capable of purifying environmental pollutant compounds by metabolism, decomposition, dechlorination, etc. preferable. Hereinafter, the biological activity will be described with reference to the purification activity of environmental pollutant compounds as an example.
In the present invention, the total number of cells of the microorganism contained in the sample,
Purifying activity and viability can be measured simultaneously. First, in order to measure these items, a dyeing process using first to third fluorescent dyes having different wavelengths is performed.

First, in order to measure the total number of cells, a staining treatment is carried out in which the first fluorescent dye is indirectly or directly bound to a microorganism of a species which can be purified by metabolism of environmental pollutant compounds. For example, in the case of a microorganism that purifies PCB as an environmental pollutant compound, it is labeled with a monoclonal antibody of a bacterial group specific to the strain and consortia of the microorganism and a red (wavelength 630 nm) fluorescent dye such as PerCP as the first fluorescent dye. The secondary antibody is allowed to act on the sample. Thereby, in the measurement described later, the total cell number of the microorganism in the sample can be measured based on the fluorescent antibody method.

Then, in order to measure the purifying activity, a second fluorescent dye, which is incorporated into a gene involved in the purifying activity of the environmental pollutant compound of the microorganism and is expressed by the gene, emits fluorescence having a wavelength different from that of the first fluorescent dye. Stain with a fluorescent dye. For example, in the case of a microorganism that purifies PCB as an environmental pollutant, as a marker for monitoring dechlorination activity, a second fluorescent dye, GFP (green fluoresc), is placed downstream of the promoters of the PCB metabolism and dechlorination enzyme genes.
ent protein) or a luciferase expression gene is introduced into the mutant strain. The specimen into which the GFP or luciferase gene has been introduced in this manner expresses the GFP or luciferase gene if it has a purifying activity. Therefore, in the measurement described later, green (530 nm)
Alternatively, the presence or absence of purifying activity can be identified by the presence or absence of yellow-green (560 nm) fluorescence. When a mutant strain into which the GFP or luciferase gene has been introduced cannot be obtained, a marker such as green that focuses on the enzyme or metabolite that specifically increases when degradation, metabolism, dechlorination activity, etc. accompanying purification are performed. Can be set.

Further, in order to measure the survival state, it is stained with a third fluorescent dye which is incorporated into dead cells of the microorganism and emits fluorescence of a wavelength different from those of the first fluorescent dye and the second fluorescent dye. To process. For example, a reddish orange color (575
Staining is performed using propidium iodide (PI), which is a third fluorescent dye that emits fluorescence of (nm). As a result, dead cells in the sample can be identified in the later-described measurement. In addition to the propidium iodide, a fluorescent dye such as ethidium homodimer can be used as the third fluorescent dye for identifying dead cells. Furthermore, the survival state may be measured by identifying living cells in the sample. In this case, a calcein derivative or the like that undergoes an enzymatic reaction in cells and emits strong fluorescence can be used.

Fluorescence in the sample dyed by the method described above is measured. The order in which the above-mentioned dyeing process is performed can be appropriately changed. For fluorescence measurement, it is preferable to use, for example, a flow cytometry (FCM) in a liquid system and a laser scanning cytometer (LSC) in a solid system. In these methods, irradiating the specimen with a laser of a predetermined wavelength,
The light generated thereby can be easily measured by an optical detector. In the present invention, for example, first, the light receiving wavelength of the light receiving unit of the measuring device is matched with the wavelengths of the first fluorescent dye, the second fluorescent dye, and the third fluorescent dye. Then, the stained sample can be flowed to a flow cytometer or installed in a laser scanning cytometer, and the fluorescence can be measured to determine the total cell number, purification activity and survival state of microorganisms that purify environmental pollutants. be able to.

As a specific example of the fluorescence measurement result, the survival state of the microorganism and the metabolic activity of the environmental pollutant compound as the purifying activity can be identified in the following states (PI: red-orange marker, GFP: Green marker). [PI ^-, GFP ^+] = There metabolic activity in living cells ^{[PI -,} GFP -] = No metabolic activity in living cells [PI ^+, GFP ^+] = There metabolic activity in dead cells [PI ^+, GFP ^-] = No metabolic activity in dead cells

The present invention can provide a kit for measuring physiological activity in order to measure the total number of cells of the above-mentioned microorganism and the physiological activity of the microorganism, and to identify live and dead cells. This physiological activity measuring kit may be provided with the above-mentioned first to third fluorescent dyes, and may contain a buffer solution, a physiological saline solution for dilution, or other reagents and instruments necessary for analysis such as a test tube. It can also be included. In addition, the first fluorescent dye, the second fluorescent dye, and the third fluorescent dye may be packaged for each required amount of one analysis, or may be sealed in a container with a scale for dilution. it can. In such a kit, the physiological activity is preferably a purifying activity for environmental pollutant compounds.

In the above embodiment, PCB is used as a concrete example of the environmental pollutant compound purified by microorganisms.
, The bioactivity measuring method and the bioactivity measuring kit of the present invention are used in a reaction device using a degrading bacterium (group) or a microorganism that decomposes other persistent environmental pollutant compounds. It can also be appropriately used for the physiological activity of the microorganism (group). In addition, the configurations described in the above embodiments can be selected or changed to other configurations without departing from the spirit of the present invention.

[0017]

As described above, according to the present invention,
It is possible to grasp the total number of cells of a microorganism having a physiological activity, in particular, an activity to purify an environmental pollutant compound in a sample, to discriminate between live and dead cells, and simultaneously measure the purifying activity of the environmental pollutant compound by the microorganism. Therefore, the situation of purification of the environmental pollutant compound can be grasped in real time. Therefore, for example, it is possible to easily search for optimal conditions for microorganisms that decompose environmental pollutants,
It becomes possible to search for a method for efficiently purifying environmental pollution.

Claims (8)

[Claims] 1. A first fluorescent dye that binds to a predetermined microorganism and emits fluorescence of a predetermined wavelength, and the first fluorescence that is incorporated into a gene involved in physiological activity in the microorganism and is expressed by the gene. A second fluorescent dye that emits fluorescence of a wavelength different from that of the dye, and a third fluorescence that is taken up by dead cells of the microorganism and emits fluorescence of a wavelength different from that of the first fluorescent dye and the second fluorescent dye. A microorganism, characterized in that a dye is allowed to act on a sample containing the microorganism, and the total number of cells of the microorganism contained in the sample, physiological activity and survival state are measured based on the wavelength difference of fluorescence generated in the sample. Method for measuring physiological activity of. 2. The total cell number is determined by the wavelength based on the first fluorescent dye, the physiological activity is determined by the wavelength based on the second fluorescent dye, and the survival state is determined by the wavelength based on the third fluorescent dye. It measures, It is characterized by the above-mentioned.
A method for measuring physiological activity of a microorganism as described. 3. The method for measuring a physiological activity of a microorganism according to claim 1, wherein the physiological activity is a purifying activity of an environmental pollutant compound. 4. The GFP (green fl) is added to the second fluorescent dye.
uorescent protein) or luciferase to introduce the expression gene of GFP or luciferase into the polychlorinated biphenyl metabolism and / or the promoter downstream of the dechlorination enzyme gene to measure the purified activity of polychlorinated biphenyl as the purifying activity. The method for measuring physiological activity of a microorganism according to claim 3, wherein 5. The method for measuring physiological activity of microorganisms according to claim 1, wherein the wavelength of the fluorescence generated by the sample is detected by a flow cytometry or a laser scanning cytometer. 6. A physiological activity measuring kit for measuring the total cell number, physiological activity and survival state of the microorganism contained in the specimen based on the wavelength of fluorescence generated by acting on the specimen containing a predetermined microorganism. A first fluorescent dye that binds to the microorganism and emits fluorescence of a predetermined wavelength, and is incorporated into a gene involved in the physiological activity in the microorganism and expressed by the gene, the first fluorescent dye. A second fluorescent dye that emits fluorescence of a wavelength different from that of the third fluorescent dye, and a third fluorescent dye that is incorporated into living cells or dead cells of the microorganism and emits fluorescence of a different wavelength from the first fluorescent dye and the second fluorescent dye. A kit for measuring physiological activity of microorganisms, comprising: 7. The kit for measuring a physiological activity of a microorganism according to claim 6, wherein the physiological activity is a purifying activity of an environmental pollutant compound. 8. The GFP (green fluorescent protein) or luciferase is provided as the second fluorescent dye in order to measure the purifying activity of polychlorinated biphenyls as the environmental pollutant. Kit for measuring physiological activity of microorganisms of.