JP4622478B2 - Microorganism measurement method - Google Patents

Description

  The present invention relates to a method for measuring microorganisms such as bacteria, yeast, mold, and viruses in the fields of food, medicine, pharmaceuticals, environment, and the like, and in particular, using a culture method, a microscope, a label such as a fluorescent label, and a gene amplification technique. The present invention relates to a method for measuring microorganisms concentrated at high magnification.

  In fields such as food, medicine, pharmaceuticals, and the environment, microbial tests for bacteria, yeasts, fungi, viruses, etc. are important. For example, it is important for food security and quality control in the food field, and for diagnosis and treatment of patients in the medical field. Furthermore, it is desired that microbial testing is quick and accurate.

  On the other hand, conventionally, in microbiological tests in the food field, the environmental field, etc., a culture method, a microcolony method, a staining method, a gene growth technique, and the like are used in order to confirm the presence of microorganisms in a sample. The culture method can selectively propagate and detect microorganisms of the target species, and has been used for over 100 years. The microcolony method is a technique developed from a culture method, and can be detected in a shorter time than the culture method by detecting the growth of microorganisms at a microscopic stage. Next, the staining method is a method for detecting microorganisms by staining them with a reagent. In recent years, staining methods using genes and antigen-antibody reactions have been performed. In addition, a PCR method (Polymerase Chain Reaction) is used in a microorganism test using a gene growth technique.

  Among these, for example, in the microbiological examination using the PCR method, in order to detect specific bacteria in the food, a solution in which food is stomatized is cultured at a medium and temperature suitable for the specific bacteria. Next, tens of microliters are sampled from the culture solution in which the bacteria are grown, and the gene is amplified together with the PCR reagent.

  In the microbiological examination using the PCR method, specific bacteria can be detected because of the gene specificity depending on the bacterial species. However, since sampling is several tens of μL, in the above inspection, only a part of the food is inspected, and the whole food is not inspected.

  As described above, in the above-described conventional microorganism test, the bacteria in a part of the sample are extracted and cultured, or the partial characteristics of the sample are merely amplified and detected. That is, only the partial characteristics of the sample are inspected, and the properties of the entire sample cannot be grasped as a whole. Therefore, an inspection using a concentration method with higher magnification and higher recovery rate is desired.

  In order to inspect the entire sample, the concentration of microorganisms in the sample has been performed in the microorganism inspection. As a method for concentrating microorganisms, centrifugation, filtration using a filter of organic polymer fibers such as cellulose, nylon, and polyester have been performed for a long time. However, these classic microbial concentration methods cannot provide a high concentration factor. Furthermore, expensive equipment such as a centrifuge and a polymer fiber filter is required, which is expensive.

As a solution to such a problem, the bacterial cell concentration method and the bacterial cell concentration reagent shown in Patent Document 1 have been proposed. In this method, first, an anion and a cation are present in an aqueous solvent containing microorganisms to produce a poorly water-soluble substance. Next, the microorganisms are adsorbed on the poorly water-soluble substance to collect the microorganisms. Furthermore, the microorganisms are recovered by dissolving a poorly water-soluble substance adsorbed by the microorganisms with a solubilizer.
JP 2000-14380 A

However, the conventional method has the following problems, and it has not been possible to efficiently measure microorganisms.
(1) By dissolving a poorly water-soluble substance adsorbed by microorganisms with a solubilizer, the volume of the phase containing the microorganisms increases, and it can be concentrated only about several tens of times.
(2) Since the recovery rate of microorganisms is about 90%, the remaining about 10% of microorganisms that could not be recovered cannot be inspected. Therefore, it cannot be used as a means for testing for negative microbial positives.
(3) Since there is no specificity in adsorption, contaminants other than microorganisms are concentrated, which causes measurement errors.
The present invention solves such a problem, and has a high magnifying power, a high recovery rate, and has a specificity that water-soluble contaminants such as starch and proteins and contaminants having a high affinity for water are not concentrated. Is used to provide a method for measuring microorganisms concentrated at high magnification. The present invention is particularly suitable for bacteria (VBNC, viable but nonculturable) that are alive but difficult to culture by conventional methods. Bacteria in such a state did not form colonies on the medium and could not be detected by conventional culture methods. Therefore, until now, it was treated as “non-existent” and excluded from hygiene management.

In order to achieve the above object, the present invention is a method for measuring microorganisms, comprising adding a surfactant and a hydrophilic organic solvent to a solution containing microorganisms to form a uniform solution, and then lowering the pH of the solution. The solution is separated into two phases, a precipitated phase in which microorganisms, a surfactant, a hydrophilic organic solvent, and water are mixed, and a supernatant phase, and the number of microorganisms concentrated in the precipitated phase is measured. It is characterized by doing.

In one embodiment of the microorganism measuring method according to the present invention, the number of microorganisms is measured using a culture method.

The microorganism concentration method according to the present invention is another embodiment, and is characterized in that the number of microorganisms is measured using a microscope.

Furthermore, the microorganism concentration method according to the present invention is characterized in that in another embodiment, the number of microorganisms is measured using a label.

Furthermore, the microorganism concentration method according to the present invention is characterized in that in another embodiment, the number of microorganisms is measured using a gene amplification method.

When the PCR method is used as a gene amplification method in the microorganism concentration method according to the present invention, the precipitated phase is filtered to capture microorganisms on the filter, washed with water, and the surfactant and the hydrophilic organic the solvent was washed stream, it is preferred that the DNA of the captured microorganisms as a template for PCR. In addition, the precipitated phase is filtered to capture microorganisms on the filter, the filter is transferred to a container together with water, and the DNA of the captured microorganism is extracted by freezing and thawing , and the DNA is used as a template for the PCR method. It is preferable to do.

For example, perfluorooctanoic acid is suitable as the surfactant. Further, as the hydrophilic organic solvent, tetrahydrofuran, dimethylformamide, ethanol or acetone is preferable.

According to the present invention, using a microorganism concentration method that has a specificity that does not concentrate water-soluble contaminants such as starch and proteins, and high-affinity contaminants such as starch and protein, at a high magnification, a high magnification. There is provided a method for measuring microorganisms concentrated in the above. As will be described later, according to the microorganism measuring method of the present invention, the following effects can be expected.
1. Since microorganisms can be concentrated at a high magnification of several tens of thousands of times, a large volume of sample can be concentrated to a small volume, and microorganisms concentrated at a high magnification can be cultured, microscopically, labeled, or gene amplified. Can be measured efficiently.
2. Microorganisms in the sample can be concentrated with a recovery rate of about 100%, accurate results can be obtained, and yin / yang determination of microorganisms can be performed.
3. Since water-soluble contaminants such as starch and proteins and contaminants with a high affinity for water are not concentrated, they can be separated from microorganisms, and accurate microorganism measurement can be performed.

  Hereinafter, the microorganism measuring method according to the present invention will be described in more detail with reference to the embodiment thereof.

The microorganism measurement method according to the present invention is a method of concentrating microorganisms using a surfactant, a hydrophilic organic solvent, and a reagent for lowering pH, and cultivating the microorganisms concentrated at a high magnification by a culture method, a microscope, a label, Or it is the method of measuring using a gene growth method, and includes the following steps at least.
1. A step of adding a surfactant and a hydrophilic organic solvent to a solution containing microorganisms to form a uniform solution (step 1).
2. Lowering the pH of the solution and separating the solution into two phases, a precipitated phase in which microorganisms, a surfactant, a hydrophilic organic solvent, and water are mixed, and a supernatant phase (step 2);
3. A step of measuring microorganisms concentrated in the precipitation phase (step 3);
The microorganism measuring method according to the present invention targets bacteria, yeasts, molds, viruses, and the like, and executes the above steps to measure microorganisms. Each step will be further described below.

Step 1
In Step 1, a surfactant and a hydrophilic organic solvent are added to a solution containing microorganisms to form a uniform solution. Such a “homogeneous solution” is a concept that includes not only a complete solution but also a pseudo-homogeneous solution in which micelles are formed. It is preferable that the surfactant and the hydrophilic organic solvent have compatibility. In Step 1, the solution containing the microorganism, the surfactant, and the hydrophilic organic solvent form a single liquid phase as a whole. As the surfactant, a fluorocarboxylic acid is preferable, and it is preferable to use a carboxylic acid in which at least one hydrogen atom bonded to each carbon atom is substituted with a fluorine atom.

  Fluorocarboxylic acids are more prone to dissociate protons than the corresponding carboxylic acids and are hydrophobic. Therefore, unless the pH of the fluorocarboxylic acid is significantly lower than that of the corresponding carboxylic acid, the proton is easily dissociated and dissolved in water, and except for some lower ones, a mineral acid is added thereto to adjust the pH. When it decreases, it will be easily dissociated and separated from the aqueous phase. Such a tendency becomes more remarkable as the degree of fluorination increases. Therefore, in the present invention, it is preferable to use a completely fluorinated carboxylic acid such as perfluorodecanoic acid or perfluorooctanoic acid.

  Among the above surfactants, for example, perfluorooctanoic acid is solid at room temperature. Therefore, when the pH of the aqueous solution containing perfluorooctanoic acid is lowered, perfluorooctanoic acid is precipitated as a solid. However, if a hydrophilic organic solvent having compatibility with acetone or tetrahydrofuran or the like is allowed to coexist in an aqueous solution containing perfluorooctanoic acid, perfluorooctanoic acid forms a precipitated phase together with the hydrophilic organic solvent and water. Separate from. One feature of the microorganism measuring method according to the present invention is to use such a phenomenon.

  In the case where the surfactant used in the present invention is a fluorocarboxylic acid represented by the following general formula (1), if it is made acidic by adding a mineral acid or an organic acid to a pH of 6 or less, it will react with microorganisms. The microorganisms can be concentrated as a precipitation phase in which the agent, the hydrophilic organic solvent, and water are mixed. That is, if fluorocarboxylic acid is used as a surfactant, the pH is reduced to about 6 or less and does not need to be lowered much. Therefore, it is suitable for concentrating microorganisms alive or concentrating genes without damaging them.

Here, R is a fluoroalkyl group, and at least one hydrogen atom bonded to each carbon atom is substituted with a fluorine atom. In general, X is a divalent organic group containing no fluorine. Specifically, it is an alkylene group having about 1 to 4 carbon atoms, an oxyalkylene group, or an alkylene group having a heteroatom such as oxygen, nitrogen or sulfur in the carbon chain. For example, —CH ₂ CH ₂ SCH ₂ CH ₂ —, —CH ₂ —O—CH ₂ — and the like can be mentioned.

  As the hydrophilic organic solvent, one or more of alcohol, ether, ketone, amide, nitrile and sulfoxide are suitable. For example, ethanol, methanol, tetrahydrofuran, dioxane, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide and the like are suitable.

  When the hydrophilic organic solvent is selected from ethers, amides, alcohols and ketones, it is possible to concentrate the microorganisms to the precipitated phase while maintaining their form. As the ether, tetrahydrofuran is particularly suitable, as the amide, particularly dimethylformamide, as the alcohol, particularly ethanol, and as the ketone, acetone is particularly suitable.

Step 2
In this step, the pH of the solution obtained in Step 1 is lowered, and this solution is separated into two phases: a precipitation phase in which microorganisms, a surfactant, a hydrophilic organic solvent, and water are mixed, and a supernatant phase. To do.

  As a reagent for lowering the pH, for example, hydrochloric acid, nitric acid, and sulfuric acid are preferably used. According to the microorganism concentration method of the present invention, when a water-soluble substance and a hydrophilic substance coexist with a microorganism, the microorganism can be separated into a precipitation phase, and the water-soluble substance and the hydrophilic substance can be separated into a supernatant phase. Thereby, microorganisms can be concentrated.

  When adding a reagent for lowering pH to a solution composed of a surfactant, a hydrophilic organic solvent, a microorganism and water, it is generally not left as it is after the addition, but it is allowed to stand after it is gently stirred. Is preferred. That is, if mixing is not performed by stirring, it takes time until the acid is uniformly diffused, and the reaction is delayed by that amount, so that phase separation is delayed. Therefore, it is preferable to provide a stirring process.

  The stirring is preferably performed by shaking or using a stirrer, vortex mixer or the like. In step 2, when the precipitation phase is formed, if the temperature of the entire reaction solution is lowered, the precipitation rate is increased. This is presumably because the solubility of the hydrophilic organic solvent that dissolves in water decreases as the temperature decreases.

  In addition, when the pH of a solution composed of a surfactant, a hydrophilic organic solvent, a microorganism and water is lowered and then centrifuged, the precipitated phase can be formed in a shorter time than the time for allowing it to stand to form a precipitated phase. Can be formed.

Step 3
In Step 3, the microorganisms concentrated in the precipitated phase from Steps 1 and 2 can be measured using various inspection methods. Examples of the inspection method include a culture method, microscopic observation, a method using a label, a method using gene amplification, and the like.

In the culture method of microorganisms, microorganisms are generally grown and detected on an agar medium. In this case, the volume of the sample smeared on the medium is limited by the area of the culture vessel bottom plate. For this reason, as shown in the present invention, in the method for culturing microorganisms, when the sample is dilute, it is preferable to sufficiently concentrate the sample. Moreover, it is preferable that microorganisms are acid resistant and organic solvent resistant. Furthermore, in the method for culturing microorganisms, it is preferable to culture at a suitable medium, temperature, and time. For example, Escherichia coli is smeared on an agar medium and then cultured at 37 ° C. for about 20 hours, and S. aureus is smeared on an agar medium and cultured at 35 ° C. for 1 to 2 days.

In the method of using a microscope for measuring microscopic observation microorganisms, it is important to identify individual microorganisms at the same time as identifying individual microorganisms. Therefore, it is preferable to use at least one of ether, amide, alcohol, and ketone as the hydrophilic organic solvent. For example, when tetrahydrofuran, dimethylformamide, ethanol, acetone or the like is used, the microorganisms are precipitated while maintaining the form, and the form can be observed using a microscope.

Examples of the microscope that can be used include a real microscope, a phase contrast microscope, a differential interference microscope, and a fluorescence microscope.
With the actual microscope, it is possible to observe stereoscopically without using a cover glass by illumination with reflected light.
In a phase contrast microscope, light transmitted through a sample with low contrast is separated into direct light and diffracted light having different phases, which are interfered with each other and observed as a bright and dark image.
In a differential interference microscope, polarized light is used, two light beams slightly shifted are interfered with each other, and a transparent sample is observed with a contrast. The depth of focus is deeper than that of a phase contrast microscope, and the surface of microorganisms and the inside can be observed in three dimensions.

  In addition, in a method using a microscope for measuring microorganisms, an observer may acquire and analyze an image using a computer in addition to directly observing the microorganisms. According to the microorganism measuring method according to the present invention, it is possible to concentrate a dilute sample and increase the microorganism concentration per unit area, which is extremely advantageous.

Methods using labels Examples of labeling methods include general staining methods, hybridization methods, and methods using antigen-antibody reactions. As the label, a fluorescent label is suitable.

  DAPI, CFDA, PI, lactophenol solution, methylene blue, Gram stain solution, Giemsa stain solution, Grocott stain solution, Victoria blue stain solution, Orsein stain solution, Tielnelsen stain solution, etc. should be used as the stain used in the staining method. Is preferred.

  In the case of staining microorganisms with DAPI, the organic phase containing microorganisms is hydrophobic. Therefore, a method in which DAPI is dissolved in an organic solvent such as ethanol and then contacted with the organic phase for staining is preferable.

  The size of the microorganism is several μm to several tens of μm for protozoa, fungi, bacteria, and the like, and several tens to several hundreds of nm for viruses. Therefore, since the resolution of the optical microscope is 0.4 to 0.8 μm due to the diffraction limit, viruses and the like cannot be observed using a microscope. In the fluorescence microscope, the above-mentioned problems can be solved. In fluorescence microscopy, various states of microorganisms can be observed using fluorescent labels. For example, the life and death of microorganisms can be observed using fluorescence microscopy.

  Alternatively, a hybridization method using a gene or an immunostaining method using an antigen-antibody reaction may be used. The labeled substance can be measured with a fluorescence microscope, a flow cytometer, a particle counter, a spectrophotometer, or the like. In the present invention, water-soluble impurities such as starch and protein, and impurities having a high affinity for water are not concentrated, and an unnecessary fluorescent signal due to impurities is generated in a fluorescence microscope or the above measuring instrument. Absent.

Hybridization means that base sequences complementary to each other form a double-stranded nucleic acid molecule between DNA and RNA. Radioisotope ⁽³³ P, ³⁵ S, etc.), and labeled DNA or RNA with a fluorescent dye or the like, and a sample are hybridized, it is possible to detect the signal using autoradiography or fluorescence microscopy, or the like.

  In addition, as a method for labeling microorganisms, an immunostaining method can be performed using an antigen-antibody reaction. The immunostaining method is a method in which an antibody to which a fluorescent dye or the like is bound is bound to an antigen to be specifically labeled and observed. The immunostaining method includes a direct method and an indirect method, and in the present invention, it is preferable to use an indirect method that has specificity and provides a high fluorescence signal.

  Fluorescent dyes are FITC, LCRed640, LCRed705, AMCA, Cascade Blue, PE / RD1, ECD, PC5 / PE-Cy5, PC7 / PE-Cy7, TRITC, Cy3, Texas Red, Cy5, APC, bodypi (Bodypy), or It is preferable to use quantum dots or the like.

  When a fluorescent labeling technique is used for measuring microorganisms, there are a method of fixing and staining microorganisms (the microorganisms are dead) and a method of staining microorganisms alive. For example, a fluorescently labeled DNA fragment is microinjected into a microorganism using a microneedle. Because there is no fixation process and microorganisms are alive, quick and detailed inspection can be performed.

In this step, microorganisms are counted using gene amplification. At this time, the gene amplification method is preferably a PCR method, a LAMP method (Loop-mediated Thermal Amplification), an ICAN method (Isothermal and Chimera-primed Amplification of Nucleic Acids), or the like.

  The PCR method uses a DNA as a template, a cycle reaction using a primer complementary to both ends of the region to be amplified and a heat-resistant DNA polymerase (the first step is annealing of the template DNA and the primer, the second step is heat-resistant DNA). The third step is a method of amplifying a target DNA region by performing an elongation reaction with a polymerase, and the third step is dissociation of double-stranded DNA. As a general test procedure, for example, bacteria are destroyed with an anionic surfactant, proteins are removed by salting out, and DNA is precipitated using isopropanol. Subsequently, a specific region of the extracted DNA is amplified using a PCR method, and it is confirmed whether the specific region of the DNA is amplified by electrophoresis using a polyacrylamide gel. As described above, the presence or absence of specific bacteria in the food is confirmed.

The LAMP method is a method for determining the presence or absence of a gene by giving chemical conditions in the gene and increasing the number of genes. Specifically, four types of primers set for six regions of the target gene (DNA or RNA) and a strand displacement type DNA polymerase are used. By utilizing the strand displacement activity of this DNA polymerase, amplification reaction can be carried out at a constant temperature, and fluorescence intensity or white precipitation of magnesium pyrophosphate can be detected in one step.
With the ICAN method, an arbitrary region of a gene up to about 4 kilobases can be amplified at a constant temperature (50 ° C. to 65 ° C.). Since the reaction can be performed at a constant temperature, gene amplification can be performed in a short time without reducing the activity of reverse transcriptase.

  In one embodiment of the method for measuring microorganisms of the present invention, when PCR is used, the microorganisms are filtered from the precipitated phase in which microorganisms, a surfactant, a hydrophilic organic solvent, and water are mixed. Only supplement and wash with water to wash away the surfactant and hydrophilic organic solvent. This is because, since the precipitated phase is hydrophobic, reagents such as probes and primers cannot come into contact with DNA, and therefore it is preferable to measure after removing organic phases other than microorganisms by filtration or the like.

  Furthermore, when tetrahydrofuran, dimethylformamide, ethanol, acetone, or the like is used as the hydrophilic organic solvent, the microorganisms are precipitated while maintaining the form, and thus it is necessary to take out DNA or RNA from the microorganisms before performing the PCR method. . As this method, it is preferable to use a freeze-thaw method, a boil method, an alkali method, or the like. In the present invention, for example, only the microorganism is captured on the filter, the filter is transferred to a container together with water, and the gene is extracted by freezing and thawing.

  Moreover, in the microorganism concentration method of the present invention, the pH can be adjusted to about 6 or less, and the gene is not damaged. For this reason, it is suitable for using the gene amplification method.

Hereinafter, the microorganism measuring method according to the present invention will be described with reference to examples.
Example 1
Escherichia coli K12, which is Escherichia coli, was used as a test strain. In the Escherichia coli separation step, Escherichia coli K12 was shaken and cultured in LB medium at 37 ° C. for 18 hours, and 10 μl of the resulting bacterial solution (about 10 ⁷ E. coli) was added to 769 ml of pure water. Next, as shown in FIG. 1 (a), 2.5 ml of 0.1 M perfluorooctanoic acid (2), 38 ml of tetrahydrofuran (3), and 192 ml of 6.0 M hydrochloric acid (4) are sequentially added to make the solution uniform. The container was closed and shaken up and down 30 times. Then, as shown in FIG.1 (b), it was left to stand at room temperature for 3 hours, and the depositing phase (5) was deposited on the container bottom face.

  Next, in the Escherichia coli extraction step, 33 ml of a 0.1 mg / ml DAPI solution dissolved in ethanol was added to the vessel containing the precipitation phase and the aqueous phase, and Escherichia coli in the precipitation phase was fluorescently stained. Next, after measuring the volume of the precipitated phase using a pipette, 5 μl each of the precipitated precipitated phase (5) and the supernatant phase (6) is taken, placed on a slide glass, and further covered with a cover glass to fluoresce. The number of bacteria was measured using a microscope.

FIG. 2 is a microscopic image of E. coli. 2A is a transmitted light image of E. coli, and FIG. 2B is a fluorescent image stained with DAPI. As a result of the analysis, the E. coli count in the supernatant phase is a 0/5 [mu] l E. coli number of precipitated phase was 5.8 × 10 ⁶ cells / 5 [mu] l. Since the volume of the deposited phase was about 20 μl, the number of E. coli in the deposited phase was determined by the following formula.

  In the E. coli separation step, the number of E. coli in the precipitation phase was almost the same as the number of E. coli added first, and E. coli was not found in the supernatant phase, so the recovery rate of E. coli was almost 100%. I understood that.

Moreover, the concentration magnification of the concentration method was calculated | required using the following formula | equation.

Example 2
In Example 2, it implemented according to the flowchart of FIG. In the same manner as in Example 1, a precipitation phase containing E. coli was deposited on the bottom of the container. The deposited phase was sucked out with a pipette and filtered onto a membrane filter having a diameter of 0.2 μm. Furthermore, about 10 ml of sterilized water was added on the membrane filter, and the precipitated phase was washed away by filtration. This washing operation was performed three times. Next, this membrane filter was lightly wrapped with tweezers, placed in a 1.5 ml microtube, 0.5 ml of sterilized water was added, and luciferase plasmid DNA was further added as an internal standard. After closing the lid of the microtube, the parafilm was wound, immersed in liquid nitrogen for 3 minutes, and then thawed in a 65 ° C. water bath for 3 minutes. This freeze-thaw operation was repeated three times to disrupt the E. coli cell membrane. Thereafter, centrifugation was performed at 12,000 rpm for 20 minutes to extract Escherichia coli DNA in the supernatant.

Next, in Example 2, the extracted DNA was detected using PCR. In PCR, the following reagents and template were added to a capillary for Light Cycler (manufactured by Roche), subjected to a centrifuge for exclusive use of Light Cycler, and then set in a Light Cycler for reaction. As for the reagent, 2 μL of 10 LC DNA master hybridization (PCR reagent manufactured by Roche), 2 μL of 1 pmol / μL probe (3′FITC), 2 μL of 1 pmol / μL (5 ′ LCRed 640), 10 pmol / μL PCR primer ECA75F (base sequence: 5′-gga agaagc ttg ctt ctt tgc tga c-3 ′) 1 μL, 10 pmol / μL PCR primer ECR619R (base sequence 5′-agc ccg ggg att tca cat ctg act tal-3 ′) Use 3.2 μL of MgCl ₂ . 1 μL of DNA template extracted from E. coli and 7.8 μL of sterilized water were added to the reaction reagent solution. As a result, E. coli DNA concentrated using perfluorooctanoic acid, tetrahydrofuran and hydrochloric acid could be detected by PCR.

It is a conceptual diagram explaining the concentration and extraction process of microorganisms in the microorganism measuring method according to the present invention. It is an image figure which shows the transmitted light image and fluorescence image which observed Escherichia coli in a deposit layer using the microscope with the microorganisms measuring method which concerns on this invention. (A) shows a transmitted light image. (B) shows a fluorescence image (DAPI staining). It is a flowchart figure explaining the microorganisms measuring method concerning the present invention.

Explanation of symbols

1 Mixed solution of E. coli and pure water 2 Perfluorooctanoic acid 3 Tetrahydrofuran 4 Hydrochloric acid 5 Precipitation phase containing E. coli 6 Supernatant phase

Claims (9)

Add a surfactant and a hydrophilic organic solvent to a solution containing microorganisms to form a uniform solution,
Then lower the pH of the solution,
The solution is separated into two phases, a precipitate phase in which microorganisms, a surfactant, a hydrophilic organic solvent, and water are mixed, and a supernatant phase,
A microorganism measuring method characterized by measuring the number of microorganisms concentrated in the precipitated phase ,
Here, the microorganism measuring method, wherein the surfactant is a fluorocarboxylic acid and the hydrophilic organic solvent is compatible with the fluorocarboxylic acid . 2. The microorganism measuring method according to claim 1, wherein the number of microorganisms is measured using a culture method. 2. The microorganism measuring method according to claim 1, wherein the number of microorganisms is measured using a microscope. 4. The microorganism measuring method according to claim 1 , wherein the number of microorganisms is measured using a label. The method for measuring microorganisms according to claim 1 or 4, wherein the number of microorganisms is measured using a gene amplification method. The gene amplification method is a PCR method,
Claims microorganisms captured on the filter by filtering the precipitated phase, a surfactant and a hydrophilic organic solvent and washing stream, characterized in that the DNA of the captured microorganisms as a template for the PCR method 5. The microorganism measuring method according to 5. The gene amplification method is a PCR method,
Filtering the precipitated phase to capture microorganisms on the filter, transferring the filter together with water into a container, extracting the DNA of the captured microorganism by freezing and thawing, and using the DNA as a template for the PCR method The microorganism measuring method according to claim 5 or 6, characterized in that. The microorganism measuring method according to claim 1, wherein the surfactant is perfluorooctanoic acid . The method for measuring microorganisms according to any one of claims 1 to 8, wherein the hydrophilic organic solvent is tetrahydrofuran, dimethylformamide, ethanol or acetone .

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