JP2005245317A - Measuring instrument for microorganism and measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a measuring instrument and a measuring method for microorganisms capable of accurately detecting and measuring target microorganisms from a specimen. <P>SOLUTION: The measuring instrument for microorganisms is provided with a micro-flow cytometer furnished with a nozzle channel flowing a sample liquid containing microorganisms as a micro-flow, a fluorescent microscope detecting the microorganisms in the nozzle channel, and a photographing means to photograph the microorganisms with the fluorescent microscope. The measuring method for microorganism contains a step to label the microorganism with fluorescent light, a step to pass the sample liquid containing the microorganism as a micro-flow, a step to photograph the microorganism in the micro-flow with a fluorescent microscope by color photography, and a step to count the number of microorganisms from the photographed image. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a microorganism measurement apparatus and measurement method, and more particularly, to a measurement apparatus and measurement method capable of multiple measurement of microorganisms.

Conventionally, as a method for accurately detecting fine particles in a short time, a microflow cytometry method in which a nozzle portion is formed using a semiconductor micromachining technology or a micromachine technology (MEMS: Micro Electro Mechanical System) has been proposed.
In this measurement method, a flow cytometry nozzle (flow path is ~ 400 nm) is produced on a glass substrate by a semiconductor processing technique, and a sample is injected into the nozzle while feeding at a flow rate of 50 to 150 nl / min. (Wavelength 632.8 nm) is irradiated, and the transmitted light is measured with a photodiode. In other words, a signal is generated when fine particles in a sample scatter laser light (for example, Non-Patent Document 1).
Che-Hsin Lin, Gwo-Bin Lee, “Micromachined flow cytometers with embedded etched optic fibers for optical detection,” Journal of Micromechanics and Microengineering, Vol. 13, No 3, pp. 447-453, May 2003

  However, in the conventional microflow cytometry method, for example, when polystyrene beads having a diameter of 20 μm are used, the signal is successfully detected. However, in the actual blood sample, the signal peaks overlap, and the number of particles is accurate. Measurement was difficult. Moreover, since measurement is performed using scattered light, it is difficult to select the type of particles, such as distinguishing blood cells (red blood cells, white blood cells, platelets, etc.).

  Therefore, an object of the present invention is to provide a microorganism measuring apparatus and a measuring method capable of accurately detecting and measuring a target microorganism from a specimen sample. In particular, an object of the present invention is to provide a measuring apparatus and a measuring method capable of multiple measurement of microorganisms having a size of 1 μm or less.

  The present invention relates to a microflow cytometer provided with a nozzle flow path through which a sample solution containing microorganisms flows as a microflow, a fluorescence microscope for detecting microorganisms in the nozzle flow path, and imaging for photographing microorganisms via the fluorescence microscope An apparatus for measuring microorganisms.

  Furthermore, the present invention provides a labeling step for fluorescently labeling microorganisms, a flow step for flowing a sample solution containing microorganisms as a microflow, a step for performing color imaging of microorganisms in the microflow via a fluorescence microscope, and a captured image And a measuring method for measuring the number of microorganisms from the microorganism.

  In the measuring apparatus according to the present invention, it is possible to measure a target organism using a very small amount of sample. In addition, multiple measurement for measuring various types of microorganisms and shape classification are possible.

  In addition, the cost of the microflow cytometer can be reduced and the size of the microflow cytometer can be reduced, and the safety of the microorganism test can be improved by disposable.

  FIG. 1 is a schematic diagram showing the principle of the microorganism measuring method according to the present embodiment. As shown in FIG. 1, in order to accurately measure the number of microorganisms contained in a microorganism sample, fine particles are caused to flow in a path having a size close to the volume of the microorganism, and the number is measured using, for example, a fluorescence microscope. Just measure. That is, since the fine particles flow in a line one by one in the path, the number of microorganisms passing therethrough is measured with a fluorescence microscope provided in the path.

  Examples of cases where microorganisms need to be detected (measured) include bacteria testing at food factories, hospitals and insurance stations, and microbiological testing in water and sewage and rivers. The type of microorganism to be detected in the present invention is not particularly limited, and bacteria such as Escherichia coli, Salmonella, Bacillus subtilis, Staphylococcus aureus, Streptococcus, Pneumoniae, Pseudomonas, Clostridium, yeast, mold, actinomycetes, Includes microorganisms in general.

  In the micro flow cytometry method, microorganisms are caused to flow using a sheath flow as in the flow cytometry. FIG. 2 is a top view of a part of a microflow cytometer used in the microflow cytometry method. The micro flow cytometer is made of a transparent polymer material such as PDMS (polydimethylsiloxane), for example, and has a structure in which three flow paths are combined with a substrate to form one flow path as shown in FIG. Yes. In such a structure, a sample flow containing microorganisms is flowed from the center, and a sheath flow (sheath flow) as a carrier is flowed from both sides thereof, thereby reducing the width of the sample flow to a micro flow (hydrodynamic focusing). .

  In the microflow cytometer according to this embodiment, for example, in order to measure microorganisms having a size of 10 μm or less, particularly about 1 μm, it is necessary to narrow the width of the sample flow to 10 μm or less by the sheath flow. The cross-sectional shape of the nozzle and each flow path may be rectangular, circular, semicircular, polygonal, etc., as long as the microsheath flow can be formed by constricting the sample flow. Here, the microsheath flow is composed of a sheath flow and a sample flow narrowed to become a microflow.

The nozzle width, which is important for narrowing the sample flow, is suitably about 10 μm to about 150 μm. If it is smaller than 10 μm, the pressure loss is large and a predetermined flow rate cannot be obtained and microorganisms may be clogged in the nozzle. If it is larger than 150 μm, if the microorganism concentration is 10,000 or more, narrowing down This is because microorganisms often overlap (do not flow in a straight line) and the measurement accuracy decreases. On the other hand, the depth of the nozzle is preferably about 10 μm.
Moreover, it is preferable that the sample channel and the sheath channel have the same cross-sectional shape and dimensions as the nozzle.

PDMS (polydimethylsiloxane: manufactured by Dow Corning) was used as the base material of the microflow cytometer, but other materials may be used as long as they are inexpensive and easily mass-produced. A transparent material that can be easily detected is desirable.
Specifically, elastomers are suitable for soft materials and plastics are suitable for hard materials. As the elastomer, transparent synthetic rubber such as silicon rubber (PDMS described above), butadiene rubber, isoprene rubber, fluorine rubber, styrene transparent rubber, and blended rubber thereof are preferable.
As properties, it is preferable that the density is about 1 g / ml, the water supply rate is 0.1% or less, the total light transmittance is 85% or more, the molding shrinkage is 1% or less, and the flexural modulus is 300 Mpa or more.

  The curable type is preferably a curable type using a chemical initiator or a crosslinking agent, a thermosetting type, or an energy ray curable type such as light.

  If it is a plastic material, it is preferable that it is a material which permeate | transmits excitation light and fluorescence. For example, polystyrene, polysulfone, acrylic, polycarbonate, polyolefin, chlorine-containing polymer, polyether, and polyester. These may be in the form of homopolymers, copolymers, blends or prepolymers. Although thermoplasticity is preferable in terms of molding, a thermosetting or energy ray curable crosslinked polymer may be used.

  Next, a method for manufacturing a microflow cytometer will be briefly described. In the manufacturing method of the microflow cytometer, first, a UV curable resist having a thickness of 10 μm is applied on, for example, a 3-inch silicon substrate. For example, SU-10 is used as the UV curable resist.

  In addition to a semiconductor material such as silicon, ceramics, glass, polymer alloy, or the like may be used for the substrate.

  Next, a mask pattern is designed, and a UV curable resist is patterned using the mask pattern. Thereby, a convex mold is formed.

  Next, about 30 ml of a transparent PDMS prepolymer is filled in the mold and held at a temperature of 37 ° C. for 40 hours to polymerize the prepolymer.

  Next, PDMS is taken out from the mold, and a micro flow cytometer replica is produced. In this replica, a groove corresponding to a micro-channel such as a sheath channel is formed.

  Finally, the surface is covered with a glass cover, and a microflow cytometer having a predetermined microchannel is completed.

  The micro flow cytometer is not limited to the replica method, and other methods such as a direct molding method may be used. Moreover, although it produced in the planar shape here, it is good also as the cylindrical shape generally used with a flow cytometer.

  FIG. 3 is a schematic diagram of a microflow cytometer according to the present embodiment, FIG. 3A is a schematic diagram of a micro flow path of the microflow cytometer, and FIG. 3B is a cross-sectional view of a detection site. FIG. 3C is a cross-sectional view of the sample liquid injection site.

  As shown in FIG. 3A, the microflow cytometer has two sheath channels having a length of 5.6 mm and a sample channel having a length of 5 mm sandwiched between them. The two sheath channels and the sample channel merge to form a nozzle. The length of the nozzle is 1.0 cm.

Further, as shown in FIG. 3 (b), the detection site is provided with a nozzle made of a rectangular cross-sectional groove having a width of 100 μm and a depth of 10 μm on a PDMS having a thickness of 6 mm. A glass cover having a thickness of about 0.5 mm is provided.
Microorganisms flowing through the nozzle are observed and measured with a fluorescence microscope through a glass cover.

Further, as shown in FIG. 3 (c), the sample liquid injection site is provided with a PDMS having a thickness of 6 mm and a sample flow path including a groove having a rectangular cross section with a width of 100 μm and a depth of 10 μm. A glass cover having a thickness of about 0.5 mm is provided on the surface.
At the end of the sample channel, a hole with a diameter of 1 mm is opened in the glass cover, and the capillary tube is connected with an epoxy resin. The sample stream is supplied from the capillary tube to the sample flow path.

  FIG. 4 is a schematic diagram of a microorganism measuring apparatus according to the present embodiment. The measurement device includes the above-described microflow cytometer. A microorganism sample is supplied to the sample flow path of the microflow cytometer via a capillary tube. Similarly, a carrier buffer is supplied to the sheath channel via a capillary tube. The microorganism sample and the carrier buffer are supplied from a microsyringe controlled by a syringe pump.

The microbial sample and the carrier buffer supplied to the sample channel and the sheath channel together flow into the nozzle.
Since the sheath channel is provided on both sides of the sample channel, the sheath stream is supplied so as to sandwich the sample stream containing microorganisms. As a result, the sample flow is narrowed in the nozzle, and the microorganisms flow in a straight line. Here, the width of the sample flow is brought close to the size of a microorganism having a size of 10 μm or less.

  A fluorescent microscope (Hg lamp) is provided on the nozzle. Microorganism optical measurement is performed using a fluorescence microscope, a high-sensitivity color CCD, and image analysis (mainly luminance distribution). The microorganism is dyed with a microorganism fluorescent labeling substance such as SYBR green.

  In the measurement apparatus according to the present embodiment, as described above, a microsyringe and a syringe pump are used to send a sample flow and a carrier flow. For example, 1 nl to 100 μl according to the size of the flow path. Other devices may be used as long as the flow rate of / ml can be controlled. For example, a micro ultrasonic pump or a micro pump may be used.

  As for the flow rates of the sheath flow and the sample flow, a flow rate of about 10 to about 200 nl / min is appropriate for the flow channel having a flow channel width of 100 μm and a depth of 10 μm as described above. In this case, the flow rates of the sample flow and sheath flow will be between about 1 and about 500 μm / second. When the flow rate is 10 nl / min or less, it becomes difficult to narrow down due to the influence of diffusion. On the other hand, when the flow rate is 200 nl / min or more, the movement of microorganisms is fast and it is often difficult to measure with a CCD.

  For the optical measurement of microorganisms, a fluorescence microscope, a high-sensitivity color CCD, and image analysis were used. However, the present invention is not limited to the combination of these. Spot measurement may be used.

  Even if a laser, a laser diode, or an LED light source is used as the excitation light, a photomultiplier tube or a photodiode is used as the light receiving side, and a filter having an arbitrary wavelength is used, the same measurement as the line measurement of the two-dimensional image is possible.

  In addition to optical measurement, for example, a thermal lens microscope or electrical or electrochemical detection using electrodes can be used. Basically, any detector having a spatial resolution in the submicron order can be used. However, when a CCD (NTSC format) is used, since the exposure time is a maximum of 33 milliseconds per frame, the sensitivity is higher than that of a cytometer that is detected in milliseconds or less.

  When using a cytometer, the type of blood cells and impurities (dust) are analyzed only from one-dimensional information (forward, backscattered light, fluorescence, etc.) for speeding up. Although there is almost no recognition capability, when a CCD is used as in the present embodiment, a two-dimensional image is obtained, so that the shape of the sample can be specified. For this reason, for example, CCD detection is effective for analysis of samples whose components are not constant, such as food materials and environmental samples.

  The type of the microorganism fluorescent labeling substance used in the present embodiment is not particularly limited as long as it can be detected. Examples include DNA stains such as SYBR Green, SYBR Gold, and DAPI, as well as phycoerythrin (PE) and FITC. Other than these, ethidium bromide, propidium iodide (Propidium iodide) Hoechst 33258/33342, Acridine orange, Chromomycin, Mithramycin, Olivomycin, Pyronin Y, Thiazole orange, Rhodamine ) 101, isothiocyanate, BCECF, BCECF-AM, C.I. SNARF-1, C.I. SNARF-1-AMA, Aequorin, Indo-1, Indo-1-AM, Fluo-3, Fluo-3-AM, Fura-2, Fura-2-AM, Oxonol, Rhodamine 123, 10-N-non-acridine orange, fluorescein, fluorescein diacetate, carboxyfluorescein (CF), carboxyfluorescein diacetate (CFDA), carboxy Examples thereof include dichlorofluorescein (CDCF) and carboxydichlorofluorescein diacetate (CDCFDA).

  Further, for example, a semiconductor fluorescent material (nanocrystal) such as a semiconductor nanoparticle such as CdS, Si, Ge, or ZnS particle may be used.

  Furthermore, if a microorganism can be specifically labeled with a fluorescently labeled antibody, each specific microorganism can be fluorescently labeled. For example, the antibody may be labeled with biotin and avidin may be bound to the fluorescent substance.

When the microorganism concentration is low, the sample solution is filtered and concentrated with a 0.45 μm membrane filter. For example, if the microorganism concentration is about 100 cells / ml, it can be detected in a short time if it is concentrated to about 1/100 to about 100 cells / 10 μl.
It is also possible to fluorescently label an antibody that can bind to a specific microorganism and fluorescently label only the target microorganism, or to collect the microorganism by binding the antibody to a magnetic bead and then detect it with a sheath flow device. The kind of the magnetic beads is not particularly limited as long as it can specifically bind the microorganism to be detected. The size of the beads is 1 μm to 10 μm.
Furthermore, by labeling these magnetic beads with fluorescent substances having different fluorescence wavelengths, various microorganisms can be simultaneously detected in color. In addition, the antibody to be used is a polyclonal antibody in the microorganism adsorption process, and the microorganism is adsorbed broadly. If a monoclonal antibody having a strict specificity is used as the fluorescently labeled antibody, the selectivity is enhanced. When detecting with fluorescent magnetic beads, since the number of beads is detected with fluorescence, it is necessary to obtain a correlation coefficient with the number of microorganisms in advance.

  Next, the measurement method according to the present embodiment will be described by taking as an example the case where the detection microorganism of the present invention is Escherichia coli.

  First, liquid feeding to the microchannel (sheath channel and sample channel) was performed using a microsyringe having a volume of 25 μl or 50 μl. Since the liquid feeding speed plays a very important role in the formation of the microsheath flow, it was controlled using a syringe pump that ensures accurate liquid feeding. The feeding speed is about 10 nl / min to about 1 μl / min.

  Sheath flow was observed at almost the entire feeding speed and solution composition. In order to narrow the sample flow, the sheath flow may be flowed faster than the sample flow. In this pattern, since the carrier flow flows from the left and right at the same speed, the sample flow is sandwiched by the sheath flow and passes through the middle of the flow path in the nozzle region.

  The channel depth was designed to be 10 μm. If the depth is less than 10 μm, the focus of the fluorescent microscope lens becomes easy, but on the other hand, the flow rate becomes faster and the number of microorganisms that can be analyzed per unit time is limited.

  An epi-illumination fluorescence microscope was used to detect microorganisms. The light source is an Hg lamp, and SYBR Green is used as a fluorescent dye, and measurement is performed using a filter having an excitation wavelength of 490 nm and a fluorescence wavelength of 520 nm.

  The objective lens is equipped with a type dedicated to fluorescence, and the magnification is 10 to 40 times. A high-sensitivity color CCD (300,000 pixels) is attached to the part of the photographic device to measure microorganisms. The observation image (NTSC format) is recorded on the VTR or directly input to the personal computer (PC) in the AVI format via the analog-digital interface. When optical measurement is performed using a photodiode or a photomultiplier tube, the signal is input to the PC as a digital signal via an AD converter.

The recorded AVI format image is taken into the moving image analysis software, and the luminance change between two points parallel to the arbitrary XY coordinates or the Y axis is measured in each video frame. As the measurement result, each frame is output to an Excel file, and the fluorescence intensity of the microorganism is graphed based on this data.
The graphed result is, for example, as shown in FIG. In FIG. 5, the horizontal axis indicates the number of video frames (frame number), and the vertical axis indicates the fluorescence intensity. As shown in the graph, the fluorescence intensity becomes a pulsed signal. A general-purpose analysis program is used to analyze the luminance change in the image.

  As described above, by using the measurement method according to the present embodiment, Escherichia coli in sewage can be detected in real time, so that optimal control of a disinfectant such as ozone is possible, which is useful for energy saving and in rivers. Real-time microorganism monitoring can be realized.

  In addition, by specifically labeling the target bacteria, it is possible to quickly grasp the status of contaminating bacteria such as Salmonella and Escherichia coli in food, shortening the waiting time for inspection in food distribution, and increasing distribution efficiency.

  In addition, the time required for infectious disease inspection in a hospital is shortened, early diagnosis is realized, and rapid bacterial inspection on-site such as examination and surgery becomes possible.

E. coli is measured using the measuring apparatus according to this embodiment.
In the measurement of E. coli, first, colonies of E. coli (JM-109) growing on an agar plate are inoculated into a nutrient medium (LB medium) and cultured for 6 hours, and an OD (optical density) of about 0.6. Collect bacteria with Subsequently, it is washed with Tris buffer (50 mM, pH 7.4), and the microorganism concentration is adjusted to about 1,000,000 cells / ml.

  Next, SYBR Green I (1 mM), which is a fluorescent reagent, is added to the E. coli solution at a volume ratio of 1/200 to 1/1000 and allowed to stand at room temperature for 5 minutes. Since SYBR Green binds to double-stranded DNA in E. coli cells and emits fluorescence, the entire E. coli emits fluorescence at 520 nm. This solution is filled into a 50 μl microsyringe and attached to a syringe pump. Such a solution becomes a sample solution.

  The carrier solution is also tris buffer (50 mM, pH 7.4), filled in a 50 μl microsyringe and attached to a syringe pump.

  Each microsyringe is connected to a microflow cytometer (microflow cell) via a capillary tube, and is sent to each channel at a flow rate of 50 nl / min (sample solution) and 150 nl / min (carrier solution). As a result, the fluorescence of microorganisms flowing in a straight line is observed by being sandwiched between carrier liquids by the sheath flow and narrowing the width of the microorganism sample flow to several microns.

  Microorganisms are photographed with a CCD camera through a fluorescence microscope. When the CCD camera capture format is NTSC format (captured at 33 frames / second), the maximum linear velocity of the microorganisms that can be observed is greatly affected by the magnification of the lens, the capture speed to the PC, etc., but about 500 μm / It is about 10 μl / min in terms of second and flow rate.

  The maximum number of microorganisms to be measured is not limited in principle if the number of frames per second of the CCD is increased or the loading into the PC is accelerated. In addition, if a photodiode is used instead of a CCD as a light receiver, measurement in milliseconds can be performed, and in this case, 10000 to 100,000 or more microorganisms can be measured in one minute. However, in the case of using a photodiode, it is necessary to make the slit of the light receiving element portion on the order of submicron to micron, or to increase the spatial resolution using a lens system.

FIG. 5 shows an output result when about 30 microorganisms are measured in 5 seconds. The horizontal axis is the number of video frames (frame number), and the vertical axis is the fluorescence intensity. As shown in FIG. 5, the number of microorganisms becomes a sharp pulse signal. The fluorescence intensity varies, for the following reason.
That is, when the peak detected in FIG. 5 is confirmed on the video screen, first, when the fluorescence intensity is 30 or more, two or more E. coli are solidified and flowed. It was found that when the fluorescence intensity was 15-30, the flow was one, and when the fluorescence intensity was 15 or less, the fluorescence staining was thin or the focus was not well achieved.

  Therefore, by counting all the peaks and further correcting the peaks in the case of two or more, the number of bacteria per unit time or unit volume can be accurately measured. FIG. 6 shows calibration curves when microorganisms are measured by this method and when they are measured by the conventional plate method (agar plate method). FIG. 6 shows the results of measuring the same microorganism sample by the conventional agar plate method and the microorganism measurement method of this example, the horizontal axis is the agar plate method, and the vertical axis is the measurement result of the microorganism measurement method. As can be seen from FIG. 6, the measurement results of both are very similar, and it can be seen that the microorganisms can be accurately measured in the microorganism measuring method according to the present embodiment.

On the other hand, when a microorganism is stained with propidium iodide instead of SYBR Green, the cell emits red fluorescence having a wavelength of around 600 nm. Propidium iodide is particularly likely to enter cells when bacterial cell membranes are damaged, and binds to DNA and emits fluorescence. For this reason, this reagent is often used to determine the viability of microorganisms in a sample.
That is, if red fluorescence is observed when a microorganism is stained with propidium iodide, the cell is likely to be damaged dead. In this embodiment, since a color CCD is used for fluorescence detection, the total number of bacteria can be measured in green, and dead bacteria can be measured in red, and microorganisms and bacteria can be determined in real time. It becomes possible.

  As described above, the measuring method according to the present embodiment can detect microorganisms in a specimen sample in a short time and with high accuracy, and further has the following advantages.

  First, it is possible to detect and measure microorganisms from a specimen sample only by concentrating or diluting to an appropriate microorganism concentration without performing pre-culture. That is, an environmental sample or a food sample can be used as it is, and it is unnecessary to proliferate the target microorganism to be detected by subjecting the sample to pre-culture and selective culture in advance.

  Second, the sample may be in a very small amount (for example, several μl), and can be accurately measured even if the number of microorganisms in the sample is 50 to 100 or less.

  Thirdly, the process from sample pretreatment to detection of microorganisms can be completed within a few minutes.

  Fourth, by using a color detector, multiple measurement is possible, and for example, the proportion of dead bacteria in a sample can be displayed in a short time.

  Fifth, since microorganisms are measured by an image, not only distinction from dust etc., but also classification of shapes (cocci, bacilli, filamentous fungi) is possible.

  Sixth, since a polymer (for example, PDMS) is used as the material of the microflow cytometer, the cost is reduced, the equipment is made compact, and the safety of microbiological examination by disposable is improved.

It is the schematic which shows the principle of the measuring method of the microorganisms concerning this Embodiment. It is a top view of a part of the microflow cytometer according to the present embodiment. It is the schematic of the micro flow cytometer concerning this Embodiment. It is the schematic of the measuring device of microorganisms concerning this Embodiment. It is the result of having measured microorganisms with the measuring method concerning this embodiment. This is a comparison between the measurement method according to the present embodiment and the conventional agar plate method.

Claims (14)

A microflow cytometer provided with a nozzle channel through which a sample liquid containing microorganisms flows as a microflow;
A fluorescence microscope for detecting the microorganism in the nozzle channel;
An apparatus for measuring microorganisms, comprising: imaging means for imaging the microorganisms through the fluorescence microscope. The micro flow cytometer
Two sheath channels, a sample channel sandwiched between the sheath channels, and a nozzle channel connected to the sheath channel and the sump channel, the sheath flow flowing through the sheath channel is The measurement apparatus according to claim 1, wherein the sample flow merges with the sample flow to form a microsheath flow in the nozzle flow path.   2. The measuring apparatus according to claim 1, wherein the width of the noise flow path is in a range of about 10 to about 150 [mu] m.   The measuring apparatus according to claim 1, wherein the photographing unit is a color CCD.   The measuring apparatus according to claim 1, wherein the microorganism is a microorganism treated with a fluorescent substance that binds to the microorganism. Two sheath channels, a sample channel sandwiched between the sheath channels, and a nozzle channel connected to the sheath channel and the sump channel are provided on the substrate,
The sheath flow flowing through the sheath flow path merges with the sample flow so as to sandwich the sample flow, and forms a microsheath flow in the nozzle flow path,
The microflow cytometer, wherein the substrate is made of one material selected from the group consisting of an elastomer and a plastic.   The microflow cytometer according to claim 6, wherein the substrate is made of polydimethylsiloxane.   7. The microflow cytometer according to claim 6, wherein the width of the noise flow path is in the range of about 10 to about 150 [mu] m. A labeling step for fluorescently labeling microorganisms;
A flow step of flowing a sample solution containing the microorganism as a microflow;
Color-photographing the microorganism in the microflow through a fluorescence microscope;
And a measuring step of measuring the number of the microorganisms from the photographed image. The measurement method according to claim 9, wherein the labeling step is a step of treating a sample containing the microorganism with a fluorescent substance that binds to the microorganism.   The flow process is a process in which a sample flow containing the microorganism is sandwiched between two sheath flows, and the sample flow is converted into a microsheath flow having a predetermined width by hydrodynamic focusing. Measurement method.   The measurement method according to claim 9, wherein the flow rate of the sample liquid is in the range of about 10 nl / min to about 200 nl / min.   The measuring method according to claim 9, wherein the measuring step is a step of measuring the number of microorganisms from the fluorescence intensity of the microorganisms photographed in the image. The said measurement process includes the process of classifying the said microorganisms image | photographed by the said image with a fluorescence wavelength, and the multiple measurement process of measuring the number of each classified microorganism. Measurement method.

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