JP2008022776A - Microorganism metering device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microorganism counting device counting live bacteria and dead bacteria by applying fluorescent dye to a sample containing microorganisms or having possibility of containing microorganisms, and improved in accuracy in comparison with a conventionally well-known technique. <P>SOLUTION: The microorganism counting device 1 enables more accurate count of the number of live bacteria through using a live/dead bacteria-dyeing reagent dyeing both of the live and dead bacteria and a dead bacteria-dyeing reagent dyeing only dead bacteria, wherein the exited wavelength of the live/dead bacteria-dyeing reagent is shorter than that of the dead bacteria-dyeing reagent to firstly discriminate microorganisms from others referring to luminous points emitting fluorescence due to the dyeing reagent dyeing the live bacteria and the dead bacteria and then discriminate the live bacteria from the dead bacteria referring to luminous points detected as those of the microorganisms. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a microorganism measuring device that rapidly detects microorganisms contained in environmental samples, food samples, etc., and measures live and dead bacteria.

  2. Description of the Related Art Conventionally, there is known a device that stains microorganisms using a fluorescent staining method, photographs fluorescent emission points as images, and inspects microorganisms or counts the number of bacteria based on the fluorescence emission wavelength and light quantity of the emission points. . There is also known an apparatus for determining a microorganism from the peak distribution of the wavelength of light emitted from a light emitting point because there are many contaminants in addition to microorganisms in the soil, water environment, and the like (for example, see Patent Document 1).

In this method, the number of microorganisms is measured by irradiating excitation light having a plurality of wavelengths, judging or recognizing microorganisms from the peak distribution of the wavelengths of fluorescent light.
International Publication No. 03/008634 Pamphlet

  However, in the conventional apparatus such as Patent Document 1 described above, if the peak wavelength is measured by finely analyzing the wavelength, for example, every 2 nm, the peak wavelength peculiar to the microorganism can be obtained and determined as a microorganism. Since it becomes a complicated device, it is difficult to put it to practical use as an actual inspection device. Therefore, it is common to measure the light quantity or the luminance value in the wavelength range using a spectral filter that transmits only a certain wavelength range (blue, green, red, etc.) of 10 to several tens of nm. It is difficult to measure the distribution. In addition, a wide variety of fluorescent staining reagents for identifying cells and microorganisms are commercially available, and various reaction mechanisms or excitation light and fluorescence are commercially available, and can be arbitrarily selected according to the purpose. However, since particulate matter (inorganic matter, organic matter, etc.) of approximately the same size as the microorganisms contained in the actual specimen emits light due to reflection or fluorescence, the microorganisms and substances other than microorganisms (foreign matter) It is necessary to distinguish easily. In addition, since the apparatus is desired to be as inexpensive as possible, it is necessary to recognize microorganisms with as simple a mechanism as possible and to distinguish between live and dead bacteria. In general, microbiological tests are performed on the presence or absence of live bacteria, dead bacteria, or specific microorganisms. It is particularly important to measure how much live bacteria are present. Particulate matter (inorganic matter, organic matter, etc.) of approximately the same size as that of microorganisms contained in the actual specimen emits light due to reflection, fluorescence, etc. As a result, there was a problem that the number of viable bacteria could not be accurately measured. In addition, when visually observed using a fluorescence microscope or the like, it is possible to discriminate between microorganisms and other foreign substances based on minute differences in fluorescent light emission, but the discrimination is performed using an optical device. Therefore, there has been a problem that the apparatus becomes very complicated and expensive.

  The present invention solves such a conventional problem, and the excitation wavelength of the first reagent is determined by using the first reagent that stains both live and dead bacteria and the second reagent that stains only dead bacteria. First, it is determined to be shorter than the second reagent. First, it is determined that the microorganism is other than the luminescent point that is fluorescent with the reagent stained with live and dead bacteria, and the luminescent point determined to be the microorganism is further determined to be live and dead. It is an object of the present invention to provide a microorganism counting device that can more accurately measure the number of viable bacteria.

  It is an object of the present invention to provide a microorganism counting apparatus that can easily and accurately distinguish microorganisms and substances other than microorganisms (foreign substances) and accurately measure the number of viable bacteria.

  In addition, fluorescent staining reagents for staining cells and microorganisms are commercially available with various reaction mechanisms. However, when using reagents with different reaction mechanisms, some may not emit light depending on the properties of the specimen. The properties include the pH of the specimen, temperature, presence / absence of a specific substance, dry state, and the like. One fluorescent staining reagent is easily affected by pH, and another fluorescent staining reagent is easily influenced by temperature. If there is a difference, it is difficult to bring the specimen into the optimum state. Very difficult to do.

  The present invention solves such a conventional problem, and both nucleic acid staining stain that binds and reacts with nucleic acid (DNA) that is hardly affected by the properties of the specimen, both of living and dead bacteria and staining reagents that stain dead bacteria. By using a reagent, and by using a fluorescent staining reagent that has only a difference in the presence or absence of cell membrane permeability in order to determine whether the cell membrane is normal to distinguish between live and dead bacteria, the properties of the specimen It is an object of the present invention to provide a microorganism counting apparatus that is very easy to perform a process for improving the pH (for example, a process for achieving an optimum PH).

  In addition, if the wavelengths of the excitation light and the fluorescence are other than visible light, it is very difficult to verify and confirm.

  The present invention solves such a conventional problem, the wavelength of the excitation light of the staining reagent for staining viable and dead bacteria is ultraviolet, and the wavelength of the excitation light of the staining reagent for staining dead bacteria at that time is blue or Make it green. Alternatively, the excitation light wavelength of the staining reagent that stains viable bacteria is blue, and the excitation light wavelength of the staining reagent that stains the dead bacteria is green, so that the fluorescence at the emission point is in the visible wavelength range. Therefore, an object of the present invention is to provide a microorganism weighing device that can be easily verified using a fluorescence microscope or the like.

  In addition, when a microorganism is stained with a live or dead bacteria staining reagent or a dead bacteria staining reagent, the degree of staining may vary greatly depending on the state of the microorganism. There was a problem that it would be wrong to judge that it was dead. For example, when the cell membrane of a microorganism is damaged by a bactericidal agent, it may be slightly stained even with a dead bacteria staining reagent, but when the microorganism is cultured, it forms a colony and may actually be a live cell. There is a problem that it is desired to accurately discriminate against such damaged bacteria.

  The present invention solves such a conventional problem, and more accurately distinguishes damaged bacteria and the like based on the ratio of the luminance value emitted by the living and dead bacteria staining reagent and the luminance value emitted by the dead bacteria staining reagent. An object of the present invention is to provide a microbial metering device.

  In addition, viable bacteria are stained only with a live / dead bacteria staining reagent, and dead bacteria are stained with both a live / dead bacteria staining reagent and a dead bacteria staining reagent. As a result, there is a problem in that the value of the color characteristic is different between the living bacteria and the dead bacteria in order to discriminate between the microorganisms and the foreign substances other than the microorganisms, and it is difficult to accurately distinguish them by one judgment.

  The present invention solves such a conventional problem, and after distinguishing between live and dead bacteria, sets a range for each color characteristic to distinguish between microorganisms and foreign substances, thereby more accurately. Another object of the present invention is to provide a microorganism weighing device that can identify microorganisms.

  In order to achieve the above object, the microorganism measuring device of the present invention reacts only with the first reagent that reacts with any of the living and dead cells and develops a color of both the living and dead cells and the dead cells as described in claim 1. In a microbiological weighing apparatus that measures both microorganisms stained by the fluorescent staining method by bringing both the second reagent that reacts only with the cell into contact with the specimen to be examined for the presence of microorganisms, excitation light in a plurality of predetermined wavelength ranges A light source that irradiates; a spectral filter that transmits only light of different wavelength ranges that is emitted by the excitation light; and a light receiving unit that receives the light, wherein the first reagent has a shorter wavelength than the second reagent Fluorescence is emitted by the light of the luminescence, and the light emission value emitted by the first reagent, the light emission value emitted by the first reagent, and the light emission by the second reagent are determined from the area, luminance value and color characteristics of the emission point emitting the fluorescence. Brightness Viable cells and dead cells are determined by sequentially integrating the luminescent points determined to be viable or dead from the microorganism judging means and the living and dead bacteria judging means. It measures at the same time.

  In the fluorescence reading apparatus according to claim 2, the first reagent and the second reagent are reagents that develop color by reacting with a nucleic acid.

  According to a third aspect of the present invention, the first reagent and the second reagent discriminate between live bacteria and dead bacteria based on a difference in permeability of a cell membrane.

  According to a fourth aspect of the present invention, there is provided a microorganism measuring apparatus comprising a first reagent whose excitation light is ultraviolet and a second reagent whose excitation light is blue or green.

  The microorganism weighing device according to claim 5 comprises a first reagent whose excitation light is blue and a second reagent whose excitation light is green.

  According to a sixth aspect of the present invention, there is provided the microorganism measuring apparatus according to the present invention, wherein the area and the luminance value of the light emitting point that emits fluorescence by the first reagent are determined for the light emitting point determined to be a microorganism by the value within the range set by the microorganism determining means. Thus, there is a viable and dead bacteria judging means for discriminating viable and dead bacteria from the ratio of the luminance values in the respective wavelength ranges in which fluorescence is emitted by the first reagent and the second reagent.

  In addition, the microorganism measuring device according to claim 7 calculates a color characteristic for each luminescent point determined to be viable or dead by the viable and dead bacteria determining means, A set value is provided, and a microorganism judging means for discriminating between live bacteria and foreign substances or dead bacteria and foreign substances from color characteristics is provided.

  According to the microorganism weighing device of the present invention, by using a fluorescent staining reagent that emits fluorescence in a short wavelength range, the shorter the wavelength, the higher the energy of light, so that some foreign substances other than microorganisms can also emit light by reflection or the like. is there. In order to calculate the color characteristics of this light emission, the larger the fluorescence wavelength range, the easier it is to recognize microorganisms. Since the foreign substance is likely to emit a wavelength different from the fluorescence wavelength of the fluorescent staining reagent, excitation with a short wavelength facilitates discrimination between the microorganism and the foreign substance, and the microorganism can be determined more accurately.

  In addition, by using a fluorescent staining reagent that reacts with nucleic acids, it is easy to optimize the properties of the specimen, thereby increasing the luminescence intensity of microorganisms, and accurately judging microorganisms and distinguishing live and dead bacteria. It can be carried out.

  In addition, by using fluorescent staining reagents having different cell membrane permeability, it is possible to distinguish between live and dead bacteria according to the degree of cell membrane damage.

  Further, by making the excitation light of the first reagent for staining viable and dead bacteria ultraviolet, the range in which the fluorescence wavelength can be analyzed within the range of the wavelength of visible light can be used, so that the microorganism can be judged more.

  In addition, by setting the wavelength of the excitation light of the first reagent for staining viable and dead bacteria to blue, it is possible to reduce the emission of light other than the microorganisms and facilitate the determination of the microorganisms.

  The invention according to claim 1 of the present invention uses a fluorescent staining reagent that stains live bacteria and dead bacteria with a short wavelength of excitation light, as compared with a fluorescent staining reagent that stains only dead bacteria. It is easy to compare the fluorescence wavelength of the emission point emitted from a foreign substance with the fluorescence wavelength of the emission point from the microorganism, and the color characteristics of the emission point (for example, comparison of light intensity and luminance value in each wavelength range, It has an effect that it is easy to distinguish microorganisms and foreign substances from the chromaticity. When detecting microorganisms by fluorescent staining, both live and dead bacteria can be detected. However, in the field of normal microorganism testing, heating and hypochlorous acid are often sterilized. Number is more important. Therefore, if it is determined that the microorganism is a foreign substance from the color characteristics of the light emission point emitted by the reagent that stains dead bacteria, it is not possible to distinguish between the live bacteria and the foreign substance. In addition, a staining reagent that stains both live and dead bacteria and a staining reagent that stains only dead bacteria are usually stained simultaneously. At that time, different excitation wavelengths and fluorescence wavelengths are always used. At least the fluorescence wavelength cannot be the same. If they are the same, you do not know which reagent is emitting fluorescence. In general, the wavelengths of light are expressed in ultraviolet, blue, green, yellow, red, and infrared, but the infrared light cannot be recognized by the human eye, so the excitation light is ultraviolet and the fluorescence wavelength is visible. It is good to use in the range of blue to red in the light range. For example, there is 1,4-diamidino-2-phenylindole (DAPI) as a reagent for staining both live and dead bacteria, and the excitation light of this reagent is ultraviolet light and emits blue fluorescence. When non-microorganisms emit light, there is a high possibility of emitting fluorescence including wavelength bands of green, yellow, and red other than blue. By calculating the wavelength of the fluorescence, that is, the color characteristic, it is possible to easily and accurately distinguish between the emission of microorganisms and the emission of foreign matter. Reagents that stain live and dead bacteria and reagents that stain dead bacteria need to use fluorescence in different wavelength ranges, so stain reagents that stain viable and dead bacteria should be reagents that emit fluorescence with excitation light in a shorter wavelength range. By using it, it is possible to increase the range for analyzing the wavelength of fluorescence, and to have an effect that it is possible to more accurately carry out discrimination between microorganisms and foreign substances. When a microorganism is detected by the microorganism weighing device, a method for confirming whether the detected substance is detecting the microorganism correctly is to visually determine the color, shape, etc. using a fluorescence microscope. Therefore, the wavelength of fluorescence is preferably in the visible light range.

  Further, the invention according to claim 2 uses a fluorescent staining reagent having the same reaction mechanism as a reagent for staining live and dead bacteria and a reagent for staining only dead bacteria. It has the characteristic that it is easy to optimize the properties. Various fluorescent staining reagents are commercially available, such as nucleic acids (DNA and RNA), intracellular enzymes (for example, esterases) and those that react with respiratory activity. Here, when two types of reagents, a reagent that reacts with nucleic acid and a reagent that reacts with an enzyme, are used, if a surfactant is contained in the sample, the reagent that reacts with the enzyme does not easily stain or emit light from microorganisms. However, some reagents that react with nucleic acids are hardly affected. It is necessary to remove the surfactant from the sample, but depending on the method of treatment, there is a possibility that the reagent that reacts with the nucleic acid may be adversely affected, and the same reaction mechanism is better. Many reagents that react with DNA or RNA nucleic acids are relatively stable and have high emission intensity. DNA and RNA are always present in cells and microorganisms, and are more likely to detect most microorganisms than staining reagents of other reaction mechanisms. For example, in the case of a reagent that reacts with an enzyme, depending on the type of microorganism and the state of activity, the enzyme is hardly present or only a small amount is present, so the amount of the reacted reagent is small, and only a level that is difficult to detect emits fluorescence. Sometimes. DNA and RNA are also present in animal cells and plant cells, but the size of microorganisms is 0.2-7 μm, and even large ones such as yeast are several tens of μm, whereas animal cells and plant cells are No matter how small it is, it is a size of several tens of μm, usually 100 μm or more, and the size of the emitted light emission point is also proportional to the area (the number of pixels when using a CCD). A microorganism can be recognized as a microorganism by distinguishing it from an animal cell or a plant cell. Therefore, by using a fluorescent staining reagent that reacts with nucleic acids, most microorganisms can be detected, and there are those that have DNA and RNA in addition to microorganisms, but the size of the emitted light emission point, that is, taken with a CCD. The light emitting point has an effect that it can be distinguished from the microorganisms and the other by the number of pixels having a luminance value higher than the set luminance value.

  The invention described in claim 3 can clearly distinguish between live and dead bacteria by using two types of fluorescent staining reagents having different cell membrane permeability as a method for distinguishing between live and dead bacteria. Has an effect. It is very difficult to define the life and death of microorganisms. To prove that it is alive reliably, for example, it can be confirmed that it is cultured to form a colony. But just because you didn't form a colony doesn't mean you are dead. There are many microorganisms that do not form colonies under normal culture conditions (medium, temperature, time, etc.). Generally, it is said that 99% or more of microorganisms present in the environment do not form colonies under normal culture conditions. In addition, when sterilized by heating or hypochlorous acid, even if the colony is not formed by the culture method, the microorganism is damaged and does not form a colony immediately, but it is not dead and should be left for a long time. In some cases, the ability to form colonies may be recovered and proliferation may occur, and it is very difficult to judge whether it is dead or alive. DAPI that stains both living and dead bacteria has a property of permeating the cell membrane, and stains microorganisms regardless of the degree of damage to the cell membrane. An example of a reagent that stains only dead bacteria is propidium iodide (PI). PI does not have the property of penetrating the cell membrane. When a microorganism dies or approaches a dead state due to sterilization or other factors, the cell membrane is damaged or denatured. When the cell membrane is normal, PI does not have cell membrane permeability and cannot enter the cell. However, when the cell membrane is damaged or denatured, it passes through the cell membrane and reacts with DNA and RNA. Although it is difficult to determine the viability of a microorganism because the cell membrane has been damaged or denatured, for example, using a certain type of microorganism, the culture conditions in which the microorganism always forms a colony are confirmed in advance, and sterilization treatment (for example, The degree of staining with PI, that is, the amount of fluorescence emitted by PI (for example, the luminance value of a luminescent spot photographed by a CCD) and the culture method for the microorganism subjected to heating, hypochlorous acid treatment, ethanol treatment, etc. By verifying the number of bacteria that formed colonies, the number of bacteria reduced or the ratio with respect to the initial number of bacteria, it is possible to determine whether live or dead. This is difficult with reagents of other reaction mechanisms, and it can be said that reagents that react with esterase and respiratory activity are alive with respect to reacted microorganisms, but there are few that react with the types of microorganisms as described above. Some of them are difficult to measure in an actual sample with an infinite number of unknown microorganisms. However, since all the microorganisms have a nucleic acid and also have a cell membrane, it is easy to judge the microorganisms. It has the action. In addition, dead bacteria shown in the present invention, while maintaining the shape of the microorganism substantially, refers to those that do not grow or very little, causing lysis, the shape of the bacteria is greatly deformed, It does not include DNA or RNA that elutes within the cell and cannot be visually confirmed under a microscope.

  In addition, the invention according to claim 4 is an excitation that particularly detects viable and dead bacteria by setting the excitation wavelength of a staining reagent that stains live and dead bacteria to ultraviolet and the excitation wavelength of the reagent that stains dead bacteria and that is blue or green. By making the wavelength ultraviolet, if the fluorescence is normally blue, measure the amount of green, yellow, and red fluorescence in the other wavelength ranges to determine the color characteristics of the emission point Since the amount of information that can be increased, it is easy to distinguish between microorganisms and foreign substances other than microorganisms. Fluorescent staining reagents use not only the above reaction mechanism but also different fluorescent dyes with the same reaction mechanism, so that the optimum excitation wavelength and the fluorescence wavelength emitted when irradiated with that excitation wavelength are very different. Are commercially available. This fluorescent staining reagent is selected by the user according to the apparatus and purpose to be used, but generally, it is rare to stain using two or more types that stain live and dead bacteria at the same time. Even specimens are often stained separately and visually checked separately. Therefore, the catalog of staining reagents rarely shows how to use staining at the same time. When DAPI is used as a reagent for staining live and dead bacteria and PI is used as a reagent for staining only dead bacteria, DAPI fluoresces blue when irradiated with ultraviolet light, and PI emits red when irradiated with green light. . When the microorganisms stained with both reagents are irradiated with ultraviolet light and emitted blue light, it is first determined that they are viable and dead bacteria, and further when irradiated with green light and fluorescent light is emitted in red, dead bacteria do not emit light in red If it is, it can be determined as a live bacteria. However, when a substance other than the microorganism emits fluorescence, it emits light with a color close to yellow or red when visually observed. This can be judged as other than microorganisms. Even in this case, there is little light emission in the blue wavelength region, and light in the blue wavelength region is often included. That is, using DAPI as a staining reagent, irradiating with ultraviolet light, using a spectral filter that transmits only light in the blue wavelength range, photographing only the light transmitted through the spectral filter with a CCD, It is difficult to judge microorganisms only by the luminance value, and the light intensity or luminance value in the wavelength range other than blue, for example, the green and red wavelength ranges is measured, and there is a certain setting for the luminance value in blue and other wavelength ranges. By calculating the chromaticity characteristic of the light emitting point such as the ratio of the luminance values or the luminance value or calculating the chromaticity of the light emitting point from each luminance value, the microorganism can be judged more accurately.

  Further, the invention according to claim 5 is an excitation wavelength for detecting particularly viable and dead bacteria by setting the excitation light of the reagent that stains dead and dead bacteria to blue and the excitation wavelength of the reagent that stains dead bacteria to green or yellow. By setting the blue to blue, it has the effect of reducing light emission other than microorganisms as much as possible. Since the light energy is higher as the wavelength is shorter, many foreign substances other than microorganisms emit fluorescence. However, if the excitation wavelength is lengthened, the emission of foreign matter is reduced, but the wavelength range for studying the color characteristics of the emission of light other than the foreign matter is shortened, and information for determining whether it is a microorganism or a foreign matter is reduced. When the excitation wavelength is blue, the emission of foreign matter is reduced, and the chromatic characteristics can be calculated from the luminance values in the green and red wavelength regions. If the excitation wavelength is green, the emission of foreign matter is somewhat less than blue, but there is no significant effect, but the fluorescence wavelength range is only the red wavelength, and to calculate the color characteristics only in that range, It is difficult to check and confirm with the naked eye because the amount of information is reduced and the fluorescent light is basically red.

  Although the wavelength range is indicated by ultraviolet, blue, green, yellow, red, etc., not only the specific peak wavelength but also the actual light should be transmitted only in the wavelength range set by the spectral filter. However, both the excitation light and the fluorescence are not sharp distributions with only peak wavelengths, but are broad distributions. Therefore, for example, the wavelength of blue in the present invention refers to a wavelength region having a certain distribution having a wavelength portion, not only light having a specific peak wavelength generally shown. The same applies to other colors.

  The invention described in claim 6 is based on the ratio of each luminescent luminance value depending on the difference in the amount or state of staining between the living and dead bacteria staining reagent and the dead bacteria staining reagent. It has the effect of being able to judge dead bacteria. However, the method of confirming live and dead bacteria is also very difficult academically. For example, for microorganisms that have been heated, colony formation does not occur under normal culture conditions (conditions for healthy microorganisms to grow), and culture conditions (medium, culture temperature, culture time, etc.) By optimizing, colonies may be formed and determined as live bacteria, and the determination of live bacteria and dead bacteria often varies depending on conditions. Therefore, the method for determining the ratio of the luminance values has an effect that it can be arbitrarily determined based on the result of the verification experiment by the culture test. The ratio shown here may be arbitrarily changed depending on, for example, the range of the absolute value of the luminance value that is fluorescently emitted by the viable and dead bacteria staining reagent. In other words, different ratios may be set for the range of low and high luminance values. Decide by comparing and verifying results with different methods such as culture tests. In addition, since microorganisms change the amount of DNA, RNA, cell membrane, etc. depending on their state, the amount of reagent uptake varies, and as a result the luminance value (light emission amount) and area (number of pixels greater than the set luminance value) Unlike the brightness value, it is used as a ratio between the area and the value multiplied by the area, and has the effect of amplifying the difference, ratio, etc. In addition, it is possible to easily distinguish between live and dead bacteria.

  In the invention according to claim 7, since the viable and dead bacteria staining reagent and the dead bacteria staining reagent have different excitation wavelength ranges and fluorescence wavelength ranges, only the live bacteria staining reagent (or the ratio of the dead bacteria staining reagent is Color characteristics may be different between microorganisms that are stained with (very small) and microorganisms that are stained with both live and dead stain reagents. By setting different color characteristics for each of live and dead bacteria, it is possible to distinguish between live and dead bacteria (microorganisms) and other foreign substances, and dead bacteria (microorganisms) and other foreign substances. , It has the effect of accurately distinguishing microorganisms.

(Embodiment 1)
First, in order to measure a sample containing microorganisms, a slide glass as a fixed part, a culture dish, a multi-well plate, or a filtration membrane, or the front side or the back side of the observation surface of a cell having a shape suitable for measurement Fix microorganisms on one side. For immobilization, a reagent such as poly-L-lysine or a polymer material having adhesiveness or adhesion such as gelatin is thinly applied to the surface, and a sample containing microorganisms is dropped and adsorbed on the surface. In the case of a membrane filter such as a membrane filter, a liquid sample is sucked from above and filtered, and microorganisms are captured and fixed on the surface of the membrane filter in a flat shape. In the present invention, it is most preferable to use such a filtration membrane because the following operations such as staining and washing can be handled easily and without losing microorganisms. Further, since the membrane filter is thin and small, it is not easy to handle as it is. Therefore, the membrane can be handled easily by using a dedicated support base, a holder with a suction port, or by connecting the holding portion to the membrane or by integrating the device.

  In the present invention, the specimen containing or possibly containing the microorganism is a liquid specimen. However, when the test object is a liquid sample such as drinking water, the specimen itself is a liquid specimen. If the object to be inspected is a solid sample such as vegetables or meat, homogenize it to obtain a liquid sample, or collect cells and microorganisms from the surface using a cotton swab, etc. It is released into a phosphate buffer or the like to make a liquid sample. When a cooking utensil such as a cutting board is an object to be inspected, microorganisms are collected from the surface using a cotton swab or the like and released into physiological saline or the like to obtain a liquid sample. Cells and microorganisms can be captured on the membrane filter by aspirating and pressure-filtering such a liquid specimen with a membrane filter, and in some cases, by vibrating and filtering using ultrasonic waves.

  In addition to the membrane filter, the fixing part is fixed to the preparation surface, the surface of a plate with high visible light transmission and high flatness, and the gap between the plates, or a sticky sheet. It is performed on the surface of a disk-shaped chip device, the surface of a flat plate medium, or the surface of a petri dish, dish, multiwell plate or the like, the surface of an electrode material or an adsorbing material. At this time, fixation is not limited to physical adsorption forces such as centrifugal force, electrostatic force, dielectrophoretic force, and hydrophobic force, but also due to an adhesive component such as gelatin, or an organism such as an antigen / antibody reaction or a ligand / receptor reaction. Bond strength can be used.

  In addition, in order to adjust the penetration of the fluorescent staining reagent, an appropriate concentration of a divalent metal complex, an aqueous solution mixed with a cationic surfactant, or the like is mixed with a liquid sample, or cells and microorganisms are mixed. The cell membrane permeability of cells and microorganisms can be kept constant by a method such as contacting from above the fixed part, filtering, or contacting from below.

  In addition, as a bivalent metal complex, ethylenediaminetetraacetic acid or the like is used in a concentration range of about 0.5 to 100 mM.

  As the cationic surfactant, those having low invasiveness to cells such as Tween 20, Tween 60, Tween 80, Triton X-100 can be used, and these are used in a concentration range of about 0.01 to 1%. .

  Next, as a fluorescent staining means, a drying prevention component is mixed, and a fixed amount of a staining reagent containing a fixed concentration of either a living or dead bacteria staining reagent or a killed bacteria staining reagent is dropped onto a fixed surface to be examined for the presence or absence of microorganisms.

  Fluorescent staining reagents react with DNA or RNA of cells or microorganisms (eg DAPI), react with respiratory activity (eg CTC), react with uptake activity (eg 2-NBDG), react with metabolic activity A variety of products (such as 6-CFDA) are commercially available. Even though the reaction mechanism is the same, there are a large number of fluorescent staining reagents with different excitation wavelengths and fluorescence wavelengths depending on the fluorescent dye, and they can be arbitrarily selected depending on the inspection purpose, the specifications of the measuring instrument, and the like. In order to identify microorganisms and determine whether they are alive or dead, it is desirable to stain the microorganisms simultaneously with both fluorescent staining reagents that stain only viable and dead bacteria. However, when the microorganism is a living microorganism, it is not stained with a reagent that stains only dead bacteria, but here, staining indicates a staining treatment.

  It is desirable to use a reagent having the same reaction mechanism as a life-and-death bacterium and a fluorescent staining reagent that stains the dying bacterium, in particular, in order to determine whether or not it is alive. Among them, it is desirable to use a nucleic acid staining reagent that reacts with DNA and RNA. Cells and microorganisms always have DNA and RNA. For example, if the cell membrane is a dead cell, the cell and the microorganism are stained with both a live cell and a dead cell reagent. If DAPI is used as a staining reagent for live and dead bacteria and PI is used as a staining reagent for dead bacteria, the light emitting point that is emitted blue by irradiating with ultraviolet light in actual observation is irradiated with green light, and then fluorescence is emitted in red. If it can be recognized as a bacterium and does not emit light, it can be determined to be a living bacterium. Microorganisms stained with a nucleic acid staining reagent, when observed under a microscope, emit light from the entire bacterium, so that when the magnification is increased, the shape of the bacterium can be recognized. The method (order) for identifying microorganisms and determining whether they are alive or dead is also important. When irradiating with ultraviolet light or green light, there is also light emission due to foreign matters other than the stained microorganisms, and it is necessary to make a judgment. First, live and dead bacteria are detected. When DAPI is used, it is determined that a light emitting point that emits blue fluorescence when irradiated with ultraviolet light is highly likely to be a viable or dead bacterium. Next, the light amount and luminance value in the wavelength region other than blue are measured. Specifically, by irradiating with ultraviolet light, first, using a spectral filter that transmits the blue wavelength range, the amount of light transmitted through the spectral filter or the luminance value of the light emitting point of the CCD that received the light is measured. To do. Next, the spectral filter that transmits light in the green wavelength range is switched, and the emission point is photographed by the CCD, and the luminance value in the green wavelength range is calculated. If necessary, measurement is performed by further dividing the wavelength in the red wavelength or green wavelength range. In this method, luminance values in a plurality of wavelength regions that can be determined as microorganisms and other than the microorganism are measured from the color characteristics of the light emission points, and the color characteristics are calculated from the luminance values to determine the microorganism. Next, irradiate with green light and confirm the point that emits red light. At this time, it is only necessary to confirm only the light emitting points that are determined to be microorganisms from the color characteristics of the light emitting points among the light emitting points that emit blue light when irradiated with ultraviolet light. Among them, the luminescent spot emitting red light can be judged as dead bacteria, and the luminescent spot not emitting light can be judged as live bacteria. When this procedure is implemented, it is possible to efficiently identify microorganisms and determine whether they are alive or dead. In order to judge that the microorganism is a microorganism or any other organism by using a staining reagent for viable and dead bacteria, the shorter the excitation wavelength, the greater the amount of information for analyzing the fluorescence wavelength and calculating the color characteristics. Since the living and dead bacteria and the dead bacteria staining reagents having the same excitation light cannot be used, it is necessary to select a fluorescent staining reagent that has a shorter excitation wavelength for staining the living and dead bacteria.

  In addition, this processing procedure is very suitable for an apparatus for automatically measuring microorganisms. A specific example will be described. A liquid specimen for inspecting the number of microorganisms is filtered with a membrane filter, and the microorganisms are captured on the membrane filter. DAPI and PI are dissolved in an optimal solution at an optimal concentration, the solution is dropped on a membrane filter and left for an optimal time, and then the solution is filtered. By this operation, the microorganisms on the membrane filter are stained with DAPI and PI. Irradiate the optimal area of the membrane filter with ultraviolet light. This area is slightly larger than the area captured once by the CCD depending on the magnification of the lens and the like, and is naturally a part to be imaged. If the area of the filter is larger than the area to be photographed, scanning is performed, and the entire filter or a plurality of photographs are taken so as to obtain an appropriate area. An image of the light emission point is taken using a spectral filter that transmits the blue wavelength. It is desirable that this spectral filter has a property of transmitting a wavelength range centered on a wavelength suitable for the fluorescence characteristics of DAPI, but it is not necessary to be concerned with this, and a wavelength range suitable for calculating a color characteristic described later. But it ’s okay. Thereafter, the same ultraviolet light is irradiated to switch to a spectral filter having a characteristic of transmitting the green wavelength range and / or the red wavelength range, and the emission point is similarly photographed. Next, green light is irradiated to the same place, and light (light emitting point) transmitted through a spectral filter having a property of transmitting the red wavelength region is photographed by the CCD. When scanning is performed, the membrane filter is moved, and an image of the light emission point is captured in the same manner as the portion other than the previously captured image. In one measurement of the membrane filter, a blue fluorescent image irradiated with ultraviolet light, a green fluorescent image and a red fluorescent image, and a red fluorescent image irradiated with green light are photographed. Image processing is performed on these images, and microorganisms are identified and viable and dead bacteria are determined. For each light emitting point of those images, position information, luminance value, area (number of pixels), etc. are stored. If the position of the light emitting point of each image is shifted due to optical characteristics such as a spectral filter, it is necessary to use corrected position information. An example of the image processing method will be described. First, for each light emitting point of the blue fluorescent image irradiated with ultraviolet light, the light emitting point within the set luminance value range and the set size (number of pixels) is recognized as a temporary living and dead bacterium. As the number of pixels, it is desirable that the size in the range of 1 to 30 can be recognized as one bacterium. If it is larger than this, the photographing area will be reduced as a result, the amount of data stored for measuring the entire filter will increase, and image processing will take a lot of time. If it is 1 or less, one bacterium cannot be recognized. Next, for the green fluorescent image and the red fluorescent image irradiated with ultraviolet light, determine the luminance value of the light emitting point at the same position as the light emitting point recognized as a temporary live and dead bacteria, and color from the luminance values of blue, green, and red. The target characteristic is calculated. Here, chromaticity is calculated as a chromatic characteristic. Although details of the method of calculating chromaticity will be described later, FIG. 1 shows an example of measurement of soil sample particles (foreign matter) and E. coli. As shown in FIG. 1, Escherichia coli has both chromaticity x and chromaticity y of 0.25 or less, but the foreign substances that are soil components have chromaticity of 0.25 or more. By calculating the chromaticity in this way, it can be distinguished from microorganisms and foreign substances other than microorganisms. Note that the value of this chromaticity may vary greatly even if the same chromaticity is measured due to characteristics such as a spectral filter. Measure the chromaticity of the bacteria to be measured and other foreign substances, and set the chromaticity setting value, range, x / y ratio, etc. that can be identified as microorganisms based on the measured values. It is necessary to determine. Of course, a set value may be provided based on chromatic characteristics other than chromaticity. Next, from the luminance value of the emission point of the red fluorescent image of the green light irradiation at the same position as the emission point of the blue fluorescent image of the ultraviolet light irradiation determined to be a microorganism from the chromaticity, the dead value above the set luminance value, the following Can be determined as viable bacteria. The light emitting points recognized as viable bacteria and dead bacteria can be integrated and measured as viable bacteria count and dead bacteria count. When an actual specimen is measured, the blue fluorescent image irradiated with ultraviolet light does not have a light emitting point, but the red fluorescent image irradiated with green light may have a light emitting point. Since this may be recognized as a foreign substance other than microorganisms, the result can be obtained more quickly without image processing. This luminescence is a phenomenon that is particularly common in ingredients contained in tea. By performing the above procedure and processing method, it is possible to identify the microorganism from the light emission point of the temporary microorganism, and distinguish between live and dead bacteria only with respect to the light emission point, so that it can be performed with minimal data storage and processing. It is possible to perform image processing efficiently and to shorten the measurement time as a result.

  For reference, FIG. 2 shows an absorption spectrum and a fluorescence spectrum of DAPI excitation light. The optimum peak wavelength of the excitation light is around 360 nm, and the fluorescence wavelength is around 460 nm. It is not a sharp distribution, but it is broad and broad around its peak wavelength. Fluorescence is emitted even with excitation light slightly different from the peak wavelength, and the fluorescence also emits a certain amount of light even in a wavelength range slightly different from the peak wavelength. The fluorescent staining reagent is not limited to DAPI, but shows a similar distribution although the peak wavelength is different. The excitation light is preferably adjusted with a light source having a peak wavelength at 360 nm or a spectral filter. However, it is sufficient that the excitation light can be irradiated with a light amount that generates fluorescence even at a slightly different wavelength. However, it is necessary to adjust the wavelengths of the excitation light and the fluorescence with a spectral filter so that the wavelengths of the excitation light and the fluorescence do not overlap. If there is an overlapping portion, the amount of light leaks from the fluorescent portion due to reflection, and there is a possibility of photographing as a light emitting point of fluorescent light emission. As one of the color characteristics of the fluorescent emission point, the peak distribution as shown in FIG. 2 is measured, compared with the peak distribution and pattern recognition, etc., and recognized as a microorganism within the set range. However, the amount of luminescence of microorganisms is very small, and if such a peak distribution is to be measured, it is a very large apparatus, becomes expensive and difficult to put into practical use. In order to calculate color characteristics for determining microorganisms easily and accurately, spectral filters having a certain degree of wavelength range (10 to 60 nm) are used, and spectral filters having different wavelength ranges are used. By using multiple, grasp the color characteristics that can distinguish microorganisms and foreign substances from the amount of light in each wavelength range or the brightness value when photographed with CCD, and determine microorganisms from the characteristics, cheap and practical equipment Can be provided.

  The fluorescent staining reagent preferably has a nucleic acid binding structure, and has an effect that it is less likely to miss a microorganism by detecting a cell having DNA or RNA. Many types of staining reagents differ in excitation wavelength and fluorescence wavelength and are commercially available. Each optimum wavelength is clearly specified and can be selected according to the purpose in a timely manner. As a reagent for dyeing viable and dead bacteria having the property of penetrating the cell membrane, DAPI (1,4-diamidino-2-phenylindole) is used as long as it emits blue fluorescence by ultraviolet excitation, and green fluorescence or yellow is used by blue excitation. It emits green and yellow fluorescence. Examples include acridine orange, oxazole yellow, thiazole orange, and polymethine cross-linked asymmetric cyanine dye compounds such as SYTO9, SYTO13, SYTO16, SYTO21, SYTO24, SYBR Green I, SYBR Green II, and SYBR Gold. Can be used. In addition, as a dead bacteria staining reagent that does not have a property of permeating with a cell membrane, it emits green fluorescence. For example, monomethine cross-linked asymmetric cyanine dye such as acridine dimer, thiazole orange dimer, oxazole yellow dimer, etc. Dimer, monomethine cross-linked asymmetric cyanine dye compounds such as SYTOX Green, TO-PRO-1, and PI (propidium iodide), hexidium bromide, ethidium bromide, LDS-751 Polymethine cross-linked asymmetric cyanine dyes such as SYTOX Orange can be used.

  In addition, these fluorescent dyes are mixed with a sample containing cells and microorganisms in advance so as to be 0.1 to 100 μM and are allowed to act simultaneously or separately, without taking time, or appropriate The action is performed at a predetermined concentration with a time interval.

  As a means for preventing the surface of the substance containing cells and microorganisms captured on the membrane filter from being dried during measurement and changing the luminescence intensity, 10 to 60% w / v glycerol or One or more sugar alcohols such as 10 to 90% v / v of D (-)-mannitol and D (-)-sorbitol are mixed.

  In addition, for the purpose of drying and solidifying, it is possible to store fluorescence emission relatively stably by mixing polyvinyl alcohol at an appropriate concentration of about 10 to 80% or covering the surface later.

  In addition, as a membrane filter suitable as a fixing | fixed part, well-known things, such as the product made from a polycarbonate with a hole diameter of 0.2 micrometer-1 micrometer, can be used, for example.

  For image detection, an excitation light source for irradiating a fluorescent dye with a specific wavelength, a spectral filter, a condensing lens for condensing the excitation light to a diameter of about 3 mm, and an excitation light component are removed. High-pass filter, light receiving filter for extracting specific wavelength components from fluorescence emitted from the sample, lens unit for enlarging, CCD and CMOS image receiving elements for converting the fluorescent image into electrical image signals Is done.

  This is the main emission wavelength of the fluorescent dye used as the fluorescent staining reagent. For example, in the case of blue excitation, when the excitation light containing a wavelength component in the vicinity of 470 nm to 510 nm is irradiated, the wavelength is in the vicinity of 510 nm to 540 nm. Fluoresce. In the case of green excitation, excitation light including a wavelength component in the vicinity of 510 nm to 550 nm is irradiated, and fluorescence having a wavelength in the vicinity of 560 to 620 nm is emitted. In the case of orange excitation, when excitation light including a wavelength component having a wavelength of 540 to 610 nm is irradiated, fluorescence having a wavelength of 560 to 630 nm is emitted.

  Therefore, when a light emitting diode is used as an excitation light source as a detection means, a blue light source can emit a wavelength of preferably around 480 nm, and a green light source preferably emits a wavelength of around 535 nm. Among those that can be produced, those that are capable of emitting a wavelength of around 560 nm are preferably used. Although it is necessary to use a spectral filter to make the wavelength range around the wavelength, the amount of light decreases when the wavelength range is narrowed. When the wavelength range is widened, problems such as the occurrence of a portion overlapping with the fluorescence wavelength also occur. Therefore, it is necessary to adjust to the optimal wavelength range.

  When a laser is used as the excitation light source, a blue light source that can emit a wavelength of preferably around 475 nm is used, and a green light source that can emit a wavelength of preferably around 535 nm is used. .

  When a halogen lamp or a mercury lamp is used as the excitation light source, an optimum interference filter can be used as an appropriate spectral filter according to the excitation wavelength of the staining reagent. In addition, the reflection angle or transmission type diffraction grating having a wavelength resolution of 0.1 to 10 nm can give an optimum angle and extract excitation light including an arbitrary wavelength.

  The condensing lens can irradiate the membrane filter on which the fluorescently stained cells and microorganisms are spread so that the irradiation range is, for example, a constant area having a diameter of about 3 mm. Furthermore, a more uniform excitation light can be irradiated by combining a diffuser plate for scattering light on the upstream side.

  The fluorescence generated by the excitation light applied to the sample passes through the high-pass filter, so that the color characteristics are not impaired, and the light component derived from the excitation light is effectively cut.

  The fluorescent light passes through a lens unit, and a charge coupled device such as a single CCD CCD as a light receiving unit, or a 3CCD including three types of RGB fluorescent filters capable of acquiring three primary colors of red (R), green (G), and blue (B). It is obtained by photographing an image composed of three colors of RGB with an exposure time of about 0.1 to 10 seconds using the unit.

  The luminance information of the color to be acquired can be used as long as it is in the fluorescence wavelength range of the fluorescent dye that is the fluorescent staining reagent. For example, in the case of SYBR Green, which is a cyanine dye, the maximum fluorescence wavelength is 521 nm, but the fluorescence spectrum extends to around 620 nm, and when used as a staining reagent for viable and dead bacteria, green (G) around 530 nm is image (a). , Red (R) in the vicinity of 610 nm can be acquired as an image (b), and microorganisms and contaminants can be discriminated using (a) and (b).

  In addition, when a single-plate monochrome CCD or CMOS is used, an image including luminance information of a necessary wavelength can be acquired by switching and using an appropriate spectral filter. At this time, as another advantage, by using the same CCD, it becomes possible to perform measurement without being affected by the difference in sensitivity characteristics between different CCDs, and the step of performing sensitivity correction can be omitted. .

  A plurality of fluorescent images acquired by these operations are processed by a microcomputer as a calculation unit or a program on an external terminal.

  The calculation unit collates and matches the luminescent spot removing unit for removing luminescent spots such as missing dots from the image, the luminescent point extracting unit for extracting luminescent points from the image, and the luminescent points of a plurality of images. A light emission point collation unit, an output unit that outputs collated numerical data, a fluorescence evaluation unit that evaluates fluorescence emission, a life / death judgment unit that distinguishes the life and death of microorganisms from the brightness of a staining reagent, and a color characteristic It comprises a microorganism determination unit that determines whether a light emitting point is a microorganism or a contaminant, and an effective area calculation unit that calculates an effective area of a measured image.

  First, there is a bright spot removing unit. This is a pixel pixel sensitivity irregularity found in a CCD or other image receiving element, or a phenomenon called dot missing due to a loss of sensitivity. If it appears above, it may be mistaken for the luminescence point of the microorganism, or the luminescence point of the microorganism cannot be obtained, which may cause an error. For this reason, it is necessary to remove such bright spots, but as a bright spot removal image, a dark field image that is not irradiated with a light source is acquired by setting the exposure time to be the same as or longer than that of the sample measurement. Get an image with only points. Then, it is possible to delete only the bright spot by subtracting the bright spot image from each image showing the light emitting spot. The image in which the bright spots are removed in this way is used below.

  About a light emission point extraction part, the thing corresponding to the set area and the range of a brightness | luminance is extracted among the light emission points contained in an image. For example, if the area is 2 to 15 and the luminance is 15 to 255, a large foreign object having an area of 16 or more can be excluded from the count in advance, and background noise (dark noise) having a luminance of 14 or less. Can be removed. Since the optimum value of this threshold value varies depending on the magnification of the lens, the intensity of the excitation light source, the exposure time, and the like, it is necessary to verify and confirm in advance the value at which microorganisms can be optimally extracted.

  In addition, when the value of (x, y) of the coordinate which showed the maximum brightness | luminance and the value of RGB are included, each brightness | luminance is simultaneously extracted as a numerical value. This processing can be executed using Image Pro Plus, which is general-purpose image processing software. Moreover, it can also be set as the program incorporating the same process.

  Next, the light emission point collating unit compares the numerical data of the extracted light emitting points with the numerical data of the light emitting points at the same position including different luminance information, collates them, and combines them.

  At this time, an image including different luminance information refers to an image acquired by a different light receiving filter, but there is a slight shift in coordinates between the images due to spectral filter characteristics and mechanical errors. If the pixel coordinates of the image are collated, they may not match. Therefore, the coordinate correction value that corrects image misalignment is added to one coordinate and collated, but especially for mechanical errors, the value of the coordinate misalignment changes with each use due to the influence of the usage environment such as temperature and humidity. May end up. Therefore, it is effective to use the optimum value for each measurement by updating and using the coordinate correction value for each measurement.

  The correction value for correcting the coordinates is acquired by reading the correction value from the position correction image acquired in advance. The position correction image is picked up using a phosphor that is reflected in all the wavelength ranges to be acquired. If the wavelength to be acquired is green and red, red fluorescent particles on the long wavelength side can be used, and the intensity of the excitation light source and the exposure time are adjusted so that the same emission intensity can be obtained. In addition, in the process of automatically calculating the correction value by the phosphor, the number of calculations increases as the number increases, and it takes time, so if it is within the range of 5 to 50 per screen 1 to several minutes, which can be obtained in a relatively short time. Adjusting to such a concentration, filtering the confirmed suspension of fluorescent particles with a fixed amount of membrane filter, and reacting with a fixed part, for position correction to obtain a position correction image Create a tip. Also, if you want to use this repeatedly for calibration for a long time, fix the beads with a polymer or by covering the metal thin film with metal vapor deposition. Instead, the position becomes constant. For calibration, it is also effective to create a fine pattern or spot by masking the fluorescent resin.

  The position correction chip created in this way is installed in the apparatus and images the image by giving the same movement as the actual measurement. This reproduces the coordinate deviation of the image that occurs due to mechanical error such as motor position control error and backlash, filter and lens manufacturing error, and optical axis shift caused by manufacturing error when assembling the device. The position accuracy can be improved by obtaining the correction value and using it in actual measurement.

  When the total of the magnifying lens system is about 200 to 300 times, the area of the object showing the luminescence point of the microorganisms seen in the image is about 1 to 20 pixels on the image receiving element. This is a value taken when the diameter of one microbial cell is about 0.6 to 5 μm. On the other hand, when two or more microbial cells are connected to each other, the area of the object of the light emission point becomes large, and some objects exceed 20 pixels. Such an object having a large light emitting point is detected as one object in most cases unless a special optical system such as a confocal optical system is used, and it is difficult to detect two objects separately. The problem at this time is that when two objects have different light emission characteristics, adjacent images are detected as the same object when the luminance is compared by comparing the light emission points by comparing the images. If the light emission luminance of microorganisms is mistakenly combined, inaccurate data that is completely different from the light emission characteristics of the original microorganisms may be formed. In order to prevent such a case, the coordinates of the light emitting point are the coordinates indicating the maximum luminance value of the object, and when collating the light emitting point between images, an error range area limited to the very vicinity from the coordinates. It is necessary to be coupled only with the light emitting point having the coordinates of the other image inside.

  Therefore, even objects extracted as objects with the same light emission point may not match when collated. In order to prevent the luminance data to be combined from being lost at that time, the luminance of the pixel of the same coordinate in the other image is detected when the light emitting point matching the other image to be compared is not detected. It is useful to extract values and combine these values. As a result, even when the light emitting point is extracted from only one image, the collation data can be created with high accuracy without losing the luminance information.

  In addition, although related to the detection accuracy of the final number of bacteria, there is a possibility that an object will be detected as dead if it does not have the above-mentioned process when living and dead bacteria are connected. However, this makes it possible to detect that live and dead bacteria are connected, leading to improved correlation with culture methods and the like.

  The collated and combined data is output as a data file by the output unit. By saving as a data file at this time, it is possible to process the subsequent processes all at once, thus improving the efficiency of the work.

  For the data file having the luminance information of the light emission point, the life-and-death determining unit classifies the light emission point as either the live bacteria group or the dead bacteria group. At this time, by giving a parameter indicating that it is a viable cell group or a dead cell group, the subsequent processing can be easily performed, and the processing can be made efficient. Note that the parameter is set by giving a variable of the data of the light emission point, such as 1 for the live bacteria group and 2 for the dead bacteria group.

  In order to determine whether the group is a live or dead group, it is desirable to use a graph as follows. First, a dot plot is created from these two values and displayed using the luminance of the living and dead bacteria staining reagent and the luminance of the dead bacteria staining reagent in the light emission point data. This is plotted for each detected emission point, with the horizontal axis representing the luminance value of the viable and dead bacteria staining reagent and the vertical axis representing the luminance value of the dead bacteria staining reagent. The dot plot is preferably displayed on the interface of a program that performs image processing, and is preferably displayed when a data file of light emission points is read.

  Next, use the cursor to create a boundary for the displayed dot plot. The boundary line can be freely created by one or a plurality of straight lines, curves, polygonal lines, etc., and is created so that the group of plots can be easily classified while viewing the plot. It should be noted that the creation process of the boundary line can be easily and accurately performed by using a grid or the like so that it can be easily performed, or by having a function of trapping the outline or plot. This boundary line indicates the ratio between the luminance value of the living and dead bacteria staining reagent and the luminance value of the death bacteria staining reagent.

  In the case of a polygonal line, it is certain that it can be created only in one direction so that the lines do not intersect.

  The created boundary line can be canceled or saved, and can be used repeatedly.

  Next, a threshold corresponding to the boundary line is calculated based on the created boundary line. The calculated threshold values are classified as dead bacteria groups on the upper and left sides of the graph and as viable bacteria groups on the opposite side, and processed by giving parameters.

  After the viable bacteria group and the dead bacteria group are determined, the following processing is performed when the microorganism determination unit separates and excludes the contaminants. Discrimination between microorganisms and contaminants is made by calculating the value of the color characteristic.

The chromatic characteristic is a value calculated with respect to the color of the light emitting point from the chromaticity, hue angle, etc., calculated from the RGB luminance values. Color systems such as Lab color system, LCh color system, and XYZ color system are used as the color system indicating the color features. Here, chromaticity based on the XYZ color system is used. Since the acquired luminance is in the RGB color space, the RGB luminance values are converted into the XYZ color system.
(Formula 1)
X = 0.3933 × R / 255 + 0.3651 × G / 255 + 0.1903 × B / 255
Y = 0.2123 × R / 255 + 0.7010 × G / 255 + 0.0858 × B / 255
Z = 0.0182 × R / 255 + 0.1117 × G / 255 + 0.9570 × B / 255
further,
x = X / (X + Y + Z)
y = Y / (X + Y + Z)
R, G, and B in the formula indicate an R luminance value, a G luminance value, and a B luminance value, respectively. As a result, the values of x and y are finally calculated as values necessary for determining whether the cells and microorganisms or impurities are present.

  It is a chromaticity value calculated for each luminescent point, but the luminescent point is given a parameter to determine whether it is a viable cell group or a dead cell group. Compared to the chromaticity threshold value set for the live bacteria group (has a certain range), if it is a dead bacteria group, the chromaticity threshold value set for the dead bacteria group Compared with (having a certain range), those showing values other than microorganisms are judged as foreign substances other than microorganisms. The luminescent point determined to be a foreign substance is excluded, and the luminescent points within the threshold range are determined as live and dead bacteria, respectively, and one luminescent point is counted as one live or one dead bacteria, Accumulated.

  Next, the total number of bacteria per unit amount (for example, 1 mL, 1 gram, etc.) contained in the actually used specimen is calculated for this count value. For this purpose, an effective area area used for image processing among the measured images is obtained by an effective area calculation unit. The effective area used for the measurement is obtained by a function using the correction value of the image as a variable.

  Assuming that the vertical length of the image is P, the horizontal length is Q, the vertical coordinate correction value is α, and the horizontal coordinate correction value is β, the number of effective area pixels M per screen is given by Equation 2. It is expressed in

(Formula 2)
M = (P−α) × (Q−β)
Also, the effective area area is obtained by calculating the area per pixel from the magnification of the lens system, the area per pixel is s, the number of fields of view is N, and the effective area area S per screen and the total effective area. Is
(Formula 3)
S = Ms
Total effective area: S × N
It becomes.

  The surface area (for example, the total area of the membrane filter) of the fixed part of the fixed part of the microorganism is divided with respect to the obtained area. By multiplying the numerical value obtained in this way by the number of counted bacteria, the final total number of living or dead microorganisms can be calculated to obtain the number of bacteria.

  By using the above method, it is possible to discriminate the life and death of microorganisms contained in the specimen and the cell culture medium, separate them from foreign substances, and measure the number.

  FIG. 3 is a block diagram showing an aspect of the microorganism weighing device for favorably implementing the present invention. The microorganism weighing apparatus 1 includes an excitation light source 2, an excitation spectral filter 3, a condensing lens 4, a high-pass filter 5, a fluorescence spectral filter 6, a lens unit 7, and a light receiving element 8 as detection means. In order to extract the target wavelength from the excitation light emitted from the excitation light source 2, the light is split by the fluorescence spectral filter 6. The split excitation light passes through a condenser lens 4 and is placed on a membrane filter 10 (which captures microorganisms stained with a nucleic acid-binding fluorescent dye on the membrane filter by a separate operation). It is focused on. The excitation light emitted from the excitation light source is condensed by the condensing lens 4, and at this time, the range irradiated with the excitation light by the condensing lens 4 is condensed on a small fixed area having a diameter of about 3 mm. The fluorescence emitted by the excitation light passes through the high-pass filter 5 to remove the excitation light component, is enlarged by the fluorescence spectral filter 6 and the lens unit 7, reaches the CCD unit 11 that is the image receiving element, and is converted into an electrical signal. The The signal thus obtained is converted into an image and subjected to image processing by the calculation unit 12.

  The excitation light source 2 can be used by switching a plurality of excitation spectral filters 3 having different wavelength ranges or a plurality of excitation spectral filters 3 having different wavelength ranges. At this time, an excitation wavelength region shorter than the excitation wavelength region of the dead bacteria staining reagent is selected. Specifically, if the excitation wavelength of the killed bacteria staining reagent is a blue wavelength, the excitation wavelength of the living bacteria staining reagent is an ultraviolet wavelength, and the excitation wavelength of the killed bacteria staining reagent is a wavelength of green or yellow. If so, the blue wavelength is used as the excitation wavelength of the viable and dead bacteria staining reagent. All microorganisms are stained when a nucleic acid-binding staining reagent is used. However, if there is a light emitting point that emits light other than microorganisms or light including electric noise, it is determined as a microorganism if the size and luminance value are equivalent to those of the microorganism. In order to determine in more detail the microorganism, it is necessary to calculate the color characteristic of the light emitting point and compare it with the value of the light emitting point of the microorganism. In order to calculate the color characteristic, the longer the wavelength range for analysis, the more advantageous. Therefore, since the calculation of the color characteristics for judging the microorganism is performed at the emission point of the excitation light of the staining reagent for viable and dead bacteria, it is necessary to carry out at a wavelength as short as possible. However, since ultraviolet light is high in energy, not only microorganisms stained with a living and dead bacteria staining reagent but also inorganic particulate substances often fluoresce due to reflection or autofluorescence. For example, if the specimen contains a large amount of aluminum oxide or the like, fluorescence may be emitted. Many things can be determined by calculating their color characteristics as substances other than microorganisms, but if there are many luminescence points of foreign substances, there is a high possibility of miscounting. The blue excitation light has a narrower wavelength range for calculating the color characteristics than ultraviolet light, but the energy is low, so that the emission of light other than microorganisms is reduced. The smaller the number, the lower the possibility of erroneous counting. There are various types of specimens that can be tested for the presence or absence of microorganisms or the number of bacteria, including food, cosmetics, pharmaceuticals, soil and river environments, and the type, quantity, and required number of bacteria other than microorganisms contained in the specimen, It is necessary to select timely depending on the sensitivity.

  In order to calculate the color characteristics, a plurality of fluorescent spectral filters 6 having different transmission wavelength ranges are used. The wavelength range is set to an optimum wavelength range in order to calculate the color characteristics. The computing unit 12 may be provided outside the microorganism weighing device 1, or a personal computer is used to take an image of the light emission point with the microorganism weighing device 1, send the image data to the personal computer, and the computer uses the image. Analysis, calculation of color characteristics, and count of microorganisms may be performed.

  FIG. 4 is a diagram showing a calculation process flow in the calculation unit 12, which is composed of a bright spot removal unit 13, a temporary microorganism determination unit 14, a microorganism determination unit 15, a live / dead bacteria determination unit 16, and an effective area calculation unit 17. ing. The calculation process flow in the calculation unit 12, the bright spot removal unit 13, the temporary microorganism determination unit 14, the microorganism determination unit 15, the viable and dead bacteria determination unit 16, the effective area calculation unit 17, and the like will be described below.

  First, a coordinate correction image is read and a coordinate correction value is calculated. Next, a variable such as a threshold value is input, and the bright spot to be removed is confirmed by the bright spot removing unit. Thereafter, the bright spot is removed from the photographed image. For the pixels of the removed bright spot portion, an average value or the like based on the luminance value of the surrounding pixels is entered. Read live and dead bacteria images (images that are stained with live and dead bacteria staining reagents, irradiate with preset excitation light, and photograph fluorescent emission points), first recognize each emission point, For those whose area value (number of pixels) is in the range of a preset value, the luminescent spot is recognized as a temporary microorganism by the temporary microorganism judging means.

  A chromaticity calculation image (fluorescence image in a wavelength range different from that of the viable and dead bacteria staining reagent) obtained by photographing a light emission point in another wavelength range for calculating the next color characteristic is read. At this time, a plurality of images having different fluorescence wavelengths may be used in order to study the color characteristics more precisely. Coordinate correction is performed for each light emission point of the chromaticity calculation image, and a luminance value at the same coordinate (even if there is no light emission point) as the light emission point determined to be a temporary microorganism is obtained.

  Calculate the color characteristics from the luminance value of the light emitting point recognized as a temporary microorganism and the luminance value of the chromaticity calculation image, and compare it with the color characteristic value calculated using a pure microorganism in advance. If the value is, the microorganism judging means 15 recognizes the light emission point as a microorganism and counts it as one microorganism (live and dead). This process is performed on all the luminescent spots determined to be temporary microorganisms, the luminescent spots determined to be microorganisms are integrated, and the number of live and dead bacteria is measured for each photographed image. Next, read the dead bacteria image (images that are stained with a dead bacteria staining reagent, irradiate with the preset excitation light, and photograph the fluorescent emission points), recognize each emission point, and correct the coordinates of the emission point To do.

  A luminance value (even if there is no light emission point) at the same coordinates as the light emission point determined to be a microorganism (live and dead) is obtained. At this time, if there is a light emitting point, it can be determined from the brightness value and the area (number of pixels) that it is not a microorganism, and the brightness value can be obtained for those within the set value range. Of course, it is necessary to subtract the luminous points determined to be other than microorganisms from the previous integrated value. With respect to the luminescent points determined to be viable and dead bacteria, the luminance values of the luminescent points of the live and dead bacteria image and the luminance values at the same position of the dead bacteria image are entered into the viable and dead bacteria determination means 16. Judging and identifying each luminescent point, assuming that the luminescent point determined to be a living bacterium is one living bacterium, determining the luminescent point determined to be a dead bacterium to be one dead bacterium, and integrating each photographic image, Count the number of viable and dead bacteria.

  The effective area in which the number of viable bacteria and the number of dead bacteria in the photographed image is calculated is calculated by the effective area calculating unit 17, and finally, the membrane filter area and its area are photographed and the membrane filter is calculated from the effective area area. The final number of bacteria in the area is calculated, and the final viable cell count and dead cell count are calculated and output.

  In FIG. The calculation result of the brightness | luminance and chromaticity of E. coli is shown. E. A water sample containing E. coli is filtered through a membrane filter and stained with SYTO9, a fluorescent dye for living and dead cells, and propidium iodide, a fluorescent dye for dead cells. It is a table | surface which shows an example of the data which acquired the G luminance image and R luminance image in. At this time, the B luminance image cannot be acquired because it overlaps the wavelength of the excitation light, and is used by substituting numerical values. This variable can be adjusted to an optimal value.

  FIG. 5B shows a specific example of the microorganism determination means 15. The chromaticity calculation process flow shown in FIG. 5B shows conversion from RGB luminance to (x, y) values in the XYZ color system. In this step, first, the RGB luminance values acquired by the means for measuring RGB luminance are converted into linear RGB and gamma correction is performed. The visual characteristics are further weighted, and the values of x and y are finally obtained as values necessary for the determination of whether the substance is a microorganism or a contaminant. At this time, for example, in a condition where blue (B) is cut by an optical filter and only green (G) and red (R) are acquired, it is assumed that blue sensitivity cannot be obtained. An optimized fixed value can be substituted and used, or a function based on a luminance value of R or G can be set and used. By comparing the obtained chromaticity value with a threshold value, it is determined whether it is a microorganism or a contaminant. The threshold value at this time is determined by experiment.

  E. The number of bacteria in the bacterial solution containing E. coli and tap water (chlorine removed) is measured. These liquid samples were dropped with a pipette from above a black membrane filter having a pore diameter of 0.45 μm and a diameter of 9 mm with a metal deposited on the surface, and suction filtered. Since the membrane filter may touch the surface as it is and is difficult to handle, the membrane filter was used by covering the periphery with a resin frame and integrating them. If the suction filtration pressure is too high, filtration cannot be performed, and if the suction filtration pressure is too low, the membrane filter may be damaged, and the membrane pressure may be broken. . When filtering on a membrane filter, it is necessary to disperse luminescent substances such as microorganisms as uniformly as possible because of ease of counting and accuracy of back calculation. Therefore, in order to make the filtration performance of the membrane filter uniform, a filter paper or the like is sandwiched in the suction port below the membrane filter, and the suction pressure is diffused to uniformly apply to the entire membrane. Separately, in order to reduce the passage resistance of the membrane filter pores, a small amount of surfactant diluent (Tween 20 0.1%) was filtered before the liquid sample was filtered. The liquid sample is E. coli. In the case of E. coli bacterial solution, 0.1 mL was filtered, and in the case of tap water, 20 mL was filtered.

  Subsequently, fluorescent staining was performed on the microorganisms collected on the membrane filter. The staining reagent used was SYTO24, which is a viable and dead bacteria staining reagent, and SYTOX Orange, which is a dead bacteria staining reagent (both are trade names). Since these staining reagents absorb light easily in the air and are easily decomposed, they were adjusted to 500 μM with dimethyl sulfoxide, and dispensed into microtubes in small amounts as stock solutions and stored. For storage, nitrogen was sealed in a microtube and stored in a dark place with a minus 20 degree freezer. The required number was thawed, and the diluent was added to 10 μL of each reagent so that the total amount was 1 mL, and mixed. This diluent must have reagent solubility, storage stability, permeability to cells, anti-drying properties, and low autofluorescence. D-sorbitol must be distilled water to satisfy these conditions. The mixture was diluted to about 50% and mixed with Tris-HCl and a small amount of surfactant (Tween 20).

  Reagents adjusted to a final concentration of 5 μM were dropped from above the membrane filter where microorganisms were collected one by one, stained at room temperature for several minutes, and excess reagents were removed by suction filtration. The order of staining is not limited, and the staining can be performed in the same manner regardless of whether the staining reagent is viable or dead.

  After staining, the excess reagent was removed as much as possible by aspiration, and then the membrane filter was placed in a microbe counting apparatus and measured.

  The microorganism counting apparatus is the one described in FIG. 3, but this time, the excitation light corresponding to the live and dead bacteria reagent is a blue LED (about 470 nm) and the excitation light corresponding to the dead bacteria reagent is a yellow LED (about 560 nm). As a spectral filter, green has transparency between 530 and 550 nm and red has transparency between 590 and 610 nm. The light source is provided with a condensing lens so that the light beam can be easily irradiated onto the imaging range.

  The membrane filter installation stage is provided with a detachable mechanism so that the stage member can fix the membrane filter from the back side to a flat surface and at a height that is in focus, eliminating the need for focus adjustment. The stage to which the membrane filter is fixed can be moved by a motor-driven XY stage, and can be continuously moved to a position designated in advance by a program.

  The fluorescence image on the surface of the membrane filter was acquired using a single-plate monochrome CCD camera with an infrared cut filter installed above the membrane filter and a magnifying lens system. When acquiring an image, the LED that is the excitation light is turned on and illuminated, and the light receiving filter can be switched to acquire an image of the desired wavelength. These cameras, light sources, filters, and stages operate as follows. It was controlled using a programmed microcomputer.

  Images are acquired at the same position using three types of images (a) blue excitation, green fluorescence, (b) blue excitation, red fluorescence, and (c) yellow excitation, red fluorescence, with an exposure time from 0.1. Images were acquired continuously in about 3 seconds, moved to the next imaging region by the stage, and images were acquired in the same manner. In addition, at the beginning of the measurement, an image was acquired without turning on the LED, a pixel portion of a bright spot having a missing dot was confirmed, and an image for removing the bright spot was acquired.

  After all the images were acquired, the bright spot was removed, the light emission points were extracted, and collation was performed by the calculation unit, and data for which the luminance value was obtained for each light emission point was created.

  In FIG. The classification method by the dot plot of the brightness | luminance of the luminescent material in E. coli and tap water and the life-and-death judgment means is shown. As shown in FIG. A plot of emission points seen in E. coli and tap water is blue excitation, which is the fluorescence wavelength of SYTO24, which is a viable and dead bacteria staining reagent, and brightness of green fluorescence and yellow excitation, which is the fluorescence wavelength of SYTOX Orange, which is a death bacteria staining reagent. The dot plot is created with the brightness at red fluorescence on two axes.

  At this time, as a boundary line that can be arbitrarily set, a straight line such that c is y = 100 and d is x = y is set, and an area that is smaller than c and smaller than d is a viable group, and other areas are dead. Designated as a fungal group, flags were set for the luminescent spots in the corresponding area, and the luminescent spots were classified. d shows the ratio of the luminance value by the living and dead bacteria staining reagent and the luminance value by the death bacteria staining reagent, and it may be judged by such a fixed ratio, or by combining the range indicated by C. Also good. Further, the ratio is not constant, and may be a ratio that varies depending on the range. When two groups are formed at the plotted points, this setting is set so that the groups are divided into them or a ratio that is considered to be optimal based on the results of the culture test.

  FIG. 6B shows a chromaticity diagram of the light emission point and a determination method by the microorganism determination means. As shown in FIG. 6 (b), a group of luminous points classified as temporary microorganisms based on their size and luminance values is shown as a graph of x and y values in the chromaticity data in the XYZ color system. Plotted above. At this time, E.I. E. coli live bacteria were distributed in the region of x <0.37 and y> 0.54, whereas luminescent materials other than microorganisms in tap water had x of 0.3 to 0.6 and y of 0.3. It was confirmed that it was distributed in a wide area of 0.6. At this time, the threshold value is E.E. The value is set with reference to the value of E. coli, x is e = 0.37, y is f = 0.54, and the group classified into the region of x <e, y> f is identified as a microorganism and included in tap water The foreign matter such as the luminescent material is discriminated. As a result, most of the detected emission points can be separated, and in tap water, 32 points are extracted from 100 points by the life / death determining means as shown in FIG. Furthermore, it was possible to determine that 8 of them were viable microorganisms by (b) of FIG. The actual program first calculates the chromaticity from the image of (b) and identifies the luminescent point identified as a microorganism with respect to the point recognized as a temporary microorganism from the size and brightness value of the luminescent point of the image of (a). Ask for. With regard to the light emission point, the brightness value obtained from the images (a) and (c) is used to determine whether the bacteria are viable or dead. By sequentially narrowing down and determining the emission points for obtaining the ratio of luminance values, etc., the calculation becomes faster and the measurement time can be shortened.

  FIG. 7 shows an example of the results of visual confirmation of the chromaticity of the luminescent spots and the luminescent spots of live bacteria, dead bacteria, and foreign substances using a fluorescence microscope for E. coli and foreign substances as in FIG. With regard to the luminescent point identified as E. coli, the value of chromaticity is different between live and dead bacteria, and by setting different color characteristic setting ranges for live and dead bacteria, Can be determined. In order to more accurately discriminate between microorganisms (live bacteria or dead bacteria), it is preferable to distinguish between live bacteria and dead bacteria after distinguishing the luminescent point from live bacteria and dead bacteria.

  This threshold is an example, but it varies depending on the type, concentration, and polarity of the solution used for staining. It is preferable to keep it.

  In addition, since there are bacteria that are difficult to culture, the appropriateness of the final number of bacteria is preferably evaluated by using a combination of appropriate culture methods and types of media.

  The present invention is an apparatus for discriminating and judging microorganisms and foreign substances other than microorganisms using fluorescent staining from a specimen containing cells and microorganisms, and judging viable and dead bacteria with respect to a luminescent point judged to be a microorganism. Therefore, it is possible to provide a microorganism weighing apparatus that can identify microorganisms and detect them with higher accuracy, and measure microorganisms in food factories, hospitals, or food service centers, or measure microorganisms in microorganism detection or environmental tests, or microorganisms. It can also be applied to detection.

Chromaticity distribution diagram stained with DAPI according to Embodiment 1 of the present invention The figure which shows the absorption spectrum and fluorescence spectrum of the excitation light of DAPI of Embodiment 1 of this invention Configuration diagram of the microorganism weighing apparatus according to Embodiment 1 of the present invention. The flowchart of the calculation process of Embodiment 1 of this invention (A) E. of the first embodiment of the present invention. The figure which shows the calculation result of the brightness | luminance and chromaticity of E. coli, (b) The figure which shows the calculation process flow of the same chromaticity (A) E. of Example 1 of the present invention. The figure which shows the dot plot of the brightness | luminance of the luminescent material in E. coli and tap water, and the classification method by the life-and-death judgment means, (b) The chromaticity diagram of the same luminescent point, and the figure which shows the judgment method by microbe judgment means E. of Example 1 of the present invention. Coli and chromaticity diagram of luminescent material in tap water and a diagram showing a method for judging viable bacteria, dead bacteria and foreign substances by means of microorganism judging means

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microorganism measuring device 2 Excitation light source 3 Excitation spectral filter 4 Condensing lens 5 High pass filter 6 Fluorescence spectral filter 7 Lens unit 8 Light receiving element 9 Inspection table 10 Membrane filter 11 CCD unit 12 Calculation unit 13 Bright spot removal unit 14 Temporary Microorganism determination means 15 Microorganism determination means 16 Life / death bacteria determination means 17 Effective area calculation unit

Claims (7)

Both the first reagent that reacts with any of the living and dead cells and develops both the living and dead cells and the second reagent that reacts only with dead cells and reacts only with dead cells are brought into contact with the specimen to be examined for the presence of microorganisms. In a microorganism weighing device that measures microorganisms stained by fluorescent staining, only a light source that emits excitation light in a plurality of predetermined wavelength ranges and light in a predetermined different wavelength range that is emitted by the excitation light are transmitted. The first reagent emits fluorescence with light having a wavelength shorter than that of the second reagent, and the area, luminance value, and color of the emission point that emits the fluorescence. Determination means for determining one microorganism from the physical characteristics, brightness value emitted by the first reagent and brightness value emitted by the second reagent, live / dead bacteria determination means for distinguishing live cells from dead cells, and the microorganism Judgment Microorganism metering device for simultaneously measuring the viable and dead bacteria by sequentially integrating the light emitting points that are determined to live cells or dead by said the stage death bacteria determination means. The microorganism measuring apparatus according to claim 1, wherein the first reagent and the second reagent are reagents that develop color by reacting with a nucleic acid. The microorganism measuring apparatus according to claim 1, wherein the first reagent and the second reagent discriminate between live bacteria and dead bacteria from the difference in permeability of the cell membrane. 2. The microorganism weighing apparatus according to claim 1, wherein the excitation wavelength of the first reagent is ultraviolet and the excitation wavelength of the second reagent is blue or green. 2. The microorganism weighing apparatus according to claim 1, wherein the excitation wavelength of the first reagent is blue and the excitation wavelength of the second reagent is green. The first reagent and the second reagent use the first reagent and the second reagent for the light emission point where the area and luminance value of the light emission point that is fluorescent by the first reagent are determined to be microorganisms based on the values within the range set by the microorganism determination means. 2. The microorganism measuring apparatus according to claim 1, further comprising viable and dead bacteria judging means for discriminating viable bacteria and dead bacteria from a ratio of luminance values in respective wavelength regions emitting fluorescence. A color characteristic is calculated for each luminescent point that has been determined to be live or dead by the viable and dead bacteria determination means, and a set value is set for each range of live and dead bacteria. The microorganism measuring device according to claim 1 or 6, further comprising a microorganism judging means for discriminating foreign matter or dead bacteria from foreign matter.

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