JP2010246442A - Lactic acid bacteria count measuring method and lactic acid bacteria count measuring apparatus - Google Patents

Abstract

An object of the present invention is to efficiently collect lactic acid bacteria from a sample containing lactic acid bacteria, and to accurately detect and measure the lactic acid bacteria.
A sample containing a lactic acid bacterium filtered and extracted by a collection filter 7 is stained with a fluorescent luminescent dye, the collection filter 7 is set on an inspection table 6 of a lactic acid bacteria count measuring apparatus, irradiated with excitation light to emit fluorescence, and a photoelectric conversion element. Analyze the area of the image taken in step 1. Area analysis distinguishes single bacteria and lactic acid bacteria lumps and foreign substances from the area and luminescence brightness of the luminescent spots, and determines the luminescent area as determined from the area and luminescence brightness of the luminescent spots determined to be lactic acid bacteria. The number of lactic acid bacteria is calculated by calculating the number of lactic acid bacteria in the size.
[Selection] Figure 2

Description

  The present invention relates to a method for measuring the number of lactic acid bacteria and a measuring apparatus for measuring the number of lactic acid bacteria in a sample containing lactic acid bacteria.

  Conventionally, this type of measurement and testing method is a smear agar culture method in which 0.1 ml of a specimen is dropped on an agar medium containing nutrient components and smeared with a congeal stick, or a mixture in which 1 ml of a specimen is mixed while the agar medium does not solidify. A culture method in which the growth activity is detected by culturing by an incubation method, a liquid culture method in which 0.1 ml or 1 ml of a specimen is added in a liquid medium, and the like is used. Examination by culture method is currently the most widely used detection means, but it can not be cultured if the culture takes 24 to 72 hours and the culture conditions (temperature, time, medium nutrients) do not match. This is a problem.

  As a method of measurement using a membrane filter, a solution containing adenosine triphosphate (hereinafter referred to as ATP) degrading enzyme is applied to the membrane filter, and then the microorganisms in the sample are collected by filtration on the dried membrane filter. If necessary, after culturing for a required time, a liquid extraction reagent for extracting ATP and a luminescence reagent, luciferin luciferase, are sprayed in the form of a mist so that luciferin and luciferase react with ATP, One photon was emitted by oxidation of luciferin, and the emitted light was captured with a high-sensitivity CCD camera to measure microorganisms (see, for example, Patent Document 1).

Japanese National Patent Publication No. 9-512713

  The conventional culture method, which is a conventional test method, requires a culture time of 24 to 48 hours or more until a test result is obtained, and waits for the test result at the stage of production of fresh food or product shipment. In other words, it is a major disadvantage in terms of time and economics. In some cases, there is a problem that the product must be shipped before the test results are known, and the time required to measure the number of lactic acid bacteria in the sample is short. It is required to be.

  In addition, in the inspection of specimens that contain fluorescent foreign substances that emit light in the same manner as lactic acid bacteria, there is no clear difference between the fluorescent foreign substances that emit light as in lactic acid bacteria and the fluorescence of lactic acid bacteria. There is a problem that the number of lactic acid bacteria cannot be measured, and it is required to accurately measure the number of lactic acid bacteria.

  The present invention solves the conventional problems, shortens the time required for measuring the number of lactic acid bacteria in a sample, discriminates lactic acid bacteria collected from a membrane filter from foreign substances, and accurately detects the number of lactic acid bacteria in a sample. An object of the present invention is to provide a method for measuring the number of lactic acid bacteria and a device for measuring the number of lactic acid bacteria that detect the number of lactic acid bacteria.

  In order to achieve the above-mentioned object, the lactic acid bacteria count method and measuring apparatus of the present invention analyze the area of an image obtained by irradiating a filter-extracted lactic acid bacteria-containing specimen with excitation light and photographing the emission point with a photoelectric conversion element, and calculating the number of lactic acid bacteria It is a method for measuring the number of lactic acid bacteria characterized by measuring.

  In addition, the area analysis is a method for measuring the number of lactic acid bacteria, which distinguishes a single bacterium, a lump of lactic acid bacteria, and foreign substances from the area of the luminescent spot and the luminescence brightness.

  In addition, a method for measuring the number of lactic acid bacteria that calculates the number of lactic acid bacteria in the size of the luminescent area is determined based on the area of the luminescent spots determined as lactic acid bacteria and the luminance of the luminescent bacteria.

  In addition, the method is a method for measuring the number of lactic acid bacteria having means for accumulating the number of lactic acid bacteria from sequentially captured images.

  In addition, the method is a method for measuring the number of lactic acid bacteria using a staining method for the lactic acid bacteria-containing specimen extracted by filtration.

  Further, the present invention is a method for measuring the number of lactic acid bacteria by adjusting the pH of the lactic acid bacteria-containing specimen extracted by filtration.

  The staining method is a method for measuring the number of lactic acid bacteria, which is a fluorescent staining method.

  In addition, the lactic acid bacteria are filtered and extracted with a collection filter, and the lactic acid bacteria are removed using one or more of the first reagent, the second reagent, the third reagent, and the fourth reagent. A light source that emits excitation light in a predetermined wavelength range after dyeing, and a light receiving means that receives light in a predetermined wavelength range that is irradiated with the excitation light and emits light in a predetermined area set by the sampling filter And an image obtained by receiving and photographing the light emitted and emitted by the light source within a set time, the received light amount being within a set threshold range and a set area range Lactic acid bacteria judging means to judge lactic acid bacteria, and from the size of one luminescent point judged to be lactic acid bacteria by lactic acid bacteria judging means, the mass of single bacteria and lactic acid bacteria is judged, and the quantity of lactic acid bacteria in the size of the lump is calculated Sequential integration A means for sequentially accumulating the quantity of lactic acid bacteria by a moving means for moving the collection filter or the light receiving means in order to confirm the light emission point of a part or the entire area of the collection filter. This is a lactic acid bacteria count measuring device for performing the lactic acid bacteria count measuring method.

  Further, the sampling filter is a lactic acid bacteria count measuring device in which the surface shape is not deformed.

  Further, the sampling filter is a lactic acid bacteria count measuring device that is a filter that does not lose color.

  Moreover, it is set as the lactic acid bacteria count measuring apparatus which carry | supported the pigment on the said filter.

  Moreover, the said collection filter is set as the lactic acid bacteria count measuring apparatus which is a dark filter.

  Moreover, it is set as the lactic acid bacteria count measuring apparatus by which the thin film containing the at least 1 sort (s) of metal component chosen from gold | metal | money, copper, chromium, platinum, and palladium was formed on the said collection filter.

  Further, the light receiving means is a lactic acid bacteria count measuring device which is a plurality of photoelectric conversion elements having an area where at least the size of lactic acid bacteria can be recognized.

  Further, the light source is of one type or a plurality of types, the predetermined wavelength range is one type or a plurality of types, and the light to be emitted emitted in the one type or a plurality of types of wavelength ranges is determined in advance. It is a lactic acid bacteria count measuring device having a light receiving means for receiving light of various types of wavelength ranges and a lactic acid bacteria judgment for judging the point or area of light emission from the ratio of the light quantity in each wavelength range as lactic acid bacteria.

  Further, a single type or a set type of light receiving means for receiving a plurality of types of light sources and a plurality of wavelength ranges is switched, and a lactic acid bacterium that detects a lactic acid bacterium with only a set type of light sources and light receiving units. This is a number measuring device.

  Also, by the lactic acid bacteria judgment means, the area of the luminescent spots recognized as other than microorganisms because of more than the set area is sequentially integrated or the area is recognized as one and the number is sequentially integrated, and the total area or total number is set This is a lactic acid bacteria count measuring device having a caution means for indicating a caution when the area or number exceeds the specified area.

  According to the present invention, it is possible to collect lactic acid bacteria on a collection filter in a sample containing lactic acid bacteria, and it is possible to detect and measure lactic acid bacteria containing streptococci in a short time.

  In addition, it is possible to discriminate between lactic acid bacteria collected from the collection filter and foreign substances, and to detect and measure the exact number of lactic acid bacteria in the specimen.

  In addition, since the filter does not deform or decolorize the filter shape, it is possible to detect and measure lactic acid bacteria with high accuracy without affecting the detection and measurement of lactic acid bacteria even when measuring by staining or fluorescent staining.

The block diagram which shows the one aspect | mode of the lactic acid bacteria count measuring apparatus of Embodiment 2 of this invention Same flowchart Same measurement flowchart Photomicrograph of fluorescence emission image of Example of the present invention

  The method for measuring the number of lactic acid bacteria according to claim 1 of the present invention is characterized in that the number of lactic acid bacteria is measured by area analysis of an image obtained by irradiating a filter-extracted lactic acid bacteria-containing specimen with excitation light and photographing the emission point with a photoelectric conversion element. The method for detecting lactic acid bacteria is to detect lactic acid bacteria efficiently and with a simple configuration.

  In addition, the area analysis has an effect that a single bacterium, a lump of lactic acid bacteria, and a foreign substance can be distinguished from the area of the luminescent spot and the luminescent brightness, and the luminescent matter in the sample can be determined and measured.

  In addition, a means for calculating the number of lactic acid bacteria by calculating the number of lactic acid bacteria in the size of the luminescent area from the area of the light emitting point determined to be lactic acid bacteria and the luminance of the luminescent bacteria and sequentially integrating the number of lactic acid bacteria, It has the effect | action that the lactic acid bacteria density | concentration in a test substance can be measured correctly.

  Moreover, it has a means for accumulating the number of lactic acid bacteria from sequentially photographed images, and has the effect of accurately measuring the concentration of lactic acid bacteria in the specimen.

  Moreover, it has the effect | action of detecting lactic acid bacteria using the staining method to the lactic acid bacteria containing test substance extracted by filtration.

  In addition, it is a method for measuring the number of lactic acid bacteria provided with means for adjusting the pH so that the pH of the lactic acid bacteria-containing specimen extracted by filtration does not change the properties of the lactic acid bacteria on the surface of the collection filter. It has the effect that lactic acid bacteria can be detected with high sensitivity.

  Further, it is a method for measuring the number of lactic acid bacteria captured on the collection filter and detecting or counting lactic acid bacteria by a staining method or a fluorescent staining method, and has an effect of optically measuring the emitted lactic acid bacteria.

  In addition, the lactic acid bacteria are filtered and extracted with a collection filter, and the lactic acid bacteria are removed using one or more of the first reagent, the second reagent, the third reagent, and the fourth reagent. A light source that emits excitation light in a predetermined wavelength range after dyeing, and a light receiving means that receives light in a predetermined wavelength range that is irradiated with the excitation light and emits light in a predetermined area set by the sampling filter And an image obtained by receiving and photographing the light emitted and emitted by the light source within a set time, the received light amount being within a set threshold range and a set area range Lactic acid bacteria judging means to judge lactic acid bacteria, and from the size of one luminescent point judged to be lactic acid bacteria by lactic acid bacteria judging means, the mass of single bacteria and lactic acid bacteria is judged, and the quantity of lactic acid bacteria in the size of the lump is calculated Sequential integration A means for sequentially accumulating the quantity of lactic acid bacteria by a moving means for moving the collection filter or the light receiving means in order to confirm the light emission point of a part or the entire area of the collection filter. It has the effect of differentiating and measuring luminescent substances and lactic acid bacteria.

  In addition, the filter is a method for measuring the number of lactic acid bacteria, which is a filter whose surface shape is not deformed. The flatness of the filter surface is improved, the detection position of lactic acid bacteria is clarified, and the apparatus configuration during detection and measurement is simplified. It has the effect of being able to.

  The filter is a method for measuring the number of lactic acid bacteria, which is a dark filter that does not lose color, and has the effect of suppressing the luminance of the background when detecting fluorescence and increasing the sensitivity.

  Further, the present invention is a method for measuring the number of lactic acid bacteria in which a thin film containing at least one metal component selected from gold, copper, chromium, platinum, and palladium is formed on the filter, and irradiated with excitation light when detecting the lactic acid bacteria. It is possible to prevent the reflection of light at the time, and to accurately detect and measure lactic acid bacteria without interfering with the detection and measurement of lactic acid bacteria.

  Further, the light source is of one type or a plurality of types, the predetermined wavelength range is one type or a plurality of types, and the light to be emitted emitted in the one type or a plurality of types of wavelength ranges is determined in advance. It has a function of detecting and detecting a light emitting point or area as a lactic acid bacterium based on the ratio of the light quantity in each wavelength range and the light receiving means for receiving light of various wavelength ranges.

  In addition, the light source and the light receiving means of one kind or a set kind of light receiving means for receiving a plurality of types of light sources and a plurality of wavelength ranges are switched, and lactic acid bacteria are detected by the light source and the light receiving means of only the set types. Has an effect.

  In addition, by the lactic acid bacteria determination means, the area of the light emitting points recognized as other than the lactic acid bacteria for the area larger than the set area is sequentially integrated, or the area is recognized as one and the number is sequentially integrated, and the total area or the total number is There is a means for detecting when the area or number exceeds the set value and indicating attention.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

(Embodiment 1)
For example, there are lactic acid bacteria and various foods containing them. When lactic acid bacteria are detected or measured, the conventional culture method is a culture time of 24 to 48 hours or more until the result of the test is obtained. It is necessary to wait for the inspection result at the stage of manufacture or product shipment, which is a great disadvantage in terms of time and economy, and in some cases, it may be necessary to ship before the inspection result is known. Therefore, quick measurement of lactic acid bacteria is necessary.

  In addition, components of dairy products include components that easily react with staining reagents and fluorescent staining reagents, and are very difficult and require skill when observed with a microscope or the like.

  Usually, in order to measure microorganisms by the membrane filter method, filtration is performed with a 0.2 to 0.4 μm filter, and the microorganisms collected on the filter are observed with a microscope, or the filter is placed on a medium. Culture. In the food field, a filter having a pore size of 0.4 μm that can collect target microorganisms and has little influence on filtration is often used.

  Filter types are PP (Polypropylene), PVC (Polyvinyl Chloride), PC (Polycarbon), PTFE (Polytetrafluorethylene), PVDF (Polyvinylidene fluoride, PE) There is.

  For example, since the surface is flat (± 2 μm or less, preferably ± 1 μm or less) as in the case of a PC material, and holes are made using radiation, and very uniform pores are formed, Since it is collected without being buried in the surface on the filter, it is usually used. Such a filter is poor in flexibility of pores and has a low aperture ratio, so that if there are particles having a particle size larger than the pores, it is difficult to filter. However, if there are no substances or components other than microorganisms, surface irregularities are small and microorganisms are not buried in the pores. Therefore, it is desirable for subsequent microscopic observations and particularly when automated and mechanically measured.

  When the lactic acid bacteria dyed or fluorescently stained are measured with a microscope after filtration and measured with an automated apparatus, it is possible to measure with high accuracy by using a dark filter that does not lose color. A dark color is black or a color close to it, but is a color that does not absorb light emitted when irradiating light of a specific wavelength according to the characteristics of the reagent, such as white light or ultraviolet light, when observing with a microscope. It is shown that. Many of the materials of the filter shown above are white or close to white, and particularly when irradiated with ultraviolet rays, reflection or autofluorescence may cause the measurement to be difficult. When the filter gets wet with water, reflection hardly occurs, but it is important as a color of the filter that the reflection is prevented when wet with water. Usually, since the material of the filter is rarely dark, it needs to be colored in black or gray that is close to black, and as its method, a pigment or ink that does not easily lose color is desirable.

  Reflection can be prevented by coating the filter surface with a metal thin film selected from gold, copper, chromium, platinum, palladium and the like by vapor deposition or the like. Gold, in particular, has a low ultraviolet reflectance. In other words, the dark color indicates a color close to black and has little light reflection.

  As a method for staining microorganisms, 4 ′, 6-diamidino-2-phenylindole dihydrochloride as a first reagent and propium iodide as a second reagent and a second reagent on a sample to which cells or microorganisms are attached are used. The reagent which mixed 6-carboxyfluorescein diacetate which is the reagent 3 and 4-methylumbelliferyl-β-D-galactoside which is the fourth reagent is brought into contact. The 4 ′, 6-diamidino-2-phenylindole dihydrochloride bound to the nucleic acid of the living and dead cells absorbs the excitation wavelength when the excitation wavelength is 359 nm and emits a fluorescence wavelength of 461 nm to cause the living and dead cells to develop color. Propidium iodide combined with dead cell nucleic acid absorbs the amount of light at the excitation wavelength when the excitation wavelength is 535 nm and changes the fluorescence wavelength to 617 nm to cause only dead cells to develop color.

  Other fluorescent reagents for live and dead cells include YTO11 to SYTO16, SYTO20 to SYTO25, SYTO40 to SYTO45, SYTO17, SYTO59 to SYTO64, SYTO80 to SYTO85, DAPI, SYBRGreen, Hoechst33342, HoechY33TO, 58 . Examples of the fluorescent reagent for dead cells include Propium Iodide, SYTOXGreen, BOBO-1, YOYO-1, YO-PRO-1, TOTO-1, POPO-3, TO-PRO3, SYTOXBlue, and SYTOXOrange. By setting the wavelength of the excitation light with the fluorescent reagent, the living and dead cells and dead cells that have reacted with the reagent can emit fluorescence.

  In addition, those that react with esterase that only live bacteria have, those that stain only live bacteria using a reagent that can detect respiratory activity, and those that die because the cell membrane is damaged because it does not penetrate the cell membrane, Permeated from there and stained with nucleic acid such as DNA to stain only dead bacteria. Similarly, it binds to nucleic acid such as DNA and has the property of permeating the cell membrane. Those that stain both bacteria, those that react with specific microorganism-derived substances that only specific microorganisms metabolize, antibodies that have fluorescent labels that react only with specific microorganisms, or those that stain only specific microorganisms with microphages, etc. There is.

  In addition, when performing a microbial test, the larger the sample amount, the better. In the conventional culture method, 0.1 ml is the maximum, so that the sample volume can be 0.1 ml or 1 ml or more by filtration, and the bacterial concentration is measured as the number per 1 ml. The greater the number, the better the sensitivity and accuracy.

(Embodiment 2)
FIG. 1 is a block diagram showing an embodiment of a lactic acid bacteria count measuring apparatus. This lactic acid bacteria count measuring apparatus includes a lens 1 as a light source condensing unit and a light receiving unit 2. In order to extract a target wavelength from the excitation light emitted from the light source 3, the light is spectrally separated by the excitation light spectral filter 4. The split excitation light passes through the prism 5 and the optical path is changed. The excitation light whose optical path has been changed is condensed on the surface of the collection filter 7 of the combined body composed of the collection filter 7 and the pedestal 8 through the lens 1 and including the collection filter 7 of lactic acid bacteria installed on the examination table 6. Is done. Therefore, the fluorescence of the lactic acid bacteria excited by the excitation light passes through the prism 5 again and reaches the light receiving unit 2. The fluorescence that reaches the light receiving unit 2 reaches the photoelectric conversion element 10 built in the light receiving unit through the fluorescence spectral filter 9 in order to extract only the target fluorescence, and is converted into a signal and recognized. Although not shown in the figure, this lactic acid bacteria count measuring device is provided with means for moving the examination table 6 and can receive all or part of the fluorescence emitted from the surface of the collection filter 7.

  In the fluorescence of 461 nm and 617 nm that reached the photoelectric conversion element 10, viable bacteria and dead bacteria emitted fluorescence of 461 nm, and dead bacteria emitted fluorescence even at 617 nm. The difference in the wavelength of light and the light wavelength is separated by the fluorescence spectral filter 9 and only the target fluorescence wavelength is extracted and reaches the photoelectric conversion element 10, which absorbs the excitation wavelength 359nm and is excited to emit fluorescence at 461nm. Are considered to be dead bacteria, and those that are excited and absorb fluorescence at 617 nm are considered dead bacteria, and those that do not emit fluorescence at both 461 nm and 617 nm are judged to be foreign substances. The fluorescence determined to be lactic acid bacteria is integrated and the quantity is measured. As the lactic acid bacteria judging means 11, there is a microcomputer programmed with the judging procedure.

  The excitation light generated from the light source 3 is condensed by the lens 1, and at this time, the range irradiated with the excitation light by the lens 1 is condensed on a small fixed area. In this case, the small fixed area indicates a range of about 0.2 μm to 7.0 μm on a side when set based on the size of the microorganism. In addition, when based on a comparison with the agar medium diffusion method, which is one of the most utilized microorganism detection means, colonies formed by a population of microorganisms cultured and grown by the agar medium diffusion method have a distance of When they are close to each other, colonies may overlap with each other, and when they are finally visually confirmed, there may be cases where the colonies are recognized as one colony. Therefore, the minute fixed area in this case indicates a range of about 100 μm to 500 μm on a side when based on a distance where colonies do not overlap each other. That is, the small fixed area is a size that can be recognized as lactic acid bacteria by the lactic acid bacteria determination means 11. The light receiving unit 2 may shoot the entire filter as a single image with a plurality of photoelectric conversion elements 10, or after shooting with a small photoelectric conversion element 10 (one side is 10 μm or less), each image is assembled. In addition, the size of lactic acid bacteria may be finally recognized.

  The irradiation time of the excitation light collected by the lens 1 depends on the quenching time and the excitation light intensity of the reagent that emits fluorescence. Depending on the type of reagent, it may be decomposed by ultraviolet light existing in nature, and it is desirable to irradiate excitation light within a range of about 2 to 300 seconds.

  When light emission is detected, if the wavelength range of the light source 3 is wide, the excitation wavelength can be adjusted and dispersed by the excitation light spectral filter 4. Since the excitation light spectral filter 4 can be changed according to the target detection target, it can cope with various fluorescent reagents.

  At the same time, when the emitted fluorescence wavelength has a wide range, it is possible to cope with various fluorescent reagents by changing the fluorescence spectral filter 9 according to the target detection target in order to detect the target emission. .

  Examples of the light source 3 include various diodes, halogen lamps, xenon lamps, cold cathode tubes, lasers, black lights, mercury lamps, and the like. Among these light sources, a diode, a cold cathode tube, a black light, or the like whose maximum excitation wavelength is relatively limited may be implemented without using the excitation light spectral filter 4 and the fluorescence spectral filter 9. For halogen lamps, mercury lamps, and the like, it may be necessary to use the excitation light spectral filter 4 and the fluorescent spectral filter 9.

  The prism 5 and the lens 1 each have a property of transmitting ultraviolet light as necessary. Quartz glass etc. are mentioned as what has the property which permeate | transmits ultraviolet light. Thereby, it can respond also to the reagent etc. which are excited by ultraviolet light. The inspection table 6 on which the site including the microorganism collection filter 7 is installed has a rotating ability. The excitation light collected by the lens 1 moves a distance corresponding to the radius from the outer peripheral portion of the microorganism collection filter 7 to the central portion or from the central portion to the outer peripheral portion. At that time, the excitation light condensed by the lens 1 is changed by changing the rotation speed of the examination table 6 when the position of the excitation light collected by the lens 1 is present at the outer peripheral portion and at the central portion. It is possible to prevent the occurrence of deviation in fluorescence, afterimage and afterglow generated by the excited reagent when the is present at the outer periphery and at the center.

  As shown in FIG. 1, the inspection table 6 has a depressed portion (device groove) for fitting a portion including the sampling filter 7, and has a shape in which the portion including the sampling filter 7 can be incorporated as it is. . At this time, for example, the inspection table 6 is provided with a metal flat plate so that the sampling filter 7 is positioned on the inspection table 6, and the sampling filter 7 is incorporated in a state of being pressed against the metal flat plate, If the collection filter 7 is held smoothly on the table 6 without unevenness, the quantification of lactic acid bacteria collected on the collection filter 7 can be made more reliable.

  It should be noted that by providing means for recognizing the condensed position, the position of the excitation light collected by the lens 1 is recognized, so that the condensed light does not deviate from the orbit, and when it deviates, it returns to the orbit again. Is set to

  Note that the minute fixed area to which the excitation light is irradiated is not limited to a polygon including a square, but may be a circle, an ellipse, or the like as long as the sample can be irradiated.

  It is also possible to use a diffraction grating or the like as means for separating excitation light or fluorescence.

  Although the fluorescence afterimage and afterglow are prevented by adjusting the rotation speed of the inspection table 6, it is also possible to prevent afterimage and afterglow by adjusting the moving speed of the lens 1 that irradiates the excitation light. It is.

  The means for recognizing the condensed position is not necessarily required to directly recognize the condensing position of the excitation light, and may be any means as long as it can grasp the trajectory on the collection filter 7.

  FIG. 2 is a measurement flowchart of the lactic acid bacteria detection method and the lactic acid bacteria count measuring device in the lactic acid bacteria-containing specimen of the present invention.

  In specimen staining, 1 ml of lactic acid bacteria-containing specimen is filtered through the collection filter 7 in Step 1. In step 2, residual components other than lactic acid bacteria on the surface of the collection filter 7 and pH adjustment are washed away with 3 ml of physiological saline and pH adjustment is performed. In step 3, 0.1 ml of a fluorescent staining reagent that stains the lactic acid bacteria collected by the collection filter 7 is dropped and spread over the entire surface of the collection filter 7 for 2 minutes. As the staining reagent, a reagent that binds to and stains DNA of lactic acid bacteria was used. In step 4, 0.1 ml of the excess reagent of the staining reagent is washed with physiological saline to complete the specimen staining.

  In the measurement, in step 5, the microorganism collection filter 7 is set on the base of the lactic acid bacteria count measuring device. About this stand, it is comprised in the state which becomes a plane by setting the collection filter 7 of microorganisms on a measurement surface, and can thereby perform the stable measurement. In step 6, the collected lactic acid bacteria are measured by the measuring device, and the measurement is completed.

  FIG. 4 shows an image of fluorescence staining and fluorescence emission. As shown in the figure, the fluorescence emission point has various sizes, and the minute size was observed with a microscope, and as a result, it was confirmed that the number of cells was one. It was also confirmed that the fluorescence emission points having a size other than the minute light-emitting substance were streptococcus with solid bacterial cells.

  FIG. 3 shows a flow chart for measuring lactic acid bacteria with respect to an image of one visual field taken by the lactic acid bacteria number measuring apparatus of FIG. When measuring a large number of fields, repeat this flow and calculate the number of lactic acid bacteria in the entire filter by multiplying the ratio of the total area of the filter and the measured area (field area x number of fields) by the number of lactic acid bacteria measured. Can do.

  In addition, although the single dyeing | staining was implemented as a fluorescent dye in the Example, double dyeing | staining can also be carried out.

  In addition, although the washing | cleaning process of the excess reagent was described in the Example, the process of washing | cleaning an excess reagent can be excluded if there is no influence on the background at the time of a measurement.

  In the specimen of the present invention, which can contain lactic acid bacteria, after irradiating with excitation light and photographing, the lactic acid bacteria number measuring method and measuring apparatus for detecting lactic acid bacteria by recognizing the shape, area and emission luminance by means of area analysis means Since no skill is required and anyone can easily measure, it can be applied to quality control nearer to the production site. In recent years, the quality level of food has been improved, and an apparatus capable of measuring the number of lactic acid bacteria more quickly, accurately, and simply has been demanded, and can be applied as a method replacing conventional inspection.

DESCRIPTION OF SYMBOLS 1 Lens 2 Light-receiving part 3 Light source 4 Excitation light spectral filter 5 Prism 6 Inspection table 7 Sampling filter 8 Base 9 Fluorescence spectral filter 10 Photoelectric conversion element 11 Lactic acid bacteria judgment means

Claims (17)

A method for measuring the number of lactic acid bacteria by irradiating an excitation light to a filtered lactic acid bacteria-containing specimen and measuring the number of lactic acid bacteria by analyzing the area of an image obtained by photographing a light emitting point with a photoelectric conversion element. 2. The method for measuring the number of lactic acid bacteria according to claim 1, wherein the area analysis distinguishes a single bacterium, a lump of lactic acid bacteria, and a foreign substance from the area of the luminescent spot and the luminescence brightness. The method for measuring the number of lactic acid bacteria according to claim 1 or 2, wherein the number of lactic acid bacteria in the size of the luminescent area is calculated from the area of the luminescent point determined to be lactic acid bacteria and the luminance of the luminescent bacteria. The method for measuring the number of lactic acid bacteria according to any one of claims 1 to 3, further comprising means for accumulating the number of lactic acid bacteria from sequentially photographed images. The method for measuring the number of lactic acid bacteria according to any one of claims 1 to 4, wherein a staining method is used for the lactic acid bacteria-containing specimen extracted by filtration. The method for measuring the number of lactic acid bacteria according to any one of claims 1 to 5, wherein the pH of the lactic acid bacteria-containing specimen extracted by filtration is adjusted. The method for measuring the number of lactic acid bacteria according to claim 5 or 6, wherein the staining method is a fluorescent staining method. Lactic acid bacteria were filtered and extracted with a collection filter, and lactic acid bacteria were stained with one or more of the first, second, third, and fourth reagents. Then, a light source that emits excitation light in a predetermined wavelength range, and a light receiving means that receives light in a predetermined wavelength range that is emitted by the excitation light and emits light in a predetermined area set by the sampling filter, An image obtained by receiving and photographing the light emitted and emitted from the light source within a set time period is a threshold range in which the received light quantity is set and a set area range is defined as lactic acid bacteria. From the size of one luminescent point judged to be lactic acid bacteria by the lactic acid bacteria judging means to judge and the lactic acid bacteria judging means, the mass of the single bacteria and lactic acid bacteria is judged, the quantity of lactic acid bacteria in the size of the mass is calculated, Accumulate sequentially The lactic acid bacteria having means for sequentially accumulating the quantity of lactic acid bacteria by moving means for moving the collection filter or the light receiving means in order to confirm the light emission point of one part or the entire area of the sampling filter An apparatus for measuring the number of lactic acid bacteria for performing a number measurement method. The lactic acid bacteria count measuring apparatus according to claim 8, wherein the collection filter is a filter whose surface shape is not deformed. The lactic acid bacteria count measuring apparatus according to claim 8 or 9, wherein the sampling filter is a filter that does not lose color. The apparatus for measuring the number of lactic acid bacteria according to any one of claims 8 to 10, wherein a pigment is supported on the filter. The lactic acid bacteria count measuring apparatus according to claim 8, wherein the collection filter is a dark filter. The lactic acid bacteria count measuring apparatus according to any one of claims 8 to 12, wherein a thin film containing at least one metal component selected from gold, copper, chromium, platinum, and palladium is formed on the collection filter. The apparatus for measuring the number of lactic acid bacteria according to any one of 8 to 13, wherein the light receiving means is a plurality of photoelectric conversion elements having an area where at least the size of the lactic acid bacteria can be recognized. There are at least one type of light source, and at least one type of wavelength range of the light source. The light receiving means receives the emitted light emitted in each wavelength range for each wavelength range, and The apparatus for measuring the number of lactic acid bacteria according to any one of claims 8 to 14, wherein a point or area of light emission is determined as a lactic acid bacterium from a ratio of light amounts. 16. A lactic acid bacterium is detected with a light source and a light receiving means of a set wavelength range by switching a light source and a light receiving means of one type or a set type of light receiving means for receiving a plurality of types of light sources and a plurality of wavelength ranges. The lactic acid bacteria count measuring apparatus in any one of. By the lactic acid bacteria judgment means, the area of the luminescent spots recognized as other than microorganisms because it is larger than the set area is sequentially integrated or the area is recognized as one and the number is sequentially integrated, and the total area or total number is the set area The lactic acid bacteria count measuring apparatus according to any one of claims 15 to 16, further comprising a caution means for indicating a caution when the number is more than the number.

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