JP2006094848A - Method for detecting microorganism and apparatus for measuring the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To accurately detect and measure microorganisms after efficiently retrieving them in an emulsion particles-containing specimen. <P>SOLUTION: In preparing a sample, an agitation and an incubation are carried out for adding a reagent A to emulsion particles to be reacted. Further agitation and incubation are carried out for adding a reagent B to the above reaction system to be reacted. Subsequently, a reagent C is added and a further agitation is carried out to complete preparing the sample. In staining the specimen, the total amount of the liquid after sample preparation is filtered through a microorganisms-retrieving filter 7 and residues other than the microorganisms on the surface of the filter are washed with filtered and sterilized water. A reagent D for staining the microorganisms is dripped on the filter 7 and spread over the whole surface of the filter 7 to carry out a staining, and after finishing the staining, an excess of the reagent D is washed off to complete staining the specimen to be measured. In measuring the specimen, the filter 7 is set on the testing table 6 of a measuring device to measure the microorganisms, thus completing the measuring operation. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a microorganism detecting method and a microorganism measuring apparatus for performing a microorganism test in, for example, raw milk or milk in a specimen that may contain emulsion particles.

Conventionally, this type of microorganism testing method uses a bleed method in which raw milk is quantitatively dropped onto a slide glass, applied to 1 cm ² , and the microorganism is stained with a staining reagent mainly composed of methylene blue. The inspection by the bleed method is quick, but since the microscope is used for a long time, the burden on the person in charge of the observation is great, and skilled skills are required for identifying components other than microorganisms.

In addition, since the amount of specimen is very small, reproducibility is low and variation is large. Furthermore, in terms of detection sensitivity, there is a problem in accuracy when measuring raw milk having a number of microorganisms of 10 ⁶ cells / ml or less due to the limit of the microscope magnification. Accordingly, in the case of raw milk having a microorganism count of 10 ⁴ cells / ml or less, it is necessary to concentrate at least 100 times before staining.

  Alternatively, Christine M Cousins (see Non-Patent Document 1) and the like separate and decompose casein and fat using a centrifuge, a nonionic surfactant (Triton X-100), and an enzyme (Aspergillus oryzae Protease). Thus, the raw milk can be filtered with a 0.6 μm membrane filter, and the microorganisms collected on the filter can be fluorescently stained to detect the microorganisms in the raw milk.

In addition, this kind of raw milk may be tested for microorganisms by mixing raw milk with triton and trypsin and filtering through a membrane having a pore of 0.6 μm (see, for example, Patent Document 1).
Japanese National Patent Publication No. 9-512713 Christine M Cousins et al., “Dairy industries international” published, April 1979, pp. 27-31.

  The conventional bleed method, which is a microbiological examination method, requires a long time for the microscope to be used by a person in charge of observation.・ It is required to accurately and easily measure the number of microorganisms.

  In addition, since the amount of the sample is very small, even if the sample is collected from the same sample, a stable test result cannot be obtained and the variation is large.

Furthermore, in terms of detection sensitivity, there is a problem in accuracy when measuring raw milk having a number of microorganisms of 10 ⁶ cells / ml or less due to the limit of the microscope magnification. Therefore, in the case of raw milk with the number of microorganisms of 10 ⁴ cells / ml or less, there is a problem that it is necessary to concentrate at least 100 times before staining, and a stable test result is obtained, and microorganisms are measured to 10 ⁴ cells / ml. It is requested.

  In addition, the detection method of Christine M Cousins (see Non-Patent Document 1) has a problem in the recovery rate of bacteria because only the supernatant is collected using a centrifuge. There is a problem that the decomposition of the components becomes insufficient, and there is a demand for enabling stable component decomposition with high microorganism recovery performance.

  Further, there is a problem that the sample processing requires about 1 hour in the component processing time, and it is required to speed up the processing time.

  In addition, there is a problem that a dark color filter is affected by decoloration or deterioration due to the influence of a surfactant, and the filter is not decolored or deteriorated.

  In addition, milk components are separated and decomposed so that they can be filtered at 0.6 μm. However, since microorganisms contained in milk components also contain microorganisms of 0.6 μm or less, the microorganisms pass through when collecting the membrane filter. Therefore, there is a demand for a filter having a pore size with a higher ability to collect microorganisms.

  In addition, since the components after separation and decomposition of milk components are 0.6 μm or less, there is a problem that components larger than microorganisms remain, and there is a problem that sufficient processing has not been performed, and microorganisms can be collected with a filter, In addition, it is required that milk components do not pass through the filter and remain.

  The present invention solves such a conventional problem, and by making milk components and emulsion particles in dairy products into fine particles by pretreatment, microorganisms can be rapidly and highly accurate without requiring skilled skills. An object of the present invention is to provide a microorganism detecting method and a microorganism measuring apparatus that can measure easily and can stably measure reproducibility using a filter that does not affect the measurement.

  In addition, there is a demand for evaluating microorganisms and cells such as streptococci and somatic cells in raw milk to manage the quality of raw milk as a dairy material and the health of dairy cows.

  This invention solves such a conventional subject, and it aims at providing the microorganisms detection method and microorganisms measuring device which can measure to the physical condition of a cow.

  In order to achieve the above object, the microorganism rapid detection method and the microorganism weighing device of the present invention detect microorganisms by making emulsion particles finer than microorganisms in a specimen that may contain emulsion particles and microorganisms. This is a microorganism detection method characterized by the above.

  In order to achieve the above-mentioned object, the microorganism rapid detection method and the microorganism weighing apparatus of the present invention are microorganism detection methods in which emulsion particles are finely divided and microorganisms are filtered and extracted.

  In order to achieve the above object, a rapid microorganism detection method and a microorganism weighing device according to the present invention are microorganism detection methods characterized by filtering and extracting microorganisms with the specimen as fine particles of 0.2 μm or less. .

  In order to achieve the above object, the microorganism rapid detection method and the microorganism weighing device of the present invention are microorganism detection methods that prevent aggregation after the emulsion particles are finely divided.

  In order to achieve the above object, the microorganism rapid detection method and microorganism weighing device of the present invention are microorganism detection methods in which a chelating agent is added to prevent aggregation.

  In order to achieve the above object, the rapid microorganism detection method and the microorganism measuring device of the present invention are a microorganism detection method characterized by adding an aqueous solution of hexametaphosphate that does not affect microorganisms as a chelating agent.

  In order to achieve the above object, the rapid microorganism detection method and the microorganism measuring device of the present invention are a microorganism detection method in which 0.01 to 10% of a hexametaphosphoric acid concentration is added to a specimen that may contain emulsion particles and microorganisms. Is.

  In order to achieve the above object, the microorganism rapid detection method and microorganism weighing device of the present invention are microorganism detection methods in which an amide-based nonionic surfactant is added to form fine particles of 0.2 μm or less.

  In order to achieve the above object, the microorganism rapid detection method and microorganism weighing device of the present invention are microorganism detection methods that use an organic solvent that enhances dispersibility as a solvent for an amide-based nonionic surfactant. is there.

  In order to achieve the above object, the microorganism rapid detection method and the microorganism weighing device of the present invention are characterized by providing a temperature for reducing the viscosity of a specimen that can contain emulsion particles and microorganisms in order to obtain fine particles of 0.2 μm or less. This is a method for detecting microorganisms.

  In order to achieve the above object, the rapid microorganism detection method and the microorganism weighing device of the present invention are microorganism detection methods characterized in that a proteolytic enzyme is added to form fine particles of 0.2 μm or less.

  In order to achieve the above object, the microorganism rapid detection method and the microorganism weighing device of the present invention are microorganism detection methods in which an amide-based nonionic surfactant and a proteolytic enzyme are mixed and added.

  In order to achieve the above object, the microorganism rapid detection method and the microorganism measuring apparatus of the present invention are characterized by using a heating means for promoting degradation after the addition of an amide-based nonionic surfactant and a proteolytic enzyme. This is a microorganism detection method.

  In order to achieve the above object, a microorganism rapid detection method and a microorganism weighing apparatus according to the present invention are microorganism detection methods characterized in that the temperature of the heating means is 40 to 70 ° C.

  In order to achieve the above object, the microorganism rapid detection method and the microorganism weighing device of the present invention add one or more kinds of ionic surfactants for reducing the viscosity to amide nonionic surfactants and proteolytic enzymes. This is a microorganism detection method.

  In order to achieve the above object, the microorganism rapid detection method and the microorganism measuring device of the present invention are characterized by using a fatty acid ester solvent with reduced water content as a solvent for the amide nonionic surfactant and the ionic surfactant. This is a microorganism detection method.

  In order to achieve the above object, the microorganism rapid detection method and the microorganism weighing device of the present invention include a microorganism collection filter for filtering and extracting microorganisms, and adjust the pH so that the properties of the microorganisms do not change on the filter surface. The present invention provides a microorganism detection method comprising means.

  In order to achieve the above object, the microorganism rapid detection method and the microorganism weighing device according to the present invention are provided with a microorganism collection filter for filtering and extracting microorganisms, and the filter is a microorganism detection method in which the surface shape is not deformed. is there.

  In order to achieve the above object, the microorganism rapid detection method and the microorganism weighing device of the present invention are microorganism detection methods in which the microorganism collection filter is a filter that does not lose color.

  In order to achieve the above object, the method for rapidly detecting microorganisms and the microorganism measuring device in dairy products of the present invention are microorganism detection methods in which a pigment is supported on a microorganism collection filter.

  In order to achieve the above object, the microorganism rapid detection method and microorganism weighing apparatus of the present invention are microorganism detection methods in which the microorganism collection filter is a dark filter.

  In order to achieve the above object, the microorganism rapid detection method and the microorganism weighing device of the present invention are microorganisms in which a thin film containing at least one metal component selected from gold, copper, chromium, platinum and palladium is formed on a microorganism collection filter. This is a detection method.

  In order to achieve the above object, the microorganism rapid detection method and the weighing apparatus of the present invention are microorganism detection methods in which microorganisms captured on a microorganism collection filter are detected or counted by a staining method.

  In order to achieve the above object, the microorganism detection method and the microorganism weighing device of the present invention are microorganism detection methods in which the staining method is a fluorescent staining method.

  In order to achieve the above object, the method for rapid detection of microorganisms and the microorganism weighing device of the present invention comprises a first reagent in which the staining method reacts only with living cells to develop color of only living cells, and reacts with only dead cells to die. A second reagent that causes only cells to develop color at a wavelength different from that of the color; a third reagent that reacts with both living and dead cells to cause both living and dead cells to develop color at a wavelength different from the color; and the microorganism of the microorganism-derived substance The microorganism detection method uses any one kind or plural kinds of reagents among at least one kind or more of the fourth reagent that develops color at a wavelength different from that of the color development by reacting with a substance unique to the above.

  In order to achieve the above object, the microorganism detection method and the microorganism weighing device of the present invention are any one or more of the first reagent, the second reagent, the third reagent, and the fourth reagent. After staining microorganisms with various types of reagents, a light source that irradiates excitation light in a predetermined wavelength range, and light in a predetermined wavelength range that emits light emitted by the excitation light is emitted from the microorganism collection filter. Light receiving means for receiving a set constant area and light emitted by the light source to emit light within a set time, and the received light quantity is within a set threshold range and the set area Microorganism judging means for judging that the range is a microorganism, and movement for moving the microorganism collecting filter or the light receiving means in order to confirm the light emission point of one part or the whole area of the microorganism collecting filter It determines the stage, and one light emitting point and one microorganism is determined that microbial by microbial determination means, by sequentially accumulating, in which the microorganism detection method having means for accumulating the number of microorganisms.

  In order to achieve the above object, the microorganism detecting method and the microorganism measuring device of the present invention are a microorganism measuring device in which the light receiving means is a plurality of photoelectric conversion elements having at least an area where the size of the microorganism can be recognized. .

  In order to achieve the above object, the method and apparatus for detecting microorganisms in dairy products according to the present invention have one or more types of light sources, and one or more types of predetermined wavelength ranges. Light receiving means for receiving light emitted in a plurality of types of wavelength ranges, and a point or area emitted from the ratio of the amount of light in each wavelength range. Is a microorganism measuring device having a microorganism judgment to judge that the microorganism is a microorganism.

  In order to achieve the above object, the microorganism detection method and the microorganism weighing device of the present invention switch the light source and light receiving means of one type or a set type of light receiving means for receiving the plurality of types of light sources and a plurality of wavelength ranges, Only the set type is a microorganism weighing device that detects microorganisms with a light source and a light receiving means in the wavelength range.

  In order to achieve the above object, the method for detecting microorganisms and the microorganism measuring apparatus in the dairy product according to the present invention sequentially integrates the areas of the luminescent spots recognized as other than the microorganisms by the microorganism judging means or more than the set area. The microorganism weighing device has a caution means for indicating a caution when the total area or total number is equal to or greater than the set area or number.

  In order to achieve the above object, the microorganism detecting method and the microorganism measuring apparatus of the present invention are a microorganism measuring apparatus having a somatic cell recognizing means for recognizing a size within a set range as the microorganism judging means as a somatic cell. is there.

  In order to achieve the above object, the microorganism detecting method and the microorganism measuring apparatus of the present invention have somatic cell recognizing means for recognizing a size within a set range as the microorganism judging means, so that both the microorganism and the somatic cell are recognized. It is a microorganism measuring device that detects

  In order to achieve the above object, the microorganism detecting method and the microorganism measuring device of the present invention determine that a microorganism is agglomerated from the size of one light emitting point determined to be a microorganism by the microorganism judging means, and determine the microorganisms in the agglomerated size. This is a microorganism measuring device having means for integrating the number of microorganisms by converting and sequentially integrating the quantities.

  In order to achieve the above object, the microorganism detecting method and the microorganism measuring apparatus of the present invention determine the microorganisms, the mass of microorganisms, and the somatic cells by the microorganism determining means, and display the number of microorganisms, the number of microorganisms converted, and the number of somatic cells. A microbial weighing apparatus comprising:

  According to the present invention, in a specimen containing emulsion particles, the emulsion particles can be collected on the filter by making the emulsion particles finer than the size of the microorganisms. It becomes possible. Moreover, the microbe detection and microbe measurement apparatus which improved the precision and sensitivity can be provided by making emulsion particles into fine particles of 0.2 μm or less.

  In addition, since the emulsion particles are 0.2 μm or less, it is easy to distinguish them from microorganisms, the work of using the microscope for a long time can be reduced, and the burden on the naked eye of the observer can be reduced. Obtainable.

  In addition, by detecting microorganisms by a staining method or fluorescent staining method, skilled skills are not necessary, and anyone can quickly and easily detect and measure microorganisms with high accuracy.

  In addition, since filtration can be performed easily, it is possible to filter a large amount of specimens, improving the measurement sensitivity of microorganisms per unit amount, and detecting stable microorganisms with little variation even when collected from the same specimen. And weigh.

  Further, unlike the method of centrifuging, since the entire amount of the sample is filtered, it is possible to detect and measure the microorganism with a very high recovery rate of the microorganism from the sample.

  Moreover, since emulsion particles can be made fine in a short time, microorganisms can be detected and measured more rapidly.

  In addition, since the filter does not deform or decolorize the filter shape even when a surfactant is added to the specimen, it can be accurately measured without affecting the detection and measurement of microorganisms, even when measured by staining or fluorescent staining. Can detect and measure microorganisms.

  In addition, microorganisms that can evaluate the health of dairy cows can be detected and measured using a test sample of raw milk.

  According to the first aspect of the present invention, in a specimen containing emulsion particles, the emulsion particles in a dairy product are filtered out into a filtrate and discharged by making the emulsion particles smaller than the size of the microorganism. This is a microorganism detection method characterized by collecting microorganisms contained in a dairy product on a filter and detecting the microorganisms, and has the effect of efficiently detecting microorganisms with a simple configuration. Making emulsion particles smaller than the size of microorganisms means separating and dissolving emulsion particles that are larger than the size of microorganisms, and making emulsion particles smaller than the size of microorganisms. Dissolve the surroundings to make the particles smaller, disperse the agglomerated particles to reduce the mass of the particles, prevent emulsion particles from becoming larger than the size of the microorganisms due to reaggregation, etc. May be reduced, or liquefaction may be used.

  In addition, the present invention is a microorganism detection method in which emulsion particles are microparticulated to extract microorganisms by filtration, and has an effect that the number of microorganisms in a sample amount can be measured by filtration.

  Further, it is a method for detecting microorganisms by filtering and extracting microorganisms with a fine particle of 0.2 μm or less, and has the effect of removing impurities and accurately measuring the number of microorganisms in the sample amount. .

  Further, it is a method for detecting microorganisms that prevents aggregation after the emulsion particles are atomized, and has an effect that the measurement can be performed even if the measurement sample that has been atomized is left for a long time.

In addition, it is a microorganism detection method in which a chelating agent is added to prevent aggregation,
It has the effect that it can be measured even if it is left for a long time without changing the properties of the micronized measurement specimen.

  In addition, it is a microorganism detection method characterized by adding an aqueous solution of hetametaphosphate that does not affect microorganisms as a chelating agent, and it does not change the properties of the micronized measurement sample, and further identifies calcium in the sample By chelating, the change in the shape of the microorganisms contained in the specimen is suppressed, and the measurement is performed even if left for a long time.

  In addition, a method for detecting microorganisms in which the concentration of hexametaphosphoric acid is added in an amount of 0.01 to 10% with respect to a specimen that may contain emulsion particles and microorganisms. Raw milk is obtained by specifying and chelating calcium in raw milk. It also suppresses changes in the shape of microorganisms contained in the specimen and has an effect of measuring even if left for a long time.

  In addition, an amide-based nonionic surfactant is added to make fine particles having a size of 0.2 μm or less, and has an effect of making components other than microorganisms in raw milk into fine particles.

  In addition, it is a microorganism detection method using an organic solvent that enhances dispersibility as a solvent for amide-based nonionic surfactants, and it improves the reactivity with the specimen and shortens the time required for micronization. Have

  In addition, the present invention provides a microorganism detection method characterized by providing a temperature at which the viscosity of a specimen that can contain emulsion particles and microorganisms is reduced in order to obtain a fine particle of 0.2 μm or less, further increasing the reactivity with the specimen. Have the effect of being able to process in a short time.

  In addition, it is a method for detecting microorganisms in which a proteolytic enzyme is added to form fine particles of 0.2 μm or less, and has an action of degrading proteins other than microorganisms and further selecting only microorganisms.

  Further, it is a microorganism detection method in which an amide-based nonionic surfactant and a proteolytic enzyme are mixed and added, and has the effect of reducing the number of samples and the number of mixing and improving workability.

  In addition, it is a microorganism detection method characterized by using a heating means that promotes degradation after the addition of an amide-based nonionic surfactant and a proteolytic enzyme. Of course, it has the effect of accelerating the degradation of proteins other than microorganisms and setting the measurement state in a short time.

  In addition, the microorganism detection method is characterized in that the temperature of the heating means is 40 to 70 ° C., enhances the enzyme reaction, promotes protein decomposition and dissolution, decreases the viscosity of the measurement sample, It has the effect of facilitating the handling of the specimen including handling.

Further, the present invention is a microorganism detection method to which a surfactant and one or more types of ionic surfactants that lower the viscosity are added, and has an effect of lowering the viscosity of the specimen.

  In addition, the present invention is a microorganism detection method characterized by using a fatty acid ester solvent with reduced moisture as a solvent for amide-based nonionic surfactants and ionic surfactants. It has the effect of reducing the reaction and adapting to various specimens.

  In addition, the microorganism detection method includes a microorganism collection filter for filtering and extracting microorganisms, and a means for adjusting pH so that the properties of the microorganisms do not change on the filter surface. It has the effect of enhancing the sensitivity and detecting microorganisms with high sensitivity.

  In addition, the microorganism detection method includes a microorganism collection filter for filtering and extracting microorganisms, and the filter is a filter whose surface shape is not deformed, and improves the flatness of the filter surface to clarify the detection position of microorganisms. The device configuration at the time of detection / measurement can be simplified.

  The microorganism collection method is a microorganism detection method in which the filter for collecting microorganisms is a dark filter that does not lose color, and has the effect of suppressing the luminance of the background during fluorescence detection and increasing the sensitivity.

Further, it is a microorganism detection method in which a thin film containing at least one metal component selected from gold, copper, chromium, platinum, and palladium is formed on a filter for collecting microorganisms. In this case, it is possible to prevent reflection of light at the time, and to accurately detect and measure microorganisms without interfering with the detection and measurement of microorganisms.

  Further, it is a microorganism detection method for detecting or counting microorganisms captured on a microorganism collection filter by a staining method or a fluorescence staining method, and has an action of optically measuring emitted microorganisms.

  A first reagent that reacts only with living cells to develop color only of living cells; a second reagent that reacts only with dead cells and develops color of only dead cells with a wavelength different from the color development; A third reagent that reacts with any of the cells and causes any of the living and dead cells to develop color at a wavelength different from that of the color development; and at least develops a color at a wavelength different from the color development by reacting with a microorganism-derived substance specific to the microorganism. One or more kinds of fourth reagents are brought into contact with the specimen, and the function of detecting both living cells and dead cells from the wavelength difference and the color development amount is provided. Have.

  In addition, after staining the microorganism with any one kind or plural kinds of reagents among the first reagent, the second reagent, the third reagent, and the fourth reagent, a predetermined wavelength is used. A light source that emits excitation light in a region; light receiving means that receives light of a predetermined wavelength region that is emitted by the excitation light and emits light; Microorganism judging means for receiving emitted light within a set time and determining that the received light quantity is within a set threshold range and within a set area range as a microorganism, and collecting the microorganism In order to confirm the light emission point of a part of the filter or the entire area, the microorganism collection filter or the moving means for moving the light receiving means, one light emitting point determined as a microorganism by the microorganism determining means, and the microorganism 1 It is determined that, by sequential accumulation, which comprises means for integrating the quantity of microorganisms has the effect that differentiate weighed luminescent substance and microorganisms.

  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. The light receiving means for receiving light of each type of wavelength range and the function of detecting and detecting the point or area emitted from the ratio of the amount of light in each wavelength range as a microorganism.

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

  In addition, by the microorganism judging means, the area of the light emitting points recognized as other than the microorganism because it is larger than the set area is sequentially accumulated, or the area is recognized as one and the number is sequentially accumulated, 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.

  Moreover, it has the effect | action of recognizing the magnitude | size within the range set with the microorganisms determination means as a somatic cell.

  Further, it has somatic cell recognition means for recognizing a size within the set range as a microorganism judging means as a somatic cell, and has an effect of detecting both the microorganism and the somatic cell.

  In addition, there is a means for accumulating the number of microorganisms by determining the mass of microorganisms from the size of one light emitting point determined to be a microorganism, converting the number of microorganisms in the size of the mass, and sequentially accumulating them. And has the effect of accurately measuring the microorganism concentration in the specimen.

  In addition, it has means for judging microorganisms and microbial masses and somatic cells by the microorganism judging means and displaying them in each item, and it can accurately measure the microorganism concentration and somatic cells in the raw milk sample simultaneously. Have

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

(Embodiment 1)
For example, there are dairy products such as milk and raw milk, as well as milk coffee and various foods that use them as raw materials. When components such as emulsion particles contained in dairy products are filtered, the filter pores are blocked and filtered. Is disturbing. In addition, proteins themselves become fluorescent substances and are difficult to distinguish from microorganisms when observed with a microscope, and also interfere with observation of microorganisms. 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. In other words, in order to filter dairy products, it is necessary to subdivide the emulsion particles contained in the dairy products and filter and extract fine particles of 0.4 μm or less or completely dissolved (ionic) microorganisms. .

  When filtering with a 0.4 μm filter, it is necessary that the raw milk components and emulsion particles be 0.4 μm or less, at least 0.2 μm or less, and preferably 0.1 μm or less. Of course, in reality, the particles are not uniformly 0.2 μm, but become fine with a certain normal distribution. Therefore, it is desirable that at least the center value of the particles is about 0.2 μm. If the maximum particle size is 0.2 μm or less, it can be applied to almost all filters, so it is more desirable if the center value is about 0.1 μm.

  Filter types are PP (Polypropylene), PVC (Polyvinyl Chloride), PC (Polycarbon), PTFE (Polytetrafluorethylene), PVDF (Polyvinylidene fluoride, PE) Depending on these filters, filtration is not always possible unless the thickness is 0.4 μm or less. For example, since MCE has flexibility in pores, some surface activity is required to improve filtration performance. Filtering is possible by dropping only the agent into the filter in advance and filtering the heated milk (50 ° C) afterwards. Become. However, in this method, the surface of the MCE is uneven, or microorganisms are buried in the pores, so that accuracy is insufficient when observing with a microscope.

  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 substances and components other than microorganisms are made into particles with a particle size smaller than the pores, filtration becomes possible, and there are few irregularities on the surface and the microorganisms are not buried in the pores. It is also desirable for mechanical measurements.

  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. By making these components also 0.2 μm or less, it becomes possible to distinguish them from microorganisms. General E. coli and Staphylococcus aureus have a size of about 0.5μm, so the components of dairy products and emulsion particles are 0.2μm or less so that even if these particles are stained, they are distinguished by size. Is possible. If the thickness is 0.1 μm, it becomes possible to distinguish more easily. However, even if it is 0.1 μm or less, if those components are covered on the filter, the entire filter will be stained, and it may be difficult to find the stained microorganisms, so that more accurate measurement is possible. It is desirable to decompose these components.

  Further, it was confirmed by microscopic observation that Escherichia coli and Staphylococcus aureus were collected by using a 0.4 μm filter. In addition, in the case where small bacteria such as Serthia are present, collection is possible by using a filter having a pore size of 0.2 μm.

  Therefore, various surfactants are mixed with raw milk in order to make emulsion particles fine, and the pore size of the PC filter is 0.4 μm. It was confirmed whether the component remained. The confirmed result is shown in FIG. In Tween 20, Tween 80, and Triton X-100, the liquid after the reaction process may or may not become a colored transparent liquid, so stable processing cannot be performed and sufficient reaction cannot be obtained. It is presumed that filtration becomes difficult and the treatment liquid becomes cloudy and large particles are present. In MEGA8, lipolane, RDA, and SDS, it was confirmed that the liquid after the reaction treatment became cloudy and filtration was difficult, and the residual effects of the components were large, greatly affecting the measurement. It is confirmed that amide-based nonionic surfactants are stable and can be filtered, the liquid after the reaction treatment is also stably processed into a transparent liquid, and there are no substances that hinder measurement. It has been found that it is effective as a surfactant for making the components in the product fine. Normally, raw milk is cloudy, but when mixed with an amide-based nonionic surfactant, it became transparent visually. Generally, when emulsion particles are mixed, the smaller the particles, the more transparent. Although it varies depending on the amount of particles, it is determined that the particle size is at least 0.2 μm or less, and since it is visually transparent, it seems that the particle size is about 0.1 μm. Ingredients contained in dairy products are often emulsions. In the present invention, a surfactant is used as a method of breaking or reducing emulsion particles. However, physical means such as homogenization and ultrasonic Even various chemical methods for changing pH and the like may be used as long as the method does not dissolve or lyse microorganisms. This is because when lysed, the nucleic acid is eluted from the cell membrane or cell wall and cannot be detected. Of course, if the method that does not damage or affect the microorganism is the best method, the staining or fluorescent staining method can distinguish the life and death of the microorganism, so that the shape of the microorganism can be maintained and the surface of the microorganism can be dissolved. Any method that does not lyse can be measured.

  In addition, it was confirmed that the amide-based nonionic surfactant had no components that interfered with the measurement, and the microbial surface was dissolved or not lysed. This is because the emulsion particles have a particle size of 0.2 μm or less, and even when visually confirmed under a microscope, it is easy to distinguish microorganisms from other particles.

  In addition, if left after reacting with the amide ion surfactant, the fatty acid in the raw milk component may recombine with calcium, reaggregate with calcium as the core, and change translucently. Transparency can be maintained even after the reaction by reacting the active agent with raw milk or adding a chelating agent that chelates calcium before and after the reaction. As a chelating agent, ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA) diethylenetriaminepentaacetic acid (DTPA), dicarboxymethyl glutamic acid tetrasodium salt (GLDA), hydroxyethylethylenediaminetriacetic acid (HEDTA), aminoethylethylene glycol ( GEDTA), triethylenetetramine hexaacetic acid (TTHA hexametaphosphoric acid, etc.), and a concentration of about 0.01 to 10% of the amount of raw milk may be used as a concentration that does not affect microorganisms in raw milk.

However, even after reacting raw milk with a chelating agent and reacting with an amide ion surfactant, if there is a lot of protein in raw milk, the protein may remain and cover the microorganism collection filter for measurement, When fluorescent staining is performed, the fluorescent reagent remains in the protein and emits light although it is thin. When the microorganism collecting filter is covered, a means for degrading protein with an enzyme or the like may be used. It is possible to measure with higher accuracy by disassembling.
Examples of proteolytic enzymes include proteinases, peptidases, trypsinogens, chymotrypsinogens, and the like, but they are capable of degrading proteins by giving a temperature although they have different active sites.

  In addition, when Triton X-100 or other surfactants are used in raw milk, many luminescent substances that appear to be other than microorganisms can be seen when judging the shape with a microscope, making it difficult to distinguish microorganisms and accurately detecting and measuring microorganisms. It can be difficult to do.

Also, depending on the type of amide-based nonionic surfactant, the viscosity may be high, and filtration may take time even if the components of the dairy product are fine particles. In water, the viscosity does not decrease, but it may become cloudy and make filtration difficult. In such a case, the addition of an ionic surfactant further reduces the viscosity and improves the filtration performance. There are five types of ionic surfactants: 1) fatty acid (anion), fatty acid sodium, fatty acid potassium, alpha sulfo fatty acid ester sodium, 2) linear alkyl benzene, linear alkyl benzene sulfonic acid Sodium, 3) Higher alcohols (anions), sodium alkyl sulfates, sodium alkyl ethers, 4) Alpha olefins, sodium alpha olefin sulfonates, 5) Normal paraffins, sodium alkyl sulfonates, etc. However, any reagent may be used as long as it does not cause a chemical reaction with the amide-based nonionic surfactant and does not dissolve or lyse microorganisms.

  Also, depending on the amount of moisture in raw milk, mixing an aqueous solution of an amide non-surfactant and an ionic surfactant may promote reaggregation and become cloudy. The amide non-surfactant and ionic surfactant It is necessary to reduce the amount of the aqueous solution. By using fatty acid ester glycerin, propylene glycol or the like instead of the aqueous solution, the influence of moisture can be reduced, re-aggregation can be prevented, and white turbidity can be prevented.

  After filtering, when dyed or fluorescently stained microorganisms are observed with a microscope 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.

  When a surfactant is used when making emulsion particles into fine particles, the color tends to be lost due to the influence of the surfactant, and as a result, the material portion of the filter is exposed and cannot be measured. 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. Further, even when a solvent that dissolves like a surfactant is used, the metal is not dissolved and the filter material is not exposed, so that measurement is not disturbed.

  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 fluorescent luminescence reagents 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.

  When conducting microbiological tests on dairy products, the larger the sample amount, the better. In the conventional bleed method, since 0.01 ml is the maximum, the amount of the sample can be 0.1 ml or 1 ml or more by filtration, and the bacteria concentration is measured as the number per 1 ml. The greater the number, the better the sensitivity and accuracy.

(Embodiment 2)
FIG. 2 is a block diagram showing an embodiment of the microorganism weighing device. This microorganism weighing device includes a lens 1 and a light receiving unit 2 as light source condensing means. In order to extract the 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 passes through the lens 1 and includes a microorganism collecting filter 7 installed on the examination table 6, that is, a combined microorganism collecting filter 7 composed of the microorganism collecting filter 7 and the pedestal 8. Focused on the surface. Therefore, the fluorescence of the microorganisms excited by the excitation light passes through the prism 5 again and reaches the light receiving unit 2. The fluorescence that has reached the light receiving unit 2 passes through the fluorescence spectroscopic filter 9 in order to extract only the target fluorescence, reaches the photoelectric conversion element 10 built in the light receiving unit, is converted into a signal, and is recognized. Although not shown in the figure, this microorganism weighing device includes means for moving the inspection table 6 and can receive all or part of the fluorescence emitted from the surface of the filter 7 for collecting microorganisms.

  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. And those that are excited and absorb fluorescence at 617 nm and emit fluorescence at 617 nm are judged as dead bacteria, and those that do not emit fluorescence at both 461 nm and 617 nm are judged as foreign substances. The fluorescence determined to be microorganisms is integrated and the quantity is measured. The microorganism judging means 11 includes a microcomputer programmed with the judging procedure and judging method.

  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 a microorganism by the microorganism 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 the microorganism 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 part including the filter 7 for collecting microorganisms has a rotating ability. The excitation light collected by the lens 1 moves a distance corresponding to the radius from the outer periphery of the microorganism collection filter 7 to the center or from the center to the outer periphery. 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. 2, the inspection table 6 has a depressed portion (apparatus groove) for fitting a portion including the microorganism collection filter 7, and the portion including the microorganism collection filter 7 can be directly incorporated therein. It can be shaped. At this time, for example, the inspection table 6 is provided with a metal flat plate so that the microorganism collecting filter 7 is positioned thereon, and the microorganism collecting filter 7 is incorporated in a state of being pressed against the metal flat plate. Thus, if the microorganism collection filter 7 is held smoothly on the inspection table 6 without unevenness, the quantification of the microorganisms collected in the microorganism 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 small fixed area for irradiating the excitation light is not limited to a polygon including a square, but may be a circle, an ellipse, or the like as long as it can irradiate the specimen.

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 that can grasp the trajectory on the microorganism collection filter 7.

Example 1
FIG. 3 is a flowchart for making emulsion particles finer than the size of microorganisms in a specimen that may contain emulsion particles and microorganisms of the present invention. Raw milk was used as a specimen that could contain emulsion particles and microorganisms.

  As a method for making emulsion particles of raw milk fine, 0.1 ml of raw milk is collected in step 1) and drawn into a reaction vessel. Furthermore, 0.5 ml of reagent A is added. Reagent A was an amide-based nonionic surfactant, Amizole. About 3% of the reagent for amizole was mixed with raw milk. As a step 2), the whole liquid is mixed with a stirrer so that the raw milk and amizole are sufficiently contacted in the liquid. As step 3), incubation is performed at a constant temperature and time in order to enhance the atomization reaction of the components by amizole. The temperature at this time is 40 ° C to 60 ° C, but is preferably around 50 ° C. The treatment time is 5 to 20 minutes, preferably around 10 minutes. By the step, the liquid in step 1) in which the components in the raw milk are finely divided and become cloudy becomes a nearly transparent liquid. As step 4), 1 ml of reagent B for further microparticulation is added to the treatment solution. Reagent B was a protease (Bacillus licheniformis Protease). For the protease concentration, a 10% aqueous solution is used. As step 5), the entire solution is mixed with a stirrer so that the treatment solution (step 3) treatment solution) and protease are sufficiently contacted. As step 6), incubation is performed at a constant temperature and time for activating the reaction by the protease. Although the temperature at this time shall be 40 to 60 degreeC, about 50 degreeC is preferable. The processing time is 5 to 10 minutes, but about 5 minutes is preferable. As step 7), 2 ml of reagent C is added in order to improve the filterability of the microorganism-collecting filter 7. As the reagent C, an ionic surfactant sodium dodecyl sulfate / SDS was used. In step 8), the treatment liquid is mixed with a stirrer to complete the micronization process. Further, the liquid that has become cloudy due to the above configuration becomes a yellow transparent liquid, and the raw milk component is finely divided.

  In specimen staining, 3.6 ml of the treatment solution is filtered through the microorganism collection filter 7 in step 9). In step 10), the remaining components other than microorganisms on the surface of the filter 7 for collecting microorganisms are washed away with 5 ml of filter sterilized water. In step 11), 0.1 ml of a reagent D which is a fluorescent staining reagent for staining the microorganisms collected by the microorganism collection filter 7 is dropped, and the reagent D is spread over the entire surface of the microorganism collection filter 7 and stained for 2 minutes. Do. Reagent D was a reagent that binds to and stains microbial DNA. In step 12), 0.1 ml of the excess reagent in reagent D is washed with sterile filtration water to complete specimen staining.

  In the measurement, the microorganism collection filter 7 is set on the base of the measuring device in step 13). About this stand, it is comprised in the state which becomes a plane by setting the filter 7 for microorganisms collection on a measurement surface, and can thereby perform the stable measurement. In step 14), the microorganisms in the collected dairy product are measured by the microorganism measuring device, and the measurement is completed.

  FIG. 4 shows a flow of measuring microorganisms with respect to an image of one field taken by the microorganism testing apparatus of FIG. When measuring a large number of fields of view, it is possible to calculate the number of microorganisms in the entire filter by repeating this flow and multiplying the ratio of the area of the entire filter and the measured area (field of view area × number of fields of view) by the number of microorganisms measured. it can.

  The results of this example are shown in FIG. It was found that there is a correlation between the conventional method and the method of the present invention.

  In addition, although the density | concentration which mixed the amizole and raw milk was 3% in the Example, even if it uses in the range of 0.03% to 6%, the same effect is acquired.

  In addition, although it does not describe in the present Example about the heating method about incubation, if a reagent and raw milk can be heated with a thermostat, a heater, an ultrasonic wave, an electron beam, etc., there will be no problem in particular. Can be used.

  Although it has been described that the treatment time is preferably about 10 minutes, the property with the mixture is not particularly changed even if the treatment is performed for a long time.

  In addition, although it described as the liquid near transparency, it is a pale or transparent thing at the visual level, and shows what became the microparticles | fine-particles of 0.2 micrometer or less.

  In the examples, an aqueous solution having a protease concentration of 10% was used, but a concentration of 1% to 15% may be used.

  In the examples, an aqueous protease solution was used, but a solvent dissolved in a solution having a pH of 4 to 8 may be used.

  In the examples, an aqueous protease solution was used, but even if propylene glycol or glycerin is used as the organic solvent, there is no difference in performance.

  In the embodiment, the cleaning removal is performed in step 10). However, the cleaning removal process can be omitted particularly when the accuracy of the microorganism measurement is set low.

  The reagent D used is a reagent that binds to and stains microbial DNA, but an antibody reaction, a metabolic enzyme reaction, or a respiratory reaction reagent may be used.

  In addition, although the single dyeing | staining was implemented as the reagent D 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 examples, raw milk was used as a specimen, but an aqueous solution of milk or dairy products can also be used.

  In addition, although it stirred using the stirrer at the time of mixing, if a reagent and a test substance can be stirred, there will be no difference in particular.

(Example 2)
FIG. 6 is a flowchart for finely pulverizing emulsion particles in a specimen that may contain the emulsion particles and microorganisms of the present invention more quickly than the size of the microorganisms. As described above, raw milk was used as a specimen that could contain emulsion particles and microorganisms.

  As a method of making emulsion particles of raw milk into fine particles, 0.1 ml of raw milk is sampled in step 1), drawn into a reaction vessel, and an aqueous reagent E of hexametaphosphoric acid diluted with distilled water as a chelating agent is added to the amount of raw milk. % Concentration is mixed and stirred in step 2).

  In step 3), reagent A is dissolved in a fatty acid ester-based glycerin aqueous solution, and 0.9 ml is added to 0.1 ml of a mixture of raw milk and reagent. In step 4, the entire liquid is mixed with a stirrer.

  At the end of step 4), the liquid in which the components in raw milk are finely divided and become cloudy becomes a nearly transparent liquid.

  As step 5), 1 ml of reagent B for further microparticulation is added to the treatment solution. Reagent B used was a protease, a protease. The whole liquid is mixed with a stirrer so that the protease can be sufficiently contacted. The liquid that has become cloudy due to the above configuration becomes a yellow transparent liquid, and the raw milk component is finely divided.

  As step 6), incubation is performed at a constant temperature and time for activating the reaction by the protease. The temperature at this time is 60 ° C.

  The processing time is 3 to 10 minutes, but about 5 minutes is preferable.

  In step 5), the treatment liquid is mixed with a stirrer to complete the micronization process.

  FIG. 7 shows an image of fluorescent emission after fluorescent staining after completion of the pretreatment. 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 medium fluorescence emission point was a streptococcus with solid bacterial cells. Furthermore, it was confirmed that a larger fluorescent emission point was a somatic cell. It was clarified that what was captured by the filter for collecting microorganisms after completion of the pretreatment can measure items that can be used as indicators of contamination in dairy cows and raw milk.

  FIG. 8 shows a flow of measuring microorganisms for an image of one field taken by the microorganism testing apparatus of FIG. When measuring a large number of fields of view, it is possible to calculate the number of microorganisms in the entire filter by repeating this flow and multiplying the ratio of the area of the entire filter and the measured area (field of view area × number of fields of view) by the number of microorganisms measured. it can. Furthermore, by using the size microorganism judging means 11, it becomes possible to separate and measure microorganisms and somatic cells.

  Although not shown in the figure, the medium fluorescent point is counted as the number of microorganisms at the medium fluorescent point by counting and counting the number of medium-sized microorganisms after determining that it is a microorganism. By converting, it is possible to measure the number of microorganisms in a lump of bacteria such as streptococci.

  In the examples, the aqueous solution reagent E of hexametaphosphoric acid was set to a concentration of 1% with respect to the amount of raw milk, but there is no problem if the concentration is 0.1% to 10%.

  Further, although the results of this example are not shown, they were the same as those shown in FIG. As in Example 1, it was confirmed that there was a correlation between the method of the present invention and the conventional method.

  The microorganism detection method and the microorganism measuring apparatus for detecting microorganisms by making emulsion particles finer than the size of microorganisms in a specimen that can contain emulsion particles and microorganisms according to the present invention do not require skilled skills, and anyone can use them. Since it can be measured easily, it can be applied to quality control near the production site of dairy products. In recent years, the quality level of raw milk, which is a raw material for dairy products, has improved, and there has been a demand for a device that can measure the number of microorganisms more quickly, accurately, and easily at a low bacterial count level. It can also be applied as a method instead of inspection.

FIG. 5 is a table showing selection of the surfactant according to the first embodiment of the present invention. The figure which shows the one aspect | mode of the microorganisms measuring device of Embodiment 2 of this invention. The flowchart which shows the reaction structure of Example 1 of this invention. Measurement flow chart of embodiment 1 of the present invention The graph which shows the correlation of the conventional method and this invention about the microbial cell in the raw milk of Example 1 of this invention The flowchart which shows the reaction structure of Example 2 of this invention. The figure which shows the fluorescence light emission image of Example 2 of this invention. Measurement flowchart of embodiment 2 of the present invention

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Lens 2 Light-receiving part 3 Light source 4 Excitation light spectral filter 5 Prism 6 Inspection stand 7 Microbe collection filter 8 Base 9 Fluorescence spectral filter 10 Photoelectric conversion element 11 Microorganism judgment means

Claims (34)

A method for detecting microorganisms, characterized in that microorganisms are detected by making emulsion particles finer than the size of microorganisms in a specimen that may contain emulsion particles and microorganisms. The microorganism detection method according to claim 1, wherein the emulsion particles are micronized and the microorganisms are filtered and extracted. The method for detecting a microorganism according to claim 1 or 2, wherein the microorganism is filtered and extracted by converting the specimen into fine particles of 0.2 µm or less. The method for detecting a microorganism according to any one of claims 1 to 3, wherein aggregation is prevented after the emulsion particles are made fine. The method for detecting a microorganism according to any one of claims 1 to 4, wherein a chelating agent is added to prevent aggregation. 6. The microorganism detection method according to claim 5, wherein an aqueous solution of hexametaphosphate that does not affect microorganisms is added as a chelating agent. The microorganism detection method according to claim 6, wherein the concentration of hetametaphosphate is 0.01 to 10% with respect to a specimen that may contain emulsion particles and microorganisms. The microorganism detection method according to any one of claims 1 to 7, wherein an amide-based nonionic surfactant is added to form fine particles of 0.2 µm or less. The microorganism detection method according to claim 8, wherein an organic solvent that enhances dispersibility is used as a solvent for the amide-based nonionic surfactant. The method for detecting a microorganism according to any one of claims 1 to 9, wherein a temperature for reducing the viscosity of a specimen that can contain emulsion particles and microorganisms is applied in order to obtain fine particles of 0.2 µm or less. The method for detecting a microorganism according to any one of claims 1 to 10, wherein a proteolytic enzyme is added to form fine particles of 0.2 µm or less. The microorganism detection method according to any one of claims 1 to 11, wherein an amide-based nonionic surfactant and a proteolytic enzyme are mixed and added. 13. The microorganism detection method according to claim 12, wherein a heating means for promoting the decomposition is used after the addition of the amide-based nonionic surfactant and the proteolytic enzyme. 14. The microorganism detection method according to claim 13, wherein the temperature of the heating means is 40 to 70 ° C. The microbial detection method according to any one of claims 1 to 14, wherein one or more ionic surfactants for reducing the viscosity are added to the amide nonionic surfactant and the proteolytic enzyme. 16. The method for detecting microorganisms according to claim 15, wherein a solvent for fatty acid esters with reduced water content is used as the solvent for the amide-based nonionic surfactant and the ionic surfactant. The microorganism detection method according to any one of claims 1 to 16, further comprising a microorganism collection filter for filtering and extracting microorganisms, and means for adjusting pH so that the properties of the microorganisms do not change on the filter surface. The microorganism detection method according to claim 17, further comprising a filter for collecting microorganisms for filtering and extracting microorganisms, wherein the filter is a filter whose surface shape is not deformed. The microorganism detection method according to claim 17 or 18, further comprising a microorganism collection filter for filtering and extracting microorganisms, wherein the filter is a filter that does not lose color. 20. The microorganism detection method according to claim 18 or 19, wherein a pigment is supported on the microorganism collection filter. The microorganism detection method according to any one of claims 18 to 20, further comprising a microorganism collection filter for filtering and extracting microorganisms, wherein the filter is a dark filter. The microorganism detection method according to any one of claims 18 to 21, wherein a thin film containing at least one metal component selected from gold, copper, chromium, platinum, and palladium is formed on the microorganism collection filter. The microorganism detection method according to any one of claims 17 to 22, wherein microorganisms captured on the microorganism collection filter are detected or counted by a staining method. The microorganism detection method according to claim 23, wherein the staining method is a fluorescence staining method. A first reagent that causes only living cells to develop color by reacting only with living cells, a second reagent that reacts only with dead cells and develops only dead cells at a wavelength different from the color, A third reagent that reacts with any of the viable and dead cells to develop a color at a wavelength different from that of the color development, and at least one kind that develops a color at a wavelength different from the color development by reacting with a microorganism-derived substance specific to the microorganism. The microorganism detection method according to claim 23 or 24, wherein any one kind or plural kinds of reagents are used among the fourth reagents. After staining the microorganism with one or more of the first reagent, the second reagent, the third reagent, and the fourth reagent, in a predetermined wavelength range A light source for irradiating excitation light; light receiving means for receiving light of a predetermined wavelength range emitted by the excitation light and receiving a predetermined area set by the microorganism collection filter; and light emitted by the light source. A microorganism judging means for receiving light within a set time and determining that the received light quantity is within a set threshold value range and a set area range as microorganisms; and In order to confirm the light emission point of one part or the entire area, it is determined that the microorganism collection filter or the light receiving means is moved, a light emission point determined as a microorganism by the microorganism determination means and one microorganism. To, by sequentially accumulating microorganisms weighing apparatus for performing the microorganism detection method of claim 25, further comprising means for integrating the quantity of microorganisms. 27. The microorganism measuring apparatus according to claim 26, wherein the light receiving means is a plurality of photoelectric conversion elements having an area where at least the size of microorganisms can be recognized. There are one type or a plurality of types of light sources, one type or a plurality of types of predetermined wavelength ranges, and a plurality of types of light that is emitted in one or more types of wavelength ranges. 28. The microorganism measuring apparatus according to claim 26 or 27, comprising: a light receiving means for receiving light in each wavelength region; and a microorganism determination for determining a light emission point or area as a microorganism from a ratio of light amounts in each wavelength region. 27. The microorganisms are detected by the light source and the light receiving means of only the set type by switching the light source and the 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 microorganism weighing device according to any one of items 28 to 28. The microorganisms judging means sequentially accumulates the areas of the light emitting points recognized as other than microorganisms because it is larger than the set area, or recognizes the area as one and sequentially accumulates the number, and the total area or the total number is the set area The microorganism weighing device according to any one of claims 26 to 29, further comprising a caution means for indicating a caution when the number is more than the number. The microorganism measuring device according to any one of claims 26 to 30, further comprising a somatic cell recognizing unit that recognizes a size within a set range as a somatic cell. 32. The microorganism weighing device according to claim 26, further comprising a somatic cell recognizing unit for recognizing a size within a set range as a somatic cell, and detecting both the microorganism and the somatic cell. Means for accumulating the quantity of microorganisms by judging the mass of microorganisms from the size of one light emitting point judged as a microorganism by the microorganism judging means, converting the number of microorganisms in the size of the mass, and sequentially integrating the quantities. A microorganism measuring device for performing the microorganism detecting method according to any one of claims 26 to 32. 34. The microorganism detection method according to claim 26, further comprising means for determining microorganisms, a mass of microorganisms and somatic cells by a microorganism determination means, and displaying the number of microorganisms, the number of microorganisms converted, and the number of somatic cells. Microbial weighing device.

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