JP2007006709A - Method for discriminating luminescent material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for discriminating a luminescent material which is a method for detecting a specimen containing a cell and a microorganism or having the possibility to contain the cell and microorganism with a fluorescent dye and has improved accuracy as compared with that of the conventionally known methods. <P>SOLUTION: The method for discrimination is carried out as follows. The cell or microorganism stained with a fluorescent dye is excited with a light source 2 and subjected to fluorescence emission. The color information such as hue angle, chromaticity, chroma and brightness of the excited and emitted fluorescence is measured with a charge coupled device unit 10 of a light receiving part 4 and discriminated with an operation part 11. The method for discrimination is a method for discriminating the luminescent material with a reduced measurement error by non-specific adsorption or contamination of an auto-luminescent material and improved accuracy and reliability. Thereby, a small-sized and inexpensive apparatus 1 for discriminating the luminescent material can be provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a measuring method used for detecting microorganisms and a method for discriminating contaminants in the detecting method.

  Conventionally, it is well known that fluorescein fluorescent dyes, which are fluorescent enzyme substrates, are useful as reagents for detecting cellular sites and microorganisms as an example of a method for determining cellular sites and microorganisms using fluorescent dyes. It is a fact that. Fluorescein-based fluorescent dyes, when permeated through cellular sites and cell membranes of microorganisms, are hydrolyzed by esterase enzymes in the cytoplasm and converted into fluorescent substances having a fluorescein skeleton (such as 5-carboxyfluorescein) to emit light Is expressed. Therefore, after contacting the fluorescein fluorescent dye on the substrate on which the cell site or microorganism is immobilized, or capturing the cell site or microorganism contacted with the fluorescein fluorescent dye on the substrate, the excitation light is not emitted. A method for determining a light spot generated by irradiation as a cell site or a microorganism has been known for a long time (for example, see Patent Document 1).

  Patent Document 1 below proposes a method using 5-sulfofluorescein diacetate and 6-sulfofluorescein diacetate, which are esterase activity index indicators, as fluorescent dyes for determining cell sites and microorganisms. This is because, as a result of culturing microbial cells present in soil in various places on an agar medium and conducting a staining evaluation test on the obtained microbial colonies using a fluorescent dye, 5-sulfofluorescein diacetate and Two types of 6-sulfofluorescein diacetate have been proposed as potential fluorescent dyes for viable bacteria detection.

  As another technique, a technique is known in which a fluorescent dye is enclosed in a bacteriophage, the target microorganism is injected with the bacteriophage, and the microorganism is detected (see, for example, Patent Document 2).

This is because the fluorescent dye can be selectively injected into a specific type of microorganism, and at the same time, nonspecific adsorption to contaminants by the free fluorescent dye can be suppressed. Further, at this time, by using a filtration filter having a pore size smaller than that of the microorganism, autofluorescence can be removed, and fine particles that can cause measurement noise and an excessive amount of bacteriophage can be removed.
JP 7-135996 A JP 2000-270892 A

  However, in the conventional method as in Patent Document 1, the fluorescent dye can be detected by reacting with cells or microorganisms contained in the specimen, but it can also be detected by reacting with impurities contained in the specimen. There is a problem that it cannot be detected accurately. As a result, the number of detected cells and the number of microorganisms are apparently larger than the number actually contained, and the detection sensitivity does not increase. This is because, mainly due to the degradability and adsorption of the fluorescent enzyme substrate, it reacts on the surface of contaminants and emits light, even if it is not a cell or a microorganism.

  Further, in the case of the technique as described in Patent Document 2, the binding property and adsorption property of the fluorescent dye can be suppressed by encapsulating the fluorescent dye in the bacteriophage. However, even if this method is used, there is a possibility that the desired detection sensitivity may not be obtained due to the influence of impurities that exhibit autofluorescence.

  The present invention solves such a conventional problem, and detects at least one color characteristic selected from the hue angle, chromaticity, saturation, and brightness of a light emitter, thereby adsorbing impurities and adsorption. An object of the present invention is to provide a discrimination method capable of discriminating from decomposed light emission.

  In addition, dyes with low emission brightness may not be distinguished because the required intensity cannot be obtained due to the cut rate of the light receiving filter, or the dyeing method with very high brightness will be saturated. However, there is a problem that color information cannot be obtained accurately, and it is required to clearly discriminate between low brightness and very high.

  The present invention solves such a conventional problem, and an object of the present invention is to provide a discrimination method capable of increasing the luminance range and detecting with high accuracy.

  In addition, when determining the emission of a fluorescent dye, when measuring the fluorescence using a single band filter or the like, autofluorescence other than the fluorescent dye, and fluorescence due to nonspecific adsorption of the fluorescent dye to impurities are the same. Therefore, there is a problem that it is difficult to discriminate, and a highly accurate discrimination method is required.

  The present invention solves such a conventional problem, and provides means capable of measuring autofluorescence and color information of fluorescent dyes using RGB luminance information and discriminating with high accuracy. is there.

  Also, when staining cells and microorganisms, if the sample scale is large, a large amount of fluorescent dye is required, and the staining power varies depending on the components contained in the sample, so that the sample can be stained stably. It is required to observe the components separately.

  Further, when cells and microorganisms live and have mobility, there is a problem that they cannot be observed for a long time because they move in the liquid, and it is required that they can be observed over time.

  The present invention is to solve such a conventional problem, by immobilizing cells and microorganisms using a membrane filter, remove the sample components, prevent the movement of the cells and microorganisms in the liquid, It is an object of the present invention to provide a discriminating method capable of stably observing luminescence over time in cells.

  In addition, depending on the base material of the collecting means, there are self-emissions with higher brightness than the light emission of the illuminant and base materials provided with the illuminant on the surface of the substrate. There is a problem that light emission other than light emission is detected and light emission cannot be detected, and it is required to absorb the light emitter.

  The present invention solves such a conventional problem, and at the same time suppresses the light emission of the base material, and at the same time, absorbs the light emission of the illuminant in contact with the base material and allows the light emission only in the detection direction. It is intended to provide.

  In addition, since dyeing has various emission wavelengths, the base material must have absorption performance corresponding to the wavelengths, and there is a problem that the types of light emitting means are limited. However, there is a demand for a substrate that suppresses light of various wavelengths.

  The present invention solves such conventional problems, and an object of the present invention is to provide a discrimination method that can be used as a substrate regardless of the emission wavelength and can be detected with high accuracy.

  Further, depending on the light emitting means, there is a problem that sufficient intensity cannot be obtained with a single cell or microorganism, and a light emitting means capable of obtaining sufficient intensity is required.

  An object of the present invention is to solve such a conventional problem, and an object of the present invention is to provide a discrimination method that can sufficiently detect even a small cell or microorganism having a small size by fluorescence.

  Further, the light emitting means has a problem that the staining performance varies every time depending on the activity of cells and microorganisms, and a chemically stable light emitting means is required.

  The present invention solves such a conventional problem, and an object of the present invention is to provide a stable discrimination method by detecting a nucleic acid which is a chemically stable biological component.

  Further, the light emitting means has a problem that the process becomes complicated when it is required to detect the activity of cells and microorganisms, and is required to be easily dyeable with a small number of dyeing steps.

  The present invention solves such a conventional problem, and an object of the present invention is to provide a simple discrimination method capable of evaluating activity with a single light emitting means.

  In addition, because the excitation light is used to heat the cell parts and microorganisms, the amount of water in the cells and microorganisms changes during observation, the fluorescent dye decomposes when dried, or the fluorescence dye changes due to changes in the polarity of the solvent. There is a problem that fluorescence emission cannot be detected due to causes such as flowing out of the cell, and it is required to prevent changes in moisture content and drying during observation.

  The present invention solves such a conventional problem, and an object of the present invention is to provide a discrimination method capable of stabilizing detection by providing an anti-drying effect.

  In addition, a water retentive solution having a high permeability to cells and microorganisms and a highly hydrophobic retentive solution have problems of cytotoxicity and cell dying, and it is required not to kill cells and microorganisms.

  The present invention solves such a conventional problem, and an object of the present invention is to provide a discrimination method capable of having a water retention effect while keeping cells and microorganisms alive.

  In addition, cells and microorganisms may be difficult to permeate the staining reagent due to the hydrophobicity of the surface, and there is a problem of lowering the luminance of light emission, and it is required that the staining reagent has sufficient permeability.

  The present invention solves such a conventional problem, and an object of the present invention is to provide a discrimination method capable of increasing cell membrane permeability and staining with high probability.

  In addition, there is a problem that sufficient intensity cannot be obtained depending on the staining method due to the influence of the pretreatment of the specimen, and a staining method capable of obtaining sufficient intensity is required.

  The present invention solves such a conventional problem, and an object of the present invention is to provide a discrimination method capable of obtaining high luminance by using a luminance sensitizer of a fluorescent dye.

  In order to achieve the above object, the discrimination method of the present invention is a method for detecting cells and microorganisms. In the method for detecting cells and microorganisms, a collecting means for collecting cells and microorganisms, a light emitting means for emitting light by staining, and Color measurement means for measuring at least one color characteristic selected from hue angle, chromaticity, saturation and brightness; and non-specific adsorption and / or non-specific adsorption other than the reactant by the luminescence means and / or the reactant by the luminescence means Equipped with luminescent material judgment means for discriminating self-luminescence other than reactants, which can identify impurities and target cells and microorganisms, improve measurement accuracy, and provide low concentration cells and microorganisms It can be measured.

  There is a discrimination method that can detect only cells and microorganisms from soil and foods containing a large amount of impurities by this means, and can discriminate and detect foreign substances that react with the detection reagent contained in the specimen. can get.

  Further, the color measuring means is characterized by using a CCD or CMOS image receiving element having a large dynamic range of optical luminance, so that cells and microorganisms can be used depending on the color regardless of the sensitivity of the light emitting means. Thus, it is possible to obtain a discriminating method capable of accurately discriminating the difference between the luminescence that has reacted with the impurities and impurities contained in the specimen other than the cells and the microorganisms.

  Further, as the color measuring means, means for measuring RGB luminances is used, and color information can be measured efficiently and accurately.

  In addition, the collection means is characterized by immobilizing cells and microorganisms using a membrane filter, which can remove the influence of the staining-inhibiting component derived from the specimen and spatially immobilize the cells or microorganisms. And can be measured stably over time.

  Moreover, the collection means is characterized by having a function of suppressing the self-emission of the component, and the background derived from the self-emission of the collection means is suppressed and the measurement sensitivity is improved. It can be carried out.

  The collecting means is characterized in that a base material that absorbs light emission is used as the material of the constituent, and a method for determining high measurement sensitivity by absorbing and removing light emission other than the light emitting means. can get.

  Further, the collection means is characterized in that a metal film is formed on the surface of the component, and is less affected by self-emission and can be measured with high sensitivity.

  In addition, a fluorescent dye is used as the light emitting means. Sensitivity can be obtained from one cell, and wavelength information can be obtained at the same time, whereby a highly accurate discrimination method can be obtained.

  In addition, the light emitting means is characterized by using a nucleic acid-staining compound as a fluorescent dye, and by detecting a chemically stable biological component, it is stable without affecting cell activity. Can be obtained.

  Moreover, it is characterized by using a compound capable of degrading intracellular esterase as a fluorescent dye, and cell activity can be measured by a simple staining process.

  Further, the light emitting means is characterized in that a drying inhibitor is mixed and used, and it is possible to stably measure by suppressing a decrease in measurement stability due to fading of fluorescence intensity.

  Moreover, in the light emitting means, sugar alcohols are mixed as an anti-drying agent, and high anti-drying performance can be obtained non-invasively to cells.

  Further, the light emitting means is characterized in that the divalent metal complex is brought into contact with cells and microorganisms, and the ion permeability of the cell membrane can be improved and the fluorescence staining performance can be enhanced.

  Further, in the light emitting means, a luminance sensitizer is mixed with a fluorescent dye and used, and the fluorescence intensity can be easily increased and the detection accuracy can be improved.

  According to the discriminating method of the present invention, by providing the light emitting means, the color feature measuring means, the light emission of the reactant, and the means for discriminating light emission other than the reactant contained in the specimen, sensitivity, accuracy, A highly reliable determination method can be provided.

  In addition, it is possible to provide an effective discrimination method capable of detecting cells and bacteria accompanied by allergies, decaying by microorganisms, and maintaining quality.

  In addition, it is possible to determine with high sensitivity, and it is possible to detect from high concentration contamination of cells and bacteria to low concentration contamination.

  In addition, it is possible to provide a discrimination method that can be detected with higher accuracy and can be easily discarded even when discarded.

  In addition, it is possible to separate impurities, detect them with high sensitivity and accuracy, and count the number of cells and bacteria.

  Moreover, the movement of a cell or bacteria can be prevented and the work at the time of discrimination can be performed efficiently.

  In addition, cells and bacteria can be observed in a live state, and by observing the reaction in real time, it can also be used for discrimination of cytotoxicity, etc.For the development of fungicides and preservatives for quality control. Can also be used.

  Moreover, the retention time of the specimen after preparation can be increased and the working efficiency can be increased by the anti-drying agent.

  In addition, by using a material having low toxicity to cells and bacteria, it is possible to reduce the environmental load, and it is possible to work safely enough for the operator.

  In addition, metal complexes can be bound to the surface of cells and bacteria, so that the penetration of reagents, etc. on the surface can be reduced, changes in osmotic pressure can be suppressed, and living cells and bacteria can be maintained as they are. Since it can be discriminated in real time, actual on-site monitoring and environmental measurement can be easily used.

In addition, a brightness sensitizer can be attached to the fluorescent dye to increase the permeability of reagents and the like on the surface, suppress changes in the osmotic pressure of the cell membrane, and maintain the activity of living cells and bacteria. Since it can be discriminated with high sensitivity and in real time, actual on-site monitoring and environmental measurement can be easily used.

  The invention according to claim 1 of the present invention is a method for detecting cells and microorganisms, a collecting means for collecting cells and microorganisms, a light emitting means for emitting light by staining, and a hue angle of a color emitted by a light emitter, Color measurement means for measuring at least one color characteristic selected from chromaticity, saturation, and lightness, and non-specific adsorption and / or other than the reactant other than the reactant by the luminescence means and / or the reactant by the luminescence means It is characterized in that it has a luminescent material judgment means for discriminating self-luminescence, and it is based on luminescence by the reactant and autoluminescence or staining derived from substances other than the target reactant mixed in the specimen Light emission can be discriminated, and the operation has a function of realizing a highly reliable measurement method.

  The invention described in claim 2 is characterized in that an image receiving element made of a CCD or CMOS having a large dynamic range of optical luminance is used as the color measuring means, and the light emission is weak. Extremely strong light emission, difference in measurement range of 2 to 4 digits or more can be measured at the same time, simplification of measurement method and discrimination method, simplification of discrimination time and discrimination means, and miniaturization It has the effect of being able to.

  The invention described in claim 3 is characterized in that means for measuring RGB brightness are used as the color measuring means, and the color information is RGB, that is, three primary colors of red, green and blue. Therefore, it is possible to obtain the color information with a simple structure.

  Further, the invention described in claim 4 is characterized in that in the collection means, cells and microorganisms are fixed using a membrane filter, measurement of contamination in the pretreatment step, etc., influence of staining inhibitory components This has the effect that a high-precision discrimination method can be realized. In addition, cells or microorganisms can be fixed in a planar manner, and the cells and microorganisms can be accurately detected one by one by scanning the surface.

  Further, the invention according to claim 5 is characterized in that the collecting means has a function of suppressing the self-luminescence of the component, and the background derived from the self-luminescence emitted by the collecting means By limiting the value to a low value, the measurement sensitivity can be improved, and various mechanisms necessary for improving the S / N can be reduced to a minimum, and a simplified discrimination method can be provided.

  The invention according to claim 6 is characterized in that in the collecting means, a base material that absorbs light emission is used as a material of the constituent, and light emission other than the light emission means such as excitation light and autofluorescence is emitted. Absorption and removal have the effect of reducing the background and realizing a discrimination method with high measurement sensitivity.

  The invention according to claim 7 is characterized in that, in the collecting means, a metal film is formed on the surface of the component, and the influence of the self-luminescence derived from the base material is reduced, with high sensitivity. It has the effect that it can be measured.

  The invention according to claim 8 is characterized in that a fluorescent dye is used as the light emitting means, and higher order information can be obtained by combining a plurality of kinds of dyes, and one cell. Therefore, it is possible to obtain a highly accurate discrimination method by obtaining sensitivity.

  The invention according to claim 9 is characterized in that in the light emitting means, a nucleic acid staining compound is used as a fluorescent dye, and by directly detecting a component contained only in a living body, It has the effect that the number can be directly counted specifically.

  The invention described in claim 10 is characterized in that, in the light emitting means, an intracellular esterase-decomposable compound is used as a fluorescent dye, and it is possible to easily detect active cells and microorganisms. Has the effect of being able to

  The invention described in claim 11 is characterized in that in the light emitting means, an anti-drying agent is mixed and used, and the influence of measurement instability due to change in fluorescence intensity resulting from drying and fading. It is possible to suppress measurement and realize highly accurate measurement.

  The invention according to claim 12 is characterized in that in the light emitting means, sugar alcohols are mixed as an anti-drying agent, and since it is liquid at room temperature, it is easy to handle, non-invasive and highly dry to cells. It is possible to obtain the prevention performance stably and to realize stable measurement at low cost.

  The invention according to claim 13 is characterized in that, in the light emitting means, the divalent metal complex is brought into contact with cells and microorganisms, the ion permeability of the cell membrane is improved, the permeability of the staining reagent, The label stability can be increased, and the measurement can be performed with high accuracy.

  The invention according to claim 14 is characterized in that, in the light emitting means, a luminance sensitizer is used by mixing with a fluorescent dye, and the fluorescent intensity is simply increased without increasing the staining step, In addition, the detection accuracy can be improved at a low cost.

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

(Embodiment 1)
First, a liquid sample is sucked and filtered from above a membrane filter serving as a collecting means, and microorganisms are two-dimensionally captured on the membrane filter surface. In the present invention, the specimen containing or possibly containing cells and microorganisms is a liquid specimen. However, when the test object is a liquid sample such as drinking water, the specimen itself is a liquid specimen. If the object to be inspected is a solid sample such as vegetables or meat, homogenize it to obtain a liquid sample, or collect cells and microorganisms from the surface using a cotton swab, etc. Release into phosphate buffer solution to make liquid sample. When a cooking utensil such as a cutting board is an object to be inspected, microorganisms are collected from the surface using a cotton swab or the like and released into physiological saline or the like to obtain a liquid sample. By sucking and filtering such a liquid specimen through a membrane filter, cells and microorganisms can be captured on the membrane filter. In addition, if necessary, an aqueous solution mixed with an appropriate concentration of metal complex is mixed with the liquid sample, or filtered from above the membrane filter on which cells and microorganisms are fixed, or contacted from below. By the technique, the cell membrane permeability of cells and microorganisms can be kept constant.

  In addition, as a metal complex, the thing corresponding to bivalence is preferable and ethylenediaminetetraacetic acid etc. are used in the density | concentration range of about 0.5-100 mM.

  Next, as a light emitting means, a drying prevention component and a brightness sensitizer are mixed, and a certain amount of a staining reagent containing a certain concentration of either or both of a live cell dead reagent and a dead cell stain reagent, which is a light emitting means, is dropped on the filter surface To do.

  The fluorescent dye preferably contains a nucleic acid binding structure, and as a living and dead cell reagent, 1,4-diamidino-2-phenylindole is used as long as it emits blue fluorescence by ultraviolet excitation, and green fluorescence or yellow by blue excitation. It emits green and yellow fluorescence. Examples include acridine orange, oxazole yellow, thiazole orange, and trimethine cross-linked asymmetric cyanine dye compounds such as SYTO9, SYTO13, SYTO16, SYTO21, SYTO24, SYBR Green I, SYBR Green II, and SYBR Gold. Can be used. The dead cell reagent emits green fluorescence. For example, monomethine cross-linked asymmetric cyanine dye dimers such as acridine dimer, thiazole orange dimer, oxazole yellow dimer, SYTOX Green, TO-PRO Monomethine cross-linked asymmetric cyanine dyes such as -1 can be used, and polymethine cross-linked asymmetric cyanine dyes such as propidium iodide, hexium bromide, ethidium bromide, LDS-751 and SYTOX Orange can be used as long as they emit red fluorescence. .

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

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

  In addition, as a membrane filter, the well-known thing with a hole diameter of 0.2 micrometer-1 micrometer can be used, for example.

  The membrane filter on which the fluorescently stained cells or microorganisms are developed is irradiated with excitation light using a condenser lens or the like so that the irradiation range is a constant area with a diameter of about 3 mm, for example, to obtain a light emitting point. .

  For example, in the case of a trimethine cross-linked asymmetric cyanine dye, when an excitation light having a wavelength of about 540 nm to 610 nm is irradiated, fluorescence having a wavelength of about 560 nm to 630 nm is emitted. When excitation light having a wavelength of about 480 nm to about 510 nm is irradiated, fluorescence having a wavelength of about 510 nm to about 540 nm is emitted.

  Therefore, in the case of a light emitting diode as an excitation light source, a blue light source preferably emits a wavelength near 480 nm, a green light source preferably emits a wavelength near 535 nm, and a yellow light source Preferably, those capable of emitting a wavelength near 560 nm are used.

  In the case of using a laser, a blue one that can emit a wavelength of preferably around 475 nm is used, and a green one that can emit a wavelength of preferably around 535 nm is used.

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

  As the fluorescent filter, a high-pass filter matched to the excitation wavelength is used so that the light component derived from the excitation light can be effectively removed without damaging the RGB information.

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

  The plurality of fluorescent images acquired by these operations include emission points derived from the fluorescence of cells and microorganisms. These emission points are set at appropriate emission intensities according to threshold values set in advance by the illuminant judging means. The size is set based on the size of the cells and microorganisms, and is determined based on values indicating chromatic features such as chromaticity and hue angle, which are calculated from the luminance values of RGB. The color measurement system and the luminescent material determination means, but the color system showing the color features is a color system such as L * a * b color system, L * C * h color system, XYZ color system, etc. Is used. Here, chromaticity based on the XYZ color system is used. First, conversion from RGB luminance values acquired by means for measuring RGB luminance to linear RGB and gamma correction are performed. This is further calculated using a constant based on the color system as a weight, and finally the values of x and y are obtained as values necessary for determining whether the cells and microorganisms or impurities. On the other hand, it is discriminated whether the cell is a cell or microorganism or a contaminant based on a preset value of the cell and microorganism and a threshold range set based on the value of the contaminant. When the luminescent matter judgment means judges the contaminants mixed in the sample with the cells and microorganisms, the total number is counted and integrated to finally determine the number of cells and microorganisms contained in the liquid sample. Weighed.

  It should be noted that the use of multiple fluorescent dyes and multiple excitation light sources can improve the discrimination accuracy and at the same time can perform life / death discrimination. desirable.

  Further, by using a linearly arranged charge coupled device of the charge coupled device unit which is a color measuring means, movement in the X axis direction (arrangement direction) becomes unnecessary, and the Y axis direction (movement direction) ), It becomes possible to carry out detection within a certain range, and weighing can be carried out quickly.

  Using the above method, cells and microorganisms contained in the original sample or cell culture medium can be discriminated and the number can be measured.

  FIG. 1 is a conceptual diagram showing a luminescent material discriminating apparatus for favorably implementing the luminescent material discriminating method of Embodiment 1 of the present invention. The luminescent object discrimination device 1 includes a light source 2, a condenser lens 3, and a light receiving unit 4. In order to extract the target wavelength from the excitation light emitted from the light source 2, the light is split by the excitation light spectral filter 5. The light source 2 only needs to be able to irradiate excitation light, and includes, for example, a light emitting diode. The split excitation light is condensed through a condenser lens 3 onto a membrane filter 7 set on an examination table 6 (which captures cells or microorganisms stained with fluorescence on the membrane filter by a separate operation). The The excitation light emitted from the light source 2 is condensed by the condensing lens 3. At this time, the range in which the excitation light is irradiated by the condensing lens 3 is condensed on a small fixed area of 1 to 3 mm square. . The fluorescently stained cells or microorganisms emit fluorescence when irradiated with excitation light, and the emitted fluorescence reaches the light receiving unit 4. Further, in order to measure the target fluorescence, it passes through the dichroic mirror 8 and the high-pass filter 9 and reaches the charge coupled device unit 10 built in the light receiving unit 4 and is signaled. The signal thus obtained is converted into an image, and image processing is performed by the calculation unit 11 which is a light emitting object determination unit. The light receiving unit 4 includes a high pass filter 9 and a charge coupled device unit 10. The illuminant determining means, that is, the calculation unit 11 is a microcomputer or the like programmed with image processing, and is also used by communicating with software operated by an externally connected terminal or the like.

  FIG. 2 is a diagram showing details of the determination method of the first embodiment. FIG. FIG. 2B is a diagram illustrating a calculation process flow of chromaticity. FIG. 2B is a diagram illustrating a calculation result of the luminance and chromaticity of E. coli.

  E. A water sample containing E. coli is filtered through a membrane filter and stained with SYTO9, a fluorescent dye for living and dead cells, and propidium iodide, a fluorescent dye for dead cells. It is a table | surface which shows an example of the data which acquired the G luminance image and R luminance image in. At this time, the B luminance image cannot be acquired because it overlaps the wavelength of the excitation light, and 1 is used instead. This variable can be adjusted to an optimal value. The luminance of RGB is calculated by the process shown in FIG. 2B, and the values of x and y are calculated. In this step, first, the RGB values obtained by the means for measuring RGB luminance are converted into linear RGB as shown in FIG. 1B by the calculation unit 11, and then gamma correction is performed. This is further calculated using a constant based on the color system as a weight, and finally the values of x and y are obtained as values necessary for determining whether the cells and microorganisms or impurities. The arithmetic unit 11 includes a microcomputer programmed based on the above flow.

  As a means for measuring RGB luminance, a charge coupled device unit such as a single-chip color CCD or a 3 CCD including three types of RGB fluorescent filters capable of acquiring three primary colors of red (R), green (G), and blue (B). and so on.

  Using such a technique, E.I. A certain amount of each of E. coli and tap water was filtered through a membrane filter, and the value was measured. FIG. 3 shows the E.I. The data of the luminous points contained in E. coli and tap water are plotted on a graph. According to this, E.I. E. coli was distributed in the region of x <0.36, y> 0.5, whereas the luminescent material in tap water had x of 0.3 to 0.6 and y of 0.3 to 0.6. It was confirmed that it was distributed over a wide area. At this time, the threshold value x is c, y is d, and the group classified into the region of x <c, y> d is E.D. It was confirmed that most of the impurities such as luminescent substances contained in tap water could be identified.

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

  The present invention is a method for detecting cells and microorganisms from a specimen containing cells and microorganisms using fluorescent staining, and performs detection with higher accuracy compared to conventionally known methods. The present invention has industrial applicability in that it can provide a discriminating method that can perform the above.

1 is a conceptual diagram showing a luminescent material discrimination device according to Embodiment 1 of the present invention. The figure which shows the detail of the discrimination method ((a) is a figure which shows the calculation result of the brightness | luminance and chromaticity of E. coli, (b) is a figure which shows the calculation process flow of chromaticity) E. The figure which shows chromaticity distribution of the luminous substance in E. coli and tap water

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Luminescent object discrimination device 2 Light source 3 Condensing lens 4 Light-receiving part 5 Excitation light spectral filter 6 Inspection stand 7 Membrane filter 8 Dichroic mirror 9 High pass filter 10 Charge-coupled element unit 11 Calculation part

Claims (14)

In the method for detecting cells and microorganisms, selected from a collecting means for collecting cells and microorganisms, a light emitting means for emitting light by staining, and a hue angle, chromaticity, saturation and lightness of the emitted color of the illuminant. Color measurement means for measuring at least one color characteristic, and luminescent matter judgment means for discriminating non-specific adsorption other than the reactant and / or reactant by the luminescence means and / or self-luminescence other than the reactant by the luminescence means A method for discriminating a luminescent material, comprising: 2. The method for discriminating a luminescent material according to claim 1, wherein an image receiving element comprising a CCD or CMOS having a large dynamic range of optical brightness is used as the color measuring means. 3. The method according to claim 1, wherein means for measuring RGB brightness is used as the color measuring means. The method for discriminating a luminescent material according to claim 1, wherein the collecting means fixes cells and microorganisms using a membrane filter. 5. The method for discriminating a luminescent material according to claim 1 or 4, wherein the collecting means has a function of suppressing self-emission of a component. 6. The method for discriminating a luminescent material according to claim 1, wherein the collecting means uses a base material that absorbs luminescence as a material of the constituent. 7. The method for discriminating a luminescent material according to claim 1, wherein the collecting means forms a metal film on the surface of the component. 2. The method for discriminating a luminescent material according to claim 1, wherein a fluorescent dye is used as the light emitting means. 9. The method for discriminating a luminescent material according to claim 1 or 8, wherein the light emitting means uses a nucleic acid-staining compound as a fluorescent dye. 10. The method for discriminating a luminescent material according to claim 1, wherein an intracellular esterase-decomposable compound is used as the fluorescent dye in the light emitting means. 11. The method for discriminating a luminescent material according to claim 1, 8, 9, or 10, wherein the light emitting means is used by mixing a drying inhibitor. 12. The method for discriminating a luminescent material according to claim 1, wherein a sugar alcohol is mixed as an anti-drying agent in the light emitting means. 13. The method for discriminating a luminescent material according to claim 1, wherein the divalent metal complex is brought into contact with cells and microorganisms in the light emitting means. 14. The method for discriminating a luminescent material according to claim 1, wherein a luminance sensitizer is mixed with a fluorescent dye in the light emitting means.

Images