JP2011223924A - Method and device for counting plankton - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a plankton counting method used for counting the number of plankton which accurately determines viability of the plankton and counting the plankton alive.SOLUTION: In the plankton counting method, a plankton identifying step of dividing the size of the plankton in advance to uniformize the size of the plankton is provided, and thereby, the amount of a light-emitting point and an light-emitting area after dyeing the plankton are set within a narrow range to some extent, and the irradiation time of an exciting light and the imaging time for imaging a light-emitting image are obtained in a fixed time with a stable light-emitting amount. Thus, the plankton counting method can easily identify the plankton and accurately count the population of plankton.

Description

  The present invention relates to a plankton counting method and a plankton counting device used for measuring the number of plankton individuals that reproduce in seawater, fresh water, etc., and for determining whether they are alive or dead.

  Conventionally, a plankton counting method is known in which this type of planton counting device is visually confirmed with a microscope or a fluorescence microscope and counts live plankton (see, for example, Non-Patent Document 1).

  Hereinafter, the plankton counting method will be described.

  There are many types of plactones, and depending on the type, it is difficult to determine whether they are alive or dead. The method of determining whether or not there is the ability to proliferate as the determination of life or death varies greatly depending on the type of plankton. The main life / death judgment methods are shown below. (1) Morphological change: Confirmation of the shape of an individual, for example, the disappearance of flagella or the ambiguity of cellular tissue by microscopic observation. (2) Existence of motility: For species with motility, the presence or absence of motility was confirmed by microscopic observation. (3) Determination of intracellular active state by staining method: Staining live cells with reagents and confirming the presence or absence of luminescent cells with a fluorescence microscope. (4) Re-growth test: The target individual is placed in the culture solution, and cell viability and proliferation are confirmed. Using one or all four methods from the above four methods, it is determined whether the planton is alive or not in the target specimen (eg, sampled seawater), that is, whether it is proliferative or not. Only the measured plankton is visually judged and counted.

  In addition, a conventional plactone counting device of this type is known in which a plankton is counted by a combination of a light blocking type particle detection means and a fluorescence detection method (see, for example, Patent Document 1).

  Hereinafter, the plankton counting method will be described.

  The particles determined to be particles by the light blocking type particle detection means are irradiated with excitation light by the fluorescence irradiation means, and the plaquenton having chlorophyll emits fluorescence, so the amount of emitted light is detected by the fluorescence detection means, and the fluorescence amount Is calculated from a calibration curve with the number of individuals of one type of plankton that is known in advance, and counted as the number of Plaqueton individuals.

  Although not a plankton counting device, a bacterial counting method using a fluorescent staining method is known as a bacterial counting method (see, for example, Patent Document 2).

  Hereinafter, a method for measuring the bacteria will be described.

  A reagent that reacts only with viable bacteria to produce a fluorescent color of only viable bacteria, and the live bacteria stained with the reagent are irradiated with irradiation light from a light source, and is generated from the wavelength difference, light intensity, and size of the fluorescence emitted light. The number of viable bacteria is counted by judging the bacteria.

JP 2003-254891 A JP 2001-286296 A

Fukushiro Yasuo et al. “Balast Water Regulations and Development Examples of Ballast Water Treatment Equipment”, NTS Publishing Co., Ltd., September 3, 2008, 29-47

  In such a conventional Plaqueton counting method, since the methods (1) to (4) are visually counted using a microscope or a fluorescence microscope, the number of individuals must be large (for example, , More than 1000), and only a part of the entire field of view of the microscope can be measured, and if there is not a certain number in the field of view, the proportion of the whole is not necessarily uniform, so the number of individuals can be counted accurately Can not. In addition, since it is a visual judgment, it greatly affects an individual's skill and experience. The presence or absence of morphological change or motility cannot be accurately counted due to differences in individual skills because it cannot be determined whether the plankton is well-known or not. In addition, regarding a method using a staining method, it is necessary to sufficiently examine the type of reagent, the staining time, and the like corresponding to the type of plankton, and there is no method that has been established yet. As for the regrowth test method, it is necessary to cultivate for at least 5 days, and it takes a long time to find out the result, and if it grew, what was the initial number of individuals? Difficult to determine. The conventional plactone counting method has the above-described problems.

  Further, in the conventional plaquen counting method using the light blocking type particle detection means and the fluorescence detection method, even if plaquenton is dead, if the chlorophyll remains in the cell by the sterilization method, it is detected. It is difficult to judge whether it is dead or alive. In addition, since the detection is performed in combination with a method of measuring particles by the light blocking method, the detection part that allows the specimen (seawater or the like) to pass is a very narrow flow path, and a large amount of specimen can be measured. difficult. Further, since the size of plankton is in a wide range of several μm to several hundreds of μm, if a specimen containing planktons having different sizes is measured, the small plankton cannot be accurately measured because it overlaps with the large plankton. The conventional Plaquenton counting method has the above-described problems.

  In addition, in the conventional bacteria counting method using the fluorescent staining method, the size of the bacteria is 0.2 μm to 1 μm at most, and the range is narrow, so usually a light emitting point with a size of 1 μm or less is detected. You can measure bacteria just by doing. Plankton has a size of several μm to several hundred μm and has a wide variety of shapes such as a circular shape, a long one, and a connected shape. As an application of this conventional counting method, it is distinguished from plankton from the size. It is very difficult. The conventional method for counting bacteria has the above-described problems.

  Therefore, the present invention solves the above-described conventional problems, can be automatically measured without depending on individual skills, and can accurately determine the determination of life and death and the number of individuals. It is an object of the present invention to provide a plankton counting method and a plankton counting apparatus capable of accurately counting even specimens containing various types of plankton with greatly different.

  In order to achieve this object, the present invention is a plankton counting method for counting plankton after staining plankton on a plankton in a specimen with a staining reagent that reacts with the physiological activity of plankton. The plankton sorting process is performed to classify the materials in advance, thereby achieving the intended purpose.

  Furthermore, in order to achieve this object, the present invention preliminarily measures the size of plankton in a plankton counting device that counts plankton after staining plankton with a staining reagent that reacts with the physiological activity of plankton. A plankton selecting means for classifying into a light source, a light source for irradiating excitation light in a predetermined wavelength region, an image photographing means for photographing light in a predetermined wavelength region emitted by the excitation light as an image, and the light source. The light emitted by the irradiated excitation light is photographed within a certain time, and when the light amount and area of the light emitting point of the photographed photographed image are within the set range, it is determined that there is one plankton and the plankton is counted. It consists of plankton counting means, thereby achieving the intended purpose.

  According to the present invention, by providing a plankton selection process that pre-sorts the size of plankton, the plankton size is uniform, so that the amount of light emitted from the plankton dye, It can be set within a narrow range to some extent, and the irradiation time of the excitation light and the shooting time for shooting the luminescent image can be obtained in a constant time, so it can be recognized as plankton. Therefore, it is possible to obtain the effect that the life and death of plankton and the number of individuals can be counted with high accuracy.

  Further, according to the present invention, the plankton selecting means for preliminarily dividing the plankton size is provided, so that the irradiation time of the excitation light and the light quantity of the light emitting point are measured for each plankton size. Since the image shooting time can be made constant, it becomes easier to set the light intensity and area of the light emission point of the shot image obtained by shooting the light emission point of the emitted plankton, and automatically based on the shot image It is possible to obtain the effect that it can be counted and counted.

The schematic diagram which shows a dyeing | staining state with a staining reagent after plankton filtration of the plankton counting method of Embodiment 2 of this invention Schematic diagram of the measurement sample Schematic diagram of plankton counting apparatus according to Embodiment 3 of the present invention

  The plankton counting method according to claim 1 of the present invention is a plankton counting method for counting plankton after staining plankton with a staining reagent that reacts with the physiological activity of plankton on the plankton in the sample, It has a configuration that it has a plankton selection process for sorting in advance.

  As a result, the size of the plankton is aligned, so that the light intensity and emission area of the emission point after the plankton staining can be set within a narrow range, and the irradiation time and emission image of the excitation light can be taken. As a result, it is possible to obtain a stable amount of emitted light within a certain period of time, so that it is possible to obtain an effect that the life and death of plankton and the number of individuals can be accurately counted.

  The staining reagent may be a reagent that reacts with intracellular esterase. As a result, it is possible to determine whether plankton is alive or not based on the presence or absence of intracellular esterase. Therefore, it is possible to easily determine whether plankton is alive or dead. By determining and counting the luminous points, there is an effect that the plankton that is alive can be counted with high accuracy.

  The staining reagent may be composed of a reagent that reacts with intracellular esterase and one or more kinds of reagents that react with physiological activities other than esterase. As a result, only light emission in one type of wavelength range is judged as plankton for light emission due to dust or foreign substances other than plankton, so by using in combination with a reagent that also reacts with physiological activities other than esterase, The effect is that the plankton's life and death and living plankton can be judged with certainty.

  In addition, after the size is classified by the plankton selection step, the plankton stained with the staining reagent may be filtered through a filter, and the plankton on the filter may be measured. As a result, the stained plankton is arranged on the filter surface, that is, at a certain height, so that it becomes possible to measure without adjusting the focus for each light emitting point, and the life and death of plankton and the living plankton can be measured. There is an effect that the judgment can be made reliably.

  Further, the plankton counting apparatus according to claim 5 of the present invention is a plankton counting apparatus that counts plankton after staining the plankton with a staining reagent that reacts with the plankton physiological activity on the plankton in the specimen. A plankton selecting means for classifying into a light source, a light source for irradiating excitation light in a predetermined wavelength region, an image photographing means for photographing light in a predetermined wavelength region emitted by the excitation light as an image, and the light source. The light emitted by the irradiated excitation light is photographed within a certain time, and when the light amount and area of the light emitting point of the photographed photographed image are within the set range, it is determined that there is one plankton and the plankton is counted. It comprises a plankton counting device comprising plankton counting means.

  As a result, the irradiation time of the excitation light and the image shooting time for measuring the light quantity of the light emission point can be made constant for each plankton size, so that the shot image of the light emission point of the emitted plankton is captured. It becomes easy to set the light amount and area of the light emitting point, and it is possible to automatically determine the plankton based on the photographed image and to count it.

  Further, according to the size of the plankton classified by the plankton selecting means, the irradiation time from the light source and the photographing time are set as preset values, and the light quantity and area of the light emitting point photographed under the set conditions are A configuration may be adopted in which one plankton is determined within the set range, and the determined plankton is counted. As a result, the conditions for emitting plankton stained for each size, the conditions for capturing the emitted image, and the conditions for determining whether the plankton is alive or dead or plankton can be made constant. Therefore, the plankton can be counted automatically.

  Embodiments of the present invention will be described below.

(Embodiment 1)
Plankton's counting method involves staining with a fluorescent staining reagent, irradiating the stained plaquenton with irradiation light of a wavelength corresponding to the fluorescent staining reagent, and counting the plankton from the light intensity and size of the emitted fluorescent wavelength range. It can be performed.

  Fluorescent staining reagents are those that react with nucleic acids (for example, DAPI, AO, PI, etc.), those that react with specific enzymes in cells, those that react with respiratory activity (for example, CTC), and substrates that are incorporated. However, any staining reagent or a combination with a plurality of staining reagents may be used. Among them, a staining reagent that reacts with a specific enzyme is suitable as a staining reagent for easily measuring the life and death of plankton. A typical example of the specific enzyme is, for example, a reagent that fluorescently stains by reacting with an esterase that only living cells have in the cell. Representative reagents are 5-carboxyfluorescein diacetate acetoxymethyl ester, 5- (6-) carboxyfluorescein diacetate, 2 ′, 7′bis- (2-carboxyethyl) -5- (6-) carboxyfluorescein acetoxy Methyl ester, 5- (6-) sulfofluorescein diacetate, fluorescein diacetate, 5-chloromethylfluorescein diacetate, 5- (6-) carboxyfluorescein diacetate succinimidyl ester, fluorescein-5-carbonylazide acetate, etc. Yes, at least one or more of these are selected and used for plankton dyeing.

  In addition, since the wavelength range of the excitation light to be irradiated and the wavelength range of the fluorescence are different depending on the type of the staining reagent, the excitation light in the wavelength range corresponding to the reagent and the fluorescence emitted when the stained cells are irradiated with the excitation light. It is necessary to measure the amount of light and the like by using a light receiving filter corresponding to the wavelength region to have fluorescence in the wavelength region. In other words, by dropping a staining reagent that stains only living cells directly onto plankton or onto a specimen on a solution containing plankton, it enters the plankton cell and is hydrolyzed by the esterase enzyme in the cell. The decomposed reagent emits fluorescence when irradiated with excitation light.

  At this time, there are two main methods for staining and counting plankton. One is a method in which the amount of a reagent for staining a specimen containing plankton is adjusted so that the concentration is optimal for staining, and the other is to collect the target plankton once with a specimen containing plankton. Filters with a filter with the optimum pore size, collects plankton on the filter, and drops an appropriate amount of the reagent adjusted to the optimal concentration for plaquen on the filter, suitable for staining In this method, the reagent is stained with the reagent on the filter for a long time, and the reagent is filtered after staining.

  The first method is to drop the solution as it is on a glass slide and measure it as it is. However, if you measure the solution as it is, there is a thickness of the solution, so focus on the position of the plankton to be measured. If it is moving, it is necessary to observe it as it moves.

  Therefore, even in the first method, it is preferable to measure the plankton collected on the filter after filtering and filtering with a filter. In order to measure the plankton dyed on the filter, the plankton can be measured with a constant focus without focusing on each plankton by making the filter a smooth surface with a constant height.

  The fluorescent light emitted by irradiating the plankton stained on this filter with excitation light passes through the light receiving filter that passes only the light of the target wavelength range corresponding to the reagent with the light receiving filter, and the amount of light in that wavelength range is passed through the photoelectric element, etc. Measured by using the light emission area within the set light intensity range, calculate the light emission area from the area of the photoelectric element, etc., determine whether it is a living plankton, light emission that was determined to be a living plankton It counts the number of bodies and counts the number of living plankton.

  In dead plankton, this esterase decreases from the inside of the cell, and depending on the type of plankton, it quickly disappears. Therefore, even if it is stained with a reagent that reacts only with the living cells, it does not fluoresce. Do not count.

  Therefore, for example, when Plakunton is killed by a device that kills Plankton, it is possible to measure the killing effect of Plakunton by measuring the number of living Plakunton before and after killing Plakunton. It is.

  By the way, plankton is a general term for living organisms floating in the water, and is broadly classified into zooplanktons such as crustaceans, jellyfish, and fish larvae, and phytoplanktons such as diatoms and cyanobacteria. There are various sizes and ranges from several μm to several hundred μm.

  In general, a large plaquenton has a high light emission intensity due to a large amount of the reagent to be stained. When the fluorescent emission point is visually observed, it can be handled by adjusting the observation time according to the plankton size. And performing image processing from the image to determine plankton, it is difficult to determine if the emission intensity differs greatly depending on the size. That is, if the light emission intensity is high, the capacity of the photoelectric element may be exceeded, so that the image may cause halation (a white image).

  Usually, small plankton has low emission intensity, so the excitation light quantity is increased, specifically, the excitation light irradiation time is lengthened, and the fluorescence emission time is lengthened, resulting in fluorescence emission. Time is received by the photoelectric element, and the integrated light quantity is taken as an image using the light emission point.

  For a large plactone, it is possible to obtain a sufficient amount of light emission for photographing a light-emitting image even with a short irradiation time.

  In other words, depending on the size of plankton, it is possible to take an image of an optimal light emission point by lengthening the irradiation time of excitation light for small plankton and shortening the irradiation time for large plaquenton. If this is combined with either, it is difficult to obtain a sufficient amount of fluorescent light to measure small plankton with a short irradiation time, and with a long irradiation time, halation or the like occurs with a large plankton to measure plankton. It is difficult to get an appropriate image.

  In order to prevent this, it is necessary to measure plankton by first classifying each plankton of a certain size and setting an optimal irradiation time for each size.

  As a method of dividing specific plankton into a certain size (plankton selection step), a solution containing plankton is filtered using prefilters having different pore sizes.

  For example, four types of pre-filters that can filter 500 μm or more, 100 μm or more, 50 μm or more, 10 μm or more are prepared, and by first filtering the specimen with a 500 μm or more prefilter, large dust other than plankton for measurement purposes is removed. Remove with this pre-filter.

  This filtered filtrate contains plankton of 500 μm or less. A part of this filtrate is filtered with a filter having a pore diameter of 0.4 μm. This filter is available in cellulosic and nylon types, so long as all filters have a uniform pore size. However, the filter surface has few irregularities and the pore size is constant, making it ideal for measuring fluorescence. A black polycarbonate isopore membrane filter is preferred.

  Plankton of several μm to 500 μm or less is collected on the filter, but the concentration of the staining reagent, the irradiation time of excitation light, and the light receiving time are those for capturing fluorescent images of plankton of 100 μm to 500 μm. Set the optimal density and time, and take a fluorescent image using an image sensor.

  In this photographed image, a luminescent image of plankton of 100 μm or less is also photographed, but only the luminescent spots determined to be plankton of 100 μm to 500 μm from the area of the luminescent image are counted. The area of the luminescent image of the luminescent spot may not match the actual plankton size. For setting the emission area, put the measured plankton on a counting plate with a scale of length, check the actual size using a microscope, verify the area of the fluorescence image, check the evaluation, The set value of the area of the fluorescence image is determined.

  Since the light emission area varies depending on the concentration of the staining reagent, the irradiation time, and the light reception time, it is necessary to set the area according to these.

  Similarly, for filtrate filtered through a prefilter of 100 μm or more, plankton of 50 μm to 100 μm, and for filtrate filtered through a prefilter of 50 μm or more, filtrate filtered through a prefilter of 10 μm to 50 μm, 10 μm or more. As for plankton of 10 μm or less, an optimal staining density, irradiation time, and light receiving time are set for each, and the number of plankton determined to be plankton at each size is counted from the area of the luminescent image at the light emission point.

  In addition, as a method of dividing plankton according to size, a pre-filter was used, but there are other methods, for example, a method using electric induction and a method using centrifugal separation. May be.

  In addition, the four types of pre-filters are used to classify the size, but it can be divided into finer or rougher areas, and the plankton emission can be made accurately within the set range. As long as a luminescent image that can be identified as plaquenton from the luminescent area of the luminescent spot can be obtained.

  However, accurate measurement is not possible unless it is divided into at least two stages such as 50 μm or more and less.

  The light source for irradiating the excitation light may be a light source that can obtain a predetermined wavelength, but the irradiation time and the light receiving time differ depending on the type of the light source.

  When a mercury lamp is used, the light intensity of the excitation light is high, so that the fluorescent light can be sufficiently emitted in a short time of 0.1 second or less. However, when an LED with low light intensity is used, irradiation is performed. It is necessary to set the time to a long time of several seconds.

  Since the irradiation time varies depending on the light source, it is set according to the light intensity of the light source. However, when using the same light source, the irradiation time for large plankton is shorter than the irradiation time for measuring small plankton. It will be time. The light reception time is the same as the irradiation time. Plankton stained with a fluorescent staining reagent emits fluorescence while being irradiated with excitation light by a light source. Since only the time for emitting the fluorescence needs to be received, the irradiation time and the light receiving time may be the same. However, even if either one is set to a different time such as a long time, there is no particular problem because a luminescent image is taken with the amount of light integrated according to the light receiving time. Therefore, if fluorescence is received after irradiation for a long time, the necessary amount of light may not be obtained only by quenching to obtain a luminescent image.

  If the field of measurement for capturing a luminescent image of stained plankton on the filter is small, the number of scans to measure the entire area of the filter increases, and if it is large, the resolution (number of pixels) of the photoelectric element and image sensor Since it is difficult to distinguish a small light emitting point from plankton, it is desirable that one side is several hundred μm to several mm.

  Since a large plankton cannot be measured from the area of the visual field, one side is preferably several mm or more, and a small plankton is preferably several mm or less. For plankton of 50 μm or less, sufficient measurement is possible if one side is about 1 mm.

  Practically, when using a 300,000-pixel image sensor and taking a light-emitting image of the filter surface with a field of view of 1 mm on a side using a magnifying lens several times, a plankton light-emitting image can be taken with high accuracy. By moving the portion to be photographed and scanning the filter surface, the total area or a part of the area of the filter is measured, and the plankton on the filter is counted.

  Recognize the light emission point of the light quantity and area within the set range from the light quantity and area emitted by one light emission image, recognize the recognized light emission point as one plankton, and integrate the light emission points As a result, the number of plankton is calculated and counted as a living plankton. With such a method, plankton can be measured automatically and accurately.

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

(Embodiment 2)
A sample containing plankton (a solution such as seawater, hereinafter referred to as a sample) is sorted by plankton sorting means for each set size. The plankton sorting means uses a prefilter A that filters 100 μm or more and a prefilter B that filters 50 μm or more. In the filtrate A filtered by the prefilter A, plaquenton of 100 μm or less exists. In the filtrate B filtered by the prefilter B, plankton of 50 μm or less exists.

  As shown in FIG. 1, the plankton of 50 μm or less (physically 0.4 μm or more) is filtered onto the membrane filter 1 by filtering the filtrate B with a membrane filter 1 having a pore diameter of 0.4 μm, which is a plankton collecting means. 2 is in a collected state. Similarly, the filtrate A is filtered through another membrane filter 1 so that plankton of 100 μm or less is collected on the membrane filter 1.

  The plankton sorting process or plankton sorting means uses a pre-filter, but any means that divides plankton according to size may be used. For example, a method of separating by the difference in the force attracted to electricity using electric induction, A method may be used in which separation is performed for each size using a density gradient separation method in which a separator is used for sorting according to the difference in weight.

  In order to stain the plankton 2 collected on the membrane filter, a staining reagent 3 adjusted to an optimum concentration is dropped on the membrane filter 1. As staining reagent 3, 0.15 mg / ml of 6CFDA (6-carboxyfluorescein diacetate) solution was used. 1 ml of filtrate B was filtered through a membrane filter 1 having a diameter of 9 mm. Drop 1 ml and leave it in that state for 10 minutes at room temperature to stain plankton with staining reagent 3.

  Thereafter, the staining reagent 3 is filtered, 0.1 ml of PBS is dropped, and the membrane filter 1 is cleaned by immediately filtering. The living plankton 2 on the membrane filter 1 is counted as a measurement specimen 4 shown in FIG.

  The plankton 2 is counted by setting the measurement specimen 4 on a fluorescence microscope, irradiating the surface of the measurement specimen 4 with excitation light (blue light) having a wavelength corresponding to the staining reagent 3, and emitting yellow-green light fluorescence. The result is visually judged as live plankton 2 and counted. At that time, not only the light emission but also the plankton is determined in consideration of the form (size, shape, etc.) of the light emission for visual observation, and the determined number is counted.

  Further, since the field of view of one measurement is narrow, the surface of the membrane filter is scanned, the plankton is counted in each field of view, and the number of individuals is integrated. If only a part of the area of the membrane filter can be measured instead of the entire area of the membrane filter, calculate the number of individuals of the entire membrane filter from the ratio of the measured area and the area of the entire membrane filter, and the amount of the sample filtered through the membrane filter If 1 ml is assumed, it can be counted as the number of living plankton individuals per ml.

  Since the plankton can be preliminarily divided into a certain size using a pre-filter, the size is the same even with visual observation. Plankton can be easily determined while the time is substantially constant and the magnification of the lens is also constant.

  The staining reagent 3 is a solution obtained by dissolving 6CFDA that reacts with intracellular esulase and stains in DMSO as a solvent. If necessary, a solution in which a solvent such as water, PBS, or EDTA is mixed, Not only a reagent that reacts with esterase, but also a staining reagent that reacts with the physiological activity of other cells may be used.

  Specifically, the nucleic acid and the reagent DAPI for staining are combined with the above 6CFDA. DAPI irradiates UV light, and blue fluorescent light is temporarily determined to be live and dead, and 6CFDA emits blue light and yellow-green fluorescent light is alive. It is determined to be plankton.

  That is, it is possible to count more accurately by counting the points issued by other than the plankton by determining the points that are fluorescent with both reagents as living plankton. The staining conditions (staining reagent concentration, staining reagent amount, staining time, staining method (staining on the filter or dropping on the specimen, etc.), etc.) are optimized depending on the object to be measured and the fluorescence detection device. And make a decision.

(Embodiment 3)
FIG. 3 is a schematic diagram of a plankton counting apparatus that automatically measures plankton of the measurement specimen 4.

  As shown in the drawing, measurement is performed through a light source 5 such as an LED or a mercury lamp, an excitation light spectral filter 6 for splitting light from the light source into an optical path irradiated from the light source, a prism 7 for changing the optical path, and a lens 8. The surface of the specimen 4 is irradiated with excitation light. The measurement sample 4 is placed on the inspection table 10 of the pedestal 9, and the pedestal automatically moves in the XY direction to move the measurement sample 4 and irradiate the membrane filter surface of the measurement sample 4 with excitation light. The structure that can move the part to be scanned and scan the entire surface.

  The plankton stained in the measurement specimen 4 emits fluorescence when irradiated with excitation light. This fluorescent light is condensed by the lens 8, passes through the prism 7, passes only light of the passed wavelength by the fluorescent spectral filter 11 that passes only a predetermined wavelength, and is emitted by the image photographing means 12 such as an image sensor. Take a picture.

  Based on the image obtained by image processing from the light intensity and area of each luminescent point of the luminescent image, the luminescent point determined to be plankton by the plankton counting means 13 is counted as one individual plankton, and for all luminescent points This operation is carried out, the luminous points determined to be plankton are integrated, and the number of individuals is counted.

  This is a processing method for one captured image, and when scanning is performed according to the area of the membrane filter 1, a captured image corresponding to the number of times of scanning is obtained. This is performed for all the photographed images, and the emission points determined to be plankton are integrated and counted. From the ratio of the total area of the photographed image and the area of the membrane filter, the total number of plankton individuals is calculated as the total number of individuals of the membrane filter area, and counted as the number of plankton individuals per amount of filtered sample.

  In addition, the area of one captured image is preferably an area of several hundred μm to several tens of mm square, and if the size of plankton to be measured is several tens of μm, it is several hundred μm to several mm square and several hundreds of μm in size. An area of several mm to several tens of mm square is preferable. The area of the luminescent spot and the actual size of the plankton do not necessarily match.

  This is because if the light intensity of the excitation light is high, the intensity of the emitted light also increases, and by increasing the light reception time when receiving light, the light intensity also increases, and a light emitting area different from the actual size can be obtained. become.

  Therefore, the actual plankton size is measured using a microscope, etc., and the plankton is used to determine the emission point under conditions such as the excitation light irradiation time, light intensity, and light reception time set at an arbitrary value. The area and the amount of light are confirmed, and the range of the light intensity and area of the light emitting point at that value is determined in advance. From this range, the luminescent point of the photographed image is determined as plankton by the plankton counting means 13 and counted.

  When counting with a normal fluorescence microscope, the magnification of the lens is increased in the case of a small emission point, depending on the size of the emission point (plankton size), and in the case of a large emission point. Although it is possible to observe with a reduced magnification, it is difficult to perform automatically. By aligning the size of plankton within a certain range, the light intensity of irradiation light, irradiation time, light reception time, and field of view of shooting can be set to a constant value. It becomes easier to set, and plankton can be easily determined.

  Since the plankton counting method and the plankton counting device according to the present invention make it possible to easily measure the number of plankton individuals and the life / death judgment by arranging the size of plankton, The present invention is useful as a plankton counting method, a plankton counting device, and the like used for counting the number of plankton individuals and verifying the effect of a device for killing plankton.

1 Membrane filter
2 Plankton
3 Staining Reagent 4 Measurement Sample 12 Image Capture Means 13 Plankton Counting Means

Claims (6)

A plankton counting method for counting plankton after staining plankton with a staining reagent that reacts with the plankton physiological activity on the plankton in the sample, the plankton counting method having a plankton sorting step for classifying the plankton size in advance . The plankton counting method according to claim 1, wherein the staining reagent is a reagent that reacts with intracellular esterase. The plankton counting method according to claim 1, wherein the staining reagent comprises a reagent that reacts with intracellular esterase and one or more reagents that react with physiological activities other than esterase. The plankton counting method according to any one of claims 1 to 3, wherein after the size is classified by the plankton selection step, the plankton stained with the staining reagent is filtered through a filter, and the plankton on the filter is counted. Plankton selection means for pre-sorting the size of plankton in a plankton counting device that counts plankton after staining plankton with a staining reagent that reacts with the plankton physiological activity on the plankton in the specimen, and a predetermined wavelength range A light source for irradiating the excitation light with the above, an image photographing means for photographing the light of a predetermined wavelength range emitted by the excitation light as an image, and the light emitted by the excitation light emitted by the light source within a certain time A plankton counting device comprising plankton counting means for determining that there is one plankton and counting the plankton when the light quantity and area of the emission point of the photographed photographed image are within the set range. The plankton counting means is configured such that the irradiation time from the light source and the photographing time are set in advance according to the size of the plankton classified by the plankton sorting means, and the light amount of the light emitting point photographed under the set conditions. 6. The plankton counting apparatus according to claim 5, wherein when the area is within the set range, the plankton is counted by determining that the number is one plankton.

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