JP2009192450A - Method of measuring phytoplankton - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of measuring phytoplankton to correctly measure the number of microphytoplankton and to perform quick, objective measurement. <P>SOLUTION: Ultrasonic waves that would not destroy the phytoplankton itself are emitted to water 5 containing the microphytoplankton forming a colony, the phytoplankton is dispersed separately into cell units, and the number of the phytoplankton of the dispersed cell units is measured by a flow cytometer 4. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a phytoplankton measurement method for measuring the amount of fine phytoplankton present in lakes and dam lakes.

  In recent years, it has been clarified that fine phytoplankton of several μm exists in lake water, and this fine phytoplankton, for example, femtoplankton (0.02-0.2 μm), picoplankton ( 0.2-2 [mu] m), nanoplankton (2-20 [mu] m), microplankton (20-200 [mu] m), etc., but it has become clear that they have an adverse effect on water quality. Fine phytoplankton is difficult to detect with an optical microscope because of its small size, and the amount of phytoplankton in the lake water is not clear. Recently, counting with a special microscope (fluorescence microscope) has been tried, but this is a pre-processing such as trapping in a filter and visual counting, and phytoplankton cannot be measured quickly and objectively. Is the current situation.

  Therefore, the inventors can disperse fine particles in a fluid, flow the fluid finely, and optically analyze the individual particles, so that flow cytometry can count about several thousand cells per second. The flow cytometer, which is a device used for flow cytometry, is considered effective for rapid counting of fine phytoplankton, and the applicability of this technology to fine phytoplankton measurement has been studied. As a result, it was found that many fine phytoplanktons in the water form clusters, and it is necessary to disperse the cells apart when measuring fine phytoplankton with a flow cytometer.

  In addition, some of the fine phytoplankton in the water are those in which multiple cells are encased in agar-like substances, and in order to accurately measure the number of cells with a flow cytometer, the agar-like substances are destroyed, It is necessary to make algae fall apart on a cell-by-cell basis.

  From the above, the inventors destroyed the agar-like substance by irradiating ultrasonic waves to such an extent that the cells are not destroyed, disaggregated into cell units, and the number of phytoplankton particles of chlorophyll and phycoerythrin It was found that the fluorescence intensity was measured with a flow cytometer. This makes it possible to more accurately count the number of fine phytoplankton, and further, when measuring with a flow cytometer, it is preferable to filter the sample with a mesh of about 50 μm to prevent clogging of the flow cell. In addition, since ultrasonic treatment disperses algae, it can also be expected to reduce the algae trapped in the mesh, and by comparing the measurement results with the flow cytometer before and after ultrasonic treatment, It has also been found that it is possible to identify colony-forming species.

  That is, the present invention solves the problems that could not be solved by the conventional technique, can more accurately measure the number of fine phytoplankton, and can perform the measurement quickly and objectively. The purpose is to provide a measurement method.

In addition, as a technique relevant to this invention, patent document 1, patent document 2, etc. are proposed until now. However, Patent Document 1 has a technical problem to count minute phytoplankton in low turbidity test water immediately and continuously, and in order to solve the problem, from a measurement cell, a particle meter, and a fluorometer. The number of particles for each particle size in the test water and the number of chlorophyll-containing particles are counted from the output of the fluorimeter, and Patent Document 2 is a technique that makes it possible to easily measure phytoplankton locally. In order to solve the problem, the sensor unit includes a sensor unit and a data analysis unit. The sensor unit includes an interference filter that transmits light of a specific wavelength and an optical sensor that detects the amount of light transmitted through the interference filter. And a plurality of types of sensor units are housed in a water pressure resistant case, and the data analysis unit is configured to store each sensor unit when the sensor unit is submerged in water. It is equipped with a means to calculate the spectral irradiance for each wavelength light based on the signal output from the unit to obtain the amount of phytoplankton, none of which is measured by a flow cytometer, The phytoplankton is not dispersed in units of cells by sonication and is technically far from the present invention.
JP 2003-254891 A JP-A-6-213802

  In order to solve the above problems, the invention according to claim 1 is directed to phytoplankton in units of cells by irradiating water containing fine phytoplankton forming a colony with ultrasonic waves to such an extent that the phytoplankton itself is not destroyed. And the number of dispersed phytoplankton per cell is measured with a flow cytometer. The invention according to claim 2 is the method according to claim 1, wherein the phytoplankton encapsulated in the agar-like substance is irradiated with ultrasonic waves at a frequency of 20 to 45 kHz and 2 to 100 w for 5 to 20 seconds, and only the agar-like substance is irradiated. It is characterized by breaking and dispersing the phytoplankton in units of cells.

  The invention described in claim 3 is characterized in that, when measuring with a flow cytometer, water is filtered with a mesh having an opening of about 50 μm in order to prevent clogging of the flow cell.

  The invention according to any one of claims 1 to 3, by irradiating water with ultrasonic waves to such an extent that the phytoplankton itself is not destroyed, destroys the agar-like substance and disperses it in cell units. Since it counts with a meter, the number of fine phytoplankton can be measured more accurately. In addition, since there is no need for visual observation, the measurement can be performed quickly and objectively. Furthermore, when measuring with a flow cytometer, water is filtered through a mesh with an opening of about 50 μm to prevent clogging of the flow cell. In this case, ultrasonic treatment disperses the algae and is trapped by the mesh. The effect of reducing algae can also be expected. Moreover, in the process of ultrasonically treating the water containing plankton, for example, a horn type or water bath type ultrasonic generation tank is used, and only the agar-like substance that encloses the algae is destroyed (algae cells themselves are not destroyed). Ultrasonic treatment is performed at a high intensity so that plankton in the water is not significantly damaged.

  In addition, the invention according to claims 1 to 3 is measured by a simple method of comparing a cytogram of an ultrasonic treatment sample with an untreated ultrasonic wave as a measurement result in relation to the measurement of phytoplankton. The type (group forming species) of the microphytoplankton in the colony forming algae can be easily identified, and after the identification, various effects can be expected using the sample.

  An embodiment of the present invention will be described with reference to the drawings.

  FIG. 1 is a drawing showing a process for explaining a phytoplankton measurement method and an outline of an apparatus for carrying out the process.

  As shown in the figure, the apparatus generally comprises a horn type ultrasonic generator 1 or a water bath type ultrasonic cleaner 2, a mesh 3 as a filtering means, and a flow cytometer 4. These instruments use commercially available products. The ultrasonic generator 1 or the ultrasonic cleaner 2 as an apparatus for generating ultrasonic waves is merely an example, and it goes without saying that other apparatuses may be used. The flow cytometer 4 includes a flow cell, an argon laser, a forward scattered light detector, a side scattered detector, a fluorescent detector (red fluorescent light, orange fluorescent light, yellow fluorescent light, green fluorescent light), an optical filter, a deflecting plate, and a sorting. It consists of components such as a regulator. Since the detailed configuration of the flow cytometer 4 and the operation in its use are well known from the past, detailed description is omitted here.

  A method for measuring phytoplankton using the above apparatus will be described for each step.

<Sonication process>
First, in the step of ultrasonically treating water containing phytoplankton, an ultrasonic generator 1 or a water bath type ultrasonic cleaner 2 having a horn-type chip 6 for containing water 5 as a sample containing microalgae is used. Use treatment to disperse phytoplankton in units of cells by irradiating water 5 with ultrasonic waves at such a strength that only the agar-like material enclosing the microalgae is destroyed (the algal cells themselves are not destroyed). Do. The ultrasonic frequency at which the dispersion effect can be expected is 20 to 45 kHz. In the horn type ultrasonic generator 1 of about 20 kHz and 2 w, irradiation is performed for about 10 to 20 seconds, and the ultrasonic cleaner 2 is about 28 to 45 kHz and 100 w. Then, phytoplankton can be dispersed by irradiation for about 5 seconds.

<Mesh processing process>
Next, in the step of mesh treatment, the water 5 is filtered through a mesh 3 having an opening of about 50 μm to remove large impurities. Note that this mesh processing step is not essential and may be added as necessary. The mesh 3 is also an example, and other filtering means may be used as long as large dust can be removed and the flow cytometer 4 can be prevented from being clogged.

<Measurement process by flow cytometry>
Next, in the measurement process by flow cytometry, the water 5 that has been meshed and transferred to another container is poured into the flow cell of the flow cytometer 4 together with the sheath liquid separately prepared in the container. The flow cells are irradiated with laser light from an argon laser while the algae in the flow cell are arranged in a row and flow in sequence one by one. The algae irradiated with laser light emit fluorescence according to the characteristics of the algae (algae have a fluorescent dye called chlorophyll or phycoerythrin), so each algae across the laser light is scattered forward. Detection is performed with a photodetector and a side scattered light detector. Further, the fluorescence generated from the algae is decomposed by wavelength using various optical filters, and the light decomposed by each wavelength is detected by a fluorescence detector (photomultiplier tube). Then, the intensity of fluorescence of each algae detected by each detector is digitized. In other words, the fluorescence detector detects 675 nm (red) reflecting chlorophyll of phytoplankton and 610 nm and 575 nm (orange, yellow) reflecting phycoerythrin and the like, and the detection result is amplified by an amplifier. Further, pulse processing and digital conversion are performed for display and analysis processing. With this analysis process, the number of phytoplankton dispersed in the water 5 can be measured.

  As described above, since the water 5 is irradiated with ultrasonic waves by the ultrasonic generator 1 or the ultrasonic cleaner 2 before measurement by the flow cytometer 4, phytoplankton can be dispersed very easily. In addition, since unnecessary impurities are removed by filtration through the mesh 3, highly pure phytoplankton can be used for measurement. Therefore, the number of fine phytoplankton can be measured more accurately by measurement using the flow cytometer 4. In addition, since measurement can be performed without relying on visual observation as before, it is quick and can guarantee an objective measurement result.

Experimental example 1
Using the surface samples of Lake Biwa, the relationship between the ultrasonic treatment time and the number detected by the flow cytometer 4 was investigated. What was used as the ultrasonic generator was a Branson ultrasonic transmitter (SONIFIER 450D) equipped with a tip (microtube) horn 7 and was used under the conditions of 20 kHz and about 2 w. 1 ml of water 5 was taken in the chip 6, and the horn 7 was directly infiltrated into the water 5 to perform ultrasonic treatment. The ultrasonic treatment time is 0 second, 2 seconds, 5 seconds, 10 seconds, and 20 seconds. The measurement was excited by an argon laser of 488 nm, and forward scatter, side scatter, and fluorescence of a fluorescent substance in algae were measured. The measured fluorescence is fluorescence of 675 nm (red) reflecting chlorophyll and 610 nm and 575 nm (orange, yellow) reflecting phycobilin and the like. As a result, it was found that the number of detected cells reached 35,000 to 40,000 and increased by ultrasonic treatment for about 10 seconds (FIG. 2).

  FIG. 3 shows a cytogram before sonication of 1 ml of Lake Biwa water containing microalgae, and FIG. 4 shows a cytogram after sonication. That is, FIG. 4 shows a part of a result obtained by performing ultrasonic treatment with the horn type ultrasonic generator 1 for about 2 w for 10 seconds and measuring the treated water with the flow cytometer 4. FIG. 4 is a numerical plot of the fluorescence intensity of each algae detected by the fluorescence detector (yellow fluorescence) and the fluorescence detector (red fluorescence). Each dot in FIG. 4 corresponds to an algae, that is, one fine phytoplankton (the same applies to FIG. 3). Since there are about 36,000 dots in FIG. 4 (the flow cytometer 4 has an automatic counting function), there are about 36,000 phytoplankton. Since the same type of phytoplankton has almost the same fluorescence intensity, if there is only one type of phytoplankton in the water, plots will be concentrated in one place when arranged as shown in FIG. . However, since FIG. 4 can be roughly divided into three regions A, B, and C, it can be seen that three or more types of phytoplankton exist. Further, in the comparison of the plots of FIG. 3 and FIG. 4, the plot of the region diagonally above region C in FIG. 4 decreases. From this, it can be seen that what was detected at this position in FIG. 3 is a colony forming algae. The change in the cytogram before and after the ultrasonic treatment is considered to be due to the fact that the microphytoplankton cluster structure is broken and becomes small particles, so that the fluorescence intensity decreases. Sort the algae in the area where the change is seen on the cytogram (the flow cytometer 4 has a function that can sort out the algae in the arbitrarily specified area on the cytogram) It is possible to identify the type of the algal colony forming algae population.

  In addition, based on the comparison of the measurement results (cytogram) of untreated ultrasonic waves (0 seconds) and ultrasonic waves (10 seconds) based on the above measurement, the algae forming the colony simultaneously emit red and yellow fluorescence. It can be seen that it is an algae (Figs. 3 and 4).

Experimental example 2
Using samples from Lake Biwa water depth, analysis was performed using water that was subjected to ultrasonic treatment for 10 seconds and untreated water. The flow cytometry measurement method is the same as in Experimental Example 1. As a result, in the vicinity of the surface layer, the number of algae detected in the sonicated sample was 40,000, which was nearly double that of the untreated sample (FIG. 5). It can be seen that there are many algae present in the community near the surface layer where the water depth is about 10 m from the water surface. That is, this Experimental Example 2 uses the ultrasonic treatment conditions determined in Experimental Example 1 (examined the optimal ultrasonic conditions), and uses ultrasonic treatment of water at different depths of Lake Biwa with and without ultrasonic treatment. The number of phytoplankton that was sonicated on the surface layer was significantly higher than the number of phytoplankton that was not sonicated, but the sonication was performed as the water depth increased. The data shows that the number of phytoplankton is at least small, in other words, that the number of phytoplankton is small with or without sonication as the water depth increases. This is considered to be because the number of fine phytoplankton forming the colony decreases as the water depth increases.

  FIG. 6 shows the results of examining the ultrasonic frequency and the effect of dispersing algae in Lake Biwa water. The untreated horizontal axis represents the number of algae (the number of detected phytoplankton / ml) measured with the flow cytometer 4 without ultrasonic treatment. The horizontal axis of 20 kHz and the horn type are processed by the ultrasonic generator 1 used in Experimental Example 1 and Experimental Example 2 in 2 w for 10 seconds. In this treatment, the number of detected phytoplankton reached nearly 15,000 per ml, and a dispersion effect was observed. Further, the samples with 28 kHz, 38 kHz, 45 kHz, and 100 kHz on the horizontal axis are processed by the ultrasonic cleaner 2 for 100 w to 120 w for 5 seconds. This 28 kHz, 38 kHz, 45 kHz ultrasonic cleaning machine 2 also showed that the number of detected phytoplankton ranges from nearly 15,000 to 20,000 per ml, and has a dispersion effect equivalent to or better than that of a 20 kHz horn type. . However, it became clear that the dispersion effect could not be expected with the ultrasonic cleaner 2 of 100 kHz.

  FIG. 7 is an image photograph of phytoplankton before ultrasonic irradiation, and FIG. 8 is an image photograph of phytoplankton after ultrasonic irradiation. From these images, it can be seen that the phytoplankton is dispersed apart by the irradiation of ultrasonic waves. Since the image shows a bar with a length of 5 μm, the size of each phytoplankton that has been broken apart can be known from comparison with this bar. Moreover, also from this microscopic observation, it turns out that the phytoplankton which has formed the colony has decreased after ultrasonic irradiation.

  As described above, the embodiment of the present invention has been described. However, this is merely an example, and the ultrasonic generator and numerical values used in the implementation are within the scope described in the claims. Thus, design changes and corrections can be made arbitrarily.

BRIEF DESCRIPTION OF THE DRAWINGS It is drawing which shows the process for demonstrating the measuring method of the phytoplankton which is one embodiment of this invention, and the apparatus outline | summary for implementing the process. It is a graph showing ultrasonic treatment time and the number of fine phytoplankton detected. It is drawing which represents the cytogram before ultrasonic processing. It is drawing which represents the cytogram after ultrasonic processing. It is a graph showing the depth direction of Lake Biwa and the number of phytoplankton. It is a graph showing an ultrasonic frequency and a phytoplankton number. It is a photograph showing the phytoplankton image before ultrasonic irradiation. It is a photograph showing the phytoplankton image after ultrasonic irradiation.

Explanation of symbols

1 Horn-type ultrasonic generator 2 Ultrasonic cleaner 3 Mesh 4 Flow cytometer 5 Water (sample)
6 Chip (micro tube)
7 Chip (microtube) horn

Claims (3)

  The water containing fine phytoplankton forming the colony is irradiated with ultrasonic waves to the extent that the phytoplankton itself is not destroyed to disperse the phytoplankton into cells, and the number of dispersed phytoplankton per cell A phytoplankton measurement method characterized by measuring with a flow cytometer.   A request to irradiate phytoplankton wrapped in agar-like material with ultrasonic waves at a frequency of 20 to 45 kHz and 2 to 100 w for 5 to 20 seconds to destroy only the agar-like material and disperse the phytoplankton in units of cells. Item 2. A phytoplankton measurement method according to Item 1.   The method for measuring phytoplankton according to claim 1 or 2, wherein when measuring with a flow cytometer, water is filtered with a mesh having an opening of about 50 µm to prevent clogging of the flow cell.