JP6221210B2 - Microorganism testing method and apparatus - Google Patents

Description

  The present invention relates to a microorganism testing method and apparatus, and more particularly to a microorganism testing method and apparatus suitable for detecting microorganisms such as plankton that are contained in ballast water and are alive.

In order to stabilize the ship, the ship that is not loaded with cargo travels with ballast water and discharges the ballast water in the sea area where the luggage is loaded.
Ballast water is usually discharged into a sea area different from the sea area on which it is mounted. Therefore, problems such as the destruction of ecosystems by transporting microbes such as plankton and bacteria contained in the ballast water to sea areas other than their original habitats. May cause.

  In order to deal with such problems, international rules regarding the regulation of ballast water have been formulated, and the “International Convention for the Regulation and Management of Ship Ballast Water and Sediment (Ballast Water Management Convention)” has been adopted. Yes.

The “Guidelines for Ballast Water Sampling (G2)” related to the above Ballast Water Management Convention is based on the “Ballast Water Emission Standard (D-2)”. For example, for a microorganism having a minimum size of 50 μm or more (hereinafter referred to as “L size organism”), the allowable number of individuals is 10 / m ³ or less, and a microorganism having a minimum size of 10 μm or more and less than 50 μm (hereinafter referred to as “S size organism”). “)” Is defined as 10 pieces / mL or less according to the minimum size of the microorganism.

  To date, a method described in Patent Document 1 is known as a method for measuring microbial cells that live in water such as the ballast water.

  According to the method described in Patent Document 1, a chemical substance that reacts with an enzyme or a coenzyme present in a living cell of a microorganism and generates a fluorescent substance in the cell is allowed to act on a measurement target sample including the microorganism, for a certain period of time. After mixed contact, the sample is irradiated with light of a wavelength necessary to excite the fluorescent substance generated in the cell, and then the light emitted from each microorganism in the sample is measured as the number of points. This is a method for measuring living cells of microbial cells.

  Thereby, in the conventional agar culture method, the measurement time required from 10 hours to several tens of hours is dramatically shortened to within 10 minutes, and the light emitted from the living bacteria is detected and measured optically and electrically. By establishing the means, it is possible to directly and automatically count viable bacteria, and there is an advantage that the sterilizer can be controlled and the product quality can be quickly managed.

  However, the method described in Patent Document 1 has a problem that the measured value varies due to differences in the type of water, temperature, type of dyeing agent, concentration, dyeing time, and the like, and improvement has been demanded.

JP 3-43069A

  In view of the above problems, the present invention does not cause a problem that the measurement value varies due to differences in the type of water, temperature, type of dyeing agent, concentration, dyeing time, etc., and inspection of microorganisms that can be stably measured It is a technical problem to provide a method and apparatus.

In order to solve the above problems, the present invention estimates the allowable number of living microorganisms contained in the ballast water discharged from the ship by the number of microorganisms present in the water in the sample container collected as a sample, A microorganism testing apparatus for measuring whether or not the ballast water drainage standard is satisfied, and a sample solution and a fluorescent staining reagent are added to a sample container formed of a light transmitting material, and the sample solution is stirred. -An agitation and mixing means for mixing; an excitation light source comprising a light source for continuously irradiating the irradiated surface of the sample container while exciting the sample solution by the agitation and mixing means; and excitation of the excitation light source A light receiving means for receiving the light fluorescently emitted by the light and converting the light into an electric signal; and noise and / or the electric power of low frequency components relating to the fluorescence emission of the sample solution itself in the electric signal converted by the light receiving means. A filtering means for filtering the noise of a high frequency component for the signal of the waveform, a predetermined receiving range on the basis of the electrical signal filtered by said filtering means detects the emission number that occurs when the microorganism has passed and counted, the light emitting number Control means for calculating the amount of microorganisms contained in the sample in the sample container, and an operation part electrically connected to the control means, and the operation part determines the size of the microorganism to be measured. Technical measures were taken to include switchable setting buttons.

  The invention according to claim 2 is characterized in that the filtering means is a band-pass filter in which a high-pass filter and a low-pass filter are connected.

  The invention according to claim 3 is arranged such that the excitation light source is irradiated with excitation light orthogonal to the irradiated surface of the sample container, while the light receiving means is excitation light of the excitation light source. A light receiving surface for receiving fluorescent light emission at an angle orthogonal to is arranged.

  The invention described in claim 4 is characterized in that a slit for regulating an observation range is provided between the light receiving means and the sample container.

  Furthermore, the invention described in claim 5 is a parallel light conversion means for converting the diffused light from the light source into parallel light that is uniformly irradiated at the same angle toward one surface between the excitation light source and the sample container. It is provided.

  The invention according to claim 6 is characterized in that the parallel light converting means is formed by drilling a threaded hole having a predetermined diameter in a flat plate having a predetermined thickness.

  The invention according to claim 7 is characterized in that the parallel light converting means is formed of a convex lens.

The invention according to claim 8 estimates the allowable number of living microorganisms contained in the ballast water discharged from the ship based on the number of microorganisms existing in the water in the sample container collected as a sample, and the ballast A method for inspecting microorganisms for measuring whether or not a water drainage standard is satisfied, a size setting step for setting the size of a microorganism to be measured, and a sample in which a sample and a fluorescent staining reagent are added to a sample container A stirring and mixing step of stirring and mixing the solution; an excitation step of continuously irradiating the irradiated surface of the sample container with the excitation light while stirring the sample solution; and receiving light emitted by the excitation light. A light receiving step for converting the signal into an electric signal, and a low frequency component noise and / or a high frequency for the waveform of the electric signal in the fluorescence of the sample solution itself in the electric signal converted by the light receiving step. A filtering step of filtering the noise components, a predetermined receiving range on the basis of the electrical signal filtered by said filtering step detects the emission number that occurs when the microorganism has passed, the sample in the sample container from the light emitting Number And a microorganism number estimation step for calculating the amount of microorganisms contained.

After the operator adds the sample and the fluorescent staining reagent to the sample container, the agitation and mixing means are activated to agitate and mix the sample solution.Then, when the excitation light source and light receiving means are activated, the microorganisms emit fluorescence. The amount of received light is converted into an electrical signal by the light receiving means and captured. The control means detects the number of luminescence based on the electric signal, and calculates the amount of microorganisms contained in the sample in the sample container from the number of luminescence. At this time, the light receiving means generates a disturbance of the electric signal due to the influence of the fluorescence emission of seawater components different from the microorganisms or the hydrolysis of the fluorescent staining reagent, and is taken into the light receiving means. There is a concern that quantity measurement errors and measurement errors may occur.
According to the present invention, since the disturbance is filtered by the filtering means before the electric signal is taken into the control means, it is possible to clearly distinguish the electric signal corresponding to the amount of received fluorescence emission of the microorganism from the disturbance. . Thereby, the measurement error of the amount of microorganisms does not occur, the problem that the measured value does not vary does not occur, and the measurement can be performed stably.

It is a perspective view showing the whole microbe inspection device concerning one embodiment of the present invention. It is a general | schematic plane sectional view of the measurement part which concerns on one Embodiment of this invention. 1 is a block diagram showing the overall configuration of a microorganism testing apparatus according to an embodiment of the present invention. It is a figure which shows an example of the circuit of the filtering means of this invention. It is a flowchart which shows the measurement flow of the microbe inspection apparatus which concerns on one Embodiment of this invention. It is a schematic sectional drawing which shows one Embodiment of a parallel light conversion means. It is an effect | action figure which shows that an observation surface narrows with the presence or absence of a slit. It is a graph which shows the correlation with the number of microorganisms and the light reception count of a photomultiplier tube (PMT). It is a graph which shows the test of whether detection is possible by the life and death of microorganisms. It is a graph which shows the waveform of the acquisition voltage before mounting | wearing with a filtering means. It is a graph which shows the waveform of the acquisition voltage after mounting | wearing with a filtering means.

  A mode for carrying out the present invention will be described with reference to the drawings. FIG. 1 is a perspective view showing an entire microorganism testing apparatus according to an embodiment of the present invention, FIG. 2 is a schematic plan sectional view of a measuring unit according to an embodiment of the present invention, and FIG. It is a block diagram which shows the whole structure of the microbe inspection apparatus which concerns on one Embodiment.

  As shown in FIGS. 1 and 2, an inspection apparatus 1 according to the present invention includes a main body 2 that incorporates a control mechanism such as a CPU board and performs information processing work such as measurement results and statistical processing work, and the main body 2 A display unit 4 formed of a liquid crystal panel or the like for displaying the measurement results, and a transparent material that transmits light (for example, glass or The main part is a measurement unit 6 that accommodates a sample container 5 formed of quartz, acrylic resin, or the like, and optically counts the number of microorganisms in the sample solution S. Reference numeral 7 denotes a rotor for stirring the sample solution S accommodated in the sample container 5, and the rotor 7 is accommodated in the sample container 5 together with the sample solution S and the luminescent reagent. When accommodated in the measuring unit 6, the magnetic stirrer 27 built in the measuring unit 6 is rotationally driven. Thereby, the number of microorganisms in the sample solution S can be counted while stirring and mixing the sample solution S composed of the sample and the luminescent reagent in the sample container 5 at a predetermined temperature, and the sample solution S can be measured without being stirred. Compared with, the microorganisms emit light brightly in a very short time, and the amount of microorganisms in the ballast water can be measured easily and in a short time.

The dimensions of the inspection apparatus 1 shown in FIG. 1 are 300 mm in width, 300 mm in depth, 100 mm in height, and about 2 to 4 kg in weight, and are accommodated in a handheld trunk (not shown) or the like. It can be carried anywhere and can be measured inside a ship or measured outdoors.
The sample container 5 formed of a transparent material that transmits light is formed in a prismatic shape having a bottom surface of 50 mm × 50 mm and a height of 60 mm, and the internal volume when the water level is 40 mm is set to 100 ml (milliliter). Has been. The sample container 5 is not limited to such a prism shape, and may be cylindrical or cubic as long as the internal volume can be secured about 100 ml (milliliter).

  As shown in FIGS. 1, 2, and 3, the measurement unit 6 includes a sample container storage unit 9 that stores and holds the sample container 5, and a light source unit 13 that emits excitation light toward the sample container 5. And a light receiving unit 19 for observing microorganisms drifting in the sample container 5 by the excitation light irradiated from the light source unit 13. The light receiving unit 19 is electrically connected to the CPU substrate 23 via the filtering unit 34. In the CPU substrate 23, the number of microorganisms in the sample solution S can be counted by an electrical signal from the light receiving unit 19, and information processing work such as measurement results, statistical processing work, and the like can be performed.

The sample container storage unit 9 is formed by holding plates 8a and 8b surrounding at least two surfaces of the sample container 5, and stores and holds the sample container 5 so as not to block light irradiation from the light source unit 13. It is.
Then, as shown in FIG. 2, the light source unit 13 is arranged so that excitation light from the normal line AP is incident on the irradiated surface G of the sample container 5. The light source unit 13 includes an LED light source 10 disposed in the vicinity of the sample container storage unit 9 and a parallel light conversion unit 11 (LED is a light source) that is disposed in front of the LED light source 10 and converts diffused light into parallel light. Since the light is diffused and irradiated in a random direction, the sample container 5 is irradiated with excitation light consisting of parallel light that is uniformly irradiated with light rays at the same angle toward one surface) and slit-shaped parallel light. And an excitation light band-pass filter 12.

  FIG. 6 is a schematic sectional view showing an embodiment of the parallel light converting means 11. In the example shown in FIG. 6A, the parallel light converting means 11 is formed by drilling a threaded hole 32 having a predetermined diameter in a flat plate 31 having a predetermined thickness, and the thickness of the flat plate 31 is adjusted to the optical path length. L and the hole diameter of the threaded hole are appropriately set. Thereby, the scattered light of the incident angle θ irradiated from the LED light source 10 is converted into parallel light when passing through the threaded hole 32. In the example shown in FIG. 6A, the optimum condition of θ and L is determined by the SN ratio test. For example, if M3 (outer diameter of screw hole) × 0.5 (pitch), θ Was 9.5 ° and L was 15 mm.

  The parallel light conversion means 11 shown in FIG. 6B is provided with a convex lens 33 on the front surface of the LED light source 10, and the scattered light emitted from the LED light source 10 passes through the convex lens 33 and is emitted to the outside. In this case, the light is converted into parallel light.

  Note that the light source unit 13 of the present embodiment uses the LED light source 10 as a light source. However, the parallel light that can be irradiated with parallel light is not limited to the LED light source 10 as long as it can excite the fluorescent substance contained in the microorganism. An LED light source, a laser light source, and a light bulb can also be employed. Needless to say, when a parallel light LED or a laser light source capable of irradiating parallel light is employed, the above-mentioned parallel light conversion means 11 is not necessary.

  As shown in FIG. 2, the light receiving unit 19 is provided such that the light receiving surface F is arranged at an angle orthogonal to the excitation light by the normal line AP from the light source unit 13. The light-receiving unit 19 is a photomultiplier tube arranged and configured to receive fluorescence with an optical axis orthogonal to the parallel light irradiated with excitation light from the LED light source 10 toward the sample container 5 ( PMT) 14, a fluorescent bandpass filter 15 disposed in front of the photomultiplier tube (PMT) 14, a condensing lens 16 disposed in front of the fluorescent bandpass filter 15, and the condensing A slit 17 disposed on the front surface of the lens 16 and a gap between the slit 17 and the sample container 5 are used to excite a fluorescent substance contained in microorganisms, thereby condensing and imaging the emitted fluorescence. And a relay lens 18.

  The slit 17 between the photomultiplier tube (PMT) 14 and the sample container 5 narrows the observation surface in a slit shape. That is, as shown in FIG. 7, (a) in the state without slits, the background on which the light receiving surface F is formed in a circle is monitored, while (b) in the state with slits, the light receiving surface F is shaded. The background formed by the vertically long slits except for is monitored. Therefore, as a result of the light receiving area of the light receiving surface F being narrowed as shown in (b), the area of background fluorescent light emission, which becomes noise, is also narrowed. This improves the detection accuracy of the fluorescence emission of microorganisms.

  Although the light receiving unit 19 uses the photomultiplier tube (PMT) 14 as the light receiving sensor, the light receiving unit 19 is not limited to this, and is not limited to this, but a silicon photodiode (SiPD) or an avalanche photodiode (APD). As in the case of a photomultiplier tube (PMT), various types of photodetectors that can detect the emission of a fluorescent substance contained in a microorganism can be employed.

  Furthermore, with reference to FIG. 3, the electrical control structure of the inspection apparatus 1 of this embodiment is demonstrated. In the center of the housing 20 forming the main body 2, an output signal converted from light to electricity by the photomultiplier tube (PMT) 14 is supplied with power from an AC power source 21 or a secondary battery 22. A CPU board 23 is provided for performing analysis, determining whether or not it is within an arbitrary luminance range, pulse counting an arbitrary luminance signal, and controlling on / off of the LED light source 10. . An AC / DC converter 24 is interposed between the AC power source 21 and the CPU board 23.

  The CPU substrate 23 is electrically connected to the photomultiplier tube (PMT) 14, the LED light source 10, a RAM 25 serving as a read / write storage unit, and a ROM 26 serving as a read-only storage unit. Further, the power button 3a, the measurement start button 3b, the external output button 3c, and the setting button 3d of the operation unit 3 shown in FIG. 1 are electrically connected. Then, on / off switching control is performed by pressing the power button 3a, measurement is started by pressing the measurement start button 3b, and data is transferred to an external printer or personal computer by pressing the external output button 3c. By pressing the setting button 3d, the measurement type is switched (L size microorganism measurement or S size microorganism measurement is switched), the judgment reference setting is changed, the threshold value setting is changed, and the measurement is performed. The time setting can be changed.

  In addition, the CPU board 23 includes a magnetic stirrer 27 that rotates the rotor 7 by magnetic force, a display unit 4 formed of a liquid crystal panel, a cooling fan 28 for a control device such as the CPU board 23, and RS-232C. An external output terminal 29 is connected.

  FIG. 4 is a diagram showing an example of a circuit of the filtering means which is a main part of the present invention. As shown in FIG. 4, an operational amplifier 35, a high-pass filter circuit 36, and a low-pass filter circuit 37 are electrically connected between the photomultiplier tube (PMT) 14 and the CPU substrate 23. The operational amplifier 35 converts the output current generated according to the amount of light received by the photomultiplier tube (PMT) 14 into a voltage, and can detect even a minute current. The high-pass filter circuit 36 is a filtering unit that reduces the frequency component lower than the predetermined frequency without attenuating the frequency component higher than the predetermined frequency in the input signal. On the other hand, the low-pass filter circuit 37 is a filtering unit that reduces the frequency component higher than the predetermined frequency without attenuating the frequency component lower than the predetermined frequency in the input signal. When the high-pass filter circuit 36 and the low-pass filter circuit 37 are connected, a band-pass filter circuit 38 that passes only a necessary frequency range is obtained.

  The operational amplifier 35 has an operational amplifier OP and a resistor R. The high-pass filter circuit 36 and the low-pass filter circuit 37 have resistors R1 and R2 and capacitors C1 and C2 that are electrically connected to each other. As a result, the output current from the photomultiplier tube (PMT) 14 is converted into a voltage by the operational amplifier 35. Next, when the signal Vin (t) is input from the input side of the band-pass filter circuit 38, disturbance is generated on the output side. A signal Vout (t) obtained by filtering the electrical signal is output. If this filtered signal Vout (t) is input to the CPU board 23, an electrical signal corresponding to the amount of received fluorescence of the microorganism and the disturbance are already clearly distinguished, so that a measurement error of the amount of microorganism occurs. Therefore, there is no problem that the measured values vary, and stable measurement is possible.

  FIG. 5 is a flowchart showing a measurement flow, and the operation of the above configuration will be described with reference to FIGS.

  First, the operator uses a pipette or the like to collect 100 ml (milliliter) as a sample from ballast water at a temperature of about 20 ° C. and put it into the sample container 5 (step 1 in FIG. 5). Next, a fluorescent staining reagent is added into the sample container 5 (step 2 in FIG. 5). As this fluorescent staining reagent, generally known calcein AM (Calcein-AM, manufactured by Promocell GMBH, Germany), FDA or the like can be used. Calcein AM tends to stain phytoplankton, while FDA tends to stain zooplankton. Therefore, staining with a staining reagent is mixed with calcein AM and FDA. When dyeing is performed with the reagent, it is possible to shorten the dyeing time of the reagent and halve the time required for dyeing. Then, after the operator puts the rotor 7 into the sample container 5, the operator accommodates it in the measurement unit 6 of the inspection apparatus 1 and attaches the lid 30 of the measurement unit 6 to complete the measurement preparation. Here, when the power button 3a is pressed, the rotor 7 is rotated by driving the magnetic stirrer 27 built in the measuring unit 6, and the sample solution S is stirred (step 3 in FIG. 5). .

  Next, when the operator depresses the measurement start button 3b of the operation unit, the LED light source 10 is turned on after a predetermined time, and the light transmitted through the excitation light band-pass filter 12 is irradiated to the sample container 5. At this time, for example, light having a wavelength of 450 nm to 490 nm is irradiated as wavelength characteristics, and the specimen (microorganism) in the sample container 5 emits fluorescence (step 4 in FIG. 5). This fluorescence passes through the fluorescence band-pass filter 15 and is detected by the photomultiplier tube (PMT) 14 (step 5 in FIG. 5).

  The photomultiplier tube (PMT) 14 converts light energy into electrical energy by utilizing the photoelectric effect, and has a current amplification function, and can detect fluorescence emission with high sensitivity. The detected electrical signal is amplified by the operational amplifier 35 and input to the band-pass filter circuit 36, and a signal obtained by filtering the electrical signal that becomes a disturbance is output (step 6 in FIG. 5). Then, the signal obtained by filtering the electric signal that becomes a disturbance is sent to the CPU board 23, and the received light waveform exceeding a certain threshold value is counted (step 7 in FIG. 5).

  Further, the CPU board 23 estimates the number of microorganisms present in 100 ml (milliliter) of water in the sample container 5 from the received light wave count value, and displays on the display unit 4 whether or not the drainage standard is satisfied. Yes (step 8 in FIG. 5).

  Examples of the present invention will be described below. First, a confirmation test of the inspection accuracy of the microorganism inspection apparatus of the above embodiment was performed.

  The correlation between the microbial population and the photomultiplier tube (PMT) light reception count was investigated. S-type scallop worms (minimum size of about 100 μm = L size organisms) are accommodated individually in a plurality of sample containers 5 (100 mL capacity) as 5 individuals, 10 individuals, 50 individuals, 100 individuals, 1000 individuals, Staining was performed with a fluorescent staining reagent FDA (concentration 0.01 [millimol / liter]). As a result, the number of waveform counts increased in accordance with the number of contained microorganisms, and 5 samples of 5, 10, 50 and 100 responded linearly (FIG. 8 (a)). FIG. 8 (b)). Therefore, the number of microorganisms present in 100 mL of ballast water can be estimated from the obtained waveform count.

  A test was conducted as to whether or not detection was possible due to the life and death of microorganisms (see FIGS. 9A to 9D). S-type rotifer (minimum size of about 100 μm = L size organism) that has been killed by heat (heated at 60 ° C. for 30 minutes) with a fluorescent staining reagent FDA (concentration 0.01 [millimol / liter]) Stain. Three samples were prepared 1 hour after the killing treatment, 20 hours after the killing treatment, and 5 days after the killing treatment. As a result, no waveform with a certain threshold value or more was found in any of the killed samples, and it was possible to discriminate between samples that contained microorganisms without killing and samples that did not contain microorganisms. Is possible.

Between the photomultiplier tube (PMT) 14 and the CPU substrate 23, the waveform of the acquired voltage before and after mounting the filtering means is compared.
FIG. 10 shows the waveform of the acquired voltage before the filtering means is attached. The waveform of the acquired voltage shown in FIG. 10 (a) is a disturbance (background component about 0.9V waveform), a clear mountain that can be identified as a living microorganism that has exceeded the threshold, and an error that does not exceed the threshold. Three with a clear mountain are mixed. In addition, the waveform of the acquired voltage shown in FIG. 10 (b) is a mixture of disturbance (background component approximately 1.4V waveform) and a clear mountain that can be identified as a living microorganism that exceeds the threshold. It has become. Furthermore, the waveform of the acquired voltage shown in FIG. 10C is a mixture of disturbance (background component approximately 0.4 V waveform) and an unclear peak that does not exceed the threshold value.

  FIG. 11 shows the waveform of the acquired voltage after the filtering means is mounted. The waveform of the acquired voltage shown in FIG. 11A is obtained when only the high-pass filter circuit 36 is attached in order to remove a background component that becomes a disturbance. Compared with the waveform shown in FIG. 10B, it can be seen that the background component of about 1.4 V is converged to 0 V, and the disturbance is removed.

  The waveform in FIG. 11B is a rising waveform when the waveform of a mountain surrounded by a broken-line circle shown in FIG. In the waveform of FIG. 11B, the vertical movement is repeated across the threshold value, which causes a measurement error to increase. Therefore, it is desirable to attach a low-pass filter circuit 37 for the purpose of removing such a high-frequency waveform.

  The waveform of the acquired voltage shown in FIG. 11C is obtained when a band-pass filter 38 in which a high-pass filter circuit 36 and a low-pass filter circuit 37 are combined is mounted in order to remove disturbance and high-frequency waveform. is there. Compared with the waveform of FIG. 11A, the high frequency noise was removed and the waveform became smooth.

  The waveform in FIG. 11 (d) is a rising waveform when the waveform of the mountain surrounded by the broken-line circle shown in FIG. 11 (c) is enlarged. In the waveform of FIG. 11 (d), the jagged waveform as shown in FIG. 11 (b) is eliminated and smoothed, and the vertical movement is not repeated across the threshold value. Thereby, it becomes possible to measure with high accuracy by making the measurement error extremely small.

As described above, according to the present embodiment, after the sample and the fluorescent staining reagent are added to the sample container 5, the sample solution is stirred and mixed by the stirring and mixing means 7, and then the sample solution is stirred while the sample solution is stirred. The excitation light is incident on the irradiated surface of the sample container, and the fluorescent light emission of the microorganisms is received by the light receiving means, so that the microorganisms emit light brightly in a very short time compared to the measurement without standing and stirring. In addition, the amount of microorganisms in the ballast water can be measured easily and in a short time. And it becomes possible to miniaturize an apparatus and to reduce manufacturing cost.
Further, since the disturbance is filtered by the filtering means before the electric signal is taken into the control means, the electric signal corresponding to the amount of received fluorescence of the microorganism and the disturbance can be clearly distinguished, and A measurement error does not occur, and there is no problem that the measurement value varies, so that stable measurement can be performed.

  The present invention can be applied to a microorganism testing apparatus for confirming whether or not the discharge standard is satisfied when discharging ballast water.

DESCRIPTION OF SYMBOLS 1 Inspection apparatus 2 Main body part 3 Operation part 4 Display part 5 Sample container 6 Measuring part 7 Rotor 8 Holding plate 9 Sample container accommodating part 10 LED light source 11 Parallel light conversion means 12 Excitation light band pass filter 13 Light source part 14 Photoelectron increase Double pipe (PMT)
DESCRIPTION OF SYMBOLS 15 Fluorescence band pass filter 16 Condensing lens 17 Slit 18 Relay lens 19 Light-receiving part 20 Case 21 AC power supply 22 Secondary battery 23 CPU board 24 AC / DC converter 25 RAM
26 ROM
27 Magnetic Stirrer 28 Fan 29 External Output Terminal 30 Lid 31 Flat Plate 32 Screw Cutting Hole 33 Cylindrical Lens 34 Filtering Means 35 Operational Amplifier 36 High Pass Filter Circuit 37 Low Pass Filter Circuit 38 Band Pass Filter Circuit

Claims (8)

Estimate the allowable number of living microorganisms contained in the ballast water discharged from the ship based on the number of microorganisms present in the water in the sample container collected as a sample, and whether or not the above drainage standard for ballast water is satisfied an inspection apparatus of a microorganism for measuring or,
A stirring and mixing means for stirring and mixing the sample solution by adding the sample and a fluorescent staining reagent to a sample container formed of a light transmitting material;
An excitation light source comprising a light source for continuously irradiating the irradiated surface of the sample container with excitation light while stirring the sample solution by the stirring and mixing means;
A light receiving means for receiving the fluorescence emitted by the excitation light of the excitation light source and converting it into an electrical signal;
Filtering means for filtering low-frequency component noise related to fluorescence emission of the sample solution itself and / or high-frequency component noise related to the waveform of the electrical signal in the electrical signal converted by the light receiving means;
Based on the electrical signal filtered by the filtering means, the number of luminescence generated when microorganisms pass through a predetermined light receiving range is detected and counted, and the amount of microorganisms contained in the sample in the sample container is calculated from the number of luminescence. Control means;
An operation unit electrically connected to the control means,
The microbe inspection apparatus, wherein the operation unit includes a setting button capable of switching a size of a microbe to be measured.   2. The microorganism testing apparatus according to claim 1, wherein the filtering means is a band-pass filter in which a high-pass filter and a low-pass filter are connected.   The excitation light source is arranged so that excitation light orthogonal to the irradiated surface of the sample container is irradiated, while the light receiving means receives fluorescence emission at an angle orthogonal to the excitation light of the excitation light source. The microorganism testing apparatus according to claim 1, wherein a light receiving surface is arranged.   4. The microorganism testing apparatus according to claim 1, wherein a slit for regulating an observation range is provided between the light receiving means and the sample container.   The parallel light converting means for converting the diffused light from the light source into parallel light that is uniformly irradiated at the same angle toward one surface is provided between the excitation light source and the sample container. The microorganism testing apparatus according to any one of the above.   6. The microorganism testing apparatus according to claim 5, wherein the parallel light converting means is formed by drilling a threaded hole having a predetermined diameter in a flat plate having a predetermined thickness.   6. The microorganism testing apparatus according to claim 5, wherein the parallel light converting means is formed of a convex lens. Estimate the allowable number of living microorganisms contained in the ballast water discharged from the ship based on the number of microorganisms present in the water in the sample container collected as a sample, and whether or not the above drainage standard for ballast water is satisfied A method for testing microorganisms for measuring
A size setting process for setting the size of the microorganism to be measured;
A stirring and mixing step of stirring and mixing the sample solution in which the sample and the fluorescent staining reagent are added to the sample container;
An excitation step of continuously irradiating the irradiated surface of the sample container with excitation light while stirring the sample solution;
A light receiving step for receiving the light emitted by the fluorescent light by the excitation light and converting it into an electrical signal;
A filtering step of filtering low-frequency component noise related to fluorescence emission of the sample solution itself and / or high-frequency component noise related to the waveform of the electric signal in the electric signal converted by the light receiving step;
Estimating the number of microorganisms by detecting the number of emitted light when a microorganism passes through a predetermined light receiving range based on the electrical signal filtered by the filtering step, and calculating the amount of microorganisms contained in the sample in the sample container from the number of emitted light And a method for inspecting microorganisms.

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