JP2013201925A - Water inspection method and water inspection system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a water inspection method and a water inspection system which are capable of accurately inspecting water in a short time to inspect whether or not microorganism is included in water as a tested object.SOLUTION: In inspecting whether or not microorganism is included in water as a tested object, the temperature of agar where the water as a tested object is smeared is kept to a constant temperature in a constant temperature bath. After the agar is held at a constant temperature in the constant temperature bath, the rising of the surface of the agar is measured using a measuring device. Based on the measurement result of the rising, it is determined whether or not the water includes the microorganism.

Description

  The present invention relates to a water inspection method and a water inspection system for inspecting whether or not microorganisms are contained in water to be inspected.

For ships such as cargo ships and tankers, when unloading cargo or crude oil, etc. at the unloading port and navigating to the loading port again, in order to balance the navigating ship, ballast water and Stores seawater called. Ballast water is generally drawn up and stored in a tank when unloading at a loading port, and discharged from the tank when loading at a loading port.
Ballast water taken from seawater at the unloading port may contain harmful microorganisms such as cholera and E. coli. When ballast water is discharged at the loading port, harmful microorganisms contaminate the sea area near the loading port. Depending on the environmental conditions, these harmful microorganisms can multiply in the sea area near the cargo port and cause serious damage such as killing organisms that originally live in the sea area. For this reason, the port management business unit is inspecting whether or not harmful microorganisms are contained in the ballast water.

Conventionally, culture methods, staining methods, and microcolony methods have been used as inspection methods for inspecting whether or not harmful microorganisms are contained in ballast water.
For example, in the culture method, a microorganism formed by spreading on an agar medium by smearing a ballast water to be inspected on an agar medium formed on a petri dish and leaving it to stand at a constant temperature condition for about one day. The above-mentioned test is performed by discriminating colonies and counting the number of colonies.
In the staining method, a reagent that reacts with the respiratory activity of microorganisms is included in ballast water. The microorganisms are stained by respiration of the microorganisms incorporating the reagent. The above-described inspection of ballast water is performed by coefficienting the stained microorganism with a microscope or the like.
In the microcolony method, as in the case of the culture method, the ballast water to be inspected is smeared on the agar medium formed on the petri dish and allowed to stand at a constant temperature for a certain period of time. Perform the above test for ballast water by examining at the microscopic stage.
For example, when inspecting whether coliform bacteria and E. coli are contained in the ballast water, a coating plate method is used. Specifically, the medium MImedium is used as the agar medium, and the ballast water is diluted with a diluent containing a phosphate buffer or the like. That is, the diluted ballast water is smeared on the flat plate on which the medium MImedium is formed. Culturing is performed by leaving it to stand for a very long time of 18 to 24 hours under a temperature condition of 33 to 47 ° C. The medium MImedium is proteose peptone no. 3. A known agar medium containing yeast extract, β-D-lactic acid and the like and agar. The number of colonies (colonies) of Escherichia coli can be obtained by counting the blue region emitted by Escherichia coli by irradiating the medium with ultraviolet light having a wavelength of 366 nm.
In 2004, since the Ballast Water Management Convention was adopted by the International Maritime Organization (IMO), ballast water treatment technology, which is a solution to the ship ballast water problem (proliferation and widening of aquatic organisms by ballast water), Development is progressing. Accordingly, there is a demand for a method for detecting a ballast water microorganism quickly and easily.

  For example, as an example of the above microcolony method, water to be examined is smeared on an agar medium formed on a petri dish, and the microcolony formed by the growth of E. coli is captured as an image with a CCD camera to identify the microcolony The method of doing is known (nonpatent literature 1).

“Rapid microbiological inspection technology”, Naohiro Noda, Takamasa Asano, Yujiro Kitade, Fuji Toki Method, Vol. 77 No. 2,2004

The above "microorganism rapid inspection technology" can automatically detect colonies and can inspect quickly compared to conventional agar medium. However, more rapid inspections are necessary for future water inspections. Is sought after. In particular, the inspection of ballast water that is performed to determine whether or not ballast water can be discharged into seawater is required to be performed quickly from the viewpoint of berthing usage fees that increase with the berthing time at the port.
In addition, in the staining method, a stained substance other than microorganisms is erroneously counted due to contaminants contained in the water to be examined, so that accurate measurement is difficult.

  Accordingly, an object of the present invention is to provide a water inspection method and a water inspection system that can accurately inspect water in a short time when inspecting whether or not microorganisms are contained in the water to be inspected. To do.

One embodiment of the present invention is a water inspection method for inspecting whether or not microorganisms are contained in water to be inspected. The method is
Maintaining the temperature of the agar medium smeared with water to be inspected at a constant temperature in a thermostatic chamber;
Measuring the height of the swell of the surface of the agar medium using a measuring device after holding the thermostat at a constant temperature; and
And a step of determining whether the determination device is water containing microorganisms based on the measurement result of the swell.

  In that case, the measuring device is preferably a digital holography microscope.

  Moreover, it is preferable that the said measuring apparatus measures the height of the rise of 0.2 micrometer-5 micrometers.

  It is preferable that the measuring device obtains a raised distribution on the surface of the agar medium.

  It is preferable that the measurement by the measurement device is performed at predetermined time intervals, and the determination device makes the determination based on the amount of swelling within the time interval at the same position of the agar medium.

  The constant temperature is preferably 20 to 40 ° C.

Another aspect of the present invention is a water inspection system that inspects whether or not microorganisms are contained in water to be inspected. The system
A thermostatic chamber that maintains the temperature of the agar medium smeared with water to be inspected at a constant temperature;
A measuring device for measuring the height of the surface of the agar medium after the agar medium is maintained at a constant temperature in the thermostat;
And a determination device that determines whether the water contains microorganisms based on the measurement result of the swell.

  The measurement device is preferably a digital holography microscope.

  In the above-described water inspection method and water inspection system, water can be accurately inspected in a short time.

It is a figure which shows the flow of the water test | inspection method of this embodiment. It is a figure which shows the outline | summary of the water test | inspection system of this embodiment. (A)-(c) is a figure explaining the culture | cultivation using the agar culture of this embodiment. It is a graph of an example which shows the increase in the microorganisms on an agar medium when it enters into the logarithmic growth phase in this embodiment. It is a photograph showing the state of start of confluence of E. coli immediately after the logarithmic growth phase. It is a figure which shows the area | region where the swelling on the agar medium of this embodiment is measured.

Hereinafter, the water inspection method and the water inspection system of the present invention will be described in detail.
FIG. 1 is a diagram illustrating a flow of a water inspection method of the present embodiment, and FIG. 2 is a diagram illustrating an outline of the water inspection system of the present embodiment. Although this embodiment demonstrates based on the example which determines whether a harmful microorganism is contained in the ballast water which the ship stored in the tank, not only ballast water but test | inspection object may be seawater or water. Good. The microorganism referred to in the present embodiment is a bacterium having a size of 0.2 to 5 μm, for example, bacteria such as Mycobacterium tuberculosis, diphtheria, shigella, Escherichia coli, Salmonella typhi, Bordetella pertussis, staphylococci, and cholera. .

The water test method of the present embodiment is a short period of time before the microorganisms grow and form colonies or microcolonys by spreading ballast water on the agar medium and growing the microorganisms contained in the ballast water. Water inspection can be performed.
First, a plurality of test dishes 14 each having an agar medium 12 formed on the petri dish 10 are prepared, and ballast water is smeared on the agar medium 12 (step S10).

  In the ballast water test, for example, it is determined whether or not microorganisms such as cholera bacteria, Escherichia coli (Coliform group), enterococci and the like are included in the ballast water beyond the allowable limit. The agar medium 12 varies depending on the type of microorganism to be grown. For example, in order to culture Vibrio cholerae, for example, a TCBS agar medium is used. The TCBS agar medium includes yeast extract, bacterial papton, sodium thiosulfate, sodium citrate, bovine bile acid, succinic acid, sodium chloride, ferric citrate, promotier blue, thymol blue, and agar. In order to culture Escherichia coli, for example, an LB agar medium containing tryptone, yeast extract, sodium chloride, agar and the like is used. For culturing enterococci, a MacConkey agar medium containing bile acids, sodium chloride, neutral red, lactose, peptone and agar is used.

  Vibrio cholerae is, for example, 0.3 to 5.0 μm in size, and Escherichia coli is 0.5 to 3.0 μm in size. In this embodiment, the bulge of the surface of the agar medium is measured using a holographic microscope. It is a size suitable for measurement using

  The petri dish (inspection petri dish 14) provided with the agar medium 12 coated with ballast water is placed in the thermostat 16, and the agar medium 14 is kept at a constant temperature in the thermostat 16 and left to stand (step S10). The thermostat 16 is not particularly limited, and is preferably 20 to 40 ° C., for example, in that microorganisms can be grown. Furthermore, it is possible to set the time for holding at a constant temperature within 8 hours, preferably within 7 hours, preferably within 6 hours, and more preferably within 5 hours. As will be described later, using a digital holographic microscope, it is possible to measure the height of the swell as much as the size of the microorganism, for example, the swell height of 0.3 μm to 5 μm, so that the microcolony can be visually recognized. The presence / absence of microorganisms can be determined in a time shorter than the time required for the process.

  Further, the holding time and holding temperature of the agar medium 14 vary depending on the type of microorganism to be grown. In the case of Vibrio cholerae, the holding temperature is, for example, 35 to 37 ° C., in the case of Escherichia coli, for example, 33 to 37 ° C., and in the case of enterococci, for example, 35 to 39 ° C.

Next, after a certain time, the agar medium 14 is taken out from the thermostatic chamber 16, and the height of the swell of the surface of the agar medium 14 (swell due to the growth of microorganisms) is measured using the holographic microscope 18 (step S14). For example, the holographic microscope 18 measures the swell of the surface of the agar medium 14 at intervals of 10 to 20 minutes after, for example, 3 hours have elapsed since the start of the culture on the agar medium 14.
FIGS. 3A to 3C are diagrams for explaining the culture using the agar culture 12. As shown in FIG. 3A, a ballast water droplet 20 is dropped on the agar culture 12, and the ballast water is smeared on the agar culture 12. As the ballast water, water from which aquatic organisms such as plankton have been removed in advance using a plankton net, a membrane filter or the like is used. The ballast water droplet 20 spreads on the agar medium 12. As shown in FIG. 3B, on the agar medium 12, microorganisms 22 such as Escherichia coli are present at the beginning of dropping, but the microorganisms 22 start to grow by the process of step S12. In particular, in the portion where active growth is carried out, as shown in FIG. 3 (c), the microorganisms grow and clump, and thus the portion 24 where the surface of the agar medium 12 is slightly raised is formed. Such growth begins, for example, 3 hours after the start of culture. That is, the microorganism 22 enters the logarithmic growth phase. FIG. 4 is a graph of an example showing an increase in the number of microorganisms per unit volume on the agar medium 12 when entering the logarithmic growth phase. In such a logarithmic growth phase, the swell of the surface of the agar medium 12 is measured 5 times during a time T, for example, 1 hour, that is, every 15 minutes (every fixed time interval). FIG. 5 shows a photograph showing the state of the start of confluence of E. coli immediately after the logarithmic growth phase.
In such a logarithmic growth phase, the surface of the agar medium 12 rapidly rises due to the denseness of the microorganisms 12. Therefore, the holographic microscope 18 measures such a rise.

  The holographic microscope 18 separates a plane wave laser beam, reflects one laser beam on the surface of the agar medium 12 and takes it into a CCD camera or the like, and the other separated laser beam through an optical system. Capture to a CCD camera (not shown). At this time, when the agar medium 12 does not rise, the optical path length of each laser beam is adjusted so that the phases of the laser beams received by the CCD camera are aligned. Thereby, the height of the rise of the surface of the agar medium 12 can be obtained by measuring the light and dark stripes on the agar medium 12 formed by the phase difference between the two laser beams. Examples of the holographic microscope 18 include a digital holographic microscope manufactured by Lynce Tec. The wavelength of laser light used in this digital holographic microscope is 683 nm. Since a light and dark image on the agar medium 12 is obtained by using the phase shift of the laser beam caused by the bulge of the surface of the agar medium 12, the holographic microscope 18 obtains the bulge of the surface of the agar medium 12. For example, the height of the swell of 0.3 μm to 5 μm can be measured. This excitement corresponds to the size of the growing bacteria.

  For example, the holographic microscope 18 can irradiate a narrow range of the agar medium 12 while scanning with a laser beam, and can measure the swell distribution on the surface. In the measurement, the information on the measurement position on the agar medium 12 is always acquired, and the swell distribution on the surface on the agar medium 12 is acquired. The reason why the measurement position on the agar medium 12 is always acquired is that information on the same measurement position is necessary in order to obtain the amount of surface swell at regular intervals, as will be described later. FIG. 6 is a diagram showing regions A to C where the swell on the agar medium 12 is measured. The holographic microscope 18 stores in advance information on the measurement positions (center positions of the areas A to C) of the areas A to C where the swell is measured in the first measurement in the measurement every certain period of time. In the second and subsequent measurements, measurement in a short time is realized by preferentially measuring the rise of the areas A to C. Of course, each time a newly raised area measured after the second time is acquired, information on the measurement position of this area can be added to the measurement positions of areas A to C.

  Information on the measured swell distribution on the surface of the agar medium 12 is sent to the determination device 26. The determination device 26 is configured by a computer, and the determination device 26 performs its function by starting a program recorded in a memory (not shown). Specifically, the determination device 26 processes the information on the bulge distribution on the surface of the agar medium 12 sent from the holographic microscope 18 and determines whether or not the ballast water contains microorganisms beyond the allowable range. Determine.

First, the determination device 26 determines whether or not the number of times that the surface swell is measured every predetermined time is equal to or greater than a preset number N (step S16). That is, in the present embodiment, the determination device 26 determines whether or not the climax has been measured N times at regular time intervals. If this determination is negative, the determination device 26 returns to step S14 and instructs the holographic microscope 18 to repeat the measurement. If the determination is affirmative, the determination device 26 proceeds to step 18.
When the determination in step S16 is affirmative, the determination device 18 determines whether or not the microorganisms are included in the ballast water beyond the allowable range using the information of the N swells on the surface of the agar medium 12 ( Step S18).

First, the determination device 26 performs a filtering process on the information on the surface swell to remove information on fine surface irregularities. The determination device 26 obtains the distribution of the amount of bulge on the surface of the agar medium 12 at regular time intervals based on the filtered surface bulge information. The amount of swell refers to the difference in the swell of the surface before and after the coincidence time elapses. In the determination device 26, the position of the bulge on the surface is fixed, the bulge amount is equal to or greater than a predetermined first threshold value at any fixed time interval, and the bulge area is preset. That the area is larger than the area is determined as the first determination condition. When any one of the rising regions (for example, the regions A to C in the case shown in FIG. 6) satisfies the first determination condition, the determination device 26 determines that the surface of the agar medium 12 is due to the growth of microorganisms. Is determined to have risen, and it is determined that the ballast water contains microorganisms exceeding the allowable range. On the other hand, the determination device 26 sets a second condition that the climax amount is equal to or smaller than a predetermined second threshold value at any time interval, and the bulge area is smaller than a preset area. When the second condition is satisfied, the determination device 26 determines that no microorganisms are included in the ballast water. If the first determination condition is not satisfied and the second determination condition is not satisfied, the determination device 26 determines that the microorganisms are not included in the ballast water. The determination result is printed out on a printer (not shown) or displayed on a display (not shown). In addition, when the determination apparatus 26 determines that the microorganisms are included in the ballast water, and further determines that the microorganisms are not included in the ballast water, the ballast water is a known ballast that is not illustrated. It is sent to a water treatment device for processing.
That is, the measurement of the swell of the surface of the agar medium 12 is performed at predetermined time intervals, and the determination device 26 makes the above determination based on the swell amount within the time interval at the same position of the agar medium 12. To do. In the example shown in FIG. 6, the amount of swell at the same measurement position is obtained at regular time intervals using information on the measurement positions of the areas A to C.

  Since ballast water is inspected using a plurality of agar media 12 as shown in FIG. 2, a plurality of determination results can be obtained for the ballast water. Therefore, when the frequency of occurrence of the determination result that the determination device 26 indicates that the ballast water includes microorganisms that exceed the allowable range for a plurality of determination results is equal to or higher than a predetermined ratio, the determination device 26 determines the individual agar medium 12. In addition to the determination result, a test result may be generated that the entire ballast water stored in the tank contains microorganisms exceeding the allowable range. Moreover, since the microorganisms which proliferate by agar medium 12 differ, it can also be determined by the kind of agar medium 12 which microorganisms are contained in ballast water exceeding tolerance. Such types of microorganisms are also printed out by a printer (not shown) or displayed on a display (not shown).

As described above, in the present embodiment, the temperature of the agar medium 12 coated with the ballast water to be inspected is maintained at a constant temperature using the thermostat 16, and then the surface of the agar medium 12 using the holographic microscope 18. Since the determination device 26 determines whether or not the water contains bacteria, based on the measurement result of the increase, for example, about 1 hour within several hours immediately after the start of the logarithmic growth phase of bacteria. Within, it can be determined whether the water contains bacteria.
In the present embodiment, since the digital holography microscope 18 is used for measuring the bulge of the surface of the agar medium 12, the bulge of the surface of the agar medium 12 due to the growth of Escherichia coli or the like can be accurately and quantitatively determined.
Since the holographic microscope 18 obtains the bulge distribution on the surface of the agar medium 12, the bulge of the surface of the agar medium 12 can be obtained no matter which region of the agar medium 12 is grown.
In the present embodiment, the measurement by the holographic microscope 13 is performed at predetermined time intervals, and the determination device 26 determines based on the amount of swell within the time interval at the same position (region) of the agar medium 12. Therefore, it is possible to obtain the presence or absence of the swell of the surface of the agar medium 12 very accurately.
In this embodiment, the constant temperature in the thermostatic chamber 16 is set to 20 to 40 ° C., and cells such as Escherichia coli, Vibrio cholerae, and enterococci can be more actively proliferated. Can be made. For this reason, the inspection of water can be performed in a short time, for example, within 8 hours.
It was necessary to leave in a thermostatic bath until colonies and microcolonys were formed on the agar medium as in the past, and it was difficult to quickly test water, but as in this embodiment, bacteria entered the logarithmic growth phase. Since the swell of the surface of the agar medium immediately after measurement can be measured very accurately, water can be tested quickly and easily.

  Although the microorganism testing method of the present invention has been described in detail above, the present invention is not limited to the above-described embodiments and modifications, and various improvements and modifications may be made without departing from the spirit of the present invention. Of course.

DESCRIPTION OF SYMBOLS 10 Petri dish 12 Agar medium 14 Inspection petri dish 16 Constant temperature bath 18 Holographic microscope 20 Droplet 22 Microorganism 24 Part 26 Judgment device

Claims (8)

A water inspection method for inspecting whether water to be inspected contains microorganisms,
Maintaining the temperature of the agar medium smeared with water to be inspected at a constant temperature in a thermostatic chamber;
Measuring the height of the swell of the surface of the agar medium using a measuring device after holding the thermostat at a constant temperature; and
And a step of determining whether the determination device is water containing microorganisms based on the measurement result of the swell.   The water test method according to claim 1, wherein the measuring device is a digital holography microscope.   The water test method according to claim 1 or 2, wherein the measuring device measures a height of swell of 0.2 µm to 5 µm.   The water test method according to any one of claims 1 to 3, wherein the measuring device calculates a swell distribution on the surface of the agar medium.   The measurement by the measurement device is performed at predetermined time intervals, and the determination device makes the determination based on the amount of swelling within the time interval at the same position of the agar medium. 5. The water inspection method according to any one of 4 above.   The said constant temperature is 20-40 degreeC, The water test | inspection method of any one of Claims 1-5. A water inspection system for inspecting whether water to be inspected contains microorganisms,
A thermostatic chamber that maintains the temperature of the agar medium smeared with water to be inspected at a constant temperature;
A measuring device for measuring the height of the surface of the agar medium after the agar medium is maintained at a constant temperature in the thermostat;
A water inspection system comprising: a determination device configured to determine whether or not the water contains microorganisms based on the measurement result of the swell. The water inspection system according to claim 7, wherein the measuring device is a digital holography microscope.

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