JPH01250043A - Methanogen measuring instrument - Google Patents

Abstract

PURPOSE:To execute image processing with high accuracy by setting an excitation light wavelength region and fluorescence wavelength region respectively at 392-418nm and 462-502nm and setting excitation light intensity at >=2,000W/m<2> and camera sensitivity at >=45muA/Lx. CONSTITUTION:The excitation light wavelength region of the light emitted from a light source 13 is set at 392-418nm by an optical filter 12 and the light intensity of this excitation light wavelength region is set at >=2,000W/m<2>. The fluorescence wavelength region is limited to 462-502nm by the optical fiber 16 and the camera sensitivity to >=45muA/Lx, then the fluorescent images of various methanogens are measured. The fluorescent image right after the irradiation of the excitation light and the fluorescent image after the lapse of the prescribed time are comparatively studied and the annihilated images are specified as the methanogen.

Description

[Detailed Description of the Invention] (Industrial Application Field) The present invention relates to a methanogen measuring device, which is one of the microbial activity measuring devices, and particularly relates to a methanogen measuring device that measures the concentration or activity of methanogens in fermentation processes, sewage treatment processes, etc. The present invention relates to a methane-producing bacteria measuring device that measures methane-producing bacteria.

[Conventional technology]

The third example is the first conventional example of a device for measuring the concentration of microorganisms.
There is one shown in the figure. This measures the absorbance of the subject.

In the figure, 1 is a fermentation tank, 8 is a specimen containing microorganisms, 2 is a light source that mainly emits visible light, 3 is a light source power source, 4 is a photomultiplier tube, 5 is the same power source, and 6 is a photomultiplier tube This is a detector that measures the photocurrent of 4.

Next, the operation will be explained.

Light emitted from the light source 2 passes through the specimen 1 containing microorganisms, this transmitted light is received by the photomultiplier tube 4, and its intensity is measured by the detector 6 as the photocurrent value of the photomultiplier tube 4. . Then, the absorbance is calculated from this value and the value corresponding to the light source.

Since a certain relationship holds between the absorbance measured in this way and the microorganism concentration, the microorganism concentration can be measured.

A second conventional example is shown in Fig. 4 (Japanese Unexamined Patent Publication No. 59-205998), which selectively measures the number of methane-producing bacteria. The principle is to measure the fluorescence intensity of fluorescent substance F420.

In the figure, 1 is a fermentation tank, 8 is an analyte contained inside the methane fermentation tank, 9 is an optical fiber for introducing and guiding light into the methane fermentation tank, and 10 is an optical fiber 9 for transmitting light emitted from a light source 13. A condenser for condensing light, 11 is a light source 13
12 is an optical filter for limiting the wavelength of the light from the light 1a13, 14 is a light source power source, 15 is a condenser for condensing the light emitted from the optical fiber 9, 16 is an optical filter, 17 is a photomultiplier tube, 18 is a power source, and 19 is a detection unit for measuring the photocurrent of the photomultiplier tube 17.

Next, the operation will be explained.

The light emitted from the light source 13 is limited to the excitation light wavelength region by the optical filter 12 . Next, the limited light is focused onto the optical fiber 9 by the condenser 10, and introduced as excitation light into the methane fermentation tank containing the specimen 8 via the optical fiber 10, and is irradiated onto the specimen 8. . Coenzyme F420 present inside the cells of the methanogen contained in the subject 8 receives this excitation light and emits fluorescence. This fluorescent light is sent to a condenser 15 via an optical fiber 9 and condensed, and its wavelength is further limited by an optical filter 16. The light whose wavelength range is limited by the optical filter 16 is received by the photomultiplier tube 17, and its intensity is measured by the detection unit 19 as a photocurrent value. Since a certain relationship exists between the fluorescence intensity thus obtained and the methanogens, the concentration of the methanogens can be evaluated by measuring the fluorescence intensity.

A third conventional example is shown in FIG.

This involves taking fluorescent images of methanogens and identifying them through image processing.

In the figure, 20 is a lens optical system for enlarging a fluorescent image emitted by methanogenic bacteria, 21 is a camera for taking a fluorescent image, and 22 is a fluorescent image processing device. is connected to.

Next, the operation will be explained.

The light emitted from the light source 13 is limited to a specific wavelength region by the optical filter 12. Next, this light with a limited wavelength is focused onto an optical fiber 9 by a condenser 10, and introduced as excitation light into the methane fermentation tank containing the subject 8 via the optical fiber 9. irradiated. Methanogens receive this excitation light and emit fluorescence from all areas of their cells. This fluorescent light is filtered by an optical filter 16 except for the wavelength range of the fluorescent light, and then magnified by a lens optical system 20, and then a fluorescent image is photographed by a camera 21.

The fluorescence image obtained in this way is sent to the image processing device 22 via the signal vA23, and the fluorescence intensity of the fluorescence image is
The shape is identified, and foreign substances other than methane-producing bacteria are removed.

Further, as a modification of this third conventional example, there is one shown in FIG.

In the figure, 24 is a conduit that guides the specimen 8 containing microorganisms to the measuring instrument, and 25 is a pipe for fixing the specimen 8 containing microorganisms during measurement. ing. 26 is the condenser 10
This is a cover glass for irradiating the subject 8 with the excitation light focused by the subject 8 and extracting the fluorescent light emitted by the subject 8 to the outside of the conduit 24.

Next, the operation will be explained.

The subject 8 sent through the conduit 24 is held and fixed by the plunger 25 between the cover glass 26 and the plunger 25. At this time, the light is emitted from the light source 2, is limited to a specific wavelength region by the optical filter 12, and is emitted from the light condenser 1.
The excitation light focused by 0 passes through the cover glass 26 and is irradiated onto the fixed subject 8 . The fixed subject 8 receives this excitation light and emits fluorescent light. This fluorescent light is extracted to the outside of the conduit 24 via the cover glass 26, magnified by the lens optical system 20, and a fluorescent image is taken by the camera 21.
Sent to 2. The image processing device 22 performs the same image processing as in the third conventional example. Therefore, it is theoretically impossible to selectively measure methanogens.

The second conventional example has a problem in measurement accuracy because it is affected by coexisting substances that overlap with the fluorescence wavelength region of F420 and F420 released outside the body.

In the third conventional example, the accuracy of the fractional measurement of methanogens has been greatly improved, but if you want to identify methanogens with high precision using normal image processing mainly based on the shape, it is difficult to identify the methanogens with high precision. Processing techniques are required, which increases processing time and requires expensive equipment.

In order to solve the above-mentioned problems and obtain a fluorescent image with less noise, it is desirable to make the wavelength width of the excitation light wavelength as narrow as possible, and also to make the wavelength width of the detected fluorescent light as narrow as possible. In the conventional example 3, the width and intensity are determined by considering the intensity of the light source, the wavelength cut filter characteristics, and the excitation and fluorescence spectral widths. However, this consideration alone is not sufficient, and it is necessary to It is necessary to investigate the fluorescent properties of methanogens and reflect them in image processing.

This invention was made to solve the above-mentioned problems, especially the drawbacks of the third conventional example.
The purpose of the methane-producing bacteria measuring device according to the present invention is to provide a methane-producing bacteria measuring device that enables highly accurate image processing by examining the relationship between intensity and fluorescent image characteristics in detail. Limiting the wavelength range to 392 to 418 nm,
The light intensity of this excitation light wavelength range is 2000 watts/m2 or more, and the fluorescent wavelength range is 462 to 502 watts/m2 or more.
nm, camera sensitivity 45. We measured the fluorescence images of various methanogens limited to crA/fx or higher, and compared and examined the fluorescence images immediately after irradiation with excitation light and the fluorescence images after a certain period of time, and found that the images that disappeared were those of methanogens. It was designed to specify that.

[Effect]

In the methane-producing bacteria measuring device of the present invention, the width of the excitation light wavelength range, the light intensity of the excitation light wavelength range, the width of the fluorescence wavelength range,
Since the camera sensitivity is limited as described above, the time decay of the fluorescence intensity of methanogens is accelerated, and images that disappear from the fluorescence image after a certain period of time can be identified as methanogens, so it is possible to identify methane-producing bacteria. Bacteria image processing is simplified and highly accurate.

〔Example〕

Since the structure of the methanogen-producing bacteria measuring device according to an embodiment of the present invention is similar to the structure of the third conventional example shown in FIG. 1, the embodiment of the present invention will be described below using this structure.

The excitation light wavelength region of the light emitted from the light source is limited to 392 to 418 nm by the optical filter 12, the light intensity of this excitation light wavelength region is set to 2000 watts/m2 or more, and the fluorescence wavelength region is limited to 462 to 50 nm by the optical filter 16.
Stable fluorescence images can be obtained by measuring the fluorescence images of various methanogens at a wavelength of 2 nm and a camera sensitivity of 45 μA/Ilx or higher. The wavelength region width of the excitation light described above is about the half-width of the excitation spectrum of F420, a fluorescent substance specific to methanogens, and such selection makes it possible to obtain a high-quality and stable fluorescence image of methanogens.

Next, a fluorescent image immediately after excitation light irradiation and a certain period of time (1 second to 5 seconds) are displayed.
2) Compare and examine the fluorescent images after the elapse of time and identify the disappeared images as methanogenic bacteria.

In addition, as other characteristics of methanogens, after fluorescence image measurement,
When the excitation wavelength range is wider than above and the light intensity is increased, the fluorescence intensity of the methanogens quickly attenuates and the fluorescence image disappears. However, the fluorescence intensity of coexisting substances such as acid-producing bacteria such as Escherichia coli does not exhibit this phenomenon.

In this way, the present invention has discovered a phenomenon in which the time decay of the fluorescence intensity of methanogens is accelerated by selecting the excitation light wavelength region, the light intensity of the excitation light wavelength region, the fluorescence wavelength region, and the camera sensitivity. This phenomenon is unique to the fluorescent substance F420 in methanogens. Incorporating this into image processing greatly facilitates the identification of methanogens.

The present invention can also be applied to the methane-producing bacteria measuring device shown in FIG. 2, which is a modification of the third conventional example. In this other embodiment of the present invention, a conduit 24 is provided to guide the specimen 8 from the fermentation tank l, and the specimen 8 is fixed during measurement. In addition, the subject 8 sandwiched between the plunger 25 and the cover glass 26
This has the effect that the capacity of the measuring device becomes constant, the measurement accuracy improves, and maintenance of the measuring device becomes easier.

〔Effect of the invention〕

As described above, according to the methane-producing bacteria measuring device of the present invention, the excitation light wavelength range is 392 to 418 nm, the light intensity of this excitation light wavelength range is 2000 watts/square meter or more,
Fluorescent wavelength range: 462-502 nm, camera sensitivity: 45
Since it is configured to measure the fluorescence images of various methanogens by limiting it to μA/j2x or more, it is possible to significantly weaken the fluorescence of coexisting substances such as acid-producing bacteria such as Escherichia coli.

This makes it easier to identify methane-producing bacteria through image processing than before, simplifies image processing, and provides an overall highly accurate and inexpensive methane-producing bacteria measuring device.

[Brief explanation of the drawing]

FIG. 1 is a configuration diagram of a methane-producing bacteria measuring device according to a third conventional example or an embodiment of the present invention, and FIG. 2 is a block diagram of a methane-producing bacteria measuring device according to a modification of the third conventional example or another embodiment of the present invention. FIG. 3 is a block diagram of a first conventional methane-producing bacteria measuring device, and FIG. 4 is a block diagram of a second conventional methane-producing bacteria measuring device. 1 is a fermentation tank, 8 is a specimen containing methanogenic bacteria, 9 is an optical fiber, 10 is a concentrator, 11 is a selector, 12.16
13 is an optical filter, 13 is a light source, 14 is a power supply, 20 is a lens optical system, 221 is a camera, 22 is an image processing device, 23 is a signal line, 24 is a conduit, 25 is a plunger, and 26 is a cover glass. Note that the same reference numerals in the figures indicate the same or equivalent parts.

Claims (1)

[Claims] (1) A light irradiation means for irradiating an object containing methanogens with excitation light in a predetermined wavelength range; , comprising means for acquiring fluorescence in a predetermined wavelength range as a fluorescence image, and an image processing circuit for processing the fluorescence image to eliminate fluorescence based on substances other than methane-producing bacteria; In a methane-producing bacteria measuring device that measures the concentration or activity of
502 nm, an excitation light intensity of 2000 watts/square meter or more, and a camera sensitivity of 45 μA/lx or more.