JP2007020528A - Method for inspecting microorganism in food by culture-combined fluorescent insitu hybridization method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for inspecting microorganisms in a food by a culture-combined fluorescent insitu hybridization method, by which the living specific microorganisms can quickly be detected/counted in high detection sensitivity and which realizes a sufficient detectable limit value, reduces a long time and enormous labors accompanied by the increase in the area of a membrane filter for filtration, and can be utilized in industrial fields such as a food production industry, a good processing industry, and the like. <P>SOLUTION: This method for inspecting the microorganisms comprises adjusting the performance conditions of the culture-combined fluorescent insitu hybridization method, such as the employment of a membrane filter having a larger or the same effective filtration area than or as the area of a circle having a diameter of ≥24 mm, to give a specific microorganism number-detectable limit value of ≤10 microorganisms (10 CFU/g) per g of a sample such as food, automatically inspecting the whole area of the membrane filter with a low magnification fluorescent light detection optical system, and then counting the number of the specific microorganisms by the utilization of an image processing technique. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for specifically detecting and counting a microorganism to be examined using an in situ hybridization method in industrial fields such as food manufacturing industry and food processing industry.

  Microbiological tests for detecting or counting specific microorganisms such as Escherichia coli, Salmonella, Listeria, Vibrio parahaemolyticus, enterobacteria, etc. are indispensable for sanitary evaluation in food and the environment. In general, these tests are performed by a culture method, and a sample is inoculated on an agar medium and cultured, and an estimation test and a confirmation test are performed on a suspicious colony, and it takes 1 to 8 days until positive determination is made.

  In recent years, a detection target microorganism in a sample is rapidly detected or counted by a fluorescence in situ hybridization method using a probe in which a fluorescent substance is labeled on an oligonucleotide having a base sequence complementary to the target base sequence of rRNA of the detection target microorganism. A method has been developed (see, for example, Patent Document 1 and Non-Patent Document 1). This method is performed as follows.

  A paraformaldehyde solution or the like is added to the sample suspension to perform immobilization (about 2 hours), and this is placed on a slide glass and dried. To this, a hybridization solution and a probe are added, kept at a specific temperature (about 3 hours), washed and dried, added with an encapsulant, and covered with a cover glass to prepare a specimen. This specimen is observed with a fluorescence microscope, and detection target microorganisms are detected by a fluorescent signal derived from the probe and counted.

  The fluorescence in situ hybridization method can detect and count microorganisms for various microorganisms by changing the type of probe to be used according to the type of microorganism to be identified. The results are obtained in about 5 hours) and are mainly used in the field of microbial ecology research.

However, this method has a drawback that the detectable limit value is about 10 ⁵ CFU / g (see, for example, Non-Patent Document 2), and life and death of the detected microorganism cannot be distinguished (for example, see Non-Patent Document 3). There is. In addition, when the number of rRNA copies in the detection target microorganism cell decreases due to the environmental conditions placed before the sample is collected, there is a problem that the detection target microorganism cannot be detected (see, for example, Non-Patent Document 2). ), It was difficult to apply to microbial tests in industrial fields such as food manufacturing and food processing.

  In order to apply the fluorescence in situ hybridization method to microorganism testing in these industrial fields, the present inventors have developed a fluorescence in situ hybridization method combined with culture and solved the above problems (for example, Patent Document 2, Non-patent document 4). This method is performed by the following steps (1) to (7).

(1) Filtration of microorganism suspension sample The sample suspension is filtered with a membrane filter (pore size 0.45 μm or less), and target bacteria are collected on the filter.
(2) Culture on solid medium The filter is placed on a solid medium (for example, an agar medium) and kept warm for a predetermined time (for example, about 6 hours).
(3) Fixing treatment The filter is placed on a filter paper containing ethanol for about 5 minutes and fixed.
(4) Hybridization A hybridization solution and a probe are added thereto and kept at a predetermined temperature.
(5) Cleaning After immersing the filter in a cleaning solution at a specified temperature for 15 minutes, rinse with distilled water.
(6) Specimen preparation The filter is placed on a slide glass, an encapsulant is added to the filter, and a cover glass is placed thereon, which is used as a specimen.
(7) Observation The specimen is observed with a fluorescence microscope.

  The combined fluorescence in situ hybridization method has three advantages over the conventional fluorescence in situ hybridization method. The first is that microbial microcolonies are formed on the membrane filter by culturing and that the microorganisms are alive. Second, even if the number of rRNA copies in the microbial cell in the sample decreases, the number of rRNA copies in the microbial cell increases due to the culture, and a strong fluorescence signal of the target microorganism can be obtained for detection and counting. To be. Thirdly, since it is only necessary to observe microcolonies larger than microorganisms, it is possible to reduce the objective lens magnification of a fluorescent microscope, which normally requires 40 times or more, to 10 times, and use a membrane filter having a diameter of 13 mm. In this case, the entire filter can be observed with a spectroscopic view of 27 fields in about 5 minutes. At this time, since the filtration amount of the membrane filter is 0.1 g in many solid foods, the detectable limit value is 100 CFU / g, and the detection sensitivity 1000 times that of the conventional fluorescence in situ hybridization method described above can be realized. Become.

Special table 2004-523223 JP 2004-16014 A Amann, R., Snaidr, J., Wagner, M., Ludwig, W. and Schleifer, K.-H. (1996) In situ visualization of high genetic diversity in a natural microbial community. Journal of Bacteriology 178, 3496- 3500. Amann, R. I., Ludwig, W. and Schleifer, K.-H. (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation.Microbiol. Rev. 59, 143-169. Tolker-Nielsen, T., Larsen, M. H., Kyed, H. and Molin, S. (1997) Effect of stress treatments on the detection of Salmonella typhimurium by in situ hybridization. International Journal of Food Microbiology, 35, 251-258. Ootsubo etal. Journal of Applied Microbiology 2003, 95, 1182-1190.

  As described above, the combined fluorescence in situ hybridization method overcomes the drawbacks of the conventional fluorescence in situ hybridization method and opens up the path of application to microbial testing in industrial fields such as the food manufacturing industry and food processing industry. However, the following technical issues must be overcome when actually trying to apply to microbiological testing of foods at industrial sites.

  In the inspection of microorganisms by a culture method that has been widely used in the industrial field such as the food manufacturing industry, detection or counting of specific living microorganisms contained in a certain amount of food sample is performed. Therefore, in order to apply the fluorescence in situ hybridization method combined with culture, not only the rapid determination of specific microorganisms but also the detectable limit value of the number of specific microorganisms must be at least equivalent to the conventional culture methods. There is. In the widely used pour plate culture method, it is usually necessary to be able to detect the number of microorganisms per unit of 0.1 g of food sample, that is, 10 microorganisms per gram (10 CFU / g). Therefore, when applying the culture combined fluorescence in situ hybridization method, it is necessary to realize a detectable limit value equivalent to this.

  For this purpose, even when the fluorescence in situ hybridization method combined with culture is applied, filtration of a suspension containing at least 0.1 g of a food sample is performed using a single membrane filter (pore size: 0.45 μm or less). It is necessary to collect all target microorganisms on the filter and accurately count each target microorganism on the filter so that the detectable limit value is 10 CFU / g or less. However, depending on the food, the membrane filter is clogged, and in many cases, a suspension containing 0.1 g of a food sample cannot be filtered. In such a case, it is appropriate to prevent clogging by increasing the size of the membrane filter and increasing the filtration area. However, in the fluorescence in situ hybridization method combined with culture, it is essential to observe the fluorescence signal of the microorganism to be detected on the membrane filter with a fluorescence microscope, so the expansion of the filtration area immediately increases the number of observation fields in proportion to the square. Tied to

  For example, if a membrane filter having a diameter of 13 mm is used and if this entire region is observed using a 10 × objective lens, careful attention should be paid so as not to miss any fluorescence signal of the detection target microorganism. In spite of this, it is necessary to observe 27 visual fields per specimen. Considering the time required for this and the fatigue of the inspector, it can be said that this alone is difficult to apply to food microbiological examinations at industrial sites. If this is made into a membrane filter having a diameter of 25 mm, which is almost doubled, it takes about 20 minutes to observe more than 110 fields of view, which cannot be said to be a practical method.

  For this reason, conventionally, the amount of suspension filtered is reduced, but the area of the membrane filter must be reduced, or a specified amount can be filtered using a membrane filter with a large area, and a part of the membrane filter must be observed. There wasn't. However, these methods do not achieve the same detectable limits as the conventional culture methods, and are not applicable to microbial testing in industrial fields such as food manufacturing and food processing. was difficult.

  The present invention requires enormous observation time and labor as the membrane filter area increases in order to achieve such a sufficient detectable limit value. If this effort is to be reduced, a sufficient detectable limit is achieved. Eliminate the dilemma that the value cannot be secured, make full use of the features of fluorescence in situ hybridization combined with culture, and quickly detect and detect specific living microorganisms that can be used in industrial fields such as the food manufacturing industry and food processing industry The purpose is to realize a microbiological test that can be counted and has high detection sensitivity.

  After a great number of trials and errors, the inventors have used a membrane filter having an effective filtration area equal to or larger than that of a circle having a diameter of at least 24 mm. It has been found that a suspension containing at least 0.1 g of a food sample can be filtered. Furthermore, by adjusting the culture time appropriately, the size of the microcolonies on the filter is set to 20 μm to 150 μm, so that the fluorescence in situ hybridization operation is performed, and the microorganisms to be detected on the membrane filter are detected. It has been found that the fluorescence signal emitted by the microcolony can be observed with a fluorescence detection optical system using a low-magnification objective lens. In addition, a fluorescent signal image is formed on the imaging surface of the solid-state imaging device, converted into a fluorescent image, and the stage equipped with the membrane filter is scanned two-dimensionally at high speed within the operating range that can cover the entire membrane filter. The fluorescence image acquired over the whole membrane filter was image-processed by the image processing means, and the number of microorganisms to be detected on the membrane filter was automatically counted in a few minutes.

  The amount of a 10-fold diluted suspension of a food sample used in the pour plate method that is a conventional culture method is 1 ml, and the weight of the food sample contained therein is equivalent to 0.1 g. Therefore, the detection limit of this method is to count one detection target microorganism contained in this 0.1 g food sample. When converted to a unit amount per 1 g food sample, 10 microorganisms are counted. This means that a considerable (10 CFU / g) number of microorganisms can be detected. Therefore, also in the fluorescence in situ hybridization method combined with culture, it is necessary to first filter a suspension containing at least 0.1 g of a food sample. When the present inventors repeated experiments with various food samples, in order to filter this, a membrane filter having an effective filtration area at least equal to or larger than the area of a circle having a diameter of 24 mm or more is required. all right. It was also found that to filter a suspension containing at least 0.1 g of a sample in many food samples more reliably, it is necessary to use a membrane filter having a diameter of 47 mm (see Table 1). (See Table 1. In the experiment of Table 1, suction filtration was performed with a transwell insert having a membrane filter with a membrane diameter of 25 mm and a pore size of 0.4 μm, or a membrane filter with a diameter of 47 mm and a pore size of 0.4 μm.) .

  When a membrane filter with a diameter of 47 mm is used, the effective filtration area is approximately a circle with a diameter of 40 mm when a filter holder for vacuum filtration (manufactured by Advantech) is used. If it is going to inspect using a microscope, even if it calculates simply from the relationship of a microscope visual field, the speculum of 250 visual fields or more is needed. Therefore, the inventors utilize the feature that it is possible to control the size of the microcolony formed on the membrane filter by controlling the culture time, which is a feature of the fluorescence in situ hybridization method combined with culture, The size of the microcolony was adjusted between 20 μm and 150 μm by adjusting the culture time between 5 hours and 6 hours at the optimum temperature of the detection target bacteria. This greatly increases the intensity and emission area of the fluorescence signal. Normally, a fluorescence signal that cannot be observed without using a fluorescence microscope optical system can be combined with a simple low-magnification fluorescence detection optical system and an inexpensive solid-state image sensor. Observable. In addition, since it has been found that it is sufficient to use about 3 to 6 times as the above optical magnification, it has a wide field of view suitable for automatic inspection of the entire membrane filter and does not require focus adjustment during the speculum. An optical system with a large depth of focus is now available.

  In this way, a low-magnification fluorescence detection optical system forms a fluorescent signal image on the imaging surface of the solid-state imaging device to form a fluorescence image, and the stage equipped with the membrane filter can be operated in an operating range that can cover the entire membrane filter. By configuring it so that it can be scanned in a high-dimensional manner and acquiring a fluorescence image across the entire membrane filter, the inspector checks the fluorescence image of the microcolony on the image monitor that displays the fluorescence image. It became possible, and it became possible to relieve fatigue much more than normal speculum work. If the presence or absence of a specific microorganism to be detected is simply confirmed, this configuration can be used as a microorganism testing method in industrial fields such as the food manufacturing industry and the food processing industry.

  However, in order to accurately count microorganisms to be detected by observing a large number of images, there is a limit to relying on the labor of the inspector, and in the industrial fields such as the food manufacturing industry and food processing industry where the number of test samples is large This part needs to be automated. Therefore, the inventors input a fluorescence image acquired over the entire membrane filter to the image processing means and performs image processing, and the number of microorganisms to be detected on the membrane filter can be automatically counted. In many fresh foods, the image processing method is cheaper because the fluorescence image of the microcolony is much better in terms of light quantity and light emission area than the fluorescence image of inclusions and the like that cause noise, and can be easily distinguished. Counting can be performed by a general particle counting process that can be realized in real time even by an image processing device.

  Further, in the present invention, since the fluorescence intensity is sufficiently strong, by applying a low magnification fluorescence detection optical system that can take a long working distance to the membrane filter surface, an expensive coaxial epifluorescence fluorescence required in a normal fluorescence microscope optical system is applied. Lighting is no longer necessary. By eliminating the need for coaxial epifluorescence illumination, the illumination optical system for fluorescence excitation can be freely configured independently of the fluorescence observation optical system, and the dichroic required for ordinary fluorescence microscope optical systems. A mirror is not required, and a fluorescent excitation illumination lamp can be freely arranged, which causes a problem with heat generation. Furthermore, since the fluorescence intensity of the microcolonies is sufficiently strong, a normally used xenon lamp or mercury lamp is not necessarily required as a fluorescent excitation illumination light source, and a metal halide lamp, a halogen lamp, or a high-intensity LED can be sufficiently observed. It was. At this time, since the illumination optical system for fluorescence excitation can be freely arranged, it is possible to irradiate illumination for fluorescence excitation from multiple directions as necessary.

  As described above, the microorganism testing method of the present invention enables rapid qualitative and quantitative detection of living specific microorganisms with high detection sensitivity equivalent to that of the conventional culture method, and realizes automation of testing. Therefore, in many industrial processes where accuracy and speed are required, such as inspection in the manufacturing process and inspection before product shipment in industrial fields such as food manufacturing industry and food processing industry. It is available and very useful for industry. In addition, since the present invention enables automatic measurement without using an expensive fluorescent microscope, it can be used regardless of the company scale.

1. SUMMARY OF THE INVENTION The present invention includes a membrane filter having an effective filtration area equal to or greater than the area of a circle having a diameter of 24 mm in a microorganism testing method such as food using a culture combined fluorescence in situ hybridization method, for example, A transwell insert having a membrane filter with a membrane diameter of 25 mm and a pore size of 0.4 μm, preferably comprising a filtration step with a membrane filter having a diameter of 47 mm and a pore size of 0.4 μm And microbiological testing methods for foods and the like.

  Further, in the present invention, in a microorganism testing method for foods using the culture combined fluorescence in situ hybridization method, a membrane filter having an effective filtration area equal to or larger than the area of a circle having a diameter of 24 mm is used. It includes a method for examining microorganisms such as food, characterized by observing a fluorescence signal using a magnification fluorescence detection optical system.

  Furthermore, the present invention uses a membrane filter having an effective filtration area equal to or greater than the area of a circle with a diameter of 24 mm in a microorganism testing method for foods and the like using the combined fluorescence in situ hybridization method. Using a low-magnification fluorescence detection optical system, the fluorescence signal of the membrane filter is observed with a solid-state imaging device, preferably the observation site can be automatically scanned over the entire membrane filter range, more preferably It includes a method for testing microorganisms such as foods, characterized by automating the counting of specific microorganisms contained in a region.

(1) Fluorescence in situ hybridization method combined with culture In the present invention, the fluorescence in situ hybridization method combined with culture is detected using a fluorescent probe for detecting microorganisms after culturing a sample containing microorganisms in a medium and growing them. To include a method.

The culture combined fluorescence in situ hybridization can be performed, for example, as follows.
(B) Sample filtration
A membrane filter is incorporated into the suction filtration filter unit, and the sample solution is filtered. When using a membrane filter with a high outer frame, place it on the filter base for suction filtration and filter the sample solution.
(B) Culture The membrane filter on the base is placed on a solid medium (gel medium or fibrous pad containing liquid medium), and the other side (back side) of the filter is brought into contact with the medium. Culture is performed under predetermined conditions, and microcolony is formed on the surface on the front side of the filter. For example, by placing a membrane filter with a high outer frame on an agar medium and culturing for about 5 to 6 hours at the optimum temperature of the bacteria to be detected, a microcolony is formed on the front side of the membrane filter. be able to.
(C) Fixation Immerse the membrane filter in an appropriate fixative (such as ethanol) and then dry it. When using a filter with a high outer frame, for example, the membrane filter can be fixed by placing it on a filter paper containing ethanol.
(D) Hybridization For example, the filter can be transferred to a dedicated container containing a hybridization buffer and a fluorescently labeled probe for detecting a specific bacterium for hybridization. When using a filter with a high outer frame, a fluorescent labeling probe for detecting a specific bacterium and a hybridization buffer are added to the filter and reacted at a predetermined temperature.
(E) Cleaning For example, the membrane filter can be transferred to a dedicated container using tweezers, and the filter can be cleaned by immersing it in a cleaning solution. When a membrane filter with a high outer frame is used, a washing solution can be added on the filter and reacted at a predetermined temperature.
(F) Specimen preparation For example, a membrane filter is placed on a slide glass, an encapsulant is added thereto, and then a cover glass is put on to form a specimen. However, when a membrane filter with a high outer frame is used, the work is not necessary.
(G) Observation with a fluorescence microscope Take out the prepared specimen or the membrane filter with an outer frame, and use this for a fluorescence observation apparatus (fluorescence microscope etc.) to detect and count specific bacteria.

  At this time, a low-magnification objective lens can be used for fluorescence microscope observation, a wide observation field can be obtained and the observation time can be shortened, and the use of the low-magnification lens increases the distance between the specimen and the lens. The membrane filter with an outer frame having a height that does not hit the objective lens can be used for observation. Thereby, a spot can be measured over the whole area of a filter, without contacting a membrane filter with an outer frame.

(2) Conditions for performing fluorescence in situ hybridization combined with high-sensitivity culture In the present invention, the detectable limit value of the number of specific microorganisms is at least equivalent to 10 (10 CFU / g) in 1 g of a sample such as food. The conditions for performing the fluorescence in situ hybridization method combined with culture are adjusted.

[Membrane filter]
Specifically, a 0.1 g sample of food or the like may be inspected at a time. For this purpose, for example, as a membrane filter, an effective filtration area having an area equal to or larger than the area of a circle having a diameter of 24 mm is required. For example, a membrane filter having a diameter of 47 mm can be used.

  The material of the membrane filter may be any commercially available material, and for example, polycarbonate or cellulose acetate can be used. The average pore size is preferably 0.2 μm to 0.5 μm so that bacteria can be collected. The bottom surface of the membrane filter may be any shape that can be placed on the suction filtration filter base, can be in close contact with the solid medium during solid culture, and can be placed on the operation console of the microscope.

  If necessary, an outer frame having a height (hereinafter referred to as an outer frame) can be provided on the outer surface of the membrane filter.

  By attaching the outer frame to the membrane filter, a certain amount of liquid can be accommodated on the membrane, and the solution for hybridization and washing can be held on the membrane filter. Further, the outer frame allows the membrane filter to be held without sagging. For example, the outer frame can be directly bonded to the membrane filter without any gaps, but a holding frame is provided around the membrane filter to hold the membrane filter so that it does not sag. You can also. The material of the outer frame may be a material that can withstand a certain tension and a certain tension for stretching the filter without slack, such as plastic, metal, and cellulose, and that does not allow liquid to permeate. The height of the outer frame is the height at which the objective lens does not come into contact with the outer frame when focusing on the filter surface with the outer frame using an objective lens (2 to 10 times). If it exists, it can be set to 3 mm-30 mm, for example. The shape of the outer frame can be arbitrarily formed, for example, in a cylindrical shape, a square, or the like, but is preferably a cylindrical outer frame. The columnar outer frame can be formed, for example, in a funnel shape or a funnel shape so that the area of the opening is larger than the area of the membrane filter. As the membrane filter with an outer frame, a commercially available product in which an outer wall is provided on the membrane filter, for example, a permeable support transwell can also be used.

  Moreover, you may provide the outer frame with the height which can mount | wear with the container containing a culture medium also on the back surface of a membrane filter depending on the case.

[Fluorescent probe]
As the fluorescent probe, various fluorescent probes can be used, and preferably, a fluorescently labeled probe can be used. Specific examples include oligonucleotide probes for fluorescently labeled rRNA, and examples of fluorescent labels include TAMRA (N, N, N ′, N′-Tetramethyl-6-6carboxyrhodamine), florescene, and rhodamine. More specifically, an enterobacteriaceae bacterial detection probe [5'-TGCTCTCGCGAGGTCGCTTCTCTT-3 'comprising an oligo DNA nucleotide fluorescently labeled with TAMRA (N, N, N', N'-Tetramethyl-6-6carboxyrhodamine) (JP 2001-136969)) or CLP-180 [5'-AATGATGATGCCATCTTTCAACA-3 'for detection of Cl. Perfringens fluorescently labeled with TAMRA (N, N, N', N'-Tetramethyl-6-6carboxyrhodamine) ] (Japanese Patent Application No. 2004-336584) can be used.

(2) Fluorescence microscope, low-magnification fluorescence detection optical system In the present invention, a microorganism for detection (also referred to as a specific microorganism) in a sample is propagated by a fluorescence in situ hybridization method combined with culture. A microscope can be used, but a low-magnification fluorescence detection optical system that is functionally equivalent and simple to a fluorescence microscope can also be used.

  Specifically, a low magnification fluorescence detection optical system having a magnification of 2 to 10 times, preferably 3 to 6 times can be used. In order to simplify the illumination method, it is preferable to use an objective lens having a working distance of 10 mm or more. By increasing the working distance of the objective lens in this way, the illumination light is not blocked by the objective lens frame, and it is possible to perform simple epi-illumination that illuminates obliquely from above without using coaxial epi-illumination. The magnification fluorescence detection optical system can be simplified and the cost can be reduced.

  As an illumination light source for fluorescence excitation of a simple low-magnification fluorescence detection optical system used for fluorescence observation in the present invention, a metal halide lamp, a halogen lamp, and a high-intensity LED can be used in addition to the xenon lamp and mercury lamp that are usually used. In this case, since the fluorescent excitation illumination optical system has a large working distance of the objective lens, it can be arranged spatially freely, and it is preferable to irradiate the fluorescence excitation illumination light from the outside of the observation optical axis. It is also possible to irradiate illumination for fluorescence excitation from multiple directions.

(3) Measuring means Automation means In the present invention, a fluorescent signal image can be formed on the imaging surface of the solid-state imaging device with a low-magnification fluorescence detection optical system, and the fluorescence image can be observed on an image monitor or the like. Furthermore, the stage on which the membrane filter is mounted can be configured so as to be capable of two-dimensional high-speed scanning over the entire membrane filter, and the fluorescence image of the entire membrane filter can be observed.

  It is also possible to input the fluorescence image signal to the image processing apparatus and easily count the number of specific microorganisms in the entire membrane filter.

2. Specific Embodiments of the Present Invention Hereinafter, embodiments of the present invention will be described with reference to the drawings.
In FIG. 1, 1 is a transwell insert (diameter 25 mm), 1a is a membrane filter fixed to the transwell insert, 2 is a sample stage, 3 is a linear stage that can be driven in a direction perpendicular to the paper surface (5). 4 is a rectilinear stage that can be driven in the direction of an arrow 6 orthogonal to 5, 7 is an objective lens, 8 is a fluorescence detection filter, 9 is an imaging lens, 10 is a solid-state imaging camera with a built-in imaging surface 11, and 12 is A light source for fluorescence excitation, 13 is a light guide, 14 is a collimator lens, 15 is a filter for fluorescence excitation, 16 is a lens for converging illumination light, and 17 is an image monitor.

  The membrane filter 1a of the transwell insert 1 mounted on the sample stage 2 is guided by the light guide 13 from the fluorescence excitation light source 12, passes through the collimator lens 14 and the fluorescence excitation filter 15, and then the illumination light convergence lens 16 The illumination light for fluorescence excitation collected in step S is irradiated obliquely from above. When TAMRA is used as the labeling fluorescent dye, an interference filter having a transmission characteristic near 550 nm is generally used as the fluorescence excitation filter 15. In addition, the fluorescent excitation light source 12 does not interfere with the use of a xenon lamp or a high-pressure mercury lamp that is usually used for fluorescent excitation, but the use of an inexpensive metal halide lamp or halogen lamp also increases the life of the light source. Long and economically effective. Depending on the type of the labeled fluorescent dye, there are cases where fluorescence can be excited with high efficiency by using a high-intensity light-emitting diode as a light source for fluorescence excitation. In this case, the output light of the light-emitting diode lamp 18 as shown in FIG. The membrane filter 1a may be directly irradiated as fluorescence excitation illumination light by the illumination light convergence lens 16 without passing through the fluorescence excitation filter 15. In this example, the case where light is guided by the light guide 13 is described. However, the effect does not change even if the illumination light from the fluorescence excitation light source 12 is directly incident on the collimator lens 14 without using the light guide. Absent.

  The image of the fluorescence excitation illumination light irradiation portion 1 b on the membrane filter 1 a is imaged on the imaging surface 11 in the solid-state imaging camera 10 by the action of the objective lens 7 and the imaging lens 9. At this time, if, for example, a fluorescence detection filter 8 such as an interference filter corresponding to the emission wavelength of the labeled fluorescent dye TAMRA is inserted in the optical path, the image obtained by the solid-state imaging camera 10 has red fluorescence specific to this probe. An image of the derived microcolony is displayed on the image monitor 17 as necessary.

  At this time, the optical magnification of the image of the fluorescence excitation illumination light irradiated portion 1b imaged on the imaging surface 11 by the objective lens 7 and the imaging lens 9 is adjusted so that the size of the microcolony is 20 μm to 150 μm. A low magnification of 3 to 6 times is sufficient, and even if a working distance of 10 mm or more is secured, sufficient observation is possible. In addition, because of its low magnification, it is possible to secure a wide field of view, and it is possible to obtain a wide field of view 10 times the diameter ratio and 100 times the area ratio compared to the case of using a normal fluorescence microscope. This also contributes to a significant reduction.

  Here, if the 3 and 4 rectilinear stages are automatically and sequentially driven step by step corresponding to the observation field, and the entire surface of the membrane filter 1a is scanned, an image of the microcolony on the entire surface of the membrane filter 1a is displayed on the image monitor 17. Is displayed, and the inspector can observe the presence or absence of the corresponding microcolony without performing a spectroscopic examination with a fluorescence microscope. At this time, since the optical magnification by the objective lens 7 and the imaging lens 9 is low, once the focus adjustment operation is performed, good imaging can be obtained over the entire surface of the membrane filter 1a, and an expensive autofocus device is not required. . In this example, the optical system is fixed and the transwell insert 1 is moved, but it is only necessary that both move relatively so that the entire surface of the membrane filter 1a can be observed. It goes without saying that the same effect as in the above example can be obtained even if the optical system is moved. Even when the optical system is moved, the optical system of the present invention can be realized at low cost because it is easy to reduce the size and weight. is there.

  Next, an example of automatically counting over the entire membrane filter will be shown based on FIG. In FIG. 3, 19 is an image processing means, 20 is a straight stage control means, and the same elements as in FIG. 1 are given the same numbers as in FIG. In addition to being displayed on the image monitor 17 in FIG. 1, the image obtained by the solid-state imaging camera 10 is also input to the image processing means 19. In the case of many fresh foods, the fluorescence image of the microcolony is much better than the fluorescence image of the autofluorescent material derived from the raw material that causes noise, and can be easily distinguished, so that it can be easily distinguished. 19 can be easily counted by following general particle counting procedures that can be realized in real time even with inexpensive image processing equipment such as image correction, noise removal, binarization, labeling, and counting, which are general image processing procedures. It is. Since the counting process in the image processing unit 19 is sequentially performed for each observation visual field over the entire surface of the membrane filter 1a by communication with the linear stage control unit 20 that controls the linear stages 3 and 4 in an integrated manner. Specifically, the number of microcolonies on the entire surface of the membrane filter 1a can be counted.

  Next, the same elements as those in FIG. 1 are denoted by the same reference numerals as those in FIG. 1, and another example in which the illumination light for fluorescence excitation is irradiated based on FIG. 4 is shown. Fluorescent light guided by the light guides 13a and 13b branched from the fluorescence excitation light source 12 and condensed by the illumination light converging lenses 16a and 16b through the collimator lenses 14a and 14b and the fluorescence excitation filters 15a and 15b, respectively. The illumination light for excitation is applied to the membrane filter 1a of the transwell insert 1 mounted on the sample stage 2 from two different obliquely upward directions. Since only a part of the output light of the fluorescence excitation light source 12 is normally incident on the light guide and used, the output light of the fluorescence excitation light source 12 can be effectively used by bifurcating in this way. By simultaneously irradiating a plurality of illumination lights for fluorescence excitation, the fluorescence intensity of the microcolony can be increased. Further, even when a member capable of shielding light is fixed to the outer periphery of the membrane filter, such as the transwell insert 1, an effect of reliably irradiating the membrane filter 1a with the illumination light for fluorescence excitation is obtained. It is done. In FIG. 4, only the case where the light guide is divided into two branches is described, but it is of course possible to increase the number of branches as three branches and four branches as necessary.

  In addition, since multiple fluorescent excitation illumination lights can be irradiated at the same time, select a labeled fluorescent dye that has an absorption wavelength band different for each type of specific microorganism to be detected but has a fluorescence emission wavelength band in the vicinity. By providing the filters 15a and 15b with narrow band transmission characteristics corresponding to the absorption wavelengths of the labeled fluorescent dyes, it is possible to simultaneously detect a plurality of types of specific microorganisms using different types of probes. In addition, by providing a shutter that can be inserted and removed in the optical path as indicated by 21a and 21b, it is possible to easily irradiate fluorescence excitation illumination light alternately, and the fluorescence excitation filters 15a and 15b By appropriately adjusting the combination of the fluorescence detection filters 8, there is an advantage that a multiband fluorescence image can be observed by multiwavelength excitation with a very simple configuration.

  The above example describes the case where the membrane filter 1a has a diameter of 25 mm, but the basic operation is the same even when the diameter is 47 mm, although the scanning area is enlarged.

First, the combined fluorescence in situ hybridization method is applied according to the following procedures (1) to (3).
(1) Test strain Escherichia coli ATCC 11775 is used as an enterobacteriaceae bacterium.
(2) Vegetables (cabbage, lettuce) and drinking water (beer, orange juice, cider) are used as test food samples. In the case of each food sample excluding drinking water, 90 ml of sterilized physiological saline is added to 10 g of sample (diluted 10 times), suspended in a stomacher, and then the test strain Escherichia coli ATCC11775 is added to prepare a food sample preparation solution. . In the case of drinking water, the sample is not diluted, and the test strain Escherichia coli ATCC 11775 is directly added to prepare a food sample preparation solution.

  (3) Transwell (corning) was used as a membrane filter. The food sample preparation solution is 0.1 g of sample (detection limit 10 CFU / g) for vegetables and orange juice, and 1 g of sample (detection limit 1 CFU / g) for carbonated drinks and beer. After suction filtration with a transwell insert having a membrane filter having a diameter of 25 mm and a pore size of 0.4 μm, the transwell insert is placed on tryptosa agar medium (Nissui Pharmaceutical Co., Ltd.), cultured at 37 ° C. for 6 hours, and filtered. Colonies were formed on the surface. After incubation, the transwell insert is placed on a filter paper containing 100% ethanol, and cell fixation is performed at room temperature for 5 minutes. Next, the transwell insert is attached to a multiple well, and 900 μl of a hybridization buffer (0.9 M NaCl, 20% formamide, 200 mM Tris-HCl (pH 7.4), 0.01% SDS) is gently injected into the transwell insert, Prehybridization is performed at 60 ° C. for 10 minutes.

  Thereafter, 50 pmol of a probe for detecting Enterobacteriaceae (Japanese Patent Laid-Open No. 2001-136969) consisting of an oligo DNA nucleotide labeled with TAMRA (N, N, N ′, N′-Tetramethyl-6-6carboxyrhodamine) was added, and 60 ° C. Hybridize for 45 minutes. Add 5 ml of washing buffer ((20 mM Tris HCl (pH 7.4), 180 mM NaCl, 0.01% SDS)) to the transwell insert and leave it at 60 ° C. for 15 minutes. The transwell insert is tilted, the washing buffer is removed, 5 ml of distilled water is added, immediately removed and washed, and then the transwell insert is placed on the sample stage of the inspection apparatus shown in FIG. 1, and colonies that emit probe-specific red fluorescence are automatically detected. Fluorescent signals were counted for automatic counting.

(4) As a comparative control of the counting results by the combined fluorescence in situ hybridization method, E. coli was measured by the culture method for the food sample preparation solution. That is, the food sample preparation solution was appropriately diluted with sterilized physiological saline, and then subjected to pour plate culture at 37 ° C. for 24 hours using a chromocartoliform agar medium (Merck). After incubation, E. coli was counted.

Result 1
The results of repeating the above test 5 times are shown in Table 2. The culture combined fluorescence in situ hybridization method using a membrane filter with a diameter of 25 mm was not significantly different from the culture method in any sample (P> 0.05), and specific bacteria were counted almost accurately. The results are shown in Table 2.

  Among the tests of Example 1 above, the transwell insert was changed to a cellulose membrane filter (Advantech) having a diameter of 47 mm and a pore size of 0.4 μm, and pork, squid, mackerel, chicken cutlet, and tofu were used as food samples, respectively. For the equivalent amount of g, the subsequent steps were repeated 5 times in the same manner as described above (detection limit 10 CFU / g). The results are shown in Table 3. In the case of using a membrane filter having a diameter of 47 mm, the fluorescence in situ hybridization method combined with culture was not significantly different from the culture method in any sample (P> 0.05), and specific bacteria were counted almost accurately.

  In this way, it is possible to automate microbial testing of living specific microorganisms with high detection sensitivity equivalent to that of conventional culture methods, with a very simple configuration that does not use an expensive fluorescence microscope device. Automatic microbiological testing equipment that can be used in industrial fields such as these can be provided at a very low cost, and can greatly contribute to the safe and reliable supply of food.

Microcolony observation method on membrane filter Alternative configuration of illumination for fluorescence excitation Configuration to automatically count the number of microorganisms Multi-branch type illumination method for fluorescence excitation

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Transwell insert 1a Membrane filter 2 Sample stand 3, 4 Straight stage 7 Objective lens 8 Fluorescence detection filter 9 Imaging lens 10 Solid-state imaging camera 11 Imaging surface 12 Fluorescence excitation light source 13 Light guide 14 Collimator lens 15 Fluorescence excitation filter 16 illumination light converging lens 17 image monitor 18 light emitting diode lamp 19 image processing means 20 rectilinear stage control means

Claims (8)

  A food or the like characterized by inspecting a sample by a fluorescence in situ hybridization method combined with culture so that a detectable limit value of the number of specific microorganisms is at least 10 per 1 g (10 CFU / g) of the sample of food or the like Microbial testing method.   The method for testing microorganisms for foods and the like according to claim 1, wherein the culture combined fluorescence in situ hybridization method includes a step of filtering a liquid containing at least 0.1 g of a sample of foods or the like through a membrane filter.   The microorganism for foods or the like according to claim 1 or 2, wherein the culture combined fluorescence in situ hybridization method uses a membrane filter having an effective filtration area equal to or larger than an area of a circle having a diameter of 24 mm. Inspection method.   4. The method for examining microorganisms for foods and the like according to any one of claims 1 to 3, wherein the specific microorganisms grown by the combined fluorescence in situ hybridization method are observed with a low-magnification optical system.   The method for testing microorganisms for foods and the like according to claim 4, wherein the low-magnification system has an optical magnification of 3 to 6 times.   The food according to any one of claims 1 to 5, wherein in the observation by the combined fluorescence in situ hybridization method, the fluorescence signal of the membrane filter is observed by irradiating fluorescence excitation illumination light from outside the observation optical axis. Microbiological testing methods such as.   2. The culture combined fluorescence in situ hybridization method, wherein a low-magnification optical system and a solid-state imaging device are combined, and the entire membrane filter is automatically scanned to observe the fluorescence signal of the membrane filter. 6. A method for testing microorganisms for foods or the like according to any one of items 1 to 6.   The method for testing microorganisms for food or the like according to any one of claims 1 to 7, wherein counting of specific microorganisms contained in the observation region is automated using the observation result of the fluorescence signal of the membrane filter.

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