JP2004344095A - Method for counting microorganisms and member for capturing microorganisms - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for rapidly counting microorganisms in a liquid specimen containing the microorganisms; and to provide a member for capturing the microorganisms for carrying out the method. <P>SOLUTION: The method for counting the microorganisms comprises capturing the microorganisms on the surfaces of filtering regions by filtering the liquid specimen containing the microorganisms by dropping the specimen from the upper part of the member for capturing the microorganisms, having the specified many filtering regions having a prescribed fine size by which the microorganisms can be captured by filtering, and formed on a plane at an interval, and counting the microorganisms in the specimen by optically examining each of the filtering regions one by one. The member for capturing the microorganisms has the specified filtering regions obtained by nipping a filtration film by two flat plates having many fine holes with prescribed sizes at the interval so that the liquid specimen may be filtered from the fine holes of one flat plate through the filtration film to the fine holes of the other flat plate to specify the filtering region. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for rapidly counting microorganisms from a liquid sample containing microorganisms (which means a concept including at least bacteria and fungi), and a microorganism capturing member used for carrying out the method. Things.
[0002]
[Prior art]
As an invention related to this kind of microorganism counting method, for example, a sample solution is filtered through a hydrophilic filtration membrane described in Patent Document 1 below, and the viable bacteria contained in the sample solution are filtered. While being sprayed with a volatile extractant having a boiling point of 120 ° C. or less for extracting adenosine-3-phosphate in living bacteria from the membrane, and then spraying the solution onto the membrane at room temperature or after heating, Is volatilized, and then a luciferin-luciferase luminescence reagent solution is sprayed to emit light, and the amount of luminescence is measured using a luminescence measuring device. ing.
[0003]
[Patent Document 1]
JP-A-5-328995
[Problems to be solved by the invention]
In the above method, since the capture of microorganisms is performed on the entire surface of the filtration membrane, it is necessary to inspect the entire surface of the filtration membrane in order to measure the amount of luminescence, for example, filtration with an effective area of 20 mm in diameter. When a membrane is used, there is a problem that several hours have to be spent for the measurement.
Therefore, an object of the present invention is to provide a method capable of rapidly counting microorganisms from a liquid sample containing microorganisms, and a microorganism capturing member used for performing the method.
[0005]
[Means for Solving the Problems]
The microorganism counting method of the present invention made in view of the above point, as described in claim 1, by separating a filtration region having a predetermined fine size capable of filtering and capturing microorganisms on a plane. After capturing the microorganisms on the surface of the filtration region by dropping and filtering a liquid sample containing microorganisms from above the microorganism capturing member whose filtration region has been identified by providing a large number, the individual filtration regions are sequentially optically filtered. And counting microorganisms in the sample.
A microorganism counting method according to a second aspect is characterized in that, in the microorganism counting method according to the first aspect, the size of each filtration region is a circle having a diameter of 1 to 1000 μm.
According to a third aspect of the present invention, in the microorganism counting method according to the first or second aspect, the ratio of the area of the entire filtration region to the area of the microorganism capturing member into which the liquid sample containing the microorganism is dropped is 10 to 75%. It is characterized by being.
Further, the microorganism counting method according to claim 4 is the microorganism counting method according to any one of claims 1 to 3, wherein a flat plate provided with a large number of pores having a predetermined size is provided on the surface of the filtration membrane. A microbe-capturing member is obtained by bringing it into close contact, and a liquid sample containing microbes is dropped from above the flat plate and filtered. It is characterized by doing.
A microorganism counting method according to a fifth aspect of the present invention is the microorganism counting method according to any one of the first to fourth aspects, wherein a fluorescent coloring reagent is added to the liquid specimen containing the microorganism and / or the microorganism captured on the surface of the filtration region. It is characterized in that the microorganisms are fluorescently colored and counted by the addition.
According to a sixth aspect of the present invention, there is provided the microorganism counting method according to any one of the first to fifth aspects, wherein the microorganism capturing member is placed on an automatic stage that can be driven by a moving program of a computer. Is characterized by auto-scanning and counting the filtration area.
Further, the microorganism trapping member of the present invention, as described in claim 7, sandwiches the filtration membrane between a large number of two flat plates provided with a plurality of pores having a predetermined size separated from each other, from the pores of one flat plate The configuration is such that the liquid sample is filtered through the filtration membrane into the pores of the other flat plate, whereby the filtration region is specified.
[0006]
BEST MODE FOR CARRYING OUT THE INVENTION
The microorganism counting method of the present invention is a microbial capturing member in which the filtration region is specified by providing a large number of filtration regions having a predetermined fine size capable of filtering and capturing microorganisms separated from each other on a plane. After capturing the microorganisms on the surface of the filtration area by dropping and filtering a liquid specimen containing microorganisms from above, the individual filtration areas are sequentially optically inspected to count the microorganisms in the specimen. It is assumed that.
According to the microorganism counting method of the present invention, the capture of microorganisms is performed in a filtration region having a predetermined fine size, so that the microorganisms are collected at a specific position. Therefore, the counting of the microorganisms may be performed only by sequentially examining the individual filtration areas, so that the measurement time can be reduced.
[0007]
The size of each filtration region in the microorganism capturing member is preferably, for example, a circle having a diameter of 1 to 1000 μm. At this time, if the size of the filtering region is selected in consideration of the size of the measuring visual region so that one filtering region exists in the measuring visual region in the optical inspection means, the measurement time is further reduced. be able to. That is, for example, when using a fluorescence microscope equipped with a 40 × objective lens as the optical inspection means, the size of the measurement field area is about 300 μm in diameter. Inspection of one filtration area can be completed by one measurement. When the size of the filtration area is larger than the size of the measurement visual field area, scanning is required for measurement of one filtration area, while when there are a plurality of filtration areas in the measurement visual field area, in the process of counting microorganisms, In order to avoid duplicate measurement of one filtration region, it is necessary to distinguish between a filtration region that has already been measured and a filtration region that has not been measured, and in any case, it may be a factor that hinders a reduction in measurement time.
[0008]
The ratio of the area of the entire filtration region to the area of the microorganism capturing member onto which the liquid specimen containing microorganisms is dropped is preferably 10 to 75%. If the area ratio is less than 10%, filtration may not be performed smoothly. On the other hand, if the area ratio exceeds 75%, the strength of the microorganism capturing member may be adversely affected. In addition, after securing a predetermined area ratio, in order to reduce the number of times of scanning for measuring each filtration region, the size of each filtration region in the microorganism capturing member is a circle having a diameter of 100 μm or more. Is preferred.
[0009]
As a specific example of the microorganism capturing member in which the filtration region is specified, a member in which a large number of flat plates are closely attached to a surface of a filtration membrane by separating pores having a predetermined size from each other is exemplified. In such a microorganism capturing member, a filtration membrane portion corresponding to a pore portion provided on a flat plate is a filtration region. The filtration membrane (membrane filter) for capturing microorganisms may be made of a material usually used as a filtration membrane such as a polycarbonate resin. The flat plate provided with a large number of fine pores having a predetermined size separated from each other may be made of a synthetic resin such as methacrylic resin. It is preferable that the filtration membrane and the flat plate are brought into close contact with an adhesive or grease in advance or at the time of use. In addition, by making the surfaces of the filtration membrane and the flat plate black, reflection of excitation light in the ultraviolet region can be prevented. In addition, instead of using a flat plate having a large number of pores having a predetermined size separated from each other, a filtration region may be provided by coating the surface of a filtration membrane in a predetermined pattern by a printing technique.
[0010]
A microorganism trapping member in which a large number of flat plates having a predetermined size are separated from each other on the surface of the filtration membrane, for example, two plates provided with a large number of separated pores having a predetermined size. It is preferable that the filtration membrane is sandwiched between flat plates, and the liquid sample is filtered from the pores of one flat plate to the pores of the other flat plate via the filtration membrane. FIG. 1 is a plan view (a) and a sectional view (b) of such a microorganism capturing member. In FIG. 1, reference numeral 1 denotes a microorganism capturing member, reference numeral 2 denotes a filtration membrane, reference numeral 3 denotes a flat plate, and reference numeral 4 denotes a pore.
[0011]
The counting of microorganisms by optical inspection of individual filtration regions in the microorganism capturing member is performed, for example, by adding microorganisms to a liquid sample containing microorganisms and / or adding microorganisms captured on the surface of the filtration region to a fluorescent coloring reagent. Let me do it.
As the fluorescent coloring reagent, various reagents known to be able to cause the microorganism to emit fluorescent light can be used. For example, fluorescent glucose (2-NBDG: 2- [N- (7-nitrobenz-2-oxa-1,3-diazole-4yl) amino] -2-deoxy-D-glucose) is taken into a living body. Thus, microorganisms can be detected on a cell-by-cell basis, and live microorganisms can be detected in a living state. The reaction time is as short as several minutes. Therefore, the method using 2-NBDG can be said to be a simple, quick and highly sensitive method, and has an advantage that real-time detection of a living microorganism can be performed. In addition, a reagent (4 ', 6-diamidino-2-phenylindole dihydrochloride, etc.) that can cause both a living microorganism and a dead microorganism to emit fluorescent light, and a wavelength that is different from the color of the dead microorganism only (Such as propidium iodide), reagents capable of causing only living microorganisms to emit fluorescence at a wavelength different from the above-mentioned color (eg, 6-carboxyfluorescein diacetate), substances derived from specific microorganisms Reagent capable of causing only specific microorganisms to emit fluorescent light at a wavelength different from the above-mentioned color by reacting with (a specific reaction with β-glucuronidase or β-galactosidase which is an enzyme protein produced by Escherichia coli or Escherichia coli group). To produce 4-methylumbelliferone -Β-D-galactoside) or the like, alone or as a mixture of two or more, counting living and dead microorganisms, counting only living microorganisms, counting only dead microorganisms, Counting of only specific microorganisms may be performed.
[0012]
FIG. 2 is a conceptual diagram showing one embodiment of a microorganism counting device for counting microorganisms captured by the microorganism capturing member. This microorganism counting apparatus includes a light source 104, a lens 110 as a light source condensing means, and a light receiving unit 111. In order to extract a target wavelength from the excitation light emitted from the light source 104, the light is split by the excitation light spectral filter 112. The split excitation light passes through the dichroic mirror 113 to change the optical path. The excitation light whose optical path has been changed is condensed on the surface of the microorganism capturing member 1 placed on the inspection table 116 via the lens 110. The inspection table 116 has a depressed portion (device groove) for fitting the microorganism capturing member 1, and has a shape in which the microorganism capturing member 1 can be directly incorporated. The fluorescence excited by the excitation light on the surface of the microorganism capturing member 1 passes through the dichroic mirror 113 again. At that time, the fluorescent light passes through the dichroic mirror 113 as it is and reaches the light receiving unit 111. The fluorescence that has reached the light receiving unit 111 passes through a fluorescence spectral filter 114 in order to extract only the target fluorescence, reaches the photoelectric conversion element 115 built in the light receiving unit 111, is converted into a signal, and is recognized. The fluorescence reaching the photoelectric conversion element 115 is determined by the microorganism determination means 105 as fluorescence derived from microorganisms or fluorescence derived from a foreign substance, and the fluorescence determined as being derived from microorganisms is added up, and the microorganisms are counted. If the inspection table 116 on which the microorganism capturing member is placed is an auto stage that can be driven by a stage by a computer moving program, and the individual filtration regions are automatically scanned and counted, the measurement time can be further reduced. be able to. At this time, if the size of the filtration area is selected in consideration of the size of the measurement visual field area so that one filtration area exists in the measurement visual field area, the auto-stage is used when measuring each filtration area. It is not necessary to control the movement of the auto stage, and the auto stage movement at the time of counting all the microorganisms in the entire filtration area only needs to be performed between the individual filtration areas. There is an advantage of not doing.
[0013]
【Example】
Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention should not be construed as being limited to the following description.
[0014]
(Production of a microorganism capture member with a specified filtration area)
The plate body and the holder, whose schematic plan view and cross-sectional view are shown in FIGS. 3 and 4, respectively, were produced using a 1 mm-thick methacrylic resin extruded plate (Delaglass A: Asahi Kasei Corporation). In the plate body and the holder, 55 fine holes having a diameter of 300 μm (area: about 0.07 mm ² ) corresponding to a filtration region were provided by cutting. In addition, in order to specify the position of the pores in order to facilitate the scanning of the individual filtration area, the center of each pore was set to a specific coordinate, and a range of a radius of 150 μm from the center was cut to form the pores. .
As the membrane filter, a black filter having a diameter of 25 mm and a pore size of 0.22 μm (Millipore) cut into a circular shape having a diameter of 8 mm was used.
After closing the pores from the upper surface of the holder using natural latex, high vacuum grease was smeared on the lower surface. Thereafter, a membrane filter was pressed against the lower surface of the holder portion in a state where the cured natural latex was removed and pores were secured, so that the holder portion and the membrane filter were brought into close contact with each other. The holder with the membrane filter in close contact was set on the plate body to form a microorganism capturing member (see FIG. 5: the ratio of the area of the entire filtration area to the effective area was 13.8%).
[0015]
(Synthesis of 2-NBDG)
0.5 g of D-glucosamine (Sigma) dissolved in 10 mL of 0.3 M sodium hydrogencarbonate solution and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-) dissolved in 20 mL of methanol. Cl) was mixed in a 100 mL Erlenmeyer flask, immediately filled with nitrogen gas, and the mouth was appropriately closed. The entire flask was covered with aluminum foil and subjected to a shaking reaction at 37 ° C. for 18 hours or more in the dark. Thereafter, methanol was removed from the reaction product using an evaporator (37 ° C.), and the total amount was adjusted to 10 mL with distilled water. A solution of this reactive product was anionic gel DEAE Sephadex A-50 @ DEAE Sephadex A-50: 2.0 g / D. W. : 300 mL (Amersham Pharmacia Co.) was loaded on an open column having an inner diameter of 30 mm and a height of 200 mm packed with eluted water using distilled water as an eluent. As a result, the eluted orange reaction product was recovered and concentrated again using an evaporator (37 ° C.). Sephadex LH-20 which has been swollen with distilled water in advance {Sephadex LH-20: 20 g / D. W. : 200 mL (Amersham Pharmacia Co.) was loaded into an open column having an inner diameter of 20 mm and a height of 250 mm filled with｝. Unreacted D-glucosamine was removed by this gel filtration, and the recovered eluate was used to obtain 2-NBDG in a dry powder form using a freeze dryer.
[0016]
(Preparation of liquid sample)
A test bacterium (E. coli K-12) was adjusted to 100 cells / 100 μL with a hemocytometer, and further diluted 2-fold at each stage, and bacterial cells of 100 cells, 50 cells, 25 cells, 12 cells, and 6 cells (in each 100 μL) Was used as a liquid sample. The diluent used was a 50 mM phosphate buffer.
[0017]
(Capture of bacteria)
11 μL of 100 μM 2-NBDG was added to 100 μL of the liquid specimen (final concentration: about 10 μM), and the mixture was placed in a water bath at 37 ° C. for 5 minutes to perform a 2-NBDG uptake reaction of cells. Then, as shown in FIG. 6, the microorganism capturing member is placed on the filter holder for reduced pressure filtration connected to the suction bottle, the liquid sample is dropped from above, and the liquid sample is filtered while aspirating with an aspirator. Bacteria were captured in 55 filtration areas of the capture member. Further, in order to remove unreacted 2-NBDG from the surface of the filtration region, a washing treatment was performed by dropping 500 μL of a phosphate buffer solution from above.
[0018]
(Counting of bacterial cells by fluorescence microscope)
The microbe-capturing member capturing the cells in the 55 filtration areas is fitted into a plate case designed to be able to maintain a moisturized state by supplying water with agar from underneath, and moving the computer. The stage was mounted on an auto stage that can be driven by a program. The individual filtration areas were sequentially visually inspected with an epifluorescence microscope (BH-2: Olympus Corporation) equipped with a 40 × focal length objective lens, and the number of bacterial cells was counted. The moving coordinates in the moving program are defined as (0, 0) at the center of the 55 pores, and the uppermost pore (0, 2100) and the lowermost pore ( 0, -2100), which was determined based on a total of three pores (unit: μm). IB excitation was used as excitation light for the fluorescence microscope. After the cells were counted as described above (the time spent from the capture of the cells to the counting by the fluorescence microscope was about 60 minutes), the membrane filter was removed from the microorganism capturing member, and the LB medium was removed from Trypton Pepton (LB medium). 2 g of Wako Pure Chemical Industries, 1 g of Yeast Extract (Difco) and 1 g of sodium chloride dissolved in 200 mL of pure water, adjusted to pH 7.0, sterilized by high pressure steam, and 1.5% Bacto agar (Difco) Was added, and cultured on an agar medium prepared to form a colony. In order to examine the correlation between the agar method and the counting method using a fluorescence microscope, 100 μL of the liquid sample at each dilution step was smeared on an agar medium and cultured at 37 ° C. for 18 hours. The results from the counting methods used were compared.
FIG. 7 is a graph showing the correlation between the agar method and the counting method using a fluorescence microscope in the liquid sample in each dilution step. ^Both, y = 0.950x-0.337, had a high correlation of R 2 = 0.946 (n = 23 ).
[0019]
(Spike test)
A sample was prepared by adding 10 μL of a liquid specimen whose number of bacteria was adjusted to 100 cells / 100 μL to 90 μL of a phosphate buffer (the number of added cells was 10 cells). To this sample, 11 μL of 100 μM 2-NBDG was added (final concentration: about 10 μM), and the mixture was placed in a water bath at 37 ° C. for 5 minutes to perform a 2-NBDG uptake reaction of cells. Thereafter, the cells were captured in the 55 filtration regions of the microorganism capturing member in the same manner as described above, and the cells were counted using a fluorescence microscope. After counting the cells as described above, the membrane filter was removed from the microorganism capturing member in the same manner as described above, and this was cultured on an agar medium to form a colony and confirm viable cells. In order to examine the correlation between the agar method and the counting method using a fluorescence microscope, 100 μL of the sample was smeared on an agar medium, cultured for 18 hours at 37 ° C., and the number of colonies formed and the counting method using a fluorescence microscope were determined. The results were compared. The result was 10.3 ± 3.7 (n = 3) in the agar method and 10.0 ± 3.5 (n = 3) in the counting using a fluorescence microscope.
[0020]
(Discussion)
From the above results, it was found that the counting method using the fluorescence microscope can quickly and accurately count all the cells contained in the liquid sample.
[0021]
【The invention's effect】
According to the present invention, there is provided a method for rapidly counting microorganisms from a liquid sample containing microorganisms, and a microorganism capturing member used for performing the method.
[Brief description of the drawings]
FIG. 1 is a plan view (a) and a sectional view (b) of one embodiment of a microorganism capturing member of the present invention.
FIG. 2 is a conceptual diagram showing one embodiment of a microorganism counting device for counting microorganisms captured by a microorganism capturing member.
FIG. 3 is a schematic plan view and a cross-sectional view of a plate main body constituting the microorganism capturing member used in the example.
FIG. 4 is a schematic plan view and a cross-sectional view of the holder section.
FIG. 5 is a schematic exploded view of the microorganism capturing member used in the example.
FIG. 6 is an explanatory diagram of a method for capturing bacterial cells in an example.
FIG. 7 is a graph showing the correlation between the agar method and the counting method using a fluorescence microscope in Examples.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Microorganism capture member 2 Filtration membrane 3 Plate 4 Pores 104 Light source 105 Microorganism judgment means 110 Lens 111 Light receiving section 112 Excitation light spectrum filter 113 Dichroic mirror 114 Fluorescence spectrum filter 115 Photoelectric conversion element 116 Inspection table

Claims (7)

A liquid sample containing microorganisms, from above the microorganism capturing member in which the filtration region is specified by providing a large number of filtration regions having a predetermined fine size capable of filtering and capturing microorganisms at a distance on a plane. A microorganism counting method comprising: capturing microorganisms on the surface of a filtration region by dropping and filtering; and sequentially optically inspecting each filtration region to count the microorganisms in the sample. 2. The method for counting microorganisms according to claim 1, wherein the size of each filtration area is a circle having a diameter of 1 to 1000 [mu] m. The microorganism counting method according to claim 1 or 2, wherein the ratio of the area of the entire filtration region to the area of the microorganism capturing member onto which the liquid specimen containing microorganisms is dropped is 10 to 75%. A microbial trapping member is formed by closely contacting a large number of flat plates provided with pores having a predetermined size separated from each other on the surface of the filtration membrane, and a liquid sample containing microorganisms is dropped from above the flat plate and filtered. The microorganism counting method according to any one of claims 1 to 3, wherein a microorganism is trapped on a surface of the filtration membrane portion corresponding to the pore portion provided in (1) as a filtration region. The microorganism according to any one of claims 1 to 4, wherein the microorganism is fluorescently colored by adding a fluorescent coloring reagent to the liquid sample containing the microorganism and / or the microorganism captured on the surface of the filtration region, and the microorganism is counted. Microbial counting method. The microorganism counting device according to any one of claims 1 to 5, wherein the microorganism capturing member is placed on an auto stage that can be driven by a stage by a moving program of a computer, and the individual filtration areas are auto-scanned and counted. Method. A filtration membrane is sandwiched between two plates provided with a large number of pores having a predetermined size and separated from each other, and the liquid sample is filtered from the pores of one plate to the pores of the other plate through the filtration membrane. A microorganism trapping member characterized in that a filtration region is specified by having such a configuration.

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