JP2011133366A - Microorganism detection method, filter, and fluorescent mark arrangement board - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To adjust the focus more quickly than hitherto on microorganisms subjected to fluorescent dyeing. <P>SOLUTION: This microorganism detection method for collecting microorganisms in a sample onto a filter by filtration, and detecting microorganisms subjected to fluorescent dyeing from the filter by using a microscope having autofocus function includes a fluorescent mark arrangement process for arranging a fluorescent mark emitting fluorescence on the filter. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a microorganism detection method, a filter, and a fluorescent mark arrangement plate.

In a microscope in which the plane movement and focusing of the stage are automated, when the microbe is photographed, the focus is adjusted by autofocus every time the visual field on the specimen is switched due to the movement of the stage. However, in order to detect all the microorganisms in the sample, it is necessary to focus on all fields of view by autofocus, so that there is a problem that it takes time to shoot. Furthermore, in the microscope, residual vibration is generated due to the movement of the stage. Therefore, autofocusing cannot be performed until this converges, and it takes time to shoot.
Therefore, as an invention for solving the delay of autofocus due to residual vibration, Japanese Patent Application Laid-Open Publication No. 2004-259542 discloses that autofocus before the residual vibration converges by calculating the frequency and estimating the focus when the vibration converges. A microscope capable of performing is disclosed.
Further, as an invention for solving the problem that it takes a long time to shoot by performing auto-focusing, Japanese Patent Application Laid-Open No. 2004-26883 discloses focusing on a real time by directly measuring the degree of focus of a target from a video signal of a CCD camera. A microscope that can be used is disclosed.

JP 2007-101623 A Special table 2001-511903 gazette

  By the way, in the above-mentioned patent document 1, the photographing time is shortened by performing auto focus before the residual vibration converges. However, when the specimen is dark, the time for exposure becomes long, so it takes time to autofocus. Therefore, when the microorganism contained in the sample is fluorescently stained, the fluorescence of the microorganism is faded during autofocusing, making it difficult to detect the microorganism. Further, even in Patent Document 2, it has been difficult to detect microorganisms for the same reason as in Patent Document 1.

  The present invention has been made in view of the above-described circumstances, and aims to focus on fluorescently stained microorganisms earlier than before.

In order to achieve the above object, in the present invention, as a first solution for the microorganism detection method, microorganisms in a sample are collected on a filter by filtration, and the fluorescence-stained microorganisms are microscopes having an autofocus function. A method for detecting microorganisms from the filter using
A means is adopted that includes a fluorescent mark arrangement step of arranging a fluorescent mark emitting fluorescence on the filter.

  In the present invention, as the second solving means relating to the microorganism detection method, the means for arranging the fluorescent mark outside the surface (filtration surface) used for the filtration of the filter in the first solving means is adopted. To do.

  In the present invention, as the third solution means related to the microorganism detection method, in the second solution means, the filtration surface is circular, and three or more fluorescent marks are arranged along the outer periphery of the filtration surface. Adopt means.

  In the present invention, as a fourth solving means related to the microorganism detection method, in the first to third solving means, a means that the fluorescent mark is composed of fluorescent beads is adopted.

  In the present invention, as a fifth solving means relating to the microorganism detection method, in any one of the first to fourth solving means, a fluorescent mark arrangement plate having a fluorescent mark arrangement hole is overlapped on the filter, and the fluorescent mark arrangement is arranged. A method of creating a fluorescent mark in the hole is adopted.

  In the present invention, as a first solving means related to the filter, a filter that collects microorganisms in a sample by filtration and that has fluorescent marks that emit fluorescence is employed.

  In the present invention, as the second solving means relating to the filter, a means is adopted in which the fluorescent mark is arranged outside the surface (filtration surface) used for filtration in the first solving means.

  In the present invention, as the third solution means related to the filter, in the second solution means, the filtration surface is circular, and three or more fluorescent marks are arranged along the outer periphery of the filtration surface. Adopt means.

  In the present invention, as the fourth solving means relating to the filter, in the first to third solving means, the means that the fluorescent mark is composed of fluorescent beads is adopted.

  In the present invention, as a first means for solving the fluorescent mark arrangement plate, a means is adopted in which a filtration hole is opened at the center and the fluorescent mark arrangement hole is opened along the outer periphery of the filtration hole.

  According to the present invention, the fluorescent mark is arranged on the filter. Thereby, the fluorescent microscope can focus on the fluorescently stained microorganism faster than before even if the field of view moves, by autofocusing with the fluorescent mark as a reference.

It is a flowchart of the Legionella detection method which concerns on embodiment of this invention. It is a schematic diagram which shows the 2nd process (fluorescent-mark arrangement | positioning process) of the Legionella bacteria detection method which concerns on embodiment of this invention. It is a schematic diagram which shows the 3rd process of the Legionella bacteria detection method which concerns on embodiment of this invention. It is a schematic diagram which shows the result of the 3rd process of the Legionella bacteria detection method which concerns on embodiment of this invention.

Embodiments of the present invention will be described below with reference to the drawings.
The Legionella detection method according to the present embodiment is a method for detecting Legionella from a sample using the FISH method, and includes first to eleventh steps as described below. Of these first to eleventh steps, the second step is the fluorescent mark placement step in the present embodiment.

The Legionella detection method will be described with reference to FIG.
[First step]
First, in a first step, a paraformaldehyde solution is added to a liquid sample containing Legionella bacteria so that the final concentration is 4%, and the liquid sample is stored overnight at a temperature of 4 ° C. A fixed specimen of the bacterium is prepared (step S1).

[Second step (fluorescent mark placement step)]
When the first step is completed, in the second step, a fluorescent mark is placed on the membrane filter used for filtering the liquid sample (step S2). Here, the second step will be specifically described with reference to FIG. First, the fluorescent mark placement plate Ab is overlaid on the membrane filter Fl so that the outer circumferences coincide (see FIG. 2A).

  The membrane filter is a polycarbonate filter having a circular shape with a diameter of 25 mm and a pore diameter of 0.22 μm. A circular region (filtration surface Fs) having a diameter of 16 mm at the center of the membrane filter is used for filtration. The fluorescent mark arrangement plate Ab is a thin silicon plate, and a hole (filtration hole Fh) having the same size as the filtration surface Fs of the membrane filter Fl is opened at the center thereof. Furthermore, the fluorescent mark arrangement | positioning board Ab has four fluorescent mark arrangement | positioning holes Ah opened at equal intervals along the outer periphery of the hole Fh for filtration. Note that the material of the fluorescent mark arrangement plate Ab is not limited to the above-described silicon, but may be any material that is flexible and can be in close contact with the membrane filter Fl. Moreover, it is preferable that the distance from the filtration hole Fh is constant, for example, 0.5 mm, in each of the fluorescent mark arrangement holes Ah.

Then, a suspension of fluorescent beads (Fluorebrite Polychromatic Red Microspheres, Polysciens, Inc) is dropped into the fluorescent mark placement hole Ah with an electrophoresis pipette. Then, as shown in FIG. 2B, four fluorescent marks Fm made of fluorescent beads are arranged on the membrane filter Fl. The fluorescent beads are used as a reference color and a reference size when extracting microorganisms and the like by image analysis, and have a size of about 0.5 μm. The fluorescent beads emit green fluorescence with a wavelength of 520 nm when irradiated with blue excitation light with a wavelength of 495 nm. The suspension of fluorescent beads is 3.64 × 10 ¹¹ Particles / ml, for example, 1/10 to 1/20, more preferably 1/100 to 1/200, more preferably limited concentration. Diluted 1/1000 to 1/2000. Then, 10 μl of this suspension is placed on the membrane filter Fl in the fluorescent mark placement hole Ah.

[Third step]
When the second step is completed, in the third step, the liquid sample is filtered by a filtration device, whereby a fixed specimen of Legionella is collected on the membrane filter (step S3). Here, the third step will be specifically described with reference to FIGS. As shown in FIG. 3, the filtration device 10 includes a filtration tower 1, a fluorescent mark arrangement plate Ab, a membrane filter Fl, and a filtration bottle 2.

  The filtration tower 1 is a cylindrical channel for introducing a liquid sample into the membrane filter Fl, and is attached on the fluorescent mark arrangement plate Ab. And the inner periphery of the filtration tower 1 and the hole Fh for filtration of the fluorescence mark arrangement | positioning board Ab correspond. Thereby, a liquid sample is guide | induced to the filtration surface Fs of the membranes filter Fl. Since the fluorescent mark arrangement plate Ab and the membrane filter Fl have been described above, description thereof will be omitted. The filtration bottle 2 is connected to a suction filter (not shown), and accumulates the liquid that has passed through the membrane filter Fl when the air inside is sucked into the suction filter.

  In such a filtering device 10, the liquid sample is filtered. When the filtration is completed, the fluorescent mark arrangement plate Ab and the membrane filter Fl are removed from the filtration tower 1 and the filtration bottle 2. Then, when the fluorescent mark arrangement plate Ab is removed from the membrane filter Fl, the specimen Sp containing Legionella is collected on the filtration surface Fs at the center of the membrane filter Fl as shown in FIG. And the four fluorescent marks Fm are arrange | positioned along the outer periphery of the filtration surface Fs.

[Fourth step]
When the third step is completed, in the fourth step, the membrane filter is dehydrated by placing it in 99.5% ethanol for 1 minute, and then dried in air at room temperature (step S4).
[Fifth step]
When the fourth step is completed, in the fifth step, a hybridization buffer is applied to the entire surface of the membrane filter (step S5). The hybridization buffer is composed of 0.9% NaCl, 20 mM Tris-Cl, 35% formamide, 2% Blocking reagent, and 0.02% SDS (Sodium Dodecyl sulphate: surfactant). is there.

[Sixth step]
When the fifth step is finished, in the sixth step, a probe is bound to Legionella on the membrane filter (step S6). The fifth step will be specifically described. First, the membrane filter is put in a container having a flat inner bottom surface such as a petri dish containing a probe solution for Legionella bacteria. Then, a container with a flat inner bottom surface such as a petri dish is incubated at a temperature of 46 ° C. for about 2 hours using an incubator. Thereby, the coupling | bonding of the probe with Legionella is accelerated | stimulated, and Legionella and a probe couple | bond together with progress of time. The probe is a probe in which a fluorescent dye is covalently bonded to a polynucleotide complementary to the nucleic acid of Legionella, which is a target microorganism (in many cases, ribosome RNA (ribosome RNA)).

[Seventh step]
When the sixth step is completed, in the seventh step, the membrane filter taken out from the probe solution is immersed in a washing buffer, and unreacted probes on the membrane filter are washed (step S7).
[Eighth step]
When the seventh step is completed, next, in the eighth step, the membrane filter is dehydrated by placing it in 99.5% ethanol for 1 minute, and then dried in air at normal temperature (step S8). ).
[Ninth step]
When the eighth step is completed, in the ninth step, after 0.05% agar is dissolved by heating, the membrane filter is immersed in an agar solution kept dissolved at about 40 ° C. Is attached so as not to contain air bubbles on the slide glass and dried (step S9). The substrate on which the membrane filter is placed may be a slide glass, but in order to increase the flatness, it is better to use a high flatness material such as a parallel flat substrate. In the case of fluorescent staining, the substrate does not need to be transparent, and a silicon wafer or the like can be used.

[Tenth step]
When the ninth step is finished, next, in a tenth step, a vector shield is dropped on the membrane filter on the slide glass, and a cover glass is placed on the membrane filter so as not to enter air bubbles (step S10). .

[Eleventh step]
When the tenth step is completed, in the eleventh step, the focus of the objective lens of the fluorescent microscope is adjusted by autofocus based on the fluorescent mark, and then Legionella on the membrane filter is detected by the fluorescent microscope. (Step S11). That is, a slide glass is attached to the fluorescence microscope, and the objective lens is disposed so as to be in contact with the cover glass. Then, after irradiating the fluorescence microscope with blue excitation light having a wavelength of 495 nm, the stage is moved so that the objective lens is directly above the fluorescent mark, and the focus of the objective lens is adjusted to the fluorescent mark by autofocus. At this time, the fluorescent mark emits fluorescence by excitation light. Thereafter, the focal point of the objective lens is adjusted to match that of Legionella based on the difference between the height of the fluorescent mark and the height of Legionella. Then, while moving the stage automatically, the specimen on the filtration surface of the membrane filter is photographed, and Legionella is detected from the photographed image based on the fluorescence.

  As described above, since adjustment is made to focus on Legionella, even if the visual field of the fluorescence microscope moves, readjustment of focus by autofocus is hardly performed. Then, when images were taken with a fluorescence microscope, in the normal method, the number of photographs focused on the entire image was about 1 in 10, but in the case of many images using the microorganism detection method according to this embodiment. An image with good focus was obtained.

  As described above, in the present embodiment, the fluorescent mark is arranged on the membrane filter. Thereby, the fluorescent microscope can focus on the fluorescently stained microorganism faster than before even if the field of view moves, by autofocusing with the fluorescent mark as a reference.

As mentioned above, although embodiment of this invention was described, this invention is not limited to the said embodiment, For example, the following modifications can be considered.
(1) Although the said embodiment applied this invention to the Legionella detection method using FISH method, this invention is not limited to this.
For example, the present invention is applied to a Legionella detection method for detecting Legionella by a fluorescent staining method other than the FISH method such as a DAPI (4 ′, 6-Diamidine-2′-phenylindole dihydrochloride) staining method or an acridine orange staining method. You may do it.

(2) In the above embodiment, four fluorescent marks are arranged on the membrane filter, but the present invention is not limited to this.
For example, any number of three or more fluorescent marks on the membrane filter may be arranged. That is, the fluorescent mark of the present invention may be three or more.

(3) In the said embodiment, although the fluorescent mark was arrange | positioned out of the filtration surface of a membrane filter, this invention is not limited to this.
For example, fluorescent marks may be arranged on the filtration surface.

(4) In the said embodiment, although the fluorescent mark was comprised with the fluorescent bead, this invention is not limited to this.
For example, the fluorescent mark may be made of fluorescent ink. That is, as long as it emits fluorescence, the fluorescent mark may be made of anything.

(5) In the embodiment described above, autofocusing is performed based on the fluorescent mark, so that even if the visual field moves, the fluorescently stained microorganism is focused earlier than before, but the present invention is limited to this. Not.
For example, when there are three or more fluorescent marks, a sample approximate surface that approximates the surface of the sample is obtained from the spatial coordinates of the fluorescent mark, and a vertical position corresponding to the horizontal position of the sample is calculated based on the sample approximate surface The vertical position may be the focal point at the horizontal position.

  DESCRIPTION OF SYMBOLS 1 ... Filtration tower, 2 ... Filtration bottle, 10 ... Filtration apparatus

Claims (10)

A microorganism detection method for collecting microorganisms in a sample on a filter by filtration, and detecting fluorescence-stained microorganisms from the filter using a microscope having an autofocus function,
A method of detecting a microorganism, comprising a fluorescent mark arranging step of arranging a fluorescent mark emitting fluorescence on the filter.   The microorganism detection method according to claim 1, wherein the fluorescent mark is arranged outside a surface (filtration surface) used for filtration of the filter.   3. The microorganism detection method according to claim 2, wherein the filtration surface is circular, and three or more fluorescent marks are arranged along the outer periphery of the filtration surface.   The microorganism detection method according to claim 1, wherein the fluorescent mark is made of fluorescent beads.   The method of detecting a microorganism according to any one of claims 1 to 4, wherein a fluorescent mark arrangement plate having a fluorescent mark arrangement hole is overlapped with the filter to create a fluorescent mark in the fluorescent mark arrangement hole. A filter that collects microorganisms in a sample by filtration,
A filter characterized in that a fluorescent mark emitting fluorescence is arranged.   The filter according to claim 6, wherein the fluorescent mark is disposed outside a surface used for filtration (filtration surface).   The filter according to claim 7, wherein the filtration surface is circular, and three or more fluorescent marks are arranged along an outer periphery of the filtration surface.   The filter according to claim 6, wherein the fluorescent mark is made of fluorescent beads. A fluorescent mark arrangement plate, wherein a filtration hole is opened in the center, and a fluorescent mark arrangement hole is formed along an outer periphery of the filtration hole.

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