JP2007181440A - Method for recovery of microorganism and vessel for recovery of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for recovery of microorganism and a vessel for recovery of the same that can easily recover microorganism at a high rate. <P>SOLUTION: The method for recovery comprises (SO1) a preparation step for preparing the recovery vessel equipped with a cylindrical structure having two openings on both ends in the axial direction, a membrane filter that divides the inside of the cylinder into the first room and the second room, and the recovering part detachably attached to the end part on the first room side; (SO2) a first injection step for injecting a liquid including microorganism (to be recovered) into the first room; (SO3) a first centrifugation step for centrifuging the liquid; (SO4) an exhausting step for exhausting the liquid passing through the membrane filter and moving; (SO5) an attaching step for attaching the recovering part to the edge part on the first room side; (SO6) a second injection step for injecting a liquid for recovering the microorganism into the second room; and (SO7) a second centrifugation step for centrifuging the liquid for recovery and recovering the solution including the microorganism for recovery to the recovery part. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a microorganism collection method and a collection container.

2. Description of the Related Art Conventionally, a microorganism is identified and quantified by binding a luminescent or fluorescent substance to a microorganism such as a bacterium (see, for example, Patent Document 1 (Luminescence Bright Point Measurement Method) below). In the method described in Patent Document 1, in order to detect microorganisms once collected by a membrane filter or the like and collect the collected microorganisms, it is necessary to collect them in a state where other fine particles are removed.
Japanese Patent No. 2594622

  The recovery of the microorganism is usually performed, for example, by washing a membrane filter that has collected the microorganism. However, in the washing, there is a problem that the microorganisms embedded in the membrane filter and the microorganisms adsorbed on the membrane filter by the interaction cannot be sufficiently recovered, and the recovery rate of the microorganisms is low. In particular, when the amount of microorganisms to be detected is small, the detection accuracy varies greatly depending on the recovery rate, so a method for recovering the microorganisms at a higher recovery rate has been demanded. In addition, there is a problem that the cleaning takes time and effort.

  The present invention has been made to solve the above problems, and an object of the present invention is to provide a microorganism recovery method and a recovery container that can easily recover microorganisms at a high recovery rate.

  The microorganism recovery method according to the present invention includes a cylindrical structure having openings at both ends in the axial direction, a cylindrical structure provided in the cylindrical part, and a first space and a second in the cylindrical part. A preparation step of preparing a collection container for microorganisms, comprising: a membrane filter that is axially partitioned into a first space; and a recovery portion that is detachable at least at an end of the cylindrical structure on the first space side; In the first injection step of injecting a liquid containing microorganisms to be collected into the first space of the body, and in the injection step so that centrifugal force is applied in the direction from the first space toward the second space. In the first centrifugation step of centrifuging the liquid injected into the first space and the first centrifugation step, the liquid that has moved from the first space to the second space through the membrane filter is discharged. At the discharge step and the end of the cylindrical structure on the first space side A mounting step for mounting the collecting portion; a second injection step for injecting a recovery liquid for recovering microorganisms into the second space from which the liquid has been discharged in the discharge step; and a first injection from the second space. The recovery liquid injected into the second space in the injection process is centrifuged so that centrifugal force is applied in the direction toward the space, and the recovery liquid containing microorganisms is recovered in the recovery part mounted in the mounting process. And a second centrifugation step.

  In the microorganism collection method according to the present invention, first, microorganisms contained in the liquid are collected by the first space side surface of the membrane filter in the first centrifugation step. Subsequently, the microorganisms collected in the second centrifugation step are removed from the membrane filter and recovered by the pressure of the recovery liquid and the centrifugal force of the centrifugation. By being collected in this way, the microorganisms that have penetrated into the membrane filter and the microorganisms that have adsorbed to the membrane filter due to the interaction can be sufficiently collected. Therefore, according to the microorganism collection method of the present invention, microorganisms can be collected at a high collection rate. In addition, the method for recovering microorganisms according to the present invention can be realized simply by performing centrifugation twice while changing the direction in which the centrifugal force is applied, so that simple recovery can be realized.

  A collection container for microorganisms according to the present invention includes a cylindrical structure having openings at both ends in the axial direction, and a first space and a second space provided in the cylindrical part of the cylindrical structure. It is characterized by comprising a membrane filter that is axially partitioned into a space, and a collection part that can be attached to and detached from at least the first space side end of the cylindrical structure. By using the collection container according to the present invention, the collection method according to the present invention can be realized.

  In the collection container according to the present invention, the membrane filter is preferably fixed by a support member from both axial sides of the cylindrical portion. With this configuration, it is possible to more easily realize a configuration that can withstand centrifugal separation from both sides in the axial direction.

  ADVANTAGE OF THE INVENTION According to this invention, the microorganisms which were immersed in the membrane filter and the microorganisms which adsorb | sucked to the membrane filter by interaction can fully be collect | recovered, and microorganisms can be collect | recovered with a high collection rate. In addition, the present invention can be realized simply by performing centrifugal separation twice by changing the direction in which the centrifugal force is applied, so that simple recovery can be realized.

  Hereinafter, preferred embodiments of a microorganism collection method and a collection container according to the present invention will be described in detail with reference to the drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted. Further, the dimensional ratios in the drawings do not necessarily match those described. First, the collection container in which the collection method is executed will be described, and then the collection method will be described. In addition, the microorganisms in this embodiment include bacteria. The microorganism may be a fine particle containing a microorganism.

  In FIG. 1, the collection container 1 of this embodiment is shown. The collection container 1 includes a cylindrical structure (membrane filter container part) 10 and a centrifuge tube body 20. The centrifuge tube body 20 is a cylindrical container having a test tube shape. As shown in FIG. 1, the tubular structure 10 can be attached to the centrifuge tube body 20 so that the axes thereof coincide with each other. The cylindrical structure 10 can be attached to the centrifuge tube body 20 from any axial direction. Here, FIG. 2 shows only the cylindrical structure 10 in the collection container 1. FIG. 3 shows an axial section of the collection container 1.

  As shown in FIGS. 1 and 2, the cylindrical structure 10 is configured to include a cylindrical portion 11 having openings at both ends in the axial direction and a membrane filter 12 provided in the cylindrical portion 11. . As shown in FIG. 2, the membrane filter 12 is provided to be fixed substantially at the center in the cylinder part 11, and the inside of the cylinder part 11 is divided into a first space 13 and a second space 14 in the axial direction. . At the time of centrifugation, a liquid is injected into one of the first space 13 and the second space 14, and the liquid passes through the membrane filter 12 and escapes to the other space. In the normal state (room temperature, atmospheric pressure state), the membrane filter 12 has a property of not allowing liquid to pass through.

  The membrane filter 12 is preferably made of a material having a hydrophobic property that does not hinder the permeation of water so that the interaction with microorganisms does not occur as much as possible. The membrane filter 12 has fine pores, and the pore diameter is preferably about 0.5 μm or less so that microorganisms can be collected during centrifugation. The pore diameter is preferably set to an appropriate value depending on the use, such as the type of microorganism to be collected. The membrane filter 12 is fixed in the cylindrical portion 11 by a plurality of support members 15, but is preferably fixed from both sides in the axial direction as shown in FIG. In the recovery method of the present embodiment, the cylindrical structure 10 is to be able to withstand the pressure of the centrifugal separation from both sides because the centrifugal separation is performed in both axial directions. Further, at that time, it is preferable that the support members 15 on both sides are respectively arranged at corresponding positions of the membrane filter 12. This is to prevent the support member 15 from interfering with the centrifugal separation as much as possible.

  The centrifuge tube body 20 can collect and hold the liquid that has passed through the membrane filter 12 of the cylindrical structure 10 by centrifugation. That is, the centrifuge tube body 20 is a collection unit in the collection container 1. As shown in FIGS. 1 and 3, the inner diameter of the centrifuge tube main body 20 is slightly larger than the outer diameter of the cylindrical portion 11 of the cylindrical structure 10 so that the cylindrical structure 10 can be attached. ing. The centrifuge tube main body 20 is preferably provided with a holding portion 22 on the inner side so that the cylindrical structure 10 can be positioned and fixed. As shown in FIGS. 1 and 3, the holding portion 22 is provided as an annular convex portion at a position in the axial direction of the tubular structure 10 in the axial direction from the opening inside the centrifuge tube body 20. Positioning of the cylindrical structure 10 is performed by the holding portion 22 serving as a stopper. In addition, when the cylindrical structure 10 can be positioned by the shape of the centrifuge tube body 20 itself, the holding portion 22 is not necessarily provided. For example, when the tapered portion of the centrifuge tube main body 20 serves as a stopper, the holding portion 22 is not necessarily provided.

  Specifically, for example, an Eppendorf tube or the like can be used for the centrifuge tube body 20. The cylinder part 11 and the centrifuge tube main body 20 of the cylindrical structure 10 are generally configured to be as large as a container used for centrifugation. Moreover, the cylindrical structure 10 and the centrifuge tube main body 20 are comprised with the material which comprises the container used for centrifugation, for example, a plastics.

  Subsequently, the microorganism collection method of the present embodiment will be described with reference to the flowchart of FIG. 4 and the schematic diagram of FIG. In the present embodiment, the collection and collection of microorganisms in the above-described microorganism identification / quantification will be described as an example.

  First, a sample containing microorganisms to be collected and recovered and the recovery container 1 described above are prepared (S01, preparation step). Specifically, the generation of the sample is performed as follows, as described in, for example, Japanese Patent No. 2594622. The microorganism to be identified and quantified is immersed in a liquid containing an antibody (label) that specifically binds to the surface antigen of the microorganism. When immersed, the microorganisms and the antibody bind to each other in the liquid (labeling). This liquid becomes a sample in this embodiment. The antibody is preliminarily bound with an enzyme that catalyzes the luminescence reaction under predetermined conditions, for example, when it reacts with a luminescence reagent. Regarding the collection container 1, the centrifuge tube body 20 is attached to the tubular structure 10 so that the first space 13 is on the opening side of the centrifuge tube body 20.

  Subsequently, as shown in FIG. 5A, the sample 30 contained in the microorganism 31 is injected into the first space 13 of the cylindrical structure 10 in the collection container 1 (S02, first injection step).

  Subsequently, as shown in FIG. 5B, the sample 30 is centrifuged so that a centrifugal force is applied in the direction from the first space 13 toward the second space 14 (direction G in FIG. 5B). (S03, first centrifugation step). Centrifugation may be usually performed using a centrifuge used for centrifugation. By this centrifugation, the microorganisms 31 are collected by the surface of the membrane filter 12 on the first space 13 side. On the other hand, the sample 30 other than the microorganisms 31 to be collected passes through the membrane filter 12 and is collected and held by the centrifuge tube body 20. At this time, the collection container 1 is in a state as shown in FIG.

  Subsequently, the cylindrical structure 10 is removed from the centrifuge tube body 20, and the remaining liquid of the centrifuged sample 30 is discharged (S04, discharge step). In the present embodiment, since the centrifuge tube body 20 is attached to the cylindrical structure 10 during the first centrifugation step, the remaining liquid (filtrate) of the sample 30 accumulates in the centrifuge body 20. . However, it is possible to perform the first centrifugation step without attaching the centrifuge tube body 20, and in that case, it is not necessary to discharge the liquid from the centrifuge tube body 20. However, considering that the centrifuge is usually contaminated, it is preferable to mount the centrifuge tube body 20 and perform the first centrifuge step.

  Subsequently, with respect to the tubular structure 10 removed as described above, the centrifuge tube body 20 is moved so that the second space 14 is on the opening side of the centrifuge tube body 20 (opposite to the direction in S01). Installing. That is, as shown in FIG. 5 (d), the centrifuge tube body 20 can collect the liquid that passes through the membrane filter 12 and falls from the first space side by centrifugation (S05, mounting step). Here, the newly installed centrifuge tube body 20 may be the one used in S01 to S04 or a new one.

  Subsequently, as shown in FIG. 5D, a recovery liquid 40 for recovering the microorganisms 31 is injected into the second space 14 of the cylindrical structure 10 in the recovery container 1 (S06, second). Injection process). Here, the recovery liquid 40 may be the same liquid as the sample 30 described above, or may be a different liquid.

  Subsequently, as shown in FIG. 5 (e), the recovery liquid 40 is applied so that centrifugal force is applied in the direction from the second space 14 toward the first space 13 (direction G in FIG. 5 (e)). Centrifugation (S07, second centrifugation step). Centrifugation may be performed in the same manner as in the first centrifugation step, but the centrifugal force may be increased or the number of times of centrifugation may be increased as compared with the first centrifugation step. By this centrifugation, the microorganisms 31 collected by the surface of the membrane filter 12 on the first space 13 side are removed from the membrane filter 12 by the pressure of the recovery liquid 40 and the centrifugal force of the centrifugation, and are removed by the centrifugal tube body 20. Collected and retained. At this time, the centrifuge tube body 20 is in a state of holding the recovery liquid 40 containing the microorganisms 31 as shown in FIG.

  Subsequently, as shown in FIG. 5 (g), by removing the cylindrical structure 10 from the recovery container 1, the recovery liquid 40 containing the microorganisms 31 can be used. In this embodiment, identification and quantification of microorganisms are performed as follows. The identification / quantification is performed, for example, by counting the microorganisms 31 with the photon counting device 50 shown in FIG. 6 (S08).

  First, the microorganisms 31 contained in the recovery liquid 40 are supplemented by a counting membrane filter 51. As shown in FIG. 6, the membrane filter 51 is arranged on the photon counting device 50 so that the counting can be performed (at this time, the microorganism 31 is arranged so as to face the photon counting camera 56 described below). . The photon counting device 50 is provided with a filter paper 52 soaked with a luminescent reagent, and the luminescent reagent is supplied from the filter paper 52 to the membrane filter 51. In the membrane filter 51 supplied with the supply reagent, the enzyme bound to the microorganism 31 via the antibody catalyzes the luminescence reaction, and as a result, light emission occurs from the portion where the microorganism 31 exists. In the photon counting device 50, the emitted light passes through the tapered image guide 53 and the fiber plate 54 and enters the photocathode 55 to generate photoelectrons. The photons generated by the photocathode 55 are photographed by the photon counting camera 56 and counted, whereby the microorganisms 31 are counted. A cap 57 is previously provided so that external light does not enter the tapered image guide 53.

  As described above, in the recovery method of this embodiment, the microorganisms 31 are removed from the membrane filter 12 by the pressure of the recovery liquid 40 and the centrifugal force of the centrifugal separation. It is possible to sufficiently recover the microorganism 31 adsorbed on the surface. Therefore, according to the collection method of the present embodiment, microorganisms can be collected at a high collection rate. In addition, since the recovery method of the present embodiment can be realized simply by performing centrifugation twice while changing the direction in which the centrifugal force is applied, simple and quick recovery can be realized. In addition, since this embodiment can be easily realized, the present embodiment can be implemented at low cost as a result.

  Moreover, if the membrane filter 12 of the cylindrical structure 10 is fixed by the support member 15 from both sides in the axial direction as in the collection container 1 of the present embodiment, it can withstand centrifugal separation from both sides in the axial direction. Can be realized more easily.

  In the present embodiment, in the collection container 1, the centrifuge tube body 20 that is a collection unit has a shape that covers the periphery of the cylindrical structure 10, but is not necessarily in such a shape (double tube type). It does not have to be. For example, as shown in FIG. 7, it may be a concave cap 25 serving as a collection portion that can be directly attached to and detached from both ends in the axial direction.

  Further, the recovery method according to the present invention is not limited to the usage method as in the above-described embodiment, and may be used, for example, when a liquid containing microorganisms is concentrated.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to a following example.

Example 1
Example 1 described below is an example relating only to the collection and recovery of microorganisms. In this example, a hydrophobic PTFE membrane filter (pore size 0.22 μm) made by Millipore filter was used as the membrane filter 12. Escherichia coli ATCC 12740 was used as the microorganism to be collected. Masuko, M., Hosoi, S. & Hayakawa, T. “Rapid Detection and counting of single bacteria in a wide field using a photon-counting TV camera.” FEMS Microbiol Lett 83, 231-238 (1992). According to the method of 1), the cells cultured aerobically were collected by centrifugation, washed once with 0.85% NaCl, centrifuged, suspended in acetone, sterilized, and immediately 0.1M phosphoric acid. The suspension was diluted with a phosphate buffer (pH 7.0), and this suspension was used as a starting material.

  0.1 M phosphate buffer (pH 7.0) was added to this cell suspension, and the cell turbidity (absorbance value at 660 nm) was adjusted to 0.2. Among these, 4.0 ml was used as the sample 30 in the present embodiment, and centrifuged at 4 ° C. and about 5000 × G for 10 minutes (first centrifugation step). The filtrate was discharged, and centrifugation for recovery of microorganisms was carried out using the recovery liquid 40 as 3.0 ml of 0.1 M phosphate buffer (pH 7.0) (second centrifugation step). The filtrate was collected and the same buffer was added to make the total volume 4.0 ml.

The turbidity of this liquid was measured, and the recovery rate of microorganisms was measured by the following formula. As a result, it was 92%.
Recovery rate = (turbidity of the cell suspension after recovery) / (turbidity of the cell suspension of the starting material) × 100
On the other hand, as a control experiment, after the first centrifugation, 1.0 ml × 3 times, the same buffer solution was added onto the membrane filter 12, washed by pipetting, and the recovered solution was adjusted to 4.0 ml. The recovery rate was 75% at maximum. This result shows that the present invention can easily recover microorganisms at a high recovery rate.

(Example 2)
Example 2 described below is an example in which microorganisms are collected and recovered, and microorganisms are identified and quantified as in this embodiment. In addition, many of the experiments in the following examples are described in Non-Patent Document 1 and Masuko, M. et al. “A Photon counting TV Camera Equipped with an Image Guide for Rapid Detection and Counting of Single Bacteria in a widefield.” Photochem Photobiol 56, 107-111 (1992). (Non-patent document 2) (The inventor is included in the author).

  Escherichia coli ATCC 12740 was used as the target microorganism for identification and quantification. According to the method of Non-Patent Document 1, aerobically cultured cells are collected by centrifugation, washed once with 0.85% NaCl, centrifuged, and then suspended and sterilized in acetone. Dilute with 1M phosphate buffer (pH 7.0) and use this suspension as starting material.

  The reagent used for labeling of E. coli was a horseradish peroxidase (hereinafter referred to as the first antibody) conjugated with an antibody against E. coli O antigen O127a (derived from rabbit) (Denka Seken Co., Ltd., hereinafter referred to as the first antibody) solution and an antibody against rabbit IgG. 2 antibody-peroxidase) solution.

  When determining the bacterial cell concentration, direct counting was performed under a phase contrast microscope using a bacterial calculation board (Sunlead Glass).

  The cell suspension concentration as a starting material is set to about 200 cells / ml. 4 ml of this suspension is used. The first antibody solution is added to this bacterial cell suspension to a final concentration of 2 μg / ml. This was left at room temperature for 30 minutes, and this was poured into the first space 13 of the cylindrical structure 10 in the collection container 1 and centrifuged at 4 ° C. and about 5000 × G for 10 minutes (first centrifugation step). . Discard the filtrate and add the second antibody-peroxidase solution (Amersham Biosciences commercial product 50 μl diluted to 4.0 ml with 0.1 M phosphate buffer (pH 7.0)) in the first space 13. And left at room temperature for 30 minutes. By this operation, peroxidase is labeled on the surface of E. coli. Then, the second antibody-peroxidase solution was removed by centrifugation. Next, 4 ml of 0.1 M phosphate buffer (pH 7.0) was injected into the first space 13 and centrifuged to wash the membrane filter 12 and E. coli.

  When the membrane filter 12 in this state is used as a material and counting is performed by the above-described counting method (S08), peroxidase adsorbed nonspecifically on the membrane filter 12 itself (not specifically bound to E. coli) is obtained. Emits light and gives a pseudo signal. Therefore, only E. coli labeled from the membrane filter 12 is recovered. Centrifugation for collecting microorganisms (labeled E. coli) was performed using 3.0 ml of the recovery liquid 40 as a 0.1 ml phosphate buffer (pH 7.0) (second centrifugation step). The filtrate was collected and the same buffer was added to make the total volume 4.0 ml.

  Suspend 0.5 ml of the above-mentioned cell suspension, which has been recovered by the recovery method of this embodiment and is sufficiently suspended, in 10 volumes of 0.1 M phosphate buffer, and filter using a commercially available filter. did. Escherichia coli was supplemented from the filtrate onto a black nitrocellulose membrane filter (pore diameter: 0.45 μm, diameter: 25 mm). Using this membrane filter as a material, counting was performed by the counting method (S08). The shooting time is 1 minute at room temperature. Refer to Non-Patent Document 2 for details of measurement.

As a result, when the recovery rate was calculated by the following formula, the recovery rate was 91%.
Recovery rate = (Bacterial concentration counted by E. coli luminescence) / (Bacterial concentration directly counted by epi-illumination microscope) × 100
The result is that when the microorganism is labeled, it is performed on the membrane filter 12, and only the microorganism is recovered from the membrane filter contaminated with the reagent and counted on another (non-contaminated) membrane filter. This shows that the concentration of the cell suspension of the starting material can be almost maintained. That is, it is shown that the present invention can easily recover microorganisms with a high recovery rate and can be effectively used for the above-described bacterial counting method.

It is a perspective view of the collection container of microorganisms concerning the embodiment of the present invention. It is a perspective view of the cylindrical structure of a collection container. It is sectional drawing of the axial direction of a collection container. It is a flowchart of the microbe collection | recovery method which concerns on embodiment of this invention. It is a schematic diagram of the microbe collection | recovery method which concerns on embodiment of this invention. It is the figure which showed the apparatus which identifies and quantifies microorganisms in embodiment. It is the figure which showed another example of the collection container.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Recovery container, 10 ... Cylindrical structure, 11 ... Tube part, 12 ... Membrane filter, 13 ... 1st space, 14 ... 2nd space, 15 ... Supporting member, 20 ... Centrifuge body, 22 ... Holding Part, 25 ... cap, 30 ... sample, 31 ... microorganism, 40 ... recovery liquid, 50 ... photon counting device, 51 ... membrane filter, 52 ... filter paper, 53 ... tapered image guide, 54 ... fiber plate, 55 ... photoelectric Cathode, 56 ... Photon counting camera, 57 ... Cap.

Claims (3)

A tubular structure having openings at both ends in the axial direction, and a membrane filter provided in the tubular portion of the tubular structure and dividing the inside of the tubular portion into a first space and a second space in the axial direction And a preparation step of preparing a collection container for microorganisms, comprising: a collection unit that can be attached to and detached from at least the first space side end of the cylindrical structure;
A first injection step of injecting a liquid containing microorganisms to be collected into the first space of the cylindrical structure;
A first centrifugation step of centrifuging the liquid injected into the first space in the injection step so that a centrifugal force is applied in a direction from the first space toward the second space;
In the first centrifugation step, a discharging step of discharging the liquid that has moved from the first space through the membrane filter to the second space;
A mounting step of mounting the collection unit on the first space side end of the cylindrical structure;
A second injection step of injecting a recovery liquid for recovering the microorganisms into the second space from which the liquid was discharged in the discharge step;
The recovery liquid injected into the second space in the injection step was centrifuged and mounted in the mounting step so that centrifugal force was applied in the direction from the second space toward the first space. A second centrifugation step for recovering a recovery liquid containing microorganisms in the recovery unit;
A method for recovering microorganisms having A cylindrical structure having openings at both ends in the axial direction;
A membrane filter provided in the cylindrical portion of the cylindrical structure and dividing the cylindrical portion into a first space and a second space in the axial direction;
A collecting part that is attachable to and detachable from at least the first space side end of the cylindrical structure;
A collection container for microorganisms.   The microbial collection container according to claim 2, wherein the membrane filter is fixed by support members from both axial sides of the cylindrical portion.

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