JP2006067901A - Separating and recovering method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of improving a recovery ratio of a target layer by cutting a cylindrical member along the lower end of the target recovery layer with a cutting means in addition to an opening in the upper part of a container and carrying out face discharge of an unnecessary layer without using a conventional one point suction method. <P>SOLUTION: The separating and recovering method comprises using a cylindrical member 1 whose one end is opened as a container for density gradient centrifugation and keeping the opened one end side of the cylindrical member 1 in upward position, directing the cylindrical member 1 in the vertical direction, stacking layers of solutions different in specific gravity in the interior of the cylindrical member 1 so that a solution having large specific gravity is positioned below the cylindrical member 1 and a solution having small specific gravity is positioned above the cylindrical member 1, putting a sample containing bacteria or foreign matters different in specific gravity into the uppermost part of the cylindrical member 1, carrying out centrifugal treatment of the solutions, and the like, in the cylindrical member, closing the upper part of the cylindrical member to keep the cylindrical member in hermetically sealed state, cutting the lower end of the target recovery layer, keeping an internal separation solution so that own weight of the internal separation solution does not exceed surface tension of the internal separation solution and preventing natural dropping of the internal separation solution and recovering the target recovery layer from the cut surface. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for separating microorganisms and foreign substances by density gradient centrifugation and a method for collecting microorganisms, and more particularly, to a method for efficiently separating and recovering only microorganisms from drinking water, foods, and foodstuffs in which microorganisms are mixed. Is.

  Conventionally, this kind of density gradient centrifugation method is known as specified in JP-T-2002-505866 (see, for example, Patent Document 1).

  Hereinafter, the Japanese translation of PCT publication No. 2002-505866 will be described with reference to FIG.

  As shown in FIG. 7, the centrifuge tube 101 changes the opening for each density gradient solution layer, injects the necessary density gradient solution for each layer, and leaves the solution containing microorganisms above the density gradient solution layer. In this method, a centrifugal process is performed using a centrifugal separator, a predetermined density gradient solution layer is collected, and only microorganisms are detected.

  In addition, the conventional separation method using density gradient is to detect the interface of each layer, put particles with a specific gravity, distinguish the specific gravity layer at the part where the particles gathered after centrifugation, and similar to microorganisms A method is generally used in which a layer having a specific gravity is provided, and then a solution contained in a food material is centrifuged with a density gradient solution and then separated, and a part where microorganisms are collected is collected and specified. As the centrifuge container used for the density gradient, it is generally used that the density gradient solution insertion part and the suction part are the same (for example, see Patent Document 1).

Hereinafter, the configuration of the centrifugal container will be described with reference to FIG. As shown in the figure, the centrifuge tube 101 is sealed with a cap 102 at the top, and a liquid sample of a food solution containing density gradient solution and microorganisms is placed in the centrifuge tube 101 from the top by an injection tube 103 disposed on the cap. Filled. After centrifugation, the capillary probe 104 is inserted into the centrifuge tube 101 along the central axis of the centrifuge tube 101 from the center point of the cap, and the target density gradient solution is aspirated and collected (see Table 2002). No. 505866: Patent Document 1).
Japanese translation of PCT publication No. 2002-505866 (FIG. 8A)

  In such a conventional separation / collection method using density gradient centrifugation, since there is only one opening at the top of the container, it is necessary to recover the target layer by single point suction using a needle, pipette, etc. Because of the single-point suction, the liquid flow is disturbed during the recovery of the separated liquid layer, and the disturbance of the liquid flow causes turbulence at the interface between the separated liquid layer and the adjacent separated liquid layer, and the recovery rate of the target recovery layer decreases. Thus, there is a demand for improving the recovery rate of the target recovery layer.

  The present invention solves such a conventional problem, and in addition to the opening of the upper part of the container, the lower part cuts the cylindrical member along the lower end of the target recovery layer by the cutting means when recovering the target recovery layer. Thus, an object of the present invention is to provide a method capable of improving the target layer recovery rate by approaching surface discharge instead of the conventional single point suction.

  In order to achieve the above object, the separation and recovery method of the present invention does not use a one-point suction method for recovering the target layer and the unnecessary liquid layer as in the case of a needle or pipette, but includes an open portion at the lower end of the cylindrical member. Thus, the surface discharge is approximated.

  By this means, the target layer recovery rate can be improved as compared with the case of single point suction, and a separation and recovery method capable of improving the accuracy of the bacteria test can be obtained.

  By providing an open portion having the same diameter as the inner diameter at the lower end of the cylindrical member by this means, the turbulence of the liquid flow can be reduced during the separation liquid layer recovery compared to the case where the inner diameter of the open portion is smaller than the inner diameter of the cylindrical member. Since the disturbance of the liquid flow is small, a separation and recovery method can be obtained that can reduce the disturbance of the boundary surface between the separation liquid layer and the adjacent separation liquid layer.

  According to the present invention, there is provided a method of improving the target layer recovery rate by providing an opening in the lower part by cutting in addition to the upper part of the container so that it is close to surface discharge instead of the conventional single point suction.

  The invention according to claim 1 of the present invention provides a cylindrical member having one end opened as a density gradient centrifugation container, with one open end of the cylindrical member facing upward, and solutions having different specific gravities inside the cylindrical member. By laminating a sample having a large specific gravity below the cylindrical member and a solution having a small specific gravity above, and putting a sample of bacteria and foreign matters having different specific gravities in the uppermost part, followed by centrifugation. Bacteria and foreign substances are separated into solution layers close to their specific gravity, and after centrifugation, the open end of the cylindrical member is the top and the vertical direction is set so that the inside of the closing means is sealed. After closing by the closing means for cutting, and by cutting means for cutting the lower end of the target recovery layer including the cylindrical member, the internal separation liquid weight exceeds the internal separation liquid surface tension with respect to the cylindrical member. Internal separation liquid The objective recovery layer can be recovered from the cut surface so that it does not fall, and after the centrifugal process, the upper part of the cylindrical member is closed by the closing means to generate a negative pressure inside the cylindrical member. Thus, when the lower end of the target recovery layer of the cylindrical member is cut by the cutting means, the natural fall of the separated liquid is minimized from the cut opening of the cylindrical member, and the unnecessary liquid layer present at the lower end of the target recovery layer is cut. By dropping, the target recovery layer can be directly recovered without disturbing the boundary layer.

  Further, it is characterized by comprising a severable material for cutting the lower end of the cylindrical member at the time of recovery of the target recovery layer, and a structure capable of closing the container opening, and a structure capable of closing the opening. Due to the fact that a negative pressure can be generated inside the cylindrical member and the material is severable, the recovery of the target layer is not a single point suction like a pipette or a needle but a surface discharge, and the separation liquid The disturbance of the liquid flow during the layer recovery can be reduced, and the disturbance of the boundary of the separation layer adjacent to the separation layer being recovered can be reduced because the disturbance of the liquid flow is small.

  In addition, a closing means is used to close the opening portion of the cylindrical member, and the opening of the upper portion of the cylindrical member is closed by the closing means, thereby reducing the inside of the closing means. By generating pressure, it is sealed and has the effect of preventing the separated liquid from falling naturally from the cut portion at the lower end of the cylindrical member.

  Further, as the closing means, the inside of the cylindrical member can be hermetically sealed by bending the cylindrical member opening portion, and the opening is closed by bending the opening portion of the upper portion of the cylindrical member, A negative pressure is generated inside the cylindrical member so as to be in a hermetically sealed state, and the natural liquid is prevented from falling naturally from a cut portion at the lower end of the cylindrical member.

  Further, as the closing means, the inside of the container can be hermetically sealed by heating the tubular member opening portion and thermally shrinking the tubular member opening portion. The opening part is heated to heat-shrink and seal the tubular member to close the opening part, and the inside of the tubular member is sealed by generating a negative pressure, and from the cut portion at the lower end of the tubular member This has the effect of preventing the natural fall of the separated liquid.

  In addition, the cylindrical member opening portion can be clipped as the closing means so that the inside of the cylindrical member can be hermetically sealed, and the opening portion on the cylindrical member upper portion can be clipped. The cylinder member is closed, and a negative pressure is generated inside the cylindrical member so as to be in a sealed state, thereby preventing the separation liquid from falling naturally from the cut portion at the lower end of the cylindrical member.

  Further, the cylindrical member can be sealed by twisting the cylindrical member opening portion as the closing means, and the cylindrical member upper portion is closed by twisting the opening portion. In addition, a negative pressure is generated inside the cylindrical member so as to be in a hermetically sealed state, and the natural liquid is prevented from falling naturally from a cut portion at the lower end of the cylindrical member.

  Also, scissors are used as the cutting means, and by cutting the lower end of the target recovery layer inside the cylindrical member, the recovery of the target layer is not a one-point suction such as a pipette or a needle. The surface discharge is performed, so that the disturbance of the liquid flow can be reduced during the recovery of the separated liquid layer, and since the disturbance of the liquid flow is small, the disturbance of the boundary surface between the separation layer and the adjacent separation layer can be reduced.

  Further, a cutter is used as the cutting means, and by cutting the lower end of the target recovery layer inside the cylindrical member, the recovery of the target layer is not a one-point suction such as a pipette or a needle. The surface discharge is performed, so that the disturbance of the liquid flow can be reduced during the recovery of the separated liquid layer, and since the disturbance of the liquid flow is small, the disturbance of the boundary surface between the separation layer and the adjacent separation layer can be reduced.

  Further, a laser is used as the cutting means, and by cutting the lower end of the target recovery layer inside the cylindrical member, the recovery of the target layer is not a one-point suction such as a pipette or a needle. The surface discharge is performed, so that the disturbance of the liquid flow can be reduced during the recovery of the separated liquid layer, and since the disturbance of the liquid flow is small, the disturbance of the boundary surface between the separation layer and the adjacent separation layer can be reduced.

  Further, the liquid layer of the separation liquid is solidified after the centrifugal treatment, and has an effect that it can be distinguished from other separation liquid layers by solidifying specific or all the separation liquid layers.

  In addition, after the centrifugal treatment, the target recovery layer can be recovered by solidifying the liquid layer in the container, and the target layer can be recovered by solidifying the target recovery layer or other separated liquid layer. Can be easily performed.

  Moreover, it is characterized by performing freezing as a method for solidifying the liquid layer, and has an effect that solidification can be easily performed by using freezing as the solidifying method.

  Further, the liquid layer is solidified by using agar as a method for solidifying, and has the effect that it can be solidified without using a freezer by using agar for the solidification method.

  In addition, the cylindrical member main body is made of a material that can withstand density gradient centrifugation, and has an effect that the cylindrical member has a strength that does not break even if centrifugal processing is performed.

  In addition, by twisting the cylindrical member, the target recovery layer stacked inside the cylindrical member can be separated from other layers, and the cylindrical member main body is recovered By twisting at the upper end and the lower end of the layer, the target layer is completely independent from the unnecessary liquid layer, and the target layer recovery operation is facilitated.

  Further, the target recovery layer stacked inside the cylindrical member is separated from the other layers by twisting the cylindrical member, and the target recovery layer is separated from the other layers by cutting at the twisted portion. The target recovery layer can be recovered, and the target member is completely independent from the unnecessary liquid layer by twisting the cylindrical member body at the upper end and the lower end of the target recovery layer. Only the target layer can be taken out by cutting at the location, making it easy to recover the target layer.

  Further, by twisting the cylindrical member, the target recovery layer laminated inside the cylindrical member is separated from the other layers, and the target recovery layer is separated from the other layers by cutting at the twisted portion. The target recovery layer can be recovered by separating and separating the target recovery layer portion separated, and the cylindrical member body is twisted at the upper end and the lower end of the target recovery layer Thus, the target layer is completely independent from the unnecessary liquid layer, and a hole is formed in the wall surface of the independent target layer to facilitate the target layer recovery operation.

  Further, as a means for laminating the liquid in the cylindrical member, after laminating the liquid having the largest specific gravity to be laminated in the lowermost layer, the cylindrical member is divided by the separating means at the upper end of the layer, It is characterized in that after the next layer is stacked and the stacking is completed by repeating this operation, the separating means is removed, and the stacking operation can be facilitated.

  Further, the cylindrical member separation portion can be clipped as the separating means so that the inside of the cylindrical member can be in a separated state, which has the effect that the laminating operation can be facilitated.

  In addition, the cylindrical member can be separated by twisting the cylindrical member separating portion as the separating means, and has the effect of facilitating the stacking operation.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings, but the separation and recovery method of the present invention is not limited to the following description.

(Embodiment 1)
As shown in FIG. 1, one end of the cylindrical member 1 is closed, and the other end is provided with an opening 2 for injecting a solution having a specific gravity different from that of a liquid specimen that may contain microorganisms. The cylindrical member 1 is cut and twisted. Is possible. The cylindrical member 1 is a cylinder or the like.

  In the above-described configuration, the opening 2 of the tubular member 1 that is a cylinder is located above, and solutions having different specific gravities are solutions below the opening 2 and below the tubular member 1 with high specific gravity, and above are solutions with low specific gravity. After confirming the injection amount with a syringe equipped with a pipette or needle, the layers were stacked, and the injection amount was determined with a syringe equipped with a pipette or needle with a sample of microorganisms and foreign matter having different specific gravities at the top. By putting in and centrifuging, microorganisms and foreign substances can be separated into solution layers close to their specific gravity, and after centrifuging, the cylindrical member 1 is placed so that the opening 2 is upward as shown in FIG. In the vertical direction, the opening 2 at the upper end is closed by a closing means such as a clip so that the inside of the cylindrical member 1 is sealed, and the unnecessary liquid layer existing below the target recovery layer is cut off by the cutting means as shown in FIG. , FIG. A separation and recovery method capable of recovering the desired collection layer as shown.

  Note that the order of closing the upper portion of the cylindrical member 1 by the closing means may be before the centrifugal treatment.

  Examples of the solution that forms a density gradient as a material include, but are not limited to, silica gel-based solution, sucrose solution, glycerin, cesium chloride solution, and other polymer materials.

  In addition, as a material, examples of the cylindrical member include vinyl, silicon rubber, polypropylene, polyolefin, polyurethane, and Teflon (registered trademark), but are not limited thereto.

  Further, the closing means includes, but is not limited to, the upper end bending of the cylindrical member 1, the sealing using heat shrinkage by heating the upper end of the cylindrical member 1, the clip, and the twist. Examples of the cylindrical member include a cylinder.

  The cutting means includes scissors, cutters, and laser cutting, but is not limited thereto.

  Moreover, although the color of the cylindrical member 1 includes transparent and translucent, it is not limited to this. The cylindrical member 1 is a cylinder or the like.

  Moreover, although the internal diameter conditions of the cylindrical member 1 include 5 mm or less and the thing of a separation liquid sucrose solution, it is not limited to this.

(Embodiment 2)
The same parts as those in the first embodiment are denoted by the same reference numerals, and detailed description thereof is omitted. As shown in FIG. 1, one end of the cylindrical member 1 is closed, and the other end is provided with an opening 2 for injecting a solution having a specific gravity different from that of a liquid specimen that may contain microorganisms. The cylindrical member 1 is cut and twisted. Is possible.

  In the above configuration, the opening 2 of the cylindrical member 1 is located above, and solutions having different specific gravities are solutions having a higher specific gravity below the cylindrical member 1 than the openings 2 and solutions having a lower specific gravity above. After the injection amount is automatically determined by the device, the layers are stacked, and the microorganisms with different specific gravity and foreign matter mixed samples are automatically injected by the device after the injection amount is confirmed by the device, and the microorganisms are centrifuged. And the foreign matter can be separated into solution layers close to their specific gravity, and after centrifugation, the cylindrical member 1 is oriented vertically so that the opening 2 is upward as shown in FIG. The cylindrical member 1 is closed by the closing means, and the unnecessary liquid layer existing below the target recovery layer is cut off by a cutting means such as scissors as shown in FIG. 3, and the target recovery is shown as shown in FIG. Layer can be recovered Is that separate recovery method. Examples of the cylindrical member 1 include a cylinder.

  In addition, the automatic device for determining the injection amount / discharge amount includes a syringe pump, but is not limited thereto.

(Embodiment 3)
The same parts as those in the first or second embodiment are denoted by the same reference numerals, and detailed description thereof is omitted. As shown in FIG. 1, one end of the cylindrical member 1 is closed, and the other end is provided with an opening 2 for injecting a solution having a specific gravity different from that of a liquid specimen that may contain microorganisms. The cylindrical member 1 is cut and twisted. Is possible.

  In the above configuration, the opening 2 of the cylindrical member 1 is located above, and solutions having different specific gravities are solutions having a higher specific gravity below the cylindrical member 1 than the openings 2 and solutions having a lower specific gravity above. After confirming the injection volume with a syringe equipped with a pipette or needle, stack the samples with microorganisms and foreign matter mixed in different specific gravities at the top and confirming the injection volume with a syringe equipped with a pipette or needle. By centrifuging, the microorganisms and foreign substances can be separated into solution layers close to their specific gravity, and after centrifuging, the cylindrical member 1 is oriented vertically so that the opening 2 is upward as shown in FIG. The upper end opening 2 is closed by a closing means such as a clip so that the inside of the cylindrical member 1 is sealed, and the boundary surface of the separation layer is visually confirmed as shown in FIG. Unnecessary liquid layer interface By dividing the cylindrical member 1 from above and dividing the target layer and other layers, the target recovery layer laminated inside the cylindrical member 1 is separated from the other layers, and the dividing portion is cut by cutting means such as scissors. Separation and recovery that can recover the target recovery layer by cutting and removing the recovery means to recover the target recovery liquid or by making a hole in the divided location where the target recovery layer is contained by means of punching means such as a needle Method.

  Further, the dividing means includes, but is not limited to, bending of the upper end of the cylindrical member 1, a clip, and twisting.

  In addition, the punching means includes a needle, but is not limited thereto.

(Embodiment 4)
The same parts as those in any of Embodiments 1 to 3 are denoted by the same reference numerals, and detailed description thereof is omitted. As shown in FIG. 1, one end of the cylindrical member 1 is closed, and the other end is provided with an opening 2 for injecting a solution having a specific gravity different from that of a liquid specimen that may contain microorganisms. The cylindrical member 1 is cut and twisted. Is possible.

  In the above configuration, the opening 2 of the cylindrical member 1 is located above, and solutions having different specific gravities are solutions having a higher specific gravity below the cylindrical member 1 than the openings 2 and solutions having a lower specific gravity above. After the injection amount is automatically determined by the device, the layers are stacked, and the microorganisms with different specific gravity and foreign matter mixed samples are automatically confirmed by the device after the discharge amount is automatically determined and centrifuged. Microorganisms and foreign substances can be separated into solution layers close to their specific gravity, and after centrifugation, the cylindrical member 1 is oriented vertically so that the opening 2 is upward as shown in FIG. 2, and the upper opening 2 is closed. 5 to close the inside of the cylindrical member 1, and as shown in FIG. 5, the boundary surface of the separation layer is confirmed at the height of the cylindrical member 1, and the target recovery layer and unnecessary liquid are separated by a dividing means such as twisting. The boundary surface of the layer is divided from the top of the cylindrical member 1, and the target layer and its layer The target recovery layer laminated inside the cylindrical member 1 is separated from the other layers by dividing the layer, and is cut by a cutting means at the dividing portion, and the target recovery liquid is recovered by removing the dividing means or by using a needle or the like A separation and recovery method in which a target recovery layer can be recovered by opening a hole in a divided portion where the target recovery layer is included by a punching means.

(Embodiment 5)
The same parts as those in any of Embodiments 1 to 4 are denoted by the same reference numerals, and detailed description thereof is omitted. As shown in FIG. 1, one end of the cylindrical member 1 is closed, and the other end is provided with an opening 2 for injecting a solution having a specific gravity different from that of a liquid specimen that may contain microorganisms. The cylindrical member 1 is cut and twisted. Is possible.

  In the above configuration, as a means for laminating the solution in the cylindrical member 1 as shown in FIG. 6, after determining the injection amount of the liquid having the largest specific gravity to be laminated in the lowermost layer with a syringe equipped with a pipette or a needle. After the lamination, the cylindrical member 1 is divided at the upper end of the layer by a dividing means such as twisting, and then the next layer is laminated after confirming the injection amount with a pipette or a syringe equipped with a needle. A separation / recovery method in which the separation means can be removed and laminated after the lamination is completed by repetition.

(Embodiment 6)
The same parts as those in any of Embodiments 1 to 5 are denoted by the same reference numerals, and detailed description thereof is omitted. As shown in FIG. 1, one end of the cylindrical member 1 is closed, and the other end is provided with an opening 2 for injecting a solution having a specific gravity different from that of a liquid specimen that may contain microorganisms. The cylindrical member 1 is cut and twisted. Is possible.

  In the above configuration, as a means for laminating the solution in the cylindrical member 1 as shown in FIG. 6, after injecting and laminating the liquid having the largest specific gravity to be laminated in the lowermost layer with an automatic injecting device, After the cylindrical member 1 is divided at the upper end of the layer by a dividing means such as a clip, the next layer is laminated by injecting and laminating the injection amount after the injection amount is determined by an automatic injection device, and the operation is repeated. A separation and recovery method in which the clip and other dividing means can be removed and stacked after the completion of the process.

(Embodiment 7)
The same parts as those in any of Embodiments 1 to 6 are denoted by the same reference numerals, and detailed description thereof is omitted. As shown in FIG. 1, one end of the cylindrical member 1 is closed, and the other end is provided with an opening 2 for injecting a solution having a specific gravity different from that of a liquid specimen that may contain microorganisms. The cylindrical member 1 is cut and twisted. Is possible.

  In the above configuration, the opening 2 of the cylindrical member 1 is located above, and solutions having different specific gravities are solutions having a higher specific gravity below the cylindrical member 1 than the openings 2 and solutions having a lower specific gravity above. After confirming the injection volume with a syringe equipped with a pipette or needle, stack the samples with microorganisms and foreign matter mixed in different specific gravities at the top and confirming the injection volume with a syringe equipped with a pipette or needle. By centrifuging, the microorganisms and foreign substances can be separated into solution layers close to their specific gravity, and after centrifuging, the cylindrical member 1 is oriented vertically so that the opening 2 is upward as shown in FIG. After solidification in the state of the cylindrical member 1 containing the separated liquid after centrifugation by solidification means such as freezing, the target recovery layer is visually confirmed, cut by cutting means such as scissors, and the target recovery layer is recovered Separation and recovery method.

  Further, the solidification means includes refrigeration (−10 ° C.), but is not limited thereto.

(Embodiment 8)
The same parts as those in any of Embodiments 1 to 7 are denoted by the same reference numerals, and detailed description thereof is omitted. As shown in FIG. 1, one end of the cylindrical member 1 is closed, and the other end is provided with an opening 2 for injecting a solution having a specific gravity different from that of a liquid specimen that may contain microorganisms. The cylindrical member 1 is cut and twisted. Is possible.

  In the above configuration, the opening 2 of the cylindrical member 1 is located above, and solutions having different specific gravities are solutions having a higher specific gravity below the cylindrical member 1 than the openings 2 and solutions having a lower specific gravity above. After the injection amount is automatically determined by the device, the layers are stacked, and the microbe and foreign matter mixed samples having different specific gravities are automatically determined at the uppermost portion by automatically determining the injection amount by the device and centrifuged. Microorganisms and foreign substances can be separated into solution layers close to their specific gravity, and after centrifugation, the cylindrical member 1 is vertically oriented so that the opening 2 is upward as shown in FIG. 2, and centrifuged by solidification means such as freezing. Separation in which the target recovery layer is recovered by solidifying in the state of the cylindrical member 1 containing the treated separation liquid, then confirming the target recovery layer using the distance in the height direction of the cylindrical member as a criterion and cutting by the cutting means Collection method.

(Embodiment 9)
The same parts as those in any of Embodiments 1 to 8 are denoted by the same reference numerals, and detailed description thereof is omitted. As shown in FIG. 1, one end of the cylindrical member 1 is closed, and the other end is provided with an opening 2 for injecting a solution having a specific gravity different from that of a liquid specimen that may contain microorganisms. The cylindrical member 1 is cut and twisted. Is possible.

  In the above configuration, the opening 2 of the cylindrical member 1 is located above, and solutions having different specific gravities are solutions having a higher specific gravity below the cylindrical member 1 than the openings 2 and solutions having a lower specific gravity above. After confirming the injection volume with a syringe equipped with a pipette or needle, stack the samples with microorganisms and foreign matter mixed in different specific gravities at the top and confirming the injection volume with a syringe equipped with a pipette or needle. By centrifuging, the microorganisms and foreign substances can be separated into solution layers close to their specific gravity, and after centrifuging, the cylindrical member 1 is oriented vertically so that the opening 2 is upward as shown in FIG. The upper end opening 2 is closed with a closing means such as a clip so that the inside of the cylindrical member 1 is hermetically sealed, and the unnecessary liquid layer existing below the target recovery layer is cut off with a cutting means such as scissors as shown in FIG. The figure Separating and recovering method can be recovered object collection layer, which can also collect the unnecessary layers as shown in FIG.

(Embodiment 10)
As shown in FIG. 1, one end of the cylindrical member 1 is closed, and the other end is provided with an opening 2 for injecting a solution having a specific gravity different from that of a liquid specimen that may contain microorganisms. The cylindrical member 1 is cut and twisted. Is possible.

  In the above configuration, the opening 2 of the cylindrical member 1 is located above, and solutions having different specific gravities are solutions having a higher specific gravity below the cylindrical member 1 than the openings 2 and solutions having a lower specific gravity above. After the injection amount is automatically determined by the device, the layers are stacked, and the microorganisms with different specific gravity and foreign matter mixed samples are automatically injected by the device after the injection amount is confirmed by the device, and the microorganisms are centrifuged. And the foreign matter can be separated into solution layers close to their specific gravity, and after centrifugation, the cylindrical member 1 is oriented vertically so that the opening 2 is upward as shown in FIG. The cylindrical member 1 is closed by the closing means, and the unnecessary liquid layer existing below the target recovery layer is cut off by a cutting means such as scissors as shown in FIG. 3, and the target recovery is shown as shown in FIG. Layer can be recovered , Separation and recovery method can be recovered unnecessary liquid layer.

(Embodiment 11)
As shown in FIG. 1, one end of the cylindrical member 1 is closed, and the other end is provided with an opening 2 for injecting a solution having a specific gravity different from that of a liquid specimen that may contain microorganisms. The cylindrical member 1 is cut and twisted. Is possible.

  In the above configuration, the opening 2 of the cylindrical member 1 is located above, and solutions having different specific gravities are solutions having a higher specific gravity below the cylindrical member 1 than the openings 2 and solutions having a lower specific gravity above. After confirming the injection volume with a syringe equipped with a pipette or needle, stack the samples with microorganisms and foreign matter mixed in different specific gravities at the top and confirming the injection volume with a syringe equipped with a pipette or needle. By centrifuging, the microorganisms and foreign substances can be separated into solution layers close to their specific gravity, and after centrifuging, the cylindrical member 1 is oriented vertically so that the opening 2 is upward as shown in FIG. The upper end opening 2 is closed with a closing means such as a clip so that the inside of the cylindrical member 1 is sealed, and the boundary surface of the separation layer is visually confirmed as shown in FIG. Recovery layer and unnecessary The boundary surface of the layer is divided from the top of the cylindrical member 1, and the target recovery layer stacked inside the cylindrical member 1 is separated from the other layers by dividing the target layer and the other layers. The target recovery layer is cut by removing the cutting means such as a clip and collecting the target recovery liquid by removing the clip or the like, or by making a hole with a needle or the like in the divided portion containing the target recovery layer by the punching means. Separation and recovery method that can recover unnecessary liquid layer.

(Embodiment 12)
As shown in FIG. 1, one end of the cylindrical member 1 is closed, and the other end is provided with an opening 2 for injecting a solution having a specific gravity different from that of a liquid specimen that may contain microorganisms. The cylindrical member 1 is cut and twisted. Is possible.

  In the above configuration, the opening 2 of the cylindrical member 1 is located above, and solutions having different specific gravities are solutions having a higher specific gravity below the cylindrical member 1 than the openings 2 and solutions having a lower specific gravity above. After the injection amount is automatically determined by the device, the layers are stacked, and the microorganisms with different specific gravity and foreign matter mixed samples are automatically confirmed by the device after the discharge amount is automatically determined and centrifuged. Microorganisms and foreign substances can be separated into solution layers close to their specific gravity, and after centrifugation, the cylindrical member 1 such as a cylinder is vertically oriented so that the opening 2 is upward as shown in FIG. 2 is closed by a closing means such as a clip, and the inside of the cylindrical member 1 is sealed, and the boundary surface of the separation layer is confirmed at the height of the cylindrical member 1 as shown in FIG. Is the boundary surface between the target recovery layer and the unnecessary liquid layer above the cylindrical member 1? Divide and divide the target layer from the other layers by dividing the target layer and other layers from the other layers, and cut them by cutting means such as scissors at the split points to divide the clips, etc. Separation and recovery, which can recover the target recovery layer by removing the means and recovering the target recovery liquid, or by making holes in the divided parts containing the target recovery layer by the punching means Method.

  By separating and recovering only microorganisms in food, cosmetics and drinking water, foreign substances other than microorganisms can be removed, and detection with fluorescent reagents, luminescent reagent staining, and antibody reagents can be detected with higher accuracy than before. It can be applied to the inspection of microorganisms in the food field, pharmaceutical field, and chemical product field.

Schematic for each component of one aspect of the separation and recovery method of Embodiments 1 to 8 of the present invention Schematic of stacking of separation liquid and sample of Embodiments 1 to 8 of the present invention Schematic of unnecessary layer cutting of Embodiments 1 and 2 of the present invention Schematic of target layer recovery according to Embodiments 1 and 2 of the present invention Schematic of boundary surface division according to Embodiments 3 and 4 of the present invention Schematic of the lamination procedure of Embodiments 5 and 6 of the present invention Sectional drawing which shows the container which inject | pours the conventional density gradient solution

Explanation of symbols

1 Cylindrical member 2 Opening

Claims (21)

As a density gradient centrifuge container, a cylindrical member whose one end is opened is placed with the opened one end side of the cylindrical member facing upward, and solutions having different specific gravities are placed inside the cylindrical member below the cylindrical member. Laminate a large solution so that a solution with a small specific gravity is placed on the top, and put samples of bacteria and foreign matter mixed in different specific gravity at the top, and centrifuge the solution layer so that the bacteria and foreign matter are close to their specific gravity. After the centrifugal separation, the one end side of the cylindrical member opened upward and closed in the vertical direction with the closing means for sealing the inside of the closing means, and the objective recovery After cutting by the cutting means for cutting the lower end of the layer including the cylindrical member, the internal separation liquid self-weight exceeds the internal separation liquid surface tension with respect to the cylindrical member so that the internal separation liquid does not fall naturally and is cut. Face Separation recovery method, characterized in that can be recovered object collection layer. The separation / recovery method according to claim 1, further comprising a severable material for cutting the lower end of the cylindrical member during the recovery of the target recovery layer and a structure capable of closing the container opening. 3. The separation and recovery method according to claim 1, wherein a closing means is used to close the open part of the cylindrical member. The separation and recovery method according to any one of claims 1 to 3, wherein the cylindrical member can be hermetically sealed by bending the cylindrical member opening portion as the closing means. The inside of the said cylindrical member can be made into a sealing state by heating the said cylindrical member opening part as said closing means, and making the said cylindrical member opening part heat-shrink. Separation and recovery method. The separation and recovery method according to any one of claims 1 to 3, wherein the cylindrical member can be hermetically sealed by clipping the cylindrical member opening portion as the closing means. The separation and recovery method according to any one of claims 1 to 3, wherein the cylindrical member can be hermetically sealed by twisting the cylindrical member opening portion as the closing means. The separation and recovery method according to claim 1, wherein scissors are used as the cutting means. The separation and recovery method according to any one of claims 1 to 7, wherein a cutter is used as the cutting means. 8. A separation and recovery method according to claim 1, wherein a laser is used as the cutting means. 4. The separation and recovery method according to claim 1, wherein the liquid layer of the separation liquid is solidified after the centrifugal treatment. The separation / recovery method according to claim 1, wherein the target recovery layer can be recovered by solidifying the liquid layer in the container after centrifugation. The separation and recovery method according to claim 12, wherein freezing is performed as a method for solidifying the liquid layer. 13. The separation and recovery method according to claim 12, wherein the liquid layer is solidified using agar. The separation and recovery method according to any one of claims 1 to 14, wherein the cylindrical member body is made of a material that can withstand density gradient centrifugation. The separation / recovery according to any one of claims 1 to 10, wherein a target recovery layer laminated inside the cylindrical member can be separated from other layers by twisting the cylindrical member. Method. The target recovery layer laminated inside the cylindrical member is separated from the other layers by twisting the cylindrical member, and the target recovery layer is separated from the other layers by cutting at the twisted portion. The separation and recovery method according to claim 16, wherein the target recovery layer can be recovered. The target recovery layer stacked inside the cylindrical member is separated from the other layers by twisting the cylindrical member, and the target recovery layer is separated from the other layers by cutting at the twisted portion. 18. The separation and recovery method according to claim 16 or 17, wherein the target recovery layer can be recovered by making a hole in the separated target recovery layer portion. As a means for laminating the liquid in the cylindrical member, after laminating the liquid having the largest specific gravity to be laminated in the lowermost layer, the cylindrical member is separated at the upper end of the layer by the separating means, and then The separation and recovery method according to any one of claims 1 to 18, wherein the separation means is removed after the layers are stacked and the stacking is completed by repeating this operation. The separation and recovery method according to claim 19, wherein the tubular member can be separated by clipping the tubular member separation portion as the separation means. 20. The separation and recovery method according to claim 19, wherein the tubular member can be separated by twisting the tubular member separation portion as the separation means.

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