JP2006280318A

JP2006280318A - Separating and recovering method

Info

Publication number: JP2006280318A
Application number: JP2005107269A
Authority: JP
Inventors: Tomoshi Matsui; 智志松井
Original assignee: Matsushita Electric Industrial Co Ltd
Current assignee: Panasonic Holdings Corp
Priority date: 2005-04-04
Filing date: 2005-04-04
Publication date: 2006-10-19

Abstract

【課題】密度勾配遠心処理後の所望する層を回収する手段に使用される方法において、密度勾配層に対応して分離可能な方法を用いることで、従来の吸引にて回収するのではなく、分離膜を介して層を分離することで所望する層ごと回収し目的層回収率向上可能な方法を提供すること。
【解決手段】密度勾配媒体を用いて、比重の異なる粒子や細胞、微生物、ウィルスと異物を含む検体を分離し濃縮後に回収する方法において、比重の異なった媒体を分離する手段である検体の微生物と異物を通過させることを可能とする孔を有する分離膜３を備えたことを特徴とするものである。
【選択図】図１In a method used as a means for recovering a desired layer after density gradient centrifugation, a method that can be separated corresponding to the density gradient layer is used, not by conventional suction, To provide a method capable of improving the target layer recovery rate by recovering the desired layers by separating the layers through a separation membrane.
In a method of separating particles, cells, microorganisms, viruses and foreign substances having different specific gravities using a density gradient medium, and collecting them after concentration, the microorganisms of the specimens are means for separating the media having different specific gravities. And a separation membrane 3 having holes that allow foreign substances to pass therethrough.
[Selection] Figure 1

Description

本発明は、密度勾配遠心分離により比重の異なった粒子や細胞、微生物、ウィルスを分離する方法及び回収する方法に関するもので、特に比重の近い物質が混在する検体から効率良く目的の粒子や細胞、微生物、ウィルスを分離し回収する方法に関するものである。 The present invention relates to a method for separating and collecting particles, cells, microorganisms, and viruses having different specific gravities by density gradient centrifugation, and a method for recovering the particles. The present invention relates to a method for separating and recovering microorganisms and viruses.

従来、この種の密度勾配遠心分離方法は、特表２００２−５０５８６６号公報に明記されたものが知られている（例えば、特許文献１参照）。 Conventionally, this kind of density gradient centrifugation method is known as specified in JP-T-2002-505866 (see, for example, Patent Document 1).

以下、その特表２００２−５０５８６６号公報について図５を参照しながら説明する。
図５は従来例の回収容器の断面を示す図である。図５に示すように、遠心チューブ１０１は上部をキャップ１０２により密閉され、キャップ上に配置された注入用チューブ１０３によって、上部より比重の異なる密度勾配溶液及び目的の粒子を含んだ溶液の液体試料を遠心チューブ１０１内に充填積層される。遠心分離後は、キャップ中心点より遠心チューブ１０１の中心軸上に沿って、遠心チューブ１０１内部に毛細管プローブ１０４を挿入し、目的の密度勾配溶液を吸引、回収するものである（特表２００２−５０５８６６号公報：特許文献１）。
特表２００２−５０５８６６号公報（図８Ａ） Hereinafter, the Japanese translation of PCT publication No. 2002-505866 will be described with reference to FIG.
FIG. 5 is a cross-sectional view of a conventional collection container. As shown in FIG. 5, the centrifuge tube 101 is sealed at the top by a cap 102, and a liquid sample of a solution containing density gradient solutions having different specific gravities and target particles from the top by an injection tube 103 disposed on the cap. Are stacked in the centrifuge tube 101. 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)

このような従来の密度勾配遠心を用いた分離回収方法では、目的の粒子を含んだ液層を回収するには、針・ピペット等を用い吸引にて回収を行う必要があり、吸引の為、所望する物質を回収する際に密度勾配液と液体試料に液流の乱れが生じ、所望する物質の回収率が低下し菌検査の精度が低下するという課題があり、積層した密度勾配層の乱れを防止し、効率良く所望する物質が含まれる液層を回収することが要求されている。 In such a conventional separation / collection method using density gradient centrifugation, in order to collect a liquid layer containing the target particles, it is necessary to collect by suction using a needle, pipette, etc. When collecting the desired substance, there is a problem that the liquid flow of the density gradient liquid and the liquid sample is disturbed, the recovery rate of the desired substance is lowered, and the accuracy of the bacteria test is lowered. It is required to recover the liquid layer containing the desired substance efficiently.

本発明は、このような従来の課題を解決するものであり、従来の吸引回収ではなく、媒体を分離する手段により境界面を含む所望する層ごと回収し、積層した密度勾配層の乱れを防止し目的層回収率を向上させ菌検査の精度を向上させることができる方法を提供することを目的としている。 The present invention solves such a conventional problem, and prevents the disturbance of the laminated density gradient layer by collecting the desired layers including the boundary surface by means of separating the medium, instead of the conventional suction collection. It is an object of the present invention to provide a method capable of improving the recovery rate of the target layer and improving the accuracy of the bacteria test.

本発明の分離回収方法は上記の目的を達成するために、目的層及び不要液層の回収方法を針・ピペット等のように吸引を用いるのでは無く、媒体を分離することで目的層もしくは不要液層を含む媒体層を境界面を含む媒体ごと回収するものである。 In order to achieve the above object, the separation and recovery method of the present invention does not use suction as in the case of a needle or pipette as a recovery method for the target layer and unnecessary liquid layer, but separates the target layer or unnecessary by separating the medium. The medium layer including the liquid layer is recovered together with the medium including the boundary surface.

この手段により吸引の場合よりも目的層回収率を向上することができ、また、菌検査の精度を向上できる分離回収方法が得られる。また他の手段は、針・ピペット等を用いずに境界面を含む所望する層ごと回収するものである。この方法により目的層回収時間を削減できる分離回収方法が得られる。 By this means, the target layer recovery rate can be improved as compared with the case of suction, and a separation and recovery method that can improve the accuracy of the bacteria test can be obtained. Another means is to collect the desired layer including the boundary surface without using a needle, pipette or the like. By this method, a separation and recovery method that can reduce the target layer recovery time is obtained.

本発明によれば、所望する粒子、細胞、微生物、ウィルスと、不要とされる異物が存在する層を分離することにより従来の吸引ではなく、境界面を含む所望する層ごと回収することで菌検査の精度を低下させることなく効率良く回収率を向上することができる効果のある方法を提供される。 According to the present invention, the desired particles, cells, microorganisms, and viruses are separated from the layer in which the unnecessary foreign matter is present, thereby collecting the desired layer including the boundary surface instead of the conventional suction. Provided is an effective method capable of improving the recovery rate efficiently without reducing the accuracy of inspection.

他の効果は針・ピペット等を用いるのではなく、境界面を含む所望する層ごと回収することで目的層回収時間を削減できる効果のある方法を提供される。 Another effect is that a desired layer recovery time can be reduced by recovering the desired layer including the boundary surface instead of using a needle or pipette.

本発明の請求項１記載の発明は、密度勾配媒体を用いて、比重の異なる粒子や細胞、微生物、ウィルスを含む検体を分離し濃縮後、回収する方法において、比重の異なった媒体を分離する手段を備えたことを特徴としたものであり、媒体を分離させることによって、従来の回収手段であるピペット等を用いた吸引回収では回収困難であった層と層の間の境界層および容器内壁付近の所望する粒子や細胞、微生物、ウィルスを媒体ごと分離し回収することによって所望する菌や異物の回収が容易になるという作用を有する。 According to the first aspect of the present invention, in a method of separating, concentrating and collecting specimens containing particles, cells, microorganisms and viruses having different specific gravities using a density gradient medium, media having different specific gravities are separated. And a boundary layer between the layers and the inner wall of the container, which are difficult to recover by suction recovery using a pipette or the like, which is a conventional recovery means, by separating the medium. By separating and collecting desired particles, cells, microorganisms and viruses in the vicinity together with the medium, the desired bacteria and foreign substances can be easily collected.

また、粒子や細胞、微生物、ウィルスを含む検体を通過させることを可能とする孔を有する形状で形成された仕切り板である分離膜を用いることを特徴としたものであり、遠心処理時のみ分離膜は微生物と異物と溶媒を通過させることにより媒体分離時に媒体漏れを防止するという作用を有する。 In addition, it is characterized by using a separation membrane, which is a partition plate formed in a shape having holes that allow a specimen containing particles, cells, microorganisms, and viruses to pass through, and is separated only during centrifugation. The membrane has the effect of preventing medium leakage during medium separation by allowing microorganisms, foreign substances, and solvents to pass therethrough.

また、前記分離膜を１枚以上用いることを特徴としたものであり、多段数の密度勾配遠心分離処理に対応するという作用を有する。 Further, it is characterized in that one or more separation membranes are used, and has an effect of corresponding to a multi-stage density gradient centrifugation process.

また、密度勾配媒体の積層後の境界面より下方に前記分離膜を位置させることを特徴としたものであり、微生物が境界面付近に存在する場合、境界面を前記分離膜で区切ることにより、上方と下方の媒体に微生物が存在することとなり未回収の微生物が発生するためこれを防止すると同時に、比重の高い下層液の方が粘性が高い場合がある為に液垂れ防止を行うという作用を有する。 In addition, the separation membrane is characterized in that the separation membrane is positioned below the boundary surface after the density gradient medium is laminated, and when microorganisms are present near the boundary surface, the boundary surface is divided by the separation membrane, This prevents microorganisms from being present in the upper and lower media, resulting in unrecovered microorganisms. At the same time, the lower layer liquid with a higher specific gravity may have a higher viscosity. Have.

また、密度勾配媒体で層を形成時に前記分離膜の下方に気泡を残さないようにすることを特徴としたものであり、気泡により境界面が崩れることを防止するという作用を有する。 Further, it is characterized in that bubbles are not left below the separation membrane when forming a layer with a density gradient medium, and has an effect of preventing the boundary surface from being collapsed by the bubbles.

また、前記分離膜を境に密度勾配媒体を積層する方法として、前記分離膜上方に比重の高い媒体を注入し遠心処理を行いその遠心力により前記分離膜下方へ比重の高い媒体を移動させると同時に前記分離膜下方の気泡を除去し比重の高い媒体の上層へ目的の粒子や細胞、微生物、ウィルスを含む検体を積層することを特徴としたものであり、遠心力を用い前記分離膜上方から前記分離膜下方へ密度勾配媒体を移動することにより前記分離膜下方に気泡の残留を低減させるという作用を有する。 Further, as a method of laminating a density gradient medium with the separation membrane as a boundary, when a medium having a high specific gravity is injected above the separation membrane and subjected to centrifugal treatment, the medium having a high specific gravity is moved below the separation membrane by the centrifugal force. At the same time, the bubbles below the separation membrane are removed, and the specimen containing the target particles, cells, microorganisms, and viruses is stacked on the upper layer of the medium with high specific gravity. By moving the density gradient medium below the separation membrane, it has the effect of reducing residual bubbles below the separation membrane.

また、粒子や細胞、微生物、ウィルスを含む検体を最上層として密度勾配層を形成後、遠心処理を行うことにより粒子や細胞、微生物、ウィルスを分離し、前記分離膜の上方もしくは下方に存在する所望する粒子や細胞、微生物、ウィルスを回収することを特徴としたものであり、前記分離膜を用いて所望する回収層を不要層と分離でき回収を容易にするという作用を有する。 In addition, after forming a density gradient layer with the specimen containing particles, cells, microorganisms, and viruses as the uppermost layer, the particles, cells, microorganisms, and viruses are separated by centrifugation, and are present above or below the separation membrane. It is characterized by collecting desired particles, cells, microorganisms, and viruses, and has an effect of facilitating recovery by separating a desired recovery layer from an unnecessary layer using the separation membrane.

また、粒子や細胞、微生物、ウィルスを含む検体を最上層として密度勾配層を形成後、遠心処理を行うことにより粒子や細胞、微生物、ウィルスを分離し、所望する粒子や細胞、微生物、ウィルスが存在する境界層を境界層の下方に設置された前記分離膜と共に分離回収すること特徴としたものであり、層と層の間の境界面を回収することは非常に困難であるが、この方法を用いることで所望する層と境界層を回収時に不要層と分離でき回収を容易にするという作用を有する。 In addition, after forming a density gradient layer with the specimen containing particles, cells, microorganisms, and viruses as the uppermost layer, the particles, cells, microorganisms, and viruses are separated by centrifugation, and the desired particles, cells, microorganisms, and viruses are removed. It is characterized by separating and collecting the existing boundary layer together with the separation membrane installed below the boundary layer, and it is very difficult to collect the boundary surface between the layers. By using this, the desired layer and the boundary layer can be separated from unnecessary layers at the time of recovery, and the recovery is facilitated.

また、所望する粒子や細胞、微生物、ウィルスを含む検体を最上層として密度勾配層を形成後、遠心処理を行うことにより粒子や細胞、微生物、ウィルスと異物を分離し、所望する粒子や細胞、微生物、ウィルスが最下層に存在する場合、前記分離膜上方に存在する不要層を前記分離膜ごと除去後、残された最下層ごと分離回収すること特徴としたものであり、層と層の間の境界面を回収することは非常に困難であるが、この方法を用いることで所望する層と境界層を回収時に不要層と分離でき回収を容易にするという作用を有する。 In addition, after forming a density gradient layer with the specimen containing the desired particles, cells, microorganisms, and viruses as the uppermost layer, the particles, cells, microorganisms, viruses and foreign substances are separated by centrifugation, and the desired particles, cells, When microorganisms and viruses are present in the lowermost layer, the unnecessary layer existing above the separation membrane is removed together with the separation membrane, and then the remaining lowermost layer is separated and recovered. However, it is very difficult to collect the boundary surface, but by using this method, the desired layer and the boundary layer can be separated from the unnecessary layer at the time of recovery, and the recovery is facilitated.

また、分離膜上方に陰圧を生じさせることを特徴としたものであり、前記分離膜を境に媒体を分離する際に陰圧により前記分離膜下方からの媒体漏れを防止することができるという作用を有する。 Further, it is characterized in that a negative pressure is generated above the separation membrane, and when the medium is separated with the separation membrane as a boundary, the medium leakage from below the separation membrane can be prevented by the negative pressure. Has an effect.

また、遠心処理後に容器を分離する手段として最上部の容器の開放口へ閉鎖手段に設けられた圧開放口より圧を抜きながら閉鎖手段を設置し容器を分離する時に前記圧開放口を密閉しながら容器を分離することを特徴としたものであり、圧を抜くことにより前記閉鎖手段を設置し易くし、閉鎖手段設置時の陰圧により分離膜より上方の媒体が分離膜より下方に移動することを防止するという作用を有する。 Further, as a means for separating the container after centrifugation, the pressure release port is sealed when the container is separated by installing a closing means while releasing pressure from the pressure release port provided in the closing means to the opening of the uppermost container. The container is separated while making it easy to install the closing means by releasing the pressure, and the medium above the separation membrane moves below the separation membrane due to the negative pressure when the closing means is installed. This has the effect of preventing this.

また、密度勾配層を形成する媒体に粘性の高い媒体を用いることを特徴としたものであり、媒体分離時に分離膜より液漏れを防止することができる作用を有する。 In addition, a medium having a high viscosity is used as a medium for forming the density gradient layer, and has an action capable of preventing liquid leakage from the separation membrane during the medium separation.

また、前記分離膜を通過不可能な大きさを持つ比重の大きい異物と比重の異なる粒子や細胞、微生物、ウィルスを含む検体との混合液でも前記分離膜を用いることにより、比重に関係なく最上層の前記分離膜により除去することができることを特徴としたものであり、分離膜を通過不能な大きさを持つ異物が混在した検体でも前ろ過処理を加えることなく、密度勾配遠心を行うことができる作用を有する。 In addition, the separation membrane can be used in a mixed solution of a foreign substance having a large specific gravity that cannot pass through the separation membrane and a specimen containing particles, cells, microorganisms, or viruses having a different specific gravity, regardless of the specific gravity. It is characterized in that it can be removed by the separation membrane in the upper layer, and density gradient centrifugation can be performed without applying pre-filtration treatment even for a sample mixed with foreign matters having a size that cannot pass through the separation membrane. It has an action that can be done.

また、請求項１乃至１３いずれかに記載の分離回収方法において、前記分離膜を境に密度勾配媒体を積層する方法として、前記分離膜下方に比重の高い媒体を前記分離膜下面に接触しない液量を入れた後に前記分離膜上方に同一の媒体を注入し遠心処理を行いその遠心力により前記分離膜下方へ同一の媒体を移動させた後に容器最上層へ比重の異なる粒子や細胞、微生物、ウィルスと異物を含む検体を積層することを特徴としたものであり、前記分離膜の下方に予め前記分離膜下面に接触しない量の媒体を注入しておくことで媒体を前記分離膜下方に全く注入せずに全て前記分離膜上方に媒体を注入し遠心力にて媒体を前記分離膜の下方に充填する時と比較して積層時間を削減できるという作用を有する。 14. The separation and recovery method according to claim 1, wherein a density gradient medium is stacked with the separation membrane as a boundary, and a liquid having a high specific gravity below the separation membrane is not in contact with the lower surface of the separation membrane. After the amount is added, the same medium is injected above the separation membrane and centrifuged, and the same medium is moved to the lower side of the separation membrane by the centrifugal force. It is characterized by laminating a specimen containing a virus and a foreign substance, and by injecting an amount of medium that does not contact the lower surface of the separation membrane in advance below the separation membrane, the medium is completely below the separation membrane. Compared with the case where the medium is completely injected above the separation membrane without being injected and the medium is filled below the separation membrane by centrifugal force, the stacking time can be reduced.

以下、本発明の実施の形態について図面を参照しながら説明するが、本発明の分離回収方法は以下の記載に限定して解釈されるものではない。 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.

（実施の形態１）
分離膜の上方に比重の大きい媒体を注入し遠心処理を行う。遠心処理を行うことにより比重の大きい媒体が分離膜の下方に移動し、その際に発生した気泡が分離手段としての分離膜の下方に存在する場合は再度遠心処理を行い、気泡がなくなるまでこの作業を繰り返す。積層が２段の場合、気泡が無くなったことを確認後、分離膜の上方に検体を注入し、遠心処理を施し比重の大きい異物を分離膜の下方に移動させ、比重の大きい異物が移動完了後、分離膜の下方に存在する層を除去する。分離膜の下方に所望する菌の比重と近似の媒体を設置し遠心処理を行う。遠心処理後、分離膜より上方の所望する菌よりも比重の小さい異物の層を除去し分離膜の下方に存在する所望する菌が存在する層を回収することにより目的の菌を回収することができる分離回収方法。方法として例えば、図１に示すように、閉鎖手段１、上方に開放口２を備え下方に分離膜３を備えた上容器４、上方に開放口５を備え下方は閉鎖された下容器６にて各々の容器と閉鎖手段１が分割可能な着脱手段を用いることにより接続し構成された分離回収容器７は、閉鎖手段１を取り外すことにより、開放口２より密度勾配媒体及び微生物と比重の異なる異物を含み得る検体を注入することが可能である。作業の詳細は図２に示す分離回収方法。 (Embodiment 1)
A medium having a large specific gravity is injected above the separation membrane and centrifuged. When the centrifugal process is performed, the medium with a large specific gravity moves below the separation membrane. If bubbles generated at that time exist below the separation membrane as the separation means, the centrifugal treatment is performed again until the bubbles disappear. Repeat the work. In the case of two layers, after confirming that air bubbles have disappeared, inject the sample above the separation membrane, and perform centrifugation to move foreign matter with a high specific gravity below the separation membrane. Then, the layer existing below the separation membrane is removed. A medium similar to the specific gravity of the desired bacterium is placed below the separation membrane and centrifuged. After centrifugation, the target bacteria can be recovered by removing the layer of foreign matter having a specific gravity smaller than the desired bacteria above the separation membrane and collecting the layer containing the desired bacteria present below the separation membrane. Possible separation and recovery methods. As a method, for example, as shown in FIG. 1, a closing means 1, an upper container 4 provided with an opening 2 above and a separation membrane 3 below, and a lower container 6 provided with an opening 5 above and closed at the bottom. The separation / collection container 7 connected to each container by using a detachable means that can be divided into the closing means 1 has a specific gravity different from that of the density gradient medium and the microorganisms from the opening 2 by removing the closing means 1. It is possible to inject a specimen that may contain foreign substances. Details of the work are the separation and recovery method shown in FIG.

なお上容器４に設置する閉鎖手段１は上容器４と下容器６の分離時に必要なもので遠心処理時については設置しなくてもよいがコンタミが予想される場合は遠心処理時も閉鎖手段１を設置しておく。 The closing means 1 to be installed in the upper container 4 is necessary when separating the upper container 4 and the lower container 6 and may not be installed at the time of centrifugation. However, if contamination is expected, the closing means is also used at the time of centrifugation. 1 is installed.

なお分離回収容器７に用いられる材料は滅菌処理であるオートクレーブ可能な材料を用いリユース可能な材料も含む。 The material used for the separation and recovery container 7 includes materials that can be reused using autoclavable materials that are sterilized.

また、材料として密度勾配を形成する媒体としては、シリカゲル系溶液、ショ糖溶液、グリセリン、塩化セシウム溶液、その他高分子材料を含むが、これに限定されるものではない。 Examples of the medium that forms a density gradient as a material include, but are not limited to, a silica gel-based solution, a sucrose solution, glycerin, a cesium chloride solution, and other polymer materials.

また、材料として、分離膜３には孔径０．１μｍ以上を持つ金網、金属フィルタ、樹脂製フィルタ、プラスチック多孔質体、ナイロンネット、セラミック多孔質体を含むが、これに限定されるものではない。 Further, as the material, the separation membrane 3 includes, but is not limited to, a wire net having a pore diameter of 0.1 μm or more, a metal filter, a resin filter, a plastic porous body, a nylon net, and a ceramic porous body. .

また、材料として、上容器４と下容器６には、ポリカーボネート、アセタール、ポリサルフォン、シリコン、ステンレスなどであるが、これに限定されるものではない。 Further, as the material, the upper container 4 and the lower container 6 are polycarbonate, acetal, polysulfone, silicon, stainless steel, etc., but are not limited thereto.

また、閉鎖手段１としては、分離膜３上方の液を分離膜３下方に液漏れしないように密閉できれば良く例えばゴム栓を含むが、これに限定されるものではない。 Moreover, as the closing means 1, what is necessary is just to be able to seal so that the liquid above the separation membrane 3 does not leak below the separation membrane 3, for example, a rubber plug is included, but is not limited thereto.

また、材料として、閉鎖手段１には、ポリカーボネート、アセタール、ポリサルフォン、シリコン、ステンレス、天然ゴム・スチレンゴム・ブタジエンゴム・クロロプレンゴム・ブチルゴム・ニトリルゴム・ハイパロン・ウレタンゴム・フッ素ゴムを含むが、これに限定されるものではない。 In addition, as a material, the closing means 1 includes polycarbonate, acetal, polysulfone, silicon, stainless steel, natural rubber, styrene rubber, butadiene rubber, chloroprene rubber, butyl rubber, nitrile rubber, hyperalon, urethane rubber, and fluorine rubber. It is not limited to.

また、上容器４と下容器６の色には透明、半透明を含むが、これに限定されるものではない。 Further, the colors of the upper container 4 and the lower container 6 include transparent and translucent, but are not limited thereto.

また、遠心処理条件として、２０００Ｇ、３０ｍｉｎ、４℃を含むがこれに限定されるものではない。 Further, the centrifugal treatment conditions include, but are not limited to, 2000G, 30 min, 4 ° C.

また、オートクレーブの条件として１２１℃、１５ｍｉｎを含むがこれに限定されるものではない。 Further, the autoclave conditions include, but are not limited to, 121 ° C. and 15 min.

また、着脱手段には、ネジ構造、勘合を含むがこれに限定されるものではない。 The attaching / detaching means includes, but is not limited to, a screw structure and fitting.

また、ピペットもしくはシリンジもしくは針を備えたシリンジにて注入量を確定した上で注入したが、シリンジポンプのような自動で注入する装置を用いても問題はない。 Moreover, although it injected, after confirming injection amount with a pipette, a syringe, or a syringe provided with a needle, there is no problem even if an automatic injection device such as a syringe pump is used.

また、上記構成において分離回収容器７に備えた下容器６に媒体移動時の遠心処理時間を削減する為に上容器４設置時に上容器４の下方に備えた分離膜３に接触しない媒体量をピペットもしくはシリンジもしくは針を備えたシリンジにて注入量を確定した上で予め注入しておいても問題は無い。 In addition, in the above configuration, in order to reduce the centrifugation time when the medium is moved to the lower container 6 provided in the separation and recovery container 7, the amount of medium that does not contact the separation membrane 3 provided below the upper container 4 when the upper container 4 is installed. There is no problem in injecting in advance after determining the injection amount with a pipette, syringe or syringe equipped with a needle.

また、下容器６に予め注入する上容器４の下方に備えた分離膜３に接触しない媒体量は上容器４設置時に上容器４の下方に備えた分離膜３からの距離１ｍｍを含むが、これに限定されるものではない。 In addition, the amount of medium that does not come into contact with the separation membrane 3 provided below the upper container 4 that is previously injected into the lower container 6 includes a distance of 1 mm from the separation membrane 3 provided below the upper container 4 when the upper container 4 is installed. It is not limited to this.

（実施の形態２）
図３の分離膜３の上方に比重の大きい媒体を注入し遠心処理を行う。遠心処理を行うことにより比重の大きい媒体が分離膜３の下方に移動し、その際に発生した気泡が分離膜３の下方に存在する場合は再度遠心処理を行い、気泡がなくなるまでこの作業を繰り返す。積層が３段以上の場合、気泡が無くなったことを確認後、分離膜ａの上方に分離膜３の下方に存在する媒体よりも比重の小さい媒体を注入し、遠心処理を行い分離膜ａを通過させることで分離膜３と分離膜ａの間を媒体で満たす。分離膜３と分離膜ａの間に気泡が存在する場合は再度遠心処理を行い気泡がなくなるまで遠心処理を繰り返す。気泡存在しないことを確認後、この作業を繰り返すことにより積層段数を増加させていくことができる分離回収方法。方法として例えば、図４に示すように、閉鎖手段１、上方に開放口２を備え下方に分離膜３を備えた上容器４、上方に開放口８を備え下方に分離膜３を備えた中容器９、上方に開放口５を備え下方は閉鎖された下容器６にて各々の容器と閉鎖手段１が分割可能な着脱手段を用いることにより接続し構成された分離回収容器１０は、閉鎖手段１を取り外すことにより、開放口２より密度勾配媒体及び微生物と比重の異なる異物を含み得る検体を注入することが可能である。作業の詳細は図３に示す分離回収方法。 (Embodiment 2)
A medium having a large specific gravity is injected above the separation membrane 3 in FIG. When the centrifugal process is performed, the medium having a large specific gravity moves below the separation membrane 3, and when bubbles generated at that time exist below the separation membrane 3, the centrifugal process is performed again, and this work is performed until the bubbles disappear. repeat. When the number of layers is three or more, after confirming that bubbles have disappeared, a medium having a specific gravity lower than that of the medium below the separation membrane 3 is injected above the separation membrane a, and the separation membrane a is subjected to centrifugal treatment. By passing through, the space between the separation membrane 3 and the separation membrane a is filled with the medium. If bubbles are present between the separation membrane 3 and the separation membrane a, the centrifugal treatment is repeated again and the centrifugal treatment is repeated until there are no more bubbles. A separation and recovery method capable of increasing the number of stacked layers by repeating this operation after confirming that no bubbles are present. As a method, for example, as shown in FIG. 4, a closing means 1, an upper container 4 having an opening 2 at the top and a separation membrane 3 at the bottom, an opening 8 at the top and a separation membrane 3 at the bottom A separation and recovery container 10 constituted by connecting a container 9 by using a detachable means in which each container and the closing means 1 can be divided by a lower container 6 having an opening 5 at the top and closed at the bottom is a closing means. By removing 1, it is possible to inject a specimen that may contain a foreign substance having a specific gravity different from that of the density gradient medium and microorganisms from the opening 2. The details of the work are the separation and recovery method shown in FIG.

なお、中容器の個数を増加させることにより4層以上の密度勾配層を処理してもよい。 Note that four or more density gradient layers may be processed by increasing the number of medium containers.

また、中容器９に用いられる材料は滅菌処理であるオートクレーブ可能な材料を用いリユース可能な材料も含む。 In addition, the material used for the inner container 9 includes a reusable material using an autoclavable material that is a sterilization process.

また、材料として、中容器９には、ポリカーボネート、アセタール、ポリサルフォン、シリコン、ステンレスなどであるが、これに限定されるものではない。 Moreover, as a material, although the inside container 9 is polycarbonate, acetal, polysulfone, silicon, stainless steel, etc., it is not limited to this.

また、中容器９の色には透明、半透明を含むが、これに限定されるものではない。 Moreover, although the color of the inside container 9 includes transparent and translucent, it is not limited to this.

また、中容器９の着脱手段には、ネジ構造、勘合を含むがこれに限定されるものではない。 Further, the attaching / detaching means for the inner container 9 includes a screw structure and a fitting, but is not limited thereto.

また、ピペットもしくはシリンジもしくは針を備えたシリンジにて注入量を確定した上で注入したが、シリンジポンプのような自動で注入する装置を用いても問題はない。 Moreover, although it injected, after confirming injection quantity with a pipette, a syringe, or a syringe provided with a needle, there is no problem even if an automatic injection device such as a syringe pump is used.

また、上記構成において下容器６に媒体移動時の遠心処理時間を削減する為に中容器９設置時に中容器９の下方に備えた分離膜３に接触しない媒体量をピペットもしくはシリンジもしくは針を備えたシリンジにて注入量を確定した上で予め注入しておいても問題はない。 In addition, in the above configuration, in order to reduce the time required for centrifugal processing when the medium is moved to the lower container 6, a pipette, a syringe or a needle is provided for the amount of medium not contacting the separation membrane 3 provided below the middle container 9 when the middle container 9 is installed. There is no problem in injecting in advance after determining the injection amount with a syringe.

また、下容器６に予め注入する中容器９の下方に備えた分離膜３に接触しない媒体量は中容器９設置時に中容器９の下方に備えた分離膜３からの距離１ｍｍを含むが、これに限定されるものではない。 Further, the amount of medium that does not come into contact with the separation membrane 3 provided below the middle container 9 previously injected into the lower container 6 includes a distance of 1 mm from the separation membrane 3 provided below the middle container 9 when the middle container 9 is installed. It is not limited to this.

また、中容器９に予め注入する上容器４の下方に備えた分離膜３に接触しない媒体量は上容器４設置時に上容器４の下方に備えた分離膜３からの距離１ｍｍを含むが、これに限定されるものではない。 Further, the amount of medium that does not come into contact with the separation membrane 3 provided below the upper container 4 previously injected into the middle container 9 includes a distance of 1 mm from the separation membrane 3 provided below the upper container 4 when the upper container 4 is installed. It is not limited to this.

また、分離膜３を２枚以上用いる時、各々は重ならないようにするためにその距離1ｍｍを含むが、これに限定されるものではない。 Further, when two or more separation membranes 3 are used, each includes a distance of 1 mm so as not to overlap, but is not limited thereto.

食品、化粧品や飲料水中の微生物のみを分離回収することにより、微生物以外の異物を除くことで、蛍光発光試薬や発光試薬染色や抗体試薬での検出が、従来に比べ精度良く検出することができ、食品分野、医薬品分野、化成品分野における微生物の検査において適用することができる。 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.

本発明の実施の形態１の分離回収方法の一態様の概略図Schematic of one aspect of the separation and recovery method of Embodiment 1 of the present invention 本発明の実施の形態１、２の分離回収方法のフローチャートFlowchart of separation and recovery method according to Embodiments 1 and 2 of the present invention 本発明の実施の形態１、２の分離膜構成の概略図Schematic diagram of separation membrane configuration of Embodiments 1 and 2 of the present invention 本発明の実施の形態２の分離回収方法の一態様の概略図Schematic of one aspect of the separation and recovery method of Embodiment 2 of the present invention 従来の密度勾配溶液を注入する容器を示す断面図Sectional drawing which shows the container which inject | pours the conventional density gradient solution

Explanation of symbols

１閉鎖手段
２開放口
３分離膜
４上容器
５開放口
６下容器
７分離回収容器
８開放口
９中容器
１０分離回収容器
ａ分離膜
ｂ分離膜 DESCRIPTION OF SYMBOLS 1 Closing means 2 Opening port 3 Separation membrane 4 Upper container 5 Opening port 6 Lower container 7 Separation and recovery container 8 Opening port 9 Middle container 10 Separation and recovery container a Separation membrane b Separation membrane

Claims

In the method of separating, concentrating and collecting specimens containing particles, cells, microorganisms, and viruses having different specific gravities using density gradient media, the separation is characterized by comprising separation means for separating media having different specific gravities Collection method.

2. The separation and recovery method according to claim 1, wherein a separation membrane, which is a partition plate formed in a shape having a hole that allows a specimen containing particles, cells, microorganisms, and viruses to pass therethrough, is used.

The separation and recovery method according to claim 1 or 2, wherein one or more separation membranes are used.

4. The separation and recovery method according to claim 1, wherein the separation membrane is positioned below a boundary surface after the density gradient medium is laminated.

5. The separation and recovery method according to claim 1, wherein bubbles are not left below the separation membrane when forming a layer with a density gradient medium.

As a method of laminating a density gradient medium with the separation membrane as a boundary, a medium having a high specific gravity is injected above the separation membrane and subjected to a centrifugal treatment, and at the same time, the medium having a high specific gravity is moved below the separation membrane by the centrifugal force. 6. The separation and recovery method according to any one of claims 1 to 5, wherein air bubbles below the separation membrane are removed, and a specimen containing target particles, cells, microorganisms, and viruses is laminated on an upper layer of a medium having a high specific gravity.

After forming a density gradient layer with the specimen containing particles, cells, microorganisms, and viruses as the top layer, the particles, cells, microorganisms, and viruses are separated by centrifugation, and particles existing above or below the separation membrane 7. The separation and recovery method according to claim 1, wherein cells, microorganisms, and viruses are recovered.

After the density gradient layer is formed with the specimen containing particles, cells, microorganisms, and viruses as the top layer, the particles, cells, microorganisms, and viruses are separated by centrifugation, and the desired particles, cells, microorganisms, and viruses are present. The separation / recovery method according to claim 1, wherein the boundary layer is separated and collected together with the separation membrane installed below the boundary layer.

After forming the density gradient layer with the specimen containing the desired particles, cells, microorganisms, and viruses as the top layer, the particles, cells, microorganisms, viruses and foreign substances are separated by centrifugation, and the desired particles, cells, microorganisms, The separation according to any one of claims 1 to 7, wherein when a virus is present in the lowermost layer, an unnecessary layer existing above the separation membrane is removed together with the separation membrane, and then the remaining lowermost layer is separated and recovered. Collection method.

10. The separation and recovery method according to claim 1, wherein a negative pressure is generated above the separation membrane.

As a means for separating the container after centrifugation, the container is closed while the pressure release port is sealed when the container is separated by installing the closing means while releasing the pressure from the pressure release port provided in the closing means to the opening of the uppermost container. The separation and recovery method according to any one of claims 1 to 10, wherein the process is separated.

The separation and recovery method according to claim 1, wherein a medium having high viscosity is used as a medium for forming the density gradient layer.

By using the separation membrane even in a mixture of a foreign substance having a specific gravity that cannot pass through the separation membrane and a specimen containing particles, cells, microorganisms, or viruses having a different specific gravity, the uppermost layer can be used regardless of the specific gravity. The separation / recovery means according to claim 1, wherein the separation / recovery means can be removed by the separation membrane.

As a method of laminating a density gradient medium with the separation membrane as a boundary, a medium having a high specific gravity is placed below the separation membrane so that a liquid volume that does not contact the lower surface of the separation membrane is injected, and then the same medium is injected above the separation membrane and centrifuged. 2. A sample containing particles, cells, microorganisms, viruses, and foreign substances having different specific gravity is stacked on the uppermost layer of the container after processing and moving the same medium below the separation membrane by centrifugal force. 14. The separation and recovery method according to any one of 13 to 13.