JPH07110301A - Method and apparatus for measuring ATP amount of cells - Google Patents
Method and apparatus for measuring ATP amount of cellsInfo
- Publication number
- JPH07110301A JPH07110301A JP27897493A JP27897493A JPH07110301A JP H07110301 A JPH07110301 A JP H07110301A JP 27897493 A JP27897493 A JP 27897493A JP 27897493 A JP27897493 A JP 27897493A JP H07110301 A JPH07110301 A JP H07110301A
- Authority
- JP
- Japan
- Prior art keywords
- container
- cells
- amount
- filtration membrane
- atp
- Prior art date
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- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000001914 filtration Methods 0.000 claims abstract description 54
- 239000012528 membrane Substances 0.000 claims abstract description 52
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 43
- 238000000605 extraction Methods 0.000 claims abstract description 29
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims description 55
- 239000007788 liquid Substances 0.000 claims description 4
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 230000000903 blocking effect Effects 0.000 claims description 2
- 238000004020 luminiscence type Methods 0.000 abstract description 13
- 238000011109 contamination Methods 0.000 abstract description 7
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 14
- 238000005259 measurement Methods 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 10
- 235000014655 lactic acid Nutrition 0.000 description 7
- 239000004310 lactic acid Substances 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 7
- 235000013305 food Nutrition 0.000 description 6
- 239000011521 glass Substances 0.000 description 6
- 230000000813 microbial effect Effects 0.000 description 6
- 238000005192 partition Methods 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 238000007689 inspection Methods 0.000 description 5
- 239000011148 porous material Substances 0.000 description 5
- 244000005700 microbiome Species 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 3
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 3
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 3
- 108060001084 Luciferase Proteins 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 3
- -1 biotechnology Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 235000015140 cultured milk Nutrition 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101150097914 FMO1 gene Proteins 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003093 cationic surfactant Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000001967 plate count agar Substances 0.000 description 1
- 238000011045 prefiltration Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
(57)【要約】
【目的】 細胞捕獲後の濾過膜を折り畳むという操作を
必要とせず、操作性が向上するのみならず、濾過膜の外
部からの汚染の危険性をも解決し、高精度にて細胞のＡ
ＴＰ量を測定することが可能な、細胞のＡＴＰ量測定方
法及び装置を提供する。
【構成】 検体中の細胞を捕獲した濾過膜１００を、折
り畳むことなく、一方が開口した皿状の容器２００内に
載置する。容器２００内に抽出試薬１０４及び発光試薬
１０７を注入し、生物化学発光反応を起こす。そして、
この容器２００の開口部と対面するようにして光電子増
倍管１０８を設置して、発光量を検出し、細胞のＡＴＰ
量を測定する。
(57) [Summary] [Purpose] Not only does the operation of folding the filtration membrane after cell capture not required, but not only the operability is improved, but the risk of contamination from the outside of the filtration membrane is also resolved and high precision At cell A
Provided are a method and an apparatus for measuring the ATP amount of cells, which can measure the TP amount. [Structure] The filtration membrane 100 capturing the cells in the sample is placed in a dish-shaped container 200 with one side open without folding. The extraction reagent 104 and the luminescent reagent 107 are injected into the container 200 to cause a biochemiluminescent reaction. And
The photomultiplier tube 108 is installed so as to face the opening of the container 200, the amount of luminescence is detected, and the ATP of the cell is detected.
Measure the quantity.
Description
【０００１】[0001]
【産業上の利用分野】本発明は、食品衛生、医学、バイ
オ、医薬品、臨床検査、超純水、環境、化粧品、酒造等
の種々の分野にて、使用する原料、原料水、或は製品等
に混在する微生物及び動物等の細胞を測定するのに利用
する測定方法及び装置に関するものであり、更に詳しく
言えば、細胞のＡＴＰ量を生物化学発光を利用して測定
し細胞個数を計測する測定方法及び装置に関するもので
ある。The present invention relates to raw materials, raw water, or products used in various fields such as food hygiene, medicine, biotechnology, pharmaceuticals, clinical tests, ultrapure water, environment, cosmetics, and brewing. The present invention relates to a measuring method and apparatus used for measuring cells such as microorganisms and animals mixed in etc., and more specifically, measuring the ATP amount of cells using biochemiluminescence to count the number of cells. The present invention relates to a measuring method and an apparatus.
【０００２】[0002]
【従来の技術】上記のような種々の分野において、大腸
菌、酵母菌、乳酸菌及びその他の生細胞の測定は、極め
て重要である。例えば、食品衛生の分野では、製品出荷
のために製品の微生物汚染の検査は必要不可欠である。
現在、細胞数の測定は、成長培地中のコロニーの計測、
血球計算盤による顕微鏡下での計測、濁度測定法等によ
って行われている。2. Description of the Related Art In various fields as described above, measurement of E. coli, yeast, lactic acid bacteria and other living cells is extremely important. For example, in the field of food hygiene, inspection of products for microbial contamination is essential for product shipping.
Currently, the number of cells is measured by counting colonies in the growth medium,
It is performed by measurement under a microscope with a hemocytometer, turbidity measurement method, and the like.
【０００３】成長培地中のコロニー計測法は、高感度
である、生細胞のみを測定できる、選択培地を用い
れば微生物細胞が固定できる等の点で優れているが、培
養を必要とするので測定に要する時間は通常１日以上で
あり、迅速に結果を得たい場合には適さない。The colony counting method in a growth medium is excellent in that it is highly sensitive, that only live cells can be measured, and that microbial cells can be fixed if a selective medium is used. It usually takes more than a day, which is not suitable for quick results.
【０００４】血球計算盤による方法は、顕微鏡を用いる
ため、操作が煩雑である、自動化が困難等の欠点を
有している。Since the method using a hemocytometer uses a microscope, it has drawbacks such as complicated operation and difficulty in automation.
【０００５】濁度測定法は、迅速で自動化が容易である
点で優れているが、感度が低い、生細胞と死細胞の
区別ができない、発酵乳等のようなベースの濁度が高
いサンプルには適用できない等の問題点を有している。The turbidity measuring method is excellent in that it is rapid and easy to automate, but it has low sensitivity, cannot distinguish live cells from dead cells, and has a high turbidity base such as fermented milk. There is a problem that it cannot be applied to.
【０００６】更に具体的に説明すれば、例えば、上記食
品衛生分野での製品の微生物汚染の検査は、前記コロニ
ー計測法で行われているが、検査に１日以上要するため
に、結果が出るまで製品を倉庫に保管しておかなければ
ならず、流通効率の点で問題があるだけでなく、牛乳等
の製品では保管時間が長いほど微生物汚染の可能性が高
くなる。More specifically, for example, the above-mentioned colony measuring method is used to inspect the product for microbial contamination in the food hygiene field, but the result is obtained because the inspection requires one day or more. Since the products must be stored in a warehouse, there is a problem in terms of distribution efficiency, and the longer the storage time of products such as milk, the higher the possibility of microbial contamination.
【０００７】又、食品で汚染を問題にする微生物濃度は
総じて低濃度であるので、高感度な検査が要求される。In addition, since the concentration of microorganisms which poses a problem of contamination in food is generally low, a highly sensitive test is required.
【０００８】このように、迅速で且つ高感度といった上
記要求を満たす微生物濃度測定法として、生きた微生物
中には必ず存在するアデノシン３リン酸（本明細書では
「ＡＴＰ」という。）を生物化学発光測定法を用いて測
定する方法が知られている。この方法は、細胞に含まれ
るＡＴＰ量が細胞数に比例することを利用しており、測
定時間が短く、高感度であるため非常に有効な方法であ
る。[0008] As described above, as a method for measuring the concentration of microorganisms that satisfies the above-mentioned requirements of being fast and highly sensitive, adenosine triphosphate (herein referred to as "ATP") which is always present in living microorganisms is biochemical. A method of measuring using a luminescence measuring method is known. This method utilizes the fact that the amount of ATP contained in cells is proportional to the number of cells, and is a very effective method because the measurement time is short and the sensitivity is high.
【０００９】生物化学発光測定法には、蛍の酵素である
ルシフェラーゼ及びルシフェリンが用いられる。つま
り、この方法を用いて細胞のＡＴＰ量を測定するために
は、細胞中に含まれるＡＴＰを抽出する試薬（本明細書
では「抽出試薬」という。）を用いて抽出した後、ルシ
フェラーゼ及びルシフェリンを用いた生物化学発光反応
試薬（本明細書では「発光試薬」という。）を作用させ
て生物化学発光に基づき生成した微小な光を高感度な発
光検出器、例えば光電子増倍管を用いて測定し、光の生
成量がＡＴＰ量に比例することを利用して、ＡＴＰを定
量する。そのＡＴＰ量から細胞量を測定することができ
る。In the biochemiluminescence assay, luciferase and luciferin, which are the enzymes of firefly, are used. That is, in order to measure the amount of ATP in cells using this method, luciferase and luciferin are extracted after extraction using a reagent for extracting ATP contained in cells (herein referred to as "extraction reagent"). By using a biochemiluminescence reaction reagent (hereinafter referred to as "luminescence reagent") that uses microbes to generate minute light generated based on biochemiluminescence using a highly sensitive luminescence detector, for example, a photomultiplier tube. ATP is quantified by measuring and utilizing the fact that the amount of light generated is proportional to the amount of ATP. The amount of cells can be measured from the amount of ATP.
【００１０】[0010]
【発明が解決しようとする課題】このように、ＡＴＰ量
を測定する方法は、細胞を測定する方法として迅速且つ
簡便な方法として有用であるが、細菌等の微生物細胞に
含まれる１細胞当たりのＡＴＰ量は、約０．００１ｐｇ
〜０．１ｐｇ（約０．００２ｆｍｏｌ〜０．２ｆｍｏ
ｌ）程度と非常に微量である。As described above, the method for measuring the amount of ATP is useful as a rapid and simple method for measuring cells, but the amount of ATP per cell contained in microbial cells such as bacteria is high. ATP amount is about 0.001 pg
~ 0.1 pg (about 0.002 fmol-0.2 fmo
1) Very small amount.
【００１１】ところが、現存の技術ではルシフェラーゼ
及びルシフェリンを用いた発光試薬の試薬ブランク及び
感度、更には、発光検出器の光検出感度などの面から、
検出できるＡＴＰ量の下限は０．１ｐｇ程度が限界であ
り、これは一般細菌数に換算すると１０3 〜１０4 個／
ｍｌに当たる。However, in the existing technology, in terms of reagent blank and sensitivity of a luminescence reagent using luciferase and luciferin, and further in terms of light detection sensitivity of a luminescence detector,
The lower limit of the amount of ATP that can be detected is about 0.1 pg, which is 10 3 to 10 4 when converted to the number of general bacteria /
Hit ml.
【００１２】しかしながら、食品衛生検査、化粧品等の
化成品細菌検査、原料水検査等の分野では、１０3 個／
ｍｌ以下の細菌数を測定する必要があるため、単純に検
体のＡＴＰ量を測定する方法では測定感度の面で不十分
であった。However, in the fields of food hygiene inspection, chemical bacteria inspection such as cosmetics, raw water inspection, etc., 10 3 pieces /
Since it is necessary to measure the number of bacteria of less than or equal to ml, the method of simply measuring the ATP amount of the sample was insufficient in terms of measurement sensitivity.
【００１３】このような問題点を解決するため、検体を
濾過膜で濾過して細胞を捕獲する方法が採用されるよう
になった。この方法は、細胞を濾過膜表面に捕獲して細
胞を濃縮し、測定する方法である。In order to solve such a problem, a method of capturing a cell by filtering a sample with a filter membrane has been adopted. This method is a method in which cells are captured on the surface of a filtration membrane to concentrate the cells and then measured.
【００１４】この方法による細胞の濃縮効率は、濾過す
る検体の液量と、後に添加する抽出試薬若しくは洗浄液
の量により決まり、１０2 個／ｍｌの細菌を含んだ検体
１００ｍｌを濾過すれば、１０4 個の細胞を捕獲するこ
とが可能になる。図７に、従来のこの種のＡＴＰ量測定
装置の一例を示す。The cell concentration efficiency by this method is determined by the amount of the sample to be filtered and the amount of the extraction reagent or the washing solution added later, and if 100 ml of the sample containing 10 2 / ml of bacteria is filtered, it becomes 10 It becomes possible to capture 4 cells. FIG. 7 shows an example of a conventional ATP amount measuring device of this type.
【００１５】つまり、従来の測定方法によれば、４７ｍ
ｍ或は２５ｍｍ直径の濾過膜１００が使用されており、
この濾過膜１００は、濾過器にて細胞を捕獲した後、小
さく折り畳まれて小型の試験管１０１に入れられる。次
いで、この試験管１０１は、遮光箱１０２の蓋１１０を
開けて、箱内に設けられた試験管ホルダ１０３にセット
され、抽出試薬１０４が試験管１０１内に注入される。That is, according to the conventional measuring method, 47 m
m or 25 mm diameter filtration membrane 100 is used,
The filtration membrane 100 captures cells with a filter, is folded into small pieces, and is put into a small test tube 101. Next, the test tube 101 is set in the test tube holder 103 provided in the box by opening the lid 110 of the light shielding box 102, and the extraction reagent 104 is injected into the test tube 101.
【００１６】試験管１０１は、モータ１０５及び回転伝
達器１０６にて振盪し、抽出試薬１０４で細胞からＡＴ
Ｐを抽出する。次いで、試験管１０１内に発光試薬１０
７を注入し、その発光量を発光検出器（光電子増倍管）
１０８で測定する。このとき、発光検出器１０８は、試
験管１０１の下方にセットするか、或は試験管１０１の
横にセットされた。The test tube 101 is shaken by the motor 105 and the rotation transmitter 106, and is extracted from the cells by the extraction reagent 104.
Extract P. Then, the luminescent reagent 10 is placed in the test tube 101.
7 is injected and the amount of emitted light is detected by a luminescence detector (photomultiplier tube).
Measure at 108. At this time, the luminescence detector 108 was set below the test tube 101 or beside the test tube 101.
【００１７】このような構成の従来の方法によれば、試
験管１０１内に濾過膜１００を入れるために、細胞捕獲
後の濾過膜１００を折り畳むという操作が必須であり、
その操作が煩わしいばかりでなく、濾過膜１００を折り
畳む過程にてこの濾過膜が外部から汚染されるという危
険性が生じる。According to the conventional method having such a configuration, in order to put the filtration membrane 100 in the test tube 101, it is essential to fold the filtration membrane 100 after cell capture,
Not only is the operation troublesome, but there is a risk that the filtration membrane will be contaminated from the outside during the process of folding the filtration membrane 100.
【００１８】更に、折り畳んだ濾過膜１００からＡＴＰ
を十分に抽出するには、抽出試薬１０４を必要以上に使
用しなければならず、そのために、濃縮効率の低下を招
いていた。Furthermore, the folded filtration membrane 100 is used to
The extraction reagent 104 must be used more than necessary in order to sufficiently extract the extract, resulting in a decrease in concentration efficiency.
【００１９】更に重要なことに、従来方法では、光電子
増倍管、即ち、発光検出器１０８は、試験管１０１の下
方或は横にセットされており、従って、試験管１０１の
ガラス管壁を通して試験管１０１内の発光量を計測する
ことが余儀なくされていた。そのために、光がガラス管
壁を通ることによる光の減衰、或は試験管１０１の汚れ
による光量の減少などが起こり、測定精度が低下する原
因となっていた。More importantly, in the conventional method, the photomultiplier tube, that is, the emission detector 108, is set below or beside the test tube 101, and therefore through the glass tube wall of the test tube 101. It was unavoidable to measure the amount of light emission in the test tube 101. Therefore, light passes through the glass tube wall and is attenuated, or the test tube 101 is contaminated to reduce the amount of light, resulting in a decrease in measurement accuracy.
【００２０】従って、本発明の目的は、細胞捕獲後の濾
過膜を折り畳むという操作を必要とせず、操作性が向上
するのみならず、濾過膜の外部からの汚染の危険性をも
解決し、高精度にて細胞のＡＴＰ量を測定することが可
能な、細胞のＡＴＰ量測定方法及び装置を提供すること
である。Therefore, the object of the present invention is not only to improve the operability without requiring the operation of folding the filter membrane after capturing the cells, but also to solve the risk of contamination from the outside of the filter membrane, An object of the present invention is to provide a method and an apparatus for measuring the amount of ATP in cells, which is capable of measuring the amount of ATP in cells with high accuracy.
【００２１】更に、本発明の他の目的は、細胞捕獲後の
濾過膜からＡＴＰを抽出するための抽出試薬の量を最小
限度とし、それによって、濃縮効率の低下を回避するこ
とができ、更には、ガラス管壁などを介することなく直
接、試料の発光量を測定することが可能となり、従っ
て、高精度にて細胞のＡＴＰ量を測定し得る、細胞のＡ
ＴＰ量測定方法及び装置を提供することである。[0021] Still another object of the present invention is to minimize the amount of extraction reagent for extracting ATP from the filtration membrane after cell capture, thereby avoiding a decrease in concentration efficiency. Enables direct measurement of the luminescence amount of the sample without going through the glass tube wall, etc. Therefore, the ATP amount of the cell can be measured with high accuracy.
A TP amount measuring method and device are provided.
【００２２】[0022]
【課題を解決するための手段】上記目的は本発明に係る
ＡＴＰ量測定方法及び装置にて達成される。要約すれ
ば、本発明は、（ａ）検体中の細胞を濾過膜に捕獲する
こと、（ｂ）前記濾過膜を、上方が開口した皿状の容器
内に載置すること、（ｃ）前記容器内に抽出試薬を注入
し、前記濾過膜に捕獲された細胞のアデノシン３リン酸
を抽出すること、（ｄ）前記容器内に発光試薬を注入
し、生物化学発光反応を起こすこと、（ｅ）前記工程
（ｄ）により発生した光を、前記容器の開口部に対面し
て配置した光電子増倍管で検出すること、を特徴とした
細胞のＡＴＰ量測定方法である。好ましくは、前記検体
中の細胞を捕獲した濾過膜は、折り畳むことなく前記容
器内に載置される。The above object can be achieved by the ATP amount measuring method and apparatus according to the present invention. In summary, the present invention provides: (a) capturing cells in a sample on a filtration membrane; (b) placing the filtration membrane in a dish-shaped container having an open top; Injecting an extraction reagent into the container to extract adenosine triphosphate of the cells captured by the filter membrane, (d) injecting a luminescent reagent into the container to cause a biochemiluminescence reaction, (e) ) A method for measuring the amount of ATP in a cell, characterized in that the light generated in the step (d) is detected by a photomultiplier tube arranged facing the opening of the container. Preferably, the filtration membrane that has captured the cells in the specimen is placed in the container without folding.
【００２３】斯かる細胞のＡＴＰ量測定方法は、正面に
開口部が形成されたハウジングと、検体中の細胞を捕獲
した濾過膜を収容することのできる上方が開口した皿状
の容器を担持し、そして前記ハウジングの正面開口部を
介して前記ハウジングに対して出入り自在とされる容器
ホルダと、前記容器ホルダに支持され、そして前記容器
を振盪するための駆動手段と、前記容器の開口部に対面
して前記ハウジングに取り付けられた光電子増倍管とを
有することを特徴とする細胞のＡＴＰ量測定装置にて好
適に実施される。好ましくは、前記容器ホルダが前記ハ
ウジングから引出された時、前記光電子増倍管の前記容
器に対面した開口部を遮蔽するためのシャッタ部材を有
する。又、好ましくは、前記容器に、抽出試薬及び発光
試薬を注入するための薬液導入手段を有する。Such a method for measuring the amount of ATP of cells carries a housing having an opening formed on the front surface and a dish-shaped container having an open upper part capable of accommodating a filtration membrane for capturing cells in a sample. And a container holder that can be moved in and out of the housing through a front opening of the housing, drive means supported by the container holder, and for shaking the container, and an opening in the container. A photomultiplier tube attached to the housing so as to face each other is preferably used for a cell ATP amount measuring device. Preferably, a shutter member is provided for blocking the opening of the photomultiplier tube facing the container when the container holder is pulled out from the housing. Further, preferably, the container has a chemical liquid introducing means for injecting an extraction reagent and a luminescent reagent.
【００２４】[0024]
【実施例】以下、本発明に係る細胞のＡＴＰ量測定方法
及び装置を図面に則して更に詳しく説明する。図１に本
発明に係る細胞のＡＴＰ量測定装置の一実施例の全体構
成を示し、図２〜図６にその詳細を示す。EXAMPLES The method and apparatus for measuring the amount of ATP in cells according to the present invention will be described below in more detail with reference to the drawings. FIG. 1 shows the entire configuration of an embodiment of the cell ATP amount measuring device according to the present invention, and FIGS. 2 to 6 show the details thereof.
【００２５】本実施例によると、細胞のＡＴＰ量測定装
置１は、遮光箱とされる断面が矩形のハウジング２を有
する。ハウジング２は、正面に開口部４を有し、中央部
には仕切り板６が設けられる。この仕切り板６は、後で
説明する容器ホルダ１０の案内支持部材として機能す
る。According to this embodiment, the cell ATP amount measuring apparatus 1 has a housing 2 which is a light-shielding box and has a rectangular cross section. The housing 2 has an opening 4 in the front and a partition plate 6 is provided in the center. The partition plate 6 functions as a guide support member for the container holder 10, which will be described later.
【００２６】ハウジング２に対して、その内部へと出入
り自在に容器ホルダ１０が設けられる。容器ホルダ１０
は、検体中の細胞を捕獲した濾過膜１００を収容するこ
とのできる皿状の容器２００を担持する支持板１２と、
この支持板１２に対して直交する態様で、即ち、Ｔ字形
状にて一体に取り付けられた閉鎖部材１４とを有する。
この閉鎖部材１４は、容器ホルダ１０がハウジング２に
挿入された時、ハウジング２の開口部４を閉鎖する扉の
役目を成す。又、この閉鎖部材１４には把手１６が固着
され、容器ホルダ１０のハウジング２への出し入れ時に
使用する。A container holder 10 is provided on the housing 2 so that it can freely move in and out. Container holder 10
Is a support plate 12 carrying a dish-shaped container 200 capable of accommodating the filtration membrane 100 in which the cells in the sample are captured,
It has a closing member 14 integrally attached in a manner orthogonal to the support plate 12, that is, in a T shape.
The closing member 14 serves as a door that closes the opening 4 of the housing 2 when the container holder 10 is inserted into the housing 2. A handle 16 is fixed to the closing member 14 and is used when the container holder 10 is taken in and out of the housing 2.
【００２７】更に説明すると、支持板１２は、仕切り板
６の上面に載置され、そしてこの仕切り板６の上面に互
いに平行に配置されたガイド１８Ａ、１８Ｂの間に適合
され、この仕切り板６上を摺動自在とされる。又、この
支持板１２の大略中央部の下面には、電動モータのよう
な駆動手段２０が取り付けられる。従って、仕切り板６
には、容器ホルダ１０をハウジング２内に装着した時
に、この電動モータ２０の進入を可能とするべく、Ｕ字
型の切欠き２２が形成されている。To further explain, the support plate 12 is placed on the upper surface of the partition plate 6, and is fitted between the guides 18A and 18B arranged parallel to each other on the upper surface of the partition plate 6, and the partition plate 6 is provided. It is slidable on the top. Further, a drive means 20 such as an electric motor is attached to the lower surface of the support plate 12 at the substantially central portion. Therefore, the partition plate 6
A U-shaped cutout 22 is formed in the housing 2 so that the electric motor 20 can enter when the container holder 10 is mounted in the housing 2.
【００２８】前記電動モータ２０の駆動軸２０Ａは、支
持板１２に穿設された貫通孔１２Ａを通って上方へと延
在し、その先端に、容器保持台２６が固定される。容器
保持台２６は、上面に凹所を有し、この凹所内に、検体
中の細胞を捕獲した濾過膜１００を収容することのでき
る容器２００が設置とされる。この容器２００は、一方
が開口した皿状の容器、即ち、シャーレなどとされる。The drive shaft 20A of the electric motor 20 extends upward through a through hole 12A formed in the support plate 12, and a container holding table 26 is fixed to the tip thereof. The container holding table 26 has a recess on the upper surface, and a container 200 capable of accommodating the filtration membrane 100 capturing the cells in the sample is installed in this recess. The container 200 is a dish-shaped container having one opening, that is, a petri dish or the like.
【００２９】容器保持台２６は、その中心をモータ２０
の駆動軸２０Ａに固定することもできるが、僅かに偏心
して取り付けることもでき、それによって、モータ２０
を駆動することにより、容器保持台２６が偏心運動を行
ない、容器保持台２６に載置された容器２００の振盪作
用をより効果的とすることができる。The container holding table 26 has its center at the motor 20.
Although it can be fixed to the drive shaft 20A of the motor 20, it can be mounted slightly eccentrically, whereby the motor 20
By driving the container holder 26, the container holder 26 performs an eccentric motion, and the shaking action of the container 200 placed on the container holder 26 can be made more effective.
【００３０】このハウジング２の上壁に光電子増倍管１
０８が設置される。光電子増倍管１０８の光検出先端部
１０８Ａは、上壁に形成された貫通孔部３からハウジン
グ２内方へと突入して配置され、前記容器ホルダ１０を
ハウジング２内に装着した時に、光電子増倍管１０８の
光検出先端部１０８Ａが、容器２００の開口部に対面す
るように構成されている。この光電子増倍管１０８は、
その支持外筒１２８を介してハウジング２上壁に固定さ
れる。The photomultiplier tube 1 is provided on the upper wall of the housing 2.
08 is installed. The photodetection tip portion 108A of the photomultiplier tube 108 is disposed so as to project into the inside of the housing 2 from the through hole portion 3 formed in the upper wall, and when the container holder 10 is mounted in the housing 2, The photodetecting tip 108A of the multiplier 108 is configured to face the opening of the container 200. This photomultiplier tube 108
It is fixed to the upper wall of the housing 2 via the supporting outer cylinder 128.
【００３１】本実施例によると、容器ホルダ１０を、図
３に一点鎖線にて示すように、ハウジング２内から引出
した時に、容器ホルダ１０の遮蔽部材１４によるハウジ
ング２の開口部４の遮光が無くなることにより、光電子
増倍管１０８の光検出先端部１０８Ａに侵入する光を抑
制するために、光電子増倍管１０８の光検出先端部１０
８Ａの下方に位置してシャッタ部材１３０が設けられ
る。このシャッタ部材１３０は、手動で操作するように
構成することも可能であるが、本実施例では、容器ホル
ダ１０のハウジング２内への装着に連動して作動するよ
うに構成される。According to this embodiment, when the container holder 10 is pulled out from the inside of the housing 2 as shown by the dashed line in FIG. 3, the shielding member 14 of the container holder 10 shields the opening 4 of the housing 2 from light. By eliminating the light, the light detecting tip portion 10A of the photomultiplier tube 108 is suppressed in order to suppress the light entering the light detecting tip portion 108A of the photomultiplier tube 108.
A shutter member 130 is provided below 8A. The shutter member 130 may be configured to be manually operated, but in this embodiment, it is configured to operate in association with the mounting of the container holder 10 in the housing 2.
【００３２】つまり、シャッタ部材１３０は、ハウジン
グ２の上壁に回転自在に設けられた回転支軸１３２に取
り付けられる。この回転支軸１３２には、先端にローラ
１３６を備えた作動レバー１３４が固定されており、
又、回転支軸１３２は、このローラ１３６が容器ホルダ
１０の支持板１２の先端部１２Ｂに当接するように、即
ち、図３にて反時計方向に、ばね部材（図示せず）にて
付勢されている。従って、今、容器ホルダ１０がハウジ
ング２内から外方へと引出されると、ローラ１３６及び
作動レバー１３４は、この作動レバー１３４がガイド部
材１８Ａに当接するまで反時計方向に回転する。そのた
めに、シャッタ部材１３０も反時計方向に回転し、図３
に一点鎖線にて示すように、光電子増倍管１０８の光検
出先端部１０８Ａの下方に位置する位置にて停止する。
これにより、ハウジング２内から引出した時に、シャッ
タ部材１３０は、光電子増倍管１０８の光検出先端部１
０８Ａに侵入する光を抑制することができる。That is, the shutter member 130 is attached to the rotation support shaft 132 rotatably provided on the upper wall of the housing 2. An operating lever 134 having a roller 136 at its tip is fixed to the rotary support shaft 132,
Further, the rotation support shaft 132 is attached by a spring member (not shown) in a counterclockwise direction in FIG. 3 so that the roller 136 comes into contact with the tip portion 12B of the support plate 12 of the container holder 10. It is energized. Therefore, when the container holder 10 is now pulled out from the inside of the housing 2, the roller 136 and the operating lever 134 rotate counterclockwise until the operating lever 134 comes into contact with the guide member 18A. Therefore, the shutter member 130 also rotates counterclockwise, and
As indicated by the alternate long and short dash line, the photomultiplier tube 108 is stopped at a position below the photodetection tip portion 108A.
Thus, when the shutter member 130 is pulled out from the inside of the housing 2, the shutter member 130 causes the photodetection tip portion 1 of the photomultiplier tube 108.
Light that enters 08A can be suppressed.
【００３３】逆に、容器ホルダ１０がハウジング２内へ
と装着される場合には、容器ホルダ１０の支持板１２の
先端がローラ１３６に当接し、ローラ１３６及び作動レ
バー１３４を、時計方向に回転する。そのために、シャ
ッタ部材１３０も時計方向に回転し、図３に実線にて示
すように、光電子増倍管１０８の光検出先端部１０８Ａ
を完全に開放し、光電子増倍管１０８の光検出先端部１
０８Ａが容器２００の開口部に対面することとなる。On the contrary, when the container holder 10 is mounted in the housing 2, the tip of the support plate 12 of the container holder 10 contacts the roller 136, and the roller 136 and the operating lever 134 are rotated clockwise. To do. Therefore, the shutter member 130 also rotates clockwise, and as shown by the solid line in FIG. 3, the photodetection tip portion 108A of the photomultiplier tube 108.
Is completely opened, and the photodetection tip 1 of the photomultiplier tube 108 is opened.
08A will face the opening of the container 200.
【００３４】光電子増倍管１０８には高圧電源１１２に
より高電圧が印加されており、光電子増倍管１０８の出
力電流は、アンプ１１１で電圧に変換され増幅されて、
Ａ／Ｄ変換器を介してコンピュータ１１３に送られる。
コンピュータ１１３には、測定結果を表示するための表
示器１１４と、それを印字するためのプリンタ１１５が
接続されている。A high voltage is applied to the photomultiplier tube 108 by a high voltage power source 112, and the output current of the photomultiplier tube 108 is converted into a voltage by an amplifier 111 and amplified,
It is sent to the computer 113 via the A / D converter.
A display 114 for displaying the measurement result and a printer 115 for printing it are connected to the computer 113.
【００３５】容器の上部には発光試薬１０７をポンプ１
１８により容器に注入するための導入管１２０と、抽出
試薬１０４をポンプ１２１により容器に注入するための
導入管１２２が設けられる。勿論、これら試薬の注入
は、装置外部においてディスペンサにより手操作で注入
しても良い。A luminescent reagent 107 is pumped to the top of the container 1
An inlet pipe 120 for injecting into the container by 18 and an inlet pipe 122 for injecting the extraction reagent 104 into the container by the pump 121 are provided. Of course, the injection of these reagents may be performed manually by a dispenser outside the device.
【００３６】上記構成とされる細胞のＡＴＰ量測定装置
を用いた細胞の測定方法を、次に、詳しく説明する。A method for measuring cells using the above-structured ATP amount measuring apparatus for cells will be described in detail below.
【００３７】従来と同様に、濾過膜１００を濾過器に設
置して検体を濾過する。濾過器は市販の濾過器を使用す
ることができる。濾過膜１００は、直径１３ｍｍ、２５
ｍｍ、４７ｍｍ程度のものが一般に市販されているが、
抽出試薬１０４の必要量と測定誤差の面から１３ｍｍ若
しくは２５ｍｍ程度の濾過膜１００を使用するのが好ま
しい。濾過膜１００の孔径は、濾過を行う細胞の種類に
より選択できるが、菌体の細胞を濾過する場合には０．
２μｍ或は０．４５μｍ程度の孔径の濾過膜１００を使
用するのが好ましい。As in the conventional case, the filtration membrane 100 is installed in the filter to filter the sample. A commercially available filter can be used as the filter. The filtration membrane 100 has a diameter of 13 mm, 25
mm and 47 mm are generally commercially available,
From the viewpoint of the required amount of the extraction reagent 104 and the measurement error, it is preferable to use the filtration membrane 100 of about 13 mm or 25 mm. The pore size of the filtration membrane 100 can be selected according to the type of cells to be filtered, but it is 0.
It is preferable to use the filtration membrane 100 having a pore size of about 2 μm or 0.45 μm.
【００３８】濾過を行う検体の量は、検体に含まれる細
胞及び細胞以外の夾雑物の量によって決まるが、検体量
が多い程細胞の濃縮効率が増加するので、細胞が少ない
場合には濾過膜１００が目詰まりしない範囲で、できる
だけ多量の検体を濾過することが好ましい。The amount of the sample to be filtered is determined by the amount of cells contained in the sample and the amount of contaminants other than the cells, but the larger the amount of sample, the more the cell concentration efficiency increases. It is preferable to filter as much sample as possible within the range in which 100 does not become clogged.
【００３９】もし、検体の夾雑物の量が多くて十分な量
の濾過が不可能な場合には、孔径の大きな濾過膜１００
を上に載せてプレフィルタとして使用することも可能で
ある。If the amount of impurities in the sample is large and a sufficient amount cannot be filtered, the filtration membrane 100 having a large pore size is used.
It is also possible to put on and use it as a prefilter.
【００４０】濾過終了後は、検体の種類により細胞以外
のＡＴＰが検体中に含まれていたり、生物化学発光反応
を妨害する物質を含む場合もあるので、滅菌水、滅菌生
理食塩水、滅菌ｐＨ緩衝液等で濾過膜１００を洗うこと
が必要である。After the completion of filtration, ATP other than cells may be contained in the sample depending on the type of sample, or a substance that interferes with the biochemiluminescence reaction may be contained. Therefore, sterile water, sterile physiological saline solution, sterile pH may be used. It is necessary to wash the filtration membrane 100 with a buffer solution or the like.
【００４１】このようにして濾過を終了した濾過膜１０
０を滅菌したピンセット等で濾過器から取り外し、容器
２００の底面上に載せる。容器２００は濾過膜１００の
大きさに応じて濾過膜１００の入る大きさのものを使用
する。濾過膜１００を載せた容器２００は、容器ホルダ
１０を引き出して、容器保持台２６上に載せた後、容器
ホルダ１０の閉鎖部材、即ち、扉１４を押して、容器ホ
ルダ１０と共にハウジング２内に押し込む。The filtration membrane 10 which has been filtered in this way
0 is removed from the filter with sterilized tweezers and placed on the bottom of the container 200. As the container 200, one having a size into which the filtration membrane 100 can be inserted is used according to the size of the filtration membrane 100. In the container 200 on which the filtration membrane 100 is placed, the container holder 10 is pulled out and placed on the container holder 26, and then the closing member of the container holder 10, that is, the door 14 is pushed to be pushed into the housing 2 together with the container holder 10. .
【００４２】これにより、シャッタ部材１３０も時計方
向に回転し、光電子増倍管１０８の光検出先端部１０８
Ａは完全に開放されて、容器２００の開口部に対面する
こととなる。このとき、光電子増倍管１０８の光検出先
端部１０８Ａと容器２００内の液面との間の距離は、１
０〜１５ｍｍ、好ましくは１１〜１３ｍｍとされる。As a result, the shutter member 130 also rotates clockwise, and the photodetection tip 108 of the photomultiplier tube 108.
A is completely opened and faces the opening of the container 200. At this time, the distance between the photodetection tip 108A of the photomultiplier tube 108 and the liquid surface in the container 200 is 1
The length is 0 to 15 mm, preferably 11 to 13 mm.
【００４３】次いで、ハウジング２の中に設置された容
器上に具備された導入管１２２から抽出試薬１０４をポ
ンプ１２１により定量だけ容器２００内に注入し、必要
に応じて、モータ２０を駆動して容器２００を振盪し、
濾過膜１００表面に捕獲された細胞からＡＴＰを抽出す
る。Next, the extraction reagent 104 is injected into the container 200 in a fixed amount by the pump 121 from the introduction pipe 122 provided on the container installed in the housing 2, and the motor 20 is driven as necessary. Shake container 200,
ATP is extracted from the cells captured on the surface of the filtration membrane 100.
【００４４】抽出試薬１０４には、界面活性剤を使用し
た抽出試薬やエタノールを用いた抽出試薬等が使用でき
るが、できるだけ生物化学発光反応を阻害しないものが
好ましく、抽出時間は１０秒から６０秒程度が適当であ
る。抽出試薬１０４の注入量は、使用する濾過膜１００
の直径によるが、１３ｍｍの濾過膜１００の場合１００
μｌ程度、２５ｍｍの濾過膜１００の場合２００μｌ程
度が適当であるが、これにこだわる必要はない。但し、
濃縮効率を考えた場合には、抽出試薬量は必要最低限と
することが好ましい。As the extraction reagent 104, an extraction reagent using a surfactant, an extraction reagent using ethanol and the like can be used, but it is preferable to use one that does not interfere with the biochemiluminescence reaction as much as possible, and the extraction time is 10 to 60 seconds. The degree is appropriate. The injection amount of the extraction reagent 104 is the filtration membrane 100 to be used.
Depending on the diameter of the filter, 100 for a 100 mm filtration membrane 100
In the case of the filtration membrane 100 having a size of about μl and 25 mm, about 200 μl is suitable, but it is not necessary to pay attention to this. However,
Considering the concentration efficiency, the amount of extraction reagent is preferably set to the minimum necessary.
【００４５】ＡＴＰを抽出後、容器２００上に配置され
た導入管１２０から発光試薬１０７をポンプ１１８によ
り定量だけ容器内に注入し、モータ２０を駆動して容器
２００を回転（振盪）させながら生物化学発光反応を起
こさせる。これによって生じた発光は、光電子増倍管１
０８でその発光量を検出する。モータ２０による容器２
００の回転は、発光試薬を良く混合するためと発光を均
一に捕らえるための補助的なもので、好ましいものでは
あるが、必須ではない。After the ATP is extracted, the luminescent reagent 107 is injected into the container from the introduction tube 120 arranged on the container 200 in a fixed amount by the pump 118, and the motor 20 is driven to rotate (shake) the container 200 for living organisms. Causes a chemiluminescent reaction. The emitted light is generated by the photomultiplier tube 1
At 08, the amount of light emission is detected. Container 2 by motor 20
The rotation of 00 is an auxiliary for well mixing the luminescent reagent and for uniformly capturing the luminescence, and is preferable but not essential.
【００４６】ポンプ１２１とポンプ１１８はコンピュー
タ１１３でコントロールされる。光電子増倍管１０８の
出力は、アンプ１１１を通してコンピユータ１１３に送
られれ、一定時間積算される。積算時間は１０〜３０秒
程度が一般的である。発光量の積算値は、既知のＡＴＰ
標準試薬で測定した発光量の積算値と比較されて、ＡＴ
Ｐ量としてコンピユータ１１３により計算され、表示器
１１４及びプリンタ１１５に結果が出力される。The pump 121 and the pump 118 are controlled by the computer 113. The output of the photomultiplier tube 108 is sent to the computer 113 through the amplifier 111 and integrated for a certain period of time. The integrated time is generally about 10 to 30 seconds. The integrated value of the amount of light emission is the known ATP
AT is compared with the integrated value of the amount of luminescence measured with a standard reagent.
The amount of P is calculated by the computer 113, and the result is output to the display 114 and the printer 115.
【００４７】上述したように、発光試薬１０７と抽出試
薬１０４は、必ずしもポンプ１２１とポンプ１１８を用
いて注入する必要はなく、装置外部においてディスペン
サにより手操作で注入することもできる。As described above, the luminescent reagent 107 and the extraction reagent 104 do not necessarily have to be injected by using the pump 121 and the pump 118, but can be manually injected by a dispenser outside the apparatus.
【００４８】以下、本発明を実施例によって更に説明す
る。The present invention will be further described below with reference to examples.
【００４９】実施例１ 本発明に従って、酵母の測定を行った。検体は、S.cere
visiaeを酵母用完全培地を用いて培養し、ｐＨ７．０の
リン酸緩衝液で希釈して、２．７×１０-2個／ｍｌ〜
２．７×１０6 個／ｍｌになるように作製した。酵母数
の測定は、酵母完全培地による標準寒天平板法により行
った。Example 1 Yeast was measured according to the present invention. Sample is S.cere
Visiae is cultivated using a complete medium for yeast, diluted with phosphate buffer of pH 7.0 to give 2.7 × 10 -2 cells / ml
It was prepared so as to be 2.7 × 10 6 pieces / ml. The number of yeasts was measured by the standard agar plate method using a complete yeast medium.
【００５０】孔径０．４５μｍ、直径１３ｍｍのニトロ
セルロース濾過膜１００（アドバンテック社製Ａ０４５
Ａ０１３Ａ）を濾過器（日本ミリポア社製ＸＦ３００１
２００）にセットし、検体１０ｍｌを濾過した。濾過膜
１００を滅菌したリン酸緩衝液１０ｍｌで洗浄後、折り
畳むことなく、内径１４ｍｍ、外径１６ｍｍ、高さ８ｍ
ｍの硝子容器とされる容器２００の底面に滅菌したピン
セットで載置した後、この容器２００を本発明の装置の
容器保持台２６に設置した。容器２００を設置した後、
容器ホルダ１０を押して容器２００をハウジング２内に
装着した。コンピユータ１１３からの信号によりポンプ
１２１が作動し、抽出試薬１０４を容器に１００μｌ注
入した。抽出試薬１０４は陽イオン界面活性剤溶液を使
用した。注入後、容器をモータで３０秒間回転振盪し、
酵母からＡＴＰを抽出した。A nitrocellulose filtration membrane 100 having a pore diameter of 0.45 μm and a diameter of 13 mm (A045 manufactured by Advantech)
A013A) filter (XF3001 manufactured by Japan Millipore)
200), and 10 ml of the sample was filtered. After washing the filtration membrane 100 with 10 ml of sterilized phosphate buffer solution, the inner diameter is 14 mm, the outer diameter is 16 mm, and the height is 8 m without folding.
After being placed on the bottom surface of the container 200, which is a glass container of m, with sterilized tweezers, the container 200 was set on the container holder 26 of the apparatus of the present invention. After installing the container 200,
The container holder 10 was pushed to mount the container 200 in the housing 2. The pump 121 was activated by the signal from the computer 113, and 100 μl of the extraction reagent 104 was injected into the container. As the extraction reagent 104, a cationic surfactant solution was used. After injection, the container was shaken by a motor for 30 seconds,
ATP was extracted from yeast.
【００５１】更に、コンピユータ１１３からの信号によ
りポンプ１１８を作動させ、発光試薬１０７（東亜電波
工業社製、商品名「ＡＴＰＡ−１Ｌ１」）を容器に１０
０μｌ注入し、生物化学発光反応を起こさせた。そし
て、光電子増倍管１０８にて発光を検出し、発光量を３
０秒間積算し、予め測定しておいた１（ｎｍｏｌ／リッ
トル）ＡＴＰ標準液の発光量の積算値と比較して酵母の
ＡＴＰ量をコンピユータ１１３にて計算し、表示器１１
４及びプリンタ１１５にて出力した。このとき、光電子
増倍管１０８の光検出先端部１０８Ａと容器２００内の
液面との間の距離は、約１３ｍｍであった。Further, the pump 118 is operated by a signal from the computer 113, and the luminescent reagent 107 (trade name "ATPA-1L1" manufactured by Toa Denpa Kogyo KK) is placed in a container.
0 μl was injected to cause a biochemiluminescent reaction. Then, light emission is detected by the photomultiplier tube 108, and the light emission amount is set to 3
The integration was performed for 0 seconds, and the ATP amount of yeast was calculated by the computer 113 by comparing with the integrated value of the luminescence amount of 1 (nmol / liter) ATP standard solution measured in advance, and the indicator 11
4 and the printer 115. At this time, the distance between the photodetection tip portion 108A of the photomultiplier tube 108 and the liquid surface in the container 200 was about 13 mm.
【００５２】図５に本発明に基づき測定した酵母数とＡ
ＴＰ量の関係を示す。比較のため、検体を濾過しないで
測定した結果を併記した。FIG. 5 shows the number of yeasts and A measured according to the present invention.
The relationship of the amount of TP is shown. For comparison, the results of measurement without filtering the sample are also shown.
【００５３】この結果から、０．１個／ｍｌ〜１０3 個
／ｍｌの酵母の測定が可能であり、濾過を行わないで直
接検体を測定した場合の１００倍の検出感度を有するこ
とが分かった。この結果は、検体１０ｍｌを濾過し１０
０μｌの抽出試薬で抽出したことによる１００倍の濃縮
の理論的な結果と良く一致している。From these results, it was found that it is possible to measure 0.1 / ml to 10 3 / ml of yeast, and the detection sensitivity is 100 times that of the case where the sample is directly measured without filtration. It was This result is obtained by filtering 10 ml of sample and
This is in good agreement with the theoretical result of 100-fold concentration by extraction with 0 μl of extraction reagent.
【００５４】実施例２ 本発明に従って、乳酸菌の測定を行った。検体は、市販
発酵乳をｐＨ７．０のリン酸緩衝液で希釈し、４．４個
／ｍｌ〜４．４×１０6 個／ｍｌの乳酸菌懸濁液を作製
した。乳酸菌数は、ＢＰＣ加プレートカウント寒天培地
（栄研化学社製）を用いて測定した。Example 2 Lactic acid bacteria were measured according to the present invention. As the sample, commercially available fermented milk was diluted with a phosphate buffer of pH 7.0 to prepare a lactic acid bacterium suspension of 4.4 cells / ml to 4.4 × 10 6 cells / ml. The number of lactic acid bacteria was measured using BPC-added plate count agar medium (manufactured by Eiken Chemical Co., Ltd.).
【００５５】孔径０．４５μｍ、直径１３ｍｍのニトロ
セルロース濾過膜１００（アドバンテック社製Ａ０４５
Ａ０１３Ａ）を濾過器（日本ミリポア社製ＸＦ３００１
２００）にセットし、検体１０ｍｌを濾過した。濾過膜
１００を滅菌したリン酸緩衝液１０ｍｌで洗浄後、折り
畳むことなく、内径１４ｍｍ、外径１６ｍｍ、高さ８ｍ
ｍの硝子容器２００の底面に滅菌したピンセットで載せ
て、実施例１と同様な操作でＡＴＰ量を測定した。A nitrocellulose filtration membrane 100 having a pore diameter of 0.45 μm and a diameter of 13 mm (A045 manufactured by Advantech)
A013A) filter (XF3001 manufactured by Japan Millipore)
200), and 10 ml of the sample was filtered. After washing the filtration membrane 100 with 10 ml of sterilized phosphate buffer solution, the inner diameter is 14 mm, the outer diameter is 16 mm, and the height is 8 m without folding.
A glass container 200 of m was placed on the bottom surface with sterilized tweezers, and the ATP amount was measured by the same operation as in Example 1.
【００５６】図６に本発明に基づき測定した乳酸菌数と
ＡＴＰ量の関係を示す。比較のため、検体を濾過しない
で測定した結果を併記した。FIG. 6 shows the relationship between the number of lactic acid bacteria and the amount of ATP measured according to the present invention. For comparison, the results of measurement without filtering the sample are also shown.
【００５７】この結果から、１０個／ｍｌ〜１０5 個／
ｍｌの乳酸菌の測定が可能であり、濾過を行わないで直
接検体を測定した場合の１００倍の検出感度を有するこ
とが分かった。この結果は、検体１０ｍｌを濾過し１０
０μｌの抽出試薬で抽出したことによる１００倍の濃縮
の理論的な結果と良く一致している。From this result, 10 pieces / ml to 10 5 pieces / ml
It was found that it is possible to measure ml of lactic acid bacterium and has a detection sensitivity 100 times that of a direct measurement of a sample without filtration. This result is obtained by filtering 10 ml of sample and
This is in good agreement with the theoretical result of 100-fold concentration by extraction with 0 μl of extraction reagent.
【００５８】[0058]
【発明の効果】以上詳しく説明したように、本発明に係
る細胞のＡＴＰ量測定方法及び装置は、検体中の細胞を
捕獲した濾過膜を一方が開口した皿状の容器内に載置
し、そして、この容器の開口部と対面するようにして光
電子増倍管１０８を設置して細胞のＡＴＰ量を測定する
構成とされるので、細胞捕獲後の濾過膜を折り畳んだ
り、濾過膜上の細胞を洗い流す等の面倒な操作を必要と
せず、操作性が向上するのみならず、濾過膜の外部から
の汚染の危険性をも解決し、高精度にて細胞のＡＴＰ量
を測定することができる。又、細胞捕獲後の濾過膜から
ＡＴＰを抽出するための抽出試薬の量を最小限度とし、
それによって、濃縮効率の低下を回避することができ、
更には、ガラス管壁などを介することなく直接、試料の
発光量を測定することができ、従って、高精度にて細胞
のＡＴＰ量を測定することができる。本発明は、これら
の効果により、細胞の測定下限を大幅に向上でき、従っ
て、食品衛生、バイオ等の微生物検査に大きく貢献する
ことができる。As described in detail above, the method and apparatus for measuring the ATP amount of cells according to the present invention places the filtration membrane, which captures the cells in the sample, in a dish-shaped container having one opening, Since the photomultiplier tube 108 is installed so as to face the opening of this container and the ATP amount of the cells is measured, the filter membrane after cell capture can be folded or the cells on the filter membrane can be folded. It does not require tedious operations such as washing away the cells, not only improves operability, but also solves the risk of contamination from the outside of the filtration membrane and enables highly accurate measurement of the ATP amount of cells. . In addition, the amount of extraction reagent for extracting ATP from the filtration membrane after cell capture is minimized,
As a result, it is possible to avoid a decrease in concentration efficiency,
Furthermore, the amount of luminescence of the sample can be measured directly without going through the glass tube wall or the like, and therefore the amount of ATP of cells can be measured with high accuracy. Due to these effects, the present invention can greatly improve the lower limit of measurement of cells, and thus can greatly contribute to food hygiene, biotechnology, and other microbial tests.
【図１】本発明に係る細胞のＡＴＰ量測定装置の一実施
例の全体構成図である。FIG. 1 is an overall configuration diagram of an embodiment of a cell ATP amount measuring device according to the present invention.
【図２】本発明に係る細胞のＡＴＰ量測定装置の断面図
である。FIG. 2 is a cross-sectional view of a cell ATP amount measuring device according to the present invention.
【図３】図２の線III-III にとった断面図である。3 is a cross-sectional view taken along the line III-III in FIG.
【図４】図２の線IV-IV にとった断面図である。本発明
に係る細胞のＡＴＰ量測定装置の一実施例の全体構成図
である。4 is a cross-sectional view taken along line IV-IV of FIG. 1 is an overall configuration diagram of an embodiment of a cell ATP amount measuring device according to the present invention.
【図５】酵母数とＡＴＰ量の関係図である。FIG. 5 is a relationship diagram between the number of yeasts and the amount of ATP.
【図６】乳酸菌数とＡＴＰ量の関係図である。FIG. 6 is a relationship diagram between the number of lactic acid bacteria and the amount of ATP.
【図７】従来の細胞のＡＴＰ量測定装置の全体構成図で
ある。FIG. 7 is an overall configuration diagram of a conventional cell ATP amount measuring device.
１ 細胞のＡＴＰ量測定装置 ２ ハウジング １０ 容器ホルダ ２０ 駆動手段（電動モータ） ２６ 容器保持台 １００ 濾過膜 １０８ 光電子増倍管 １２０、１２２ 薬液導入管 １３０ シャッタ部材 ２００ 容器 1 Cell ATP Amount Measuring Device 2 Housing 10 Container Holder 20 Driving Means (Electric Motor) 26 Container Holding Base 100 Filtration Membrane 108 Photomultiplier Tubes 120, 122 Chemical Solution Introducing Tube 130 Shutter Member 200 Container
Claims (5)
こと、（ｂ）前記濾過膜を、上方が開口した皿状の容器
内に載置すること、（ｃ）前記容器内に抽出試薬を注入
し、前記濾過膜に捕獲された細胞のアデノシン３リン酸
を抽出すること、（ｄ）前記容器内に発光試薬を注入
し、生物化学発光反応を起こすこと、（ｅ）前記工程
（ｄ）により発生した光を、前記容器の開口部に対面し
て配置した光電子増倍管で検出すること、を特徴とした
細胞のＡＴＰ量測定方法。1. (a) Capturing cells in a sample on a filtration membrane, (b) placing the filtration membrane in a dish-shaped container having an open top, (c) in the container Injecting an extraction reagent to extract adenosine triphosphate of the cells captured by the filtration membrane; (d) injecting a luminescent reagent into the container to cause a biochemiluminescent reaction; (e) the step A method for measuring the amount of ATP in cells, characterized in that the light generated in (d) is detected by a photomultiplier tube arranged facing the opening of the container.
折り畳むことなく前記容器内に載置される請求項１の細
胞のＡＴＰ量測定方法。2. The filtration membrane capturing the cells in the specimen,
The method for measuring an ATP amount of a cell according to claim 1, which is placed in the container without being folded.
と、検体中の細胞を捕獲した濾過膜を収容することので
きる上方が開口した皿状の容器を担持し、そして前記ハ
ウジングの正面開口部を介して前記ハウジングに対して
出入り自在とされる容器ホルダと、前記容器ホルダに支
持され、そして前記容器を振盪するための駆動手段と、
前記容器の開口部に対面して前記ハウジングに取り付け
られた光電子増倍管とを有することを特徴とする細胞の
ＡＴＰ量測定装置。3. A housing having an opening formed on a front surface thereof, and a dish-shaped container having an upper opening capable of accommodating a filtration membrane trapping cells in a sample, and the front opening portion of the housing. A container holder that can be moved in and out of the housing through a drive means supported by the container holder and for shaking the container;
An ATP amount measuring device for cells, comprising: a photomultiplier tube attached to the housing so as to face the opening of the container.
出された時、前記光電子増倍管の前記容器に対面した光
検出端部を遮蔽するためのシャッタ部材を有することを
特徴とする請求項３の細胞のＡＴＰ量測定装置。4. The shutter member for blocking the photodetection end of the photomultiplier tube facing the container when the container holder is pulled out from the housing. ATP amount measuring device for cells.
入するための薬液導入手段を有することを特徴とする請
求項４の細胞のＡＴＰ量測定装置。5. The cell ATP amount measuring device according to claim 4, wherein said container has a liquid medicine introducing means for injecting an extraction reagent and a luminescent reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27897493A JPH07110301A (en) | 1993-10-12 | 1993-10-12 | Method and apparatus for measuring ATP amount of cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27897493A JPH07110301A (en) | 1993-10-12 | 1993-10-12 | Method and apparatus for measuring ATP amount of cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH07110301A true JPH07110301A (en) | 1995-04-25 |
Family
ID=17604668
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP27897493A Pending JPH07110301A (en) | 1993-10-12 | 1993-10-12 | Method and apparatus for measuring ATP amount of cells |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07110301A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006059359A1 (en) * | 2004-11-30 | 2006-06-08 | Dml Co., Ltd. | Measuring kit for microbe in liquid sample, and relevant measuring method and measuring apparatus |
| JP2008111680A (en) * | 2006-10-27 | 2008-05-15 | Dkk Toa Corp | ATP amount measuring method and apparatus |
| CN102016538A (en) * | 2008-02-25 | 2011-04-13 | 比兹帕克(澳大利亚)有限公司 | Punch sampling apparatus and method |
| WO2014046183A1 (en) | 2012-09-20 | 2014-03-27 | 東亜ディーケーケー株式会社 | Quantification method, quantification device, and quantification kit |
-
1993
- 1993-10-12 JP JP27897493A patent/JPH07110301A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006059359A1 (en) * | 2004-11-30 | 2006-06-08 | Dml Co., Ltd. | Measuring kit for microbe in liquid sample, and relevant measuring method and measuring apparatus |
| JP2008111680A (en) * | 2006-10-27 | 2008-05-15 | Dkk Toa Corp | ATP amount measuring method and apparatus |
| CN102016538A (en) * | 2008-02-25 | 2011-04-13 | 比兹帕克(澳大利亚)有限公司 | Punch sampling apparatus and method |
| JP2011513698A (en) * | 2008-02-25 | 2011-04-28 | ビズパック （オーストラリア） プロプライエタリー リミテッド | Apparatus and method for punch sampling |
| WO2014046183A1 (en) | 2012-09-20 | 2014-03-27 | 東亜ディーケーケー株式会社 | Quantification method, quantification device, and quantification kit |
| JP2014062785A (en) * | 2012-09-20 | 2014-04-10 | Dkk Toa Corp | Quantitative determination method, quantitative determination apparatus and kit for quantitative determination |
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