JP3734131B2

JP3734131B2 - Microbe count measuring apparatus and microbe count measuring method

Info

Publication number: JP3734131B2
Application number: JP05219199A
Authority: JP
Inventors: 竜一八浪
Original assignee: Panasonic Corp; Matsushita Electric Industrial Co Ltd
Current assignee: Panasonic Corp; Panasonic Holdings Corp
Priority date: 1999-03-01
Filing date: 1999-03-01
Publication date: 2006-01-11
Anticipated expiration: 2019-03-01
Also published as: JP2000245434A

Description

【０００１】
【発明の属する技術分野】
本発明は溶液中の微生物数を測定するための微生物数測定装置および微生物数測定方法に関するものである。
【０００２】
【従来の技術】
従来、溶液中の微生物数を測定する方法として特開昭５７−５０６５２に記載されたもの等の多数の技術が知られている。
【０００３】
しかし、従来の技術による微生物数の測定方法は、試料液に専用の薬剤、例えば酵素や色素を投入して生化学反応を起こさせ、その反応経過または結果を蛍光や発光によって測定するものであり、測定感度は比較的高いが微生物分野及び生化学分野に関する専門知識が必要であったり、また専用で高価な大型の測定装置が必要となり、さらには専任者による作業が必要となる等、とても一般的かつ簡易に微生物数を測定することができるものではなかった。
【０００４】
そこで、特開昭５９−９１９００に記載されたものをはじめとする、物理的手段のみを使い、薬剤を一切用いないで、小型で、試料系に組み込んでの自動測定が可能な、簡易な微生物数検出装置が提案されたが、微生物数が１０の８乗ｃｅｌｌｓ／ｍｌ（１ｍｌ中に微生物数が１億個）以上にならないと検出できないためその応用範囲に著しい制限が加えられていた。
【０００５】
【発明が解決しようとする課題】
このように、従来の技術による微生物数測定装置で測定感度を上げるためには、何らかの薬剤の使用や、専用の測定装置，専門知識を持った専任者による操作が必要であった。また薬剤を使用しない簡易型の装置では、専任者を必要とせず測定が可能になるが、微生物数が非常に多くないと測定が難しく、低感度の測定器しか得られないし、微生物を移動させて局部的に濃度を上げて感度を向上させたくても簡易でメンテナンスフリーな手段がないという問題があった。
【０００６】
そこで、本発明者は、本出願に先立って、被測定液中の微生物を誘電泳動して集め、微生物数の濃度が上がった段階で測定して検量し、被測定液中の微生物数を測定する新方式の微生物数測定装置を提案した。これは電界が集中した個所に電界集中部への引力で微生物を集めるものであるが、電界集中部への引力を生じさせる印加電圧の周波数帯は微生物の種類や試料の導電率により大小様々で、微生物によっては電界集中部への引力をあまり利用できないものもあった。従って、測定する微生物の種類によっては、測定できたりできなかったりして、どのような微生物に対しても簡単に微生物数が測定できる実用性の高い測定装置を測定することは困難であった。
【０００７】
そこでこれらの問題を解決するため本発明は、薬剤や特別な装置を必要とすることなく、簡単な構造で且つ高感度で、どのような微生物を含有する試料であっても迅速に微生物数を自動で測定できるメンテナンスフリーの微生物数測定装置を提供することを目的とする。
【０００８】
さらに本発明は、どのような微生物を含有する試料であっても高感度で、迅速に微生物数を測定できる微生物数測定方法を提供することを目的とする。
【０００９】
【課題を解決するための手段】
上記の目的を達成するために本発明の微生物数測定装置は、微生物含有の液体を内部に導入することができ、内壁面に円筒形状の電極が設けられた測定セルと、前記測定セル内に設けられ前記微生物含有の液体に浸漬された円盤状の頭部をもつ板状の泳動電極と、前記泳動電極と前記円筒形状の電極に接続され前記微生物を誘電泳動するため交流電圧を印加する泳動電源回路と、前記交流電圧の周波数を変化させることができ、前記微生物に対して電界集中部からの斥力を生じさせることができる所定の周波数を選択する周波数変化手段と、前記微生物の数を測定する測定部と、
前記周波数変化手段が選択した周波数の交流電圧が前記泳動電極に印加されたとき、前記測定部に微生物の数の測定を行わせる制御手段を備え、前記泳動電極は、前記交流電圧の印加時にその頭部の円周方向端面に電界集中部が形成されるように配置され、前記測定部は、前記微生物に対する電界集中部からの斥力によって、弱電界部分となる前記泳動電極の表面に集まった前記微生物の数を測定することを特徴とする。
【００１０】
これにより、薬剤や特別な装置を必要とすることなく、簡単な構造で且つ高感度で、どのような微生物を含有する試料であっても迅速に微生物数を自動で測定できる。
【００１１】
さらに本発明の微生物数測定方法は、内壁面に円筒形状の電極を備えた測定セル内に微生物含有の液体を導入し、該測定セル内の液体に円盤状の頭部をもつ板状の泳動電極を浸漬するとともに、該泳動電極と前記円筒形状の電極間に交流電圧を印加し、前記微生物を誘電泳動により収集して前記微生物の数を測定する微生物数測定方法であって、前記泳動電極は、前記交流電圧の印加時にその頭部の円周方向端面に電界集中部が形成されるように配置され、前記泳動電極と前記円筒形状の電極間に形成された前記電界集中部と前記微生物との間に前記電界集中部への引力を生じさせる周波数の交流電圧を印加して、前記電界集中部分に前記微生物を収集し、さらに前記電界集中部からの斥力を生じさせる周波数の交流電圧を印加して、弱電界部分となる前記泳動電極の表面に微生物を収集し、収集された微生物の数を測定して前記液体中の微生物数を算出することを特徴とする。
【００１２】
これにより、どのような微生物を含有する試料であっても高感度で、迅速に微生物数を測定できる。
【００１３】
【発明の実施の形態】
請求項１に記載された発明は、微生物含有の液体を内部に導入することができ、内壁面に円筒形状の電極が設けられた測定セルと、前記測定セル内に設けられ前記微生物含有の液体に浸漬された円盤状の頭部をもつ板状の泳動電極と、前記泳動電極と前記円筒形状の電極に接続され前記微生物を誘電泳動するため交流電圧を印加する泳動電源回路と、前記交流電圧の周波数を変化させることができ、前記微生物に対して電界集中部からの斥力を生じさせることができる所定の周波数を選択する周波数変化手段と、前記微生物の数を測定する測定部と、前記周波数変化手段が選択した周波数の交流電圧が前記泳動電極に印加されたとき、前記測定部に微生物の数の測定を行わせる制御手段を備え、前記泳動電極は、前記交流電圧の印加時にその頭部の円周方向端面に電界集中部が形成されるように配置され、前記測定部は、前記微生物に対する電界集中部からの斥力によって、弱電界部分となる前記泳動電極の表面に集まった前記微生物の数を測定することを特徴とする微生物数測定装置であるから、泳動電極と測定セル内の電界集中点との間で電界集中部からの斥力を作用させることになり、大量の試料の微生物を泳動電極の表面に集めることができ、簡単な構造で且つ高感度で、どのような微生物を含有する試料であっても迅速に微生物数を自動で測定できる。
【００１４】
請求項２に記載された発明は、微生物含有の液体を内部に導入することができ、内壁面に円筒形状の電気的に接地された電極が設けられた測定セルと、前記測定セル内に設けられ前記微生物含有の液体に浸漬された円盤状の頭部をもつ板状の泳動電極と、一出力端が前記泳動電極に接続されるとともに他出力端が接地され、前記微生物を誘電泳動するための交流電圧を印加する泳動電源回路と、前記交流電圧の周波数を変化させることができ、前記微生物に対して電界集中部からの斥力を生じさせることができる所定の周波数を選択する周波数変化手段と、前記微生物の数を測定する測定部と、前記周波数変化手段が選択した周波数の交流電圧が前記泳動電極に印加されたとき、前記測定部に微生物の数の測定を行わせる制御手段を備え、前記泳動電極は、前記交流電圧の印加時にその頭部の円周方向端面に電界集中部が形成されるように配置され、前記測定部は、前記微生物に対する電界集中部からの斥力によって、弱電界部分となる前記泳動電極の表面に集まった前記微生物の数を測定することを特徴とする微生物数測定装置であるから、一方の出力端を接地するので簡単な構成となる。
【００１５】
請求項３に記載された発明は、前記測定部が光源と受光部を備え、前記光源から前記微生物に照射された光の反射強度により微生物の数を求めることを特徴とする請求項１または２記載の微生物数測定装置であるから、低濃度の微生物でも簡単且つ高感度に微生物数を測定できる。
【００１６】
請求項４に記載された発明は、前記周波数変化手段が前記電界集中部への引力を生じる所定の周波数を選択することができることを特徴とする請求項１〜３のいずれかに記載の微生物数測定装置であるから、一旦電界集中点に鎖状に集めた微生物の塊を再び電界集中部からの斥力で泳動電極の表面に集めることになり、より多くの微生物を集中させることができる。
【００１７】
請求項５に記載された発明は、内壁面に円筒形状の電極を備えた測定セル内に微生物含有の液体を導入し、該測定セル内の液体に円盤状の頭部をもつ板状の泳動電極を浸漬するとともに、該泳動電極と前記円筒形状の電極間に交流電圧を印加し、前記微生物を誘電泳動により収集して前記微生物の数を測定する微生物数測定方法であって、前記泳動電極は、前記交流電圧の印加時にその頭部の円周方向端面に電界集中部が形成されるように配置され、前記泳動電極と前記円筒形状の電極間に形成された前記電界集中部と前記微生物との間に前記電界集中部からの斥力を生じさせる周波数の交流電圧を印加し、弱電界部分となる前記泳動電極の表面に前記微生物を収集し、収集された微生物の数を測定して前記液体中の微生物数を算出することを特徴とする微生物数測定方法であるから、泳動電極と測定セル内の電界集中点との間で電界集中部からの斥力を作用させることになり、大量の試料の微生物を泳動電極の表面に集めることができ、どのような微生物を含有する試料であっても高感度で、迅速に微生物数を測定できる。
【００１８】
請求項６に記載された発明は、内壁面に円筒形状の電極を備えた測定セル内に微生物含有の液体を導入し、該測定セル内の液体に円盤状の頭部をもつ板状の泳動電極を浸漬するとともに、該泳動電極と前記円筒形状の電極間に交流電圧を印加し、前記微生物を誘電泳動により収集して前記微生物の数を測定する微生物数測定方法であって、前記泳動電極は、前記交流電圧の印加時にその頭部の円周方向端面に電界集中部が形成されるように配置され、前記泳動電極と前記円筒形状の電極間に形成された前記電界集中部と前記微生物との間に前記電界集中部への引力を生じさせる周波数の交流電圧を印加して、前記電界集中部分に前記微生物を収集し、さらに前記電界集中部からの斥力を生じさせる周波数の交流電圧を印加して、弱電界部分となる前記泳動電極の表面に微生物を収集し、収集された微生物の数を測定して前記液体中の微生物数を算出することを特徴とする微生物数測定方法であるから、一旦電界集中点に鎖状に集めた微生物の塊を再び電界集中部からの斥力で泳動電極の表面に集めることになり、より多くの微生物を集中させることができる。
【００１９】
以下、本発明の実施の形態について、図１〜図６と後述する（数１）〜（数３）を用いて説明する。
【００２０】
（実施の形態１）
本発明の一実施の形態である微生物数測定装置について図面を参照しながら詳細に説明する。図１は本発明の実施の形態１における微生物数測定装置の全体構成図、図２（ａ）は本発明の実施の形態１における電極で電界集中部への引力を作用させた状態図、図２（ｂ）は本発明の実施の形態１における電極で電界集中部への引力を作用させた後電界集中部からの斥力を作用させる状態図、図３（ａ）は電界集中部からの斥力が生じる周波数帯が狭い誘電泳動力−周波数図、図３（ｂ）は電界集中部からの斥力が生じる周波数帯が広い誘電泳動力−周波数図、図４は反射光量と微生物数の関係図である。
【００２１】
図１において、１は測定セル、１ａは測定セル内壁面上に形成された円筒形状の電極、２は円盤状の頭部をもつ板状の泳動電極である。測定セル１の電極１ａは金属等の導電性の材料から構成される。３は電磁弁、４は試料系配管、５は測定セル１の電極１ａと泳動電極２に接続され、測定セル１内に導入された微生物を誘電泳動するために交流電圧を印加する泳動電源回路、６は泳動電極２の頭部の円周方向端面に形成される電界集中部への引力を生じさせるための周波数と、逆にこの電界集中部からの斥力を生じさせるための周波数を変化選択できる周波数変化手段、７は電界集中部からの斥力により弱電界部分となる泳動電極２の頭部の表面に集まった微生物の数を測定する測定部である。この周波数と誘電泳動力の関係については後述する。周波数変化手段６は少なくとも電界集中部からの斥力を発生させる周波数を選択でき、さらに電界集中部への引力を発生させる周波数も選択できるものである。周波数変化手段６としてはインバーターを構成するハードまたはソフトの回路等がある。７ａは測定部７が測定するための光を発する光源、７ｂは光源７ａからの光を受光する受光部である。８は、周波数変化手段６が引力を引き起こす周波数を交流電圧に与えるとともに、同じく電界集中部からの斥力を引き起す周波数も交流電圧に与えたとき、測定部７に微生物数を測定するように指示する制御手段である。これらのうち少なくとも測定部７と制御手段８はマイクロプロセッサ等で構成される。周波数変化手段６が電界集中部への引力を発生する周波数を選択しない場合には、制御手段８は電界集中部への引力を起こさせることはなく、電界集中部からの斥力を起こす周波数を交流電圧に与えるものである。９は測定結果を表示するＬＣＤ等の表示手段、１０は微生物である。
【００２２】
実施の形態１における電極1ａは円筒形状をしており、表面は滑らかである。また泳動電極２は板状であり、その板厚は約０．１ｍｍである。０．１ｍｍという板厚は後述するギャップの間隙１ｃｍに比較して非常に小さいため泳動電極２は事実上厚みを無視できる２次元的な広がりのみをもった電極と考えることが出来る。そこで、泳動電極２の円周方向端面を境界端と、そして泳動電極２の円形の電極広がり面を表面と、以下称することにする。また泳動電極２の表面は鏡面になっており、光を良く反射できるようになっている。
【００２３】
電極１ａ及び泳動電極２は極端に抵抗が高くない限りどのような材料から作成されてもよいが、液体中での使用、特に本実施の形態１のように水中で使用されることを想定すると、なるべくイオン化傾向が低い金属が望ましい。誘電泳動時には電極間に強い電界が生じるため、印加する電圧の周波数と水中の電解質濃度によっては電気分解が生じることがあるからである。電気分解が生じるとイオン化傾向の大きな金属から構成された電極では、電極の溶解が生じ電極形状の崩れや極端な場合には電極の破断等を生じてしまう。そこで、本実施の形態１では泳動電極２の主材料として白金を使用している。また、内壁面の電極１ａの材料は同様の理由からイオン化傾向が低い金属であるのが望ましいが、場合によってはアルミで本体を形成し、表面に白金等のメッキを施すのがコスト等の面から適当である。
【００２４】
なお、本実施の形態１では、泳動電源回路５の一出力端を泳動電極２に接続し、他出力端を測定セル１内の電極１ａに接続している。しかし、この電極１ａを接地するとともに、泳動電源回路５の他出力端も接地することにより、回路構成を簡単化でき、コストを下げることができる。
【００２５】
さて、本実施の形態１の電極１ａと泳動電極２の境界端のギャップとなる間隙は１ｃｍに設定している。誘電泳動の効果を大きくするためには電極１ａと泳動電極２を近づけて電界の変化を大きくするほうが良いが、あまりに近づけすぎると導電率の高い試料を測定する際に電気分解が発生しやすくなるため望ましくない。そこで本実施の形態１においては、電極１ａと泳動電極２の境界端のギャップとなる間隙は１ｃｍに設定しているが、この値は試料の導電率に対応して０．１〜１０ｃｍの範囲で適宜調節されることが望ましい。
【００２６】
なお、ここで本発明において検出対象としている微生物について説明する。本発明で言う微生物とは一般に細菌、真菌、放線菌、リケッチア、マイコプラズマ、ウイルスとして分類されている、いわゆる微生物学の対象となっている微生物のほかに、原生動物や原虫のうちの小型のもの、生物体の幼生、分離または培養した動植物細胞、***、血球、核酸、蛋白質等も含む広い意味での生体または生体由来の微粒子である。また本発明では、測定対象として液体中に存在する微生物を想定している。
【００２７】
さらに本発明で起こる微生物の誘電泳動について詳細に説明する。さらなる説明が必要であれば文献Ｊ．ｔｈｅｏｒ．Ｂｉｏｌ（１９７２）ｖｏｌ．３７，１−１３を参照されたい。
【００２８】
さて、液中の電極間に微生物に対して引力を作用させる周波数帯域の交流電圧を印加すると、これによって発生する交流電界の作用で、測定セル１内の微生物はその誘電的な性質によって最も電場が強くかつ不均一な部分、すなわち電界集中部に泳動される。なお、ここで交流電圧というのは、正弦波のほか、ほぼ一定の周期で流れの向きを変える電圧のことであり、かつ両方向の電流の平均値が等しいものである。
【００２９】
この時に微生物の誘電体微粒子としての双極子モーメントをμとすると、誘電泳動力Ｆは電場Ｅとの間に（数１）の関係を持つ。
【００３０】
【数１】

さらに、微生物の細胞質の比誘電率をε₂、微生物を含んでいる液体の比誘電率をε₁、微生物を球体と見なしたときの半径をａ、円周率をπとすると、誘電泳動力Ｆは（数２）のように書き換えることができる。
【００３１】
【数２】

（数２）は誘電泳動力Ｆが電位勾配、媒質と誘電体微粒子としての微生物の比誘電率の差などの影響を受けることを示している。ここで、実施の形態１では誘電泳動のために交流電圧を印加するので、比誘電率εは周波数依存性をもち、たとえばε₁に対して交流に対する応答を考慮したものをε₁’とすると、ε₁’は（数３）のようにε₁に虚数単位ｊと媒質の電気伝導率σ₁と角周波数ωを用いた項が付加された形になる。
【００３２】
【数３】

ε₂に関しても同様であり、交流に対する応答を考慮したものをε₂’とすると、ε₂’はε₂に虚数単位ｊと媒質の電気伝導率σ₂と角周波数ωを用いた項が付加されたものとなり、Ｆはε₁をε₁’、ε₂をε₂’に置換したもので表現される。
【００３３】
このように、誘電泳動力Ｆは媒質の電気伝導率の項も含むことになる。
【００３４】
さて、この媒質の電気伝導率が具体的に誘電泳動力に対してどのように作用するのかを説明する。今、媒質を水とし、微生物としての一般的な比誘電率等の値を用いて誘電泳動力と周波数の関係をグラフに表したのが図３（ａ）（ｂ）である。図３（ａ）（ｂ）から明らかなように誘電泳動はある周波数領域では電界集中部への引力として、そしてある周波数領域では電界集中部からの斥力として作用する。図３（ａ）は液の導電率が低い場合を示し、電界集中部への引力が１０ＫＨｚ〜１０ＭＨｚという広い周波数帯で発生するものである。逆に図３（ｂ）は液の導電率が高いすなわちイオン濃度が高い場合で、電界集中部への引力が５００ＫＨｚ〜１０ＭＨｚという狭い周波数帯で発生するものである。いずれも１０ＫＨｚ以下では常に比較的大きな電界集中部からの斥力を発生し、１０ＭＨｚ以上ではどのような微生物に対しても安定して電界集中部からの斥力を発生することが分かる。このように変化の大きい引力よりも安定した電界集中部からの斥力を利用すれば、微生物を安定して操作することができる。
【００３５】
この電界集中部への引力を作用させた状態を示すのが図２（ａ）である。微生物１０が泳動電極２の境界端に放射状に鎖を延ばしたような形に並んでいる。これは泳動電極２の境界端に電界が集中し、微生物１０はこの電気力線に沿って並ぶからである。微生物１０の鎖はいわばこのとき形成されている電気力線を示している。ただ、この放射状に延びた鎖状の状態は均一な密度に凝集したものでなく、光学的な測定にはあまり適当ではない。
【００３６】
次に、電界集中部からの斥力を作用させたときの状態を示すのが図２（ｂ）である。この状態は、光学的な測定のためには、鎖状となった電界集中部への引力の場合より測定が容易な凝集状態となっている。ただ、このとき必ずしも先に電界集中部への引力で微生物を集めた後に電界集中部からの斥力を作用させなければならないわけではないが、まず電界集中部への引力で微生物を収集した後に電界集中部からの斥力を作用させる方が一旦境界端に鎖状に集めた微生物１０を電界集中部からの斥力で泳動電極２の表面に移動させることになり、効率的に多くの微生物１０を集中させることができる。電界集中部からの斥力だけでは泳動電極２の付近の微生物１０のみが凝集できるだけであるが、電界集中部への引力まで加えると測定セル１内の広い範囲の微生物１０まで凝集させることができる。ただ、この場合、電界集中部への引力を利用できる微生物１０でなければならないという制約があり、利用できる場合が限られる。このように電界集中部からの斥力を作用させると、ギャップ内の微生物１０は泳動電極２の表面上に塊状に凝集し、測定部７により光学的な測定が精度よく行えるものである。しかも、微生物１０の種類によらずどのような微生物１０に対しても同じように操作でき、微生物数を測定することができる。
【００３７】
ここで、微生物１０を含む試料は、微生物１０と同時にイオンを含んでいることが多く、一般に導電率は高い。したがって、このような試料に対して引力を作用させるためには先に説明したように印加する周波数を慎重に選ばねばならない。しかしながら、斥力は試料の導電率にあまり依存することが無いので安定した操作ができる。これは導電率が高い一般的な微生物含有試料に対して何ら前処理することなく測定を行えることを意味している。
【００３８】
続いて、測定部７について説明する。実施の形態１の光源７ａは、近赤外域に発光波長をもつレーザ−ダイオードである。受光部７ｂは、泳動電極２の頭部表面上に塊状になった微生物１０が反射する光源７ａからの光を捉えるためのもので、実施の形態１ではフォトダイオード使用されている。そして、光源７ａにはレンズや偏光板等から構成される光源光学系が設けられ、受光部７ｂにもレンズや偏光板等から構成される受光光学系が設けられている。これらは微生物によって反射される光を効率よく受光部７ｂに導入するためのものである。測定部７は図示しないマイクロプロセッサ等から構成され、受光部７ｂで検出された光の強度信号とその時間変化を測定、算出する。図４によれば、微生物１０の数が増加すると初期光量から反射光量は漸減していく。従って受光部７ｂで所定時間経過時点の反射光量を測定してやれば泳動電極２上の微生物数が測定、算出でき、被測定液中の微生物数（濃度）が測定できることになる。この経過時間における微生物数の測定では時間がかかるため、反射光量の時間変化（勾配）を測定することで微生物数を算出することも可能である。これによって測定時間の短縮が可能となる。
【００３９】
次に、被測定液を測定セル１内に導入してから、微生物の移動、測定、洗浄する一連の流れを説明する。初期状態では試料系配管４と測定セル４内を遮断するための電磁弁３は開放状態にされており、試料配管４内の液体は測定セル１内を自由に通過できる。所定のタイミングで測定動作に入ると、制御手段８は電磁弁３を閉鎖し、測定セル１を試料系から遮断して閉鎖系を構成する。その後、制御手段８は測定セル１内の流動が収まると予想される所定時間が経過すると、測定部７に光源７ａの点灯を指示する。光源７ａが点灯すると、受光部７ｂが測定初期状態の光量を測定し、測定部７がメモリ内に格納する。
【００４０】
次いで、制御手段８は周波数変化手段６と泳動電源回路５に誘電泳動をさせるための交流電圧を電極１ａと泳動電極２の間に印加するように指令する。実施の形態１においては、微生物１０に電界集中部からの斥力を発生する前に予め電界集中部への引力を発生させるための選択と条件の入力が周波数変化手段６に対してなされており、周波数変化手段６は電界集中部への引力を発生する周波数、例えば１ＭＨｚの周波数を泳動電源回路５から供給される交流電圧に与える。微生物１０に対して電界集中部である泳動電極２の境界端への引力が発生する。この交流電圧を所定の時間印加することで、図２（ａ）で示すように、泳動電極２付近の微生物１０が泳動電極２の周囲に集められる。この時間が経過すると再び制御手段８からの信号で、周波数変化手段６は電界集中部からの斥力を発生する周波数、例えば１ＫＨｚの周波数を泳動電源回路５から供給される交流電圧に与える。すると、図２（ｂ）の泳動電極２の周りに鎖状に集まった微生物１０は斥力により泳動電極２の頭部表面上に塊状になって凝集する。
【００４１】
このとき制御手段８は測定部７に測定を開始するように指令する。測定部７は光源７ａを発光させ、受光部７ｂに反射光量を測定させる。この反射光量と測定時間を測定部７はメモリに格納し、所定時間刻みで発光と受光を繰り返す。測定部７は格納されたこれらのデータから、反射光量の時間変化率を計算し、予め記憶されている微生物数と時間変化のテーブルでこの時間変化率を比較し、微生物数（濃度）を割り出す。あるいは反射光量の時間変化のテーブルから微生物数（濃度）を割り出しても良い。この測定結果を制御手段８は表示手段９に表示させ、泳動電源回路５と周波数変化手段６に交流電圧の印加を停止させる。次いで制御手段８は測定セル１内の洗浄のため電磁弁３を開放させる。電磁弁３が開放されると、試料配管４から液体が流れ込み、測定セル１内の被測定液も排出され、一連の測定動作が終了する。
【００４２】
以上の測定動作は、第１ステップとして電界集中部への引力を作用させ、第２ステップで電界集中部からの斥力を作用させたが、電界集中部への引力を作用させないで電界集中部からの斥力を作用させるだけで測定するのでもよいのはいうまでもない。この場合、微生物１０の種類や試料の導電率によらず同一の周波数で測定できるので、測定部７にデフォルトでこの測定を行うようにしておくのが適当である。
【００４３】
このように、本実施の形態１においては、反射光量を測定することで微生物数（濃度）の測定が可能になる。また電界集中部からの斥力を用いて小さな泳動電極の頭部表面上に微生物を集中させることができ、低濃度の微生物であっても測定することができる。さらに、電界集中部からの斥力を利用することで、微生物の種類や条件によらず所定の周波数で測定が可能になるものである。
【００４４】
（実施の形態２）
次に本発明の実施の形態２の微生物数測定装置及び微生物数測定方法の説明をする。図５は散乱光量と微生物数の関係図、図６は本発明の実施の形態２における微生物数測定装置の全体構成図である。本実施の形態２の微生物数測定装置はその受光部が微生物による散乱光を測定するものである点以外、基本的に実施の形態１の微生物数測定装置と同一であるから、詳細な説明は実施の形態１の説明に譲って省略する。
【００４５】
実施の形態２の受光部７ｂは光源７ａからの光軸と鋭角をなす方向に受光部７ｂが置かれている。この受光部７ｂは光源７ａから照射された光が微生物１０によって散乱された光量を測定するものである。図５に示すように、散乱光量は微生物数とともに増加し、微生物数が多くなると頭打ちになるものである。従って微生物数が少ない状態でも比較的鋭敏な応答をするから、反射光量を測定するより比較的短時間で測定することが可能なものである。
【００４６】
【発明の効果】
本発明によれば、薬剤や特別な装置を必要とすることなく、簡単な構造で且つ高感度で、どのような微生物を含有する試料であっても迅速に微生物数を自動で測定できるメンテナンスフリーの微生物数測定装置を提供することができ、また、どのような微生物を含有する試料であっても高感度で、迅速に微生物数を測定できる微生物数測定方法を提供することができる。
【図面の簡単な説明】
【図１】本発明の実施の形態１における微生物数測定装置の全体構成図
【図２】（ａ）本発明の実施の形態１における電極で電界集中部への引力を作用させた状態図
（ｂ）本発明の実施の形態１における電極で電界集中部への引力を作用させた後電界集中部からの斥力を作用させる状態図
【図３】（ａ）電界集中部からの斥力が生じる周波数帯が狭い誘電泳動力ー周波数図
（ｂ）電界集中部からの斥力が生じる周波数帯が広い誘電泳動力ー周波数図
【図４】反射光量と微生物数の関係図
【図５】散乱光量と微生物数の関係図
【図６】本発明の実施の形態２における微生物数測定装置の全体構成図
【符号の説明】
１測定セル
１ａ電極
２泳動電極
３電磁弁
４試料系配管
５泳動電源回路
６周波数変化手段
７測定部
７ａ光源
７ｂ受光部
８制御手段
９表示手段
１０微生物[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a microorganism count measuring apparatus and a microorganism count measuring method for measuring the number of microorganisms in a solution.
[0002]
[Prior art]
Conventionally, many techniques such as those described in JP-A-57-50652 are known as methods for measuring the number of microorganisms in a solution.
[0003]
However, the conventional method for measuring the number of microorganisms is a method in which a dedicated chemical such as an enzyme or a dye is introduced into a sample solution to cause a biochemical reaction, and the reaction process or result is measured by fluorescence or luminescence. The measurement sensitivity is relatively high, but it requires specialized knowledge in the microbiological and biochemical fields, requires a dedicated and expensive large-sized measuring device, and requires work by a dedicated person. It was not possible to measure the number of microorganisms simply and easily.
[0004]
Therefore, simple microorganisms that use only physical means, such as those described in JP-A-59-91900, do not use any drugs, are small, and can be automatically measured in a sample system. Although a number detection device has been proposed, since the number of microorganisms cannot be detected unless the number of microorganisms is 10 8 cells / ml (100 million microorganisms in 1 ml) or more, the application range has been significantly limited.
[0005]
[Problems to be solved by the invention]
As described above, in order to increase the measurement sensitivity with the conventional microorganism count measuring apparatus, it is necessary to use some kind of medicine, a dedicated measuring apparatus, and an operation by a dedicated person having specialized knowledge. In addition, simple devices that do not use drugs can be measured without the need for dedicated personnel.However, if the number of microorganisms is not very large, measurement is difficult, and only low-sensitivity measuring instruments can be obtained. However, there is a problem that there is no simple and maintenance-free means even if it is desired to increase the concentration locally to improve the sensitivity.
[0006]
Therefore, prior to the present application, the present inventor collects the microorganisms in the solution to be measured by dielectrophoresis, measures and calibrates the microorganisms when the concentration of the microorganisms increases, and measures the number of microorganisms in the solution to be measured. A new method for measuring the number of microorganisms was proposed. In this method, microorganisms are collected by the attractive force to the electric field concentration part where the electric field is concentrated, but the frequency band of the applied voltage that generates the attractive force to the electric field concentration part varies depending on the type of microorganism and the conductivity of the sample. Some microorganisms cannot utilize the attractive force to the electric field concentration part. Therefore, depending on the type of microorganism to be measured, it was difficult to measure, and it was difficult to measure a highly practical measuring apparatus that can easily measure the number of microorganisms for any microorganism.
[0007]
Therefore, in order to solve these problems, the present invention can quickly count the number of microorganisms in any sample containing microorganisms with a simple structure and high sensitivity without the need for drugs or special equipment. An object of the present invention is to provide a maintenance-free microbial count measuring apparatus capable of automatic measurement.
[0008]
Furthermore, an object of the present invention is to provide a method for measuring the number of microorganisms which can rapidly measure the number of microorganisms with high sensitivity regardless of the sample containing any microorganism.
[0009]
[Means for Solving the Problems]
In order to achieve the above object, the microorganism count measuring apparatus of the present invention can introduce a microorganism-containing liquid into the inner wall surface. Cylindrical A measuring cell provided with electrodes, and immersed in the microorganism-containing liquid provided in the measuring cell Plate-shaped with a disk-shaped head Electrophoretic electrode, electrophoretic electrode and Cylindrical electrode Connected to Said An electrophoretic power supply circuit for applying an alternating voltage for dielectrophoresis of microorganisms, and a predetermined frequency capable of changing the frequency of the alternating voltage and generating repulsive force from the electric field concentration part to the microorganisms are selected. Frequency changing means to Said A measuring unit for measuring the number of microorganisms;
When the alternating voltage of the frequency selected by the frequency changing unit is applied to the electrophoresis electrode, the control unit includes a control unit that causes the measurement unit to measure the number of microorganisms, The electrophoretic electrode is disposed so that an electric field concentration portion is formed on a circumferential end surface of the head when the alternating voltage is applied, By repulsion from the electric field concentration part against microorganisms , Become weak electric field part On the surface of the electrophoresis electrode Gathered said Microbe of It is characterized by measuring numbers.
[0010]
Thus, the number of microorganisms can be automatically and quickly measured for any sample containing microorganisms with a simple structure and high sensitivity without the need for drugs or special devices.
[0011]
Furthermore, the method for measuring the number of microorganisms of the present invention is provided on the inner wall surface. Cylindrical A microorganism-containing liquid is introduced into a measurement cell equipped with electrodes, and the liquid in the measurement cell Plate-shaped with a disk-shaped head While immersing the electrophoresis electrode, the electrophoresis electrode and the Cylindrical An alternating voltage is applied between the electrodes, and the microorganisms are collected by dielectrophoresis. Said Microbe of A method for measuring the number of microorganisms for measuring a number, The migration electrode is arranged such that an electric field concentration portion is formed on the circumferential end face of the head when the alternating voltage is applied, The electrophoresis electrode and The cylindrical shape Formed between the electrodes Said Electric field concentration Part And between the microorganisms Said Apply an alternating voltage with a frequency that causes attraction to the electric field concentration part, Said In the electric field concentration part Said Collect microorganisms and further Said Apply an alternating voltage with a frequency that generates repulsive force from the electric field concentration part, The surface of the electrophoresis electrode Collect microorganisms and collect microorganisms of The number of microorganisms in the liquid is calculated by measuring the number.
[0012]
Thereby, even if it is a sample containing what kind of microorganisms, the number of microorganisms can be measured rapidly with high sensitivity.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
The invention described in claim 1 can introduce a microorganism-containing liquid into the interior, Cylindrical A measuring cell provided with electrodes, and immersed in the microorganism-containing liquid provided in the measuring cell Plate-shaped with a disk-shaped head Electrophoretic electrode, electrophoretic electrode and Cylindrical electrode Connected to Said An electrophoretic power supply circuit for applying an alternating voltage for dielectrophoresis of microorganisms, and a predetermined frequency capable of changing the frequency of the alternating voltage and generating repulsive force from the electric field concentration part to the microorganisms are selected. Frequency changing means to Said A measurement unit that measures the number of microorganisms, and a control unit that causes the measurement unit to measure the number of microorganisms when an alternating voltage of a frequency selected by the frequency changing unit is applied to the migration electrode, The electrophoretic electrode is disposed so that an electric field concentration portion is formed on a circumferential end face of the head when the alternating voltage is applied, By repulsion from the electric field concentration part against microorganisms , Become weak electric field part On the surface of the electrophoresis electrode Gathered said Microbe of Since the microbe count measuring device is characterized by measuring the number, a repulsive force from the electric field concentration part acts between the migration electrode and the electric field concentration point in the measurement cell, and a large number of sample microorganisms are removed. It can be collected on the surface of the electrophoresis electrode, has a simple structure and high sensitivity, and can automatically measure the number of microorganisms quickly even for any sample containing microorganisms.
[0014]
The invention described in claim 2 can introduce a microorganism-containing liquid into the inside. Wall In Cylindrical Electrically grounded electrode A measuring cell provided with A plate-like electrophoresis electrode having a disk-like head provided in the measurement cell and immersed in the microorganism-containing liquid; One output end is connected to the migration electrode and the other output end is grounded, Said An electrophoretic power supply circuit for applying an alternating voltage for dielectrophoresis of microorganisms, and a predetermined frequency capable of changing the frequency of the alternating voltage and generating repulsive force from the electric field concentration part to the microorganisms. Frequency changing means to select; Said When an alternating voltage having a frequency selected by the frequency change means and a measuring unit that measures the number of microorganisms is applied to the migration electrode, Number Equipped with control means to make measurements, The electrophoretic electrode is disposed so that an electric field concentration portion is formed on a circumferential end surface of the head when the alternating voltage is applied, By repulsion from the electric field concentration part against microorganisms , Become weak electric field part On the surface of the electrophoresis electrode Gathered said Microbe of Since the microorganism count measuring apparatus is characterized by measuring the number, since one output terminal is grounded, the configuration is simple.
[0015]
The invention described in claim 3 Said The measuring unit includes a light source and a light receiving unit. Said Microorganisms by the reflection intensity of the light irradiated to the microorganisms of The microorganism count measuring apparatus according to

claim

1 or 2, characterized in that the number of microorganisms can be easily and highly sensitively measured even with a low concentration of microorganisms.
[0016]
The invention described in claim 4 Said Frequency changing means Said The microorganism frequency measuring device according to any one of claims 1 to 3, wherein a predetermined frequency that generates an attractive force to the electric field concentration portion can be selected, and is once collected in a chain form at the electric field concentration point The mass of microorganisms is again collected on the surface of the electrophoresis electrode by the repulsive force from the electric field concentration part, and more microorganisms can be concentrated.
[0017]
The invention described in claim 5 is provided on the inner wall surface. Cylindrical A microorganism-containing liquid is introduced into a measurement cell equipped with electrodes, and the liquid in the measurement cell Plate-shaped with a disk-shaped head While immersing the electrophoresis electrode, the electrophoresis electrode and the Cylindrical An alternating voltage is applied between the electrodes, and the microorganisms are collected by dielectrophoresis. Said Microbe of A method for measuring the number of microorganisms for measuring a number, The migration electrode is arranged such that an electric field concentration portion is formed on the circumferential end face of the head when the alternating voltage is applied, The electrophoresis electrode and the Cylindrical Formed between the electrodes Said Electric field concentration Part And between the microorganisms Said Apply an alternating voltage with a frequency that generates repulsive force from the electric field concentration part, and weak electric field part The surface of the electrophoresis electrode In Said Collecting microorganisms, collected microorganisms of Since the number of microorganisms is calculated by measuring the number of microorganisms in the liquid, a repulsive force from the electric field concentration portion is applied between the migration electrode and the electric field concentration point in the measurement cell. That is, a large amount of sample microorganisms can be collected on the surface of the electrophoresis electrode, and any number of microorganism-containing samples can be rapidly measured with high sensitivity.
[0018]
The invention described in claim 6 is provided on the inner wall surface. Cylindrical A microorganism-containing liquid is introduced into a measurement cell equipped with electrodes, and the liquid in the measurement cell Plate-shaped with a disk-shaped head While immersing the electrophoresis electrode, the electrophoresis electrode and the Cylindrical An alternating voltage is applied between the electrodes, and the microorganisms are collected by dielectrophoresis. Said Microbe of A method for measuring the number of microorganisms for measuring a number, The migration electrode is arranged such that an electric field concentration portion is formed on the circumferential end face of the head when the alternating voltage is applied, The electrophoresis electrode and The cylindrical shape Formed between the electrodes Said Electric field concentration Part And between the microorganisms Said Apply an alternating voltage with a frequency that causes attraction to the electric field concentration part, Said In the electric field concentration part Said Collect microorganisms and further Said Apply an alternating voltage with a frequency that generates repulsive force from the electric field concentration part, The surface of the electrophoresis electrode Collect microorganisms and collect microorganisms of Since the method of measuring the number of microorganisms is characterized in that the number of microorganisms in the liquid is calculated by measuring the number of the microorganisms, the lump of microorganisms once collected in a chain form at the electric field concentration point is migrated again by the repulsive force from the electric field concentration part. It will be collected on the surface of the electrode, and more microorganisms can be concentrated.
[0019]
Hereinafter, embodiments of the present invention will be described with reference to FIGS. 1 to 6 and (Equation 1) to (Equation 3) described later.
[0020]
(Embodiment 1)
A microorganism count measuring apparatus according to an embodiment of the present invention will be described in detail with reference to the drawings. FIG. 1 is an overall configuration diagram of a microorganism count measuring apparatus according to Embodiment 1 of the present invention, and FIG. 2A is a state diagram in which an attractive force is applied to an electric field concentration portion by an electrode according to Embodiment 1 of the present invention. 2 (b) is a state diagram in which a repulsive force is applied from the electric field concentration portion after an attractive force is applied to the electric field concentration portion by the electrode in Embodiment 1 of the present invention, and FIG. 3 (a) is a repulsive force from the electric field concentration portion. Fig. 3B is a diagram showing the relationship between the amount of reflected light and the number of microorganisms. Fig. 3B is a diagram showing the relationship between the amount of reflected light and the number of microorganisms. is there.
[0021]
In FIG. 1, 1 is a measurement cell, 1a is a cylindrical electrode formed on the inner wall surface of the measurement cell, and 2 is a plate-shaped migration electrode having a disk-shaped head. The electrode 1a of the measurement cell 1 is made of a conductive material such as metal. 3 is a solenoid valve, 4 is a sample system pipe, 5 is connected to the electrode 1a and the migration electrode 2 of the measurement cell 1, and an electrophoresis power supply circuit for applying an alternating voltage to perform dielectrophoresis of microorganisms introduced into the

measurement cell

1 , 6 are selected to change the frequency for generating an attractive force to the electric field concentration portion formed on the circumferential end face of the head of the electrophoresis electrode 2 and conversely the frequency for generating a repulsive force from the electric field concentration portion. A frequency changing means 7 is a measuring unit for measuring the number of microorganisms gathered on the surface of the head of the migration electrode 2 which becomes a weak electric field part due to repulsive force from the electric field concentration part. The relationship between this frequency and the dielectrophoretic force will be described later. The frequency changing means 6 can select at least a frequency that generates a repulsive force from the electric field concentration portion, and can also select a frequency that generates an attractive force to the electric field concentration portion. As the frequency changing means 6, there is a hardware or software circuit constituting an inverter. Reference numeral 7a denotes a light source that emits light for measurement by the measuring unit 7, and 7b denotes a light receiving unit that receives light from the light source 7a. 8 indicates that the frequency changing means 6 gives the frequency causing the attractive force to the AC voltage, and also instructs the measuring unit 7 to measure the number of microorganisms when the frequency causing the repulsive force from the electric field concentration portion is also given to the AC voltage. Control means. Among these, at least the measurement unit 7 and the control means 8 are constituted by a microprocessor or the like. When the frequency changing means 6 does not select the frequency that generates the attractive force to the electric field concentration part, the control means 8 does not cause the attractive force to the electric field concentration part, and the frequency causing the repulsive force from the electric field concentration part is changed to AC. It is given to voltage. Reference numeral 9 is a display means such as an LCD for displaying the measurement result, and 10 is a microorganism.
[0022]
The electrode 1a in Embodiment 1 has a cylindrical shape, and the surface is smooth. The migration electrode 2 is plate-shaped and has a thickness of about 0.1 mm. Since the plate thickness of 0.1 mm is much smaller than the gap 1 cm described later, the migration electrode 2 can be considered as an electrode having only a two-dimensional expansion in which the thickness can be virtually ignored. Therefore, the circumferential end surface of the migration electrode 2 is referred to as a boundary end, and the circular electrode spreading surface of the migration electrode 2 is referred to as a surface hereinafter. Further, the surface of the electrophoresis electrode 2 is a mirror surface so that light can be well reflected.
[0023]
The electrode 1a and the migration electrode 2 may be made of any material as long as the resistance is not extremely high. However, assuming that the electrode 1a and the migration electrode 2 are used in a liquid, particularly in water as in the first embodiment. A metal having a low ionization tendency is desirable. This is because a strong electric field is generated between the electrodes during dielectrophoresis, and electrolysis may occur depending on the frequency of the applied voltage and the concentration of the electrolyte in water. When electrolysis occurs, an electrode composed of a metal having a high ionization tendency causes dissolution of the electrode, resulting in a collapse of the electrode shape or, in an extreme case, breakage of the electrode. Therefore, in the first embodiment, platinum is used as the main material of the migration electrode 2. Further, the material of the electrode 1a on the inner wall surface is preferably a metal having a low ionization tendency for the same reason. However, in some cases, it is costly to form a main body with aluminum and to plate the surface with platinum or the like. Is appropriate.
[0024]
In the first embodiment, one output end of the migration power supply circuit 5 is connected to the migration electrode 2, and the other output end is connected to the electrode 1 a in the measurement cell 1. However, by grounding the electrode 1a and grounding the other output terminal of the electrophoretic power supply circuit 5, the circuit configuration can be simplified and the cost can be reduced.
[0025]
Now, the gap that becomes the gap at the boundary between the electrode 1a and the migration electrode 2 of the first embodiment is set to 1 cm. In order to increase the dielectrophoretic effect, it is better to bring the electrode 1a and the electrophoretic electrode 2 closer to increase the electric field change. However, if the electrode 1a and the electrophoretic electrode 2 are too close to each other, electrolysis tends to occur when measuring a sample with high conductivity. Therefore, it is not desirable. Therefore, in the first embodiment, the gap serving as the gap between the electrode 1a and the migration electrode 2 is set to 1 cm, but this value is in the range of 0.1 to 10 cm corresponding to the conductivity of the sample. It is desirable to adjust appropriately.
[0026]
Here, the microorganisms to be detected in the present invention will be described. The microorganisms referred to in the present invention are generally classified as bacteria, fungi, actinomycetes, rickettsia, mycoplasma, viruses, so-called microbiological microorganisms, and small protozoa and protozoa. These are living organisms or living organism-derived microparticles in a broad sense including larvae of living organisms, isolated or cultured animal and plant cells, sperm, blood cells, nucleic acids, proteins, and the like. In the present invention, a microorganism present in a liquid is assumed as a measurement target.
[0027]
Furthermore, the dielectrophoresis of microorganisms that occurs in the present invention will be described in detail. If further explanation is required, reference J.I. theor. Biol (1972) vol. 37, 1-13.
[0028]
Now, when an AC voltage in a frequency band that causes attraction to microorganisms is applied between the electrodes in the liquid, the microorganisms in the measurement cell 1 are most affected by the dielectric properties due to the action of the AC electric field generated thereby. Is migrated to a strong and non-uniform portion, that is, an electric field concentration portion. Here, the AC voltage is not only a sine wave but also a voltage that changes the direction of the flow at a substantially constant period, and the average value of the currents in both directions is equal.
[0029]
At this time, if the dipole moment as the dielectric fine particles of the microorganism is μ, the dielectrophoretic force F has a relationship of (Equation 1) with the electric field E.
[0030]
[Expression 1]

Furthermore, the relative permittivity of the cytoplasm of the microorganism is expressed as ε ₂ , The relative dielectric constant of the liquid containing microorganisms ε ₁ The dielectrophoretic force F can be rewritten as (Equation 2), where a is the radius when the microorganism is regarded as a sphere, and π is the circumference.
[0031]
[Expression 2]

(Equation 2) indicates that the dielectrophoretic force F is affected by a potential gradient, a difference in relative permittivity between microorganisms as a medium and dielectric fine particles, and the like. Here, in the first embodiment, since an AC voltage is applied for dielectrophoresis, the relative permittivity ε has frequency dependence, for example, ε ₁ That takes into account the response to AC ₁ 'Then ε ₁ 'Is ε like (Equation 3) ₁ Imaginary unit j and electrical conductivity σ of the medium ₁ And a term using the angular frequency ω.
[0032]
[Equation 3]

ε ₂ The same applies to ε, and ε ₂ 'Then ε ₂ 'Is ε ₂ Imaginary unit j and electrical conductivity σ of the medium ₂ And the term using the angular frequency ω are added, and F is ε ₁ Ε ₁ ', Ε ₂ Ε ₂ It is expressed by replacing with '.
[0033]
As described above, the dielectrophoretic force F includes a term of electric conductivity of the medium.
[0034]
Now, how the electrical conductivity of this medium specifically acts on the dielectrophoretic force will be described. FIGS. 3A and 3B are graphs showing the relationship between the dielectrophoretic force and frequency using water as a medium and values such as a general relative dielectric constant as a microorganism. As is apparent from FIGS. 3A and 3B, dielectrophoresis acts as an attractive force to the electric field concentration portion in a certain frequency region and as a repulsive force from the electric field concentration portion in a certain frequency region. FIG. 3A shows a case where the electrical conductivity of the liquid is low, and the attractive force to the electric field concentration part is generated in a wide frequency band of 10 KHz to 10 MHz. On the other hand, FIG. 3B shows a case where the conductivity of the liquid is high, that is, the ion concentration is high, and the attractive force to the electric field concentration portion is generated in a narrow frequency band of 500 KHz to 10 MHz. It can be seen that repulsive force from a relatively large electric field concentration portion is always generated at 10 KHz or lower, and repulsive force from the electric field concentration portion is stably generated against any microorganism at 10 MHz or higher. Thus, if the repulsive force from the electric field concentration part which is more stable than the attractive force having a large change is used, the microorganism can be stably operated.
[0035]
FIG. 2A shows a state where an attractive force is applied to the electric field concentration portion. The microorganisms 10 are arranged in such a manner that chains are radially extended at the boundary end of the migration electrode 2. This is because the electric field concentrates on the boundary edge of the migration electrode 2 and the microorganisms 10 are lined up along the lines of electric force. The chain of the microorganism 10 indicates electric lines of force formed at this time. However, this radially extending chain state is not agglomerated to a uniform density and is not very suitable for optical measurement.
[0036]
Next, FIG. 2B shows a state when a repulsive force from the electric field concentration portion is applied. This state is an agglomerated state that is easier to measure than the case of the attractive force to the chained electric field concentration portion for optical measurement. However, at this time, it is not always necessary to apply the repulsive force from the electric field concentration part after collecting the microorganisms first by the attractive force to the electric field concentration part. When the repulsive force from the concentrated portion is applied, the microorganisms 10 once collected in a chain form at the boundary end are moved to the surface of the migration electrode 2 by the repulsive force from the electric field concentrated portion, and many microorganisms 10 are efficiently concentrated. Can be made. Only the microorganisms 10 in the vicinity of the migration electrode 2 can be aggregated only by the repulsive force from the electric field concentration part. However, if an attractive force is applied to the electric field concentration part, the microorganisms 10 in a wide range in the measurement cell 1 can be aggregated. However, in this case, there is a restriction that the microorganism 10 must be able to use the attractive force to the electric field concentration part, and the case where it can be used is limited. When the repulsive force from the electric field concentration portion is applied in this way, the microorganisms 10 in the gap are aggregated in a lump on the surface of the migration electrode 2, and the measurement unit 7 can perform optical measurement with high accuracy. In addition, any microorganism 10 can be operated in the same manner regardless of the type of microorganism 10, and the number of microorganisms can be measured.
[0037]
Here, the sample containing the microorganism 10 often contains ions at the same time as the microorganism 10, and generally has high conductivity. Therefore, in order to apply an attractive force to such a sample, the frequency to be applied must be carefully selected as described above. However, since the repulsive force does not depend much on the conductivity of the sample, a stable operation can be performed. This means that measurement can be performed without any pretreatment on a general microorganism-containing sample having high conductivity.
[0038]
Next, the measurement unit 7 will be described. The light source 7a of Embodiment 1 is a laser-diode having an emission wavelength in the near infrared region. The light-receiving unit 7b is for capturing light from the light source 7a reflected by the microorganisms 10 in the shape of a lump on the surface of the head of the migration electrode 2. In the first embodiment, a photodiode is used. The light source 7a is provided with a light source optical system including a lens and a polarizing plate, and the light receiving unit 7b is also provided with a light receiving optical system including a lens and a polarizing plate. These are for efficiently introducing the light reflected by the microorganisms into the light receiving portion 7b. The measuring unit 7 is composed of a microprocessor (not shown) and the like, and measures and calculates the intensity signal of light detected by the light receiving unit 7b and its change over time. According to FIG. 4, the amount of reflected light gradually decreases from the initial amount of light as the number of microorganisms 10 increases. Accordingly, if the amount of reflected light at the time when a predetermined time elapses is measured by the light receiving unit 7b, the number of microorganisms on the electrophoresis electrode 2 can be measured and calculated, and the number of microorganisms (concentration) in the liquid to be measured can be measured. Since the measurement of the number of microorganisms during this elapsed time takes time, it is possible to calculate the number of microorganisms by measuring the temporal change (gradient) in the amount of reflected light. This makes it possible to shorten the measurement time.
[0039]
Next, a series of flow for introducing, measuring, and washing microorganisms after introducing the liquid to be measured into the measurement cell 1 will be described. In the initial state, the solenoid valve 3 for shutting off the sample system pipe 4 and the measurement cell 4 is opened, and the liquid in the sample pipe 4 can freely pass through the measurement cell 1. When the measurement operation is started at a predetermined timing, the control means 8 closes the electromagnetic valve 3 and shuts off the measurement cell 1 from the sample system to constitute a closed system. Thereafter, the control means 8 instructs the measuring unit 7 to turn on the light source 7a when a predetermined time expected to stop the flow in the measuring cell 1 has elapsed. When the light source 7a is turned on, the light receiving unit 7b measures the light amount in the initial measurement state, and the measuring unit 7 stores the light in the memory.
[0040]
Next, the control means 8 instructs the frequency changing means 6 and the migration power supply circuit 5 to apply an AC voltage between the electrode 1 a and the migration electrode 2 for causing dielectrophoresis. In the first embodiment, before the repulsive force from the electric field concentrating portion is generated in the microorganism 10, selection and condition input for generating the attractive force to the electric field concentrating portion are made to the frequency changing means 6 in advance. The frequency changing means 6 gives the AC voltage supplied from the electrophoretic power supply circuit 5 a frequency that generates an attractive force to the electric field concentration portion, for example, a frequency of 1 MHz. An attractive force to the boundary end of the migration electrode 2 that is an electric field concentration portion is generated with respect to the microorganism 10. By applying the AC voltage for a predetermined time, the microorganisms 10 near the migration electrode 2 are collected around the migration electrode 2 as shown in FIG. When this time elapses, the frequency changing means 6 again gives a frequency for generating a repulsive force from the electric field concentrating portion, for example, a frequency of 1 KHz to the AC voltage supplied from the electrophoretic power supply circuit 5 by a signal from the control means 8. Then, the microorganisms 10 gathered in a chain form around the migration electrode 2 in FIG. 2B aggregate and aggregate on the head surface of the migration electrode 2 due to repulsion.
[0041]
At this time, the control means 8 instructs the measurement unit 7 to start measurement. The measuring unit 7 causes the light source 7a to emit light and causes the light receiving unit 7b to measure the amount of reflected light. The measurement unit 7 stores the reflected light amount and the measurement time in a memory, and repeats light emission and light reception at predetermined time intervals. The measurement unit 7 calculates the time change rate of the amount of reflected light from these stored data, compares the time change rate with the number of microorganisms stored in advance and the time change table, and calculates the number of microorganisms (concentration). . Alternatively, the number of microorganisms (concentration) may be calculated from a table of temporal changes in the amount of reflected light. The control unit 8 displays the measurement result on the display unit 9 and stops the application of the AC voltage to the electrophoresis power supply circuit 5 and the frequency changing unit 6. Next, the control means 8 opens the electromagnetic valve 3 for cleaning the measurement cell 1. When the electromagnetic valve 3 is opened, the liquid flows from the sample pipe 4, the liquid to be measured in the measurement cell 1 is also discharged, and a series of measurement operations ends.
[0042]
In the above measurement operation, an attractive force is applied to the electric field concentrating portion as the first step and a repulsive force is applied from the electric field concentrating portion in the second step, but from the electric field concentrating portion without applying the attractive force to the electric field concentrating portion. Needless to say, it may be measured simply by applying the repulsive force. In this case, since measurement can be performed at the same frequency regardless of the type of microorganism 10 and the conductivity of the sample, it is appropriate that the measurement unit 7 perform this measurement by default.
[0043]
Thus, in Embodiment 1, the number of microorganisms (concentration) can be measured by measuring the amount of reflected light. Moreover, microorganisms can be concentrated on the head surface of a small electrophoresis electrode by using repulsive force from the electric field concentration part, and even a low concentration microorganism can be measured. Furthermore, by using the repulsive force from the electric field concentration part, measurement can be performed at a predetermined frequency regardless of the type and condition of the microorganism.
[0044]
(Embodiment 2)
Next, a microorganism count measuring apparatus and a microorganism count measuring method according to Embodiment 2 of the present invention will be described. FIG. 5 is a diagram showing the relationship between the amount of scattered light and the number of microorganisms, and FIG. 6 is an overall configuration diagram of the microorganism count measuring apparatus according to Embodiment 2 of the present invention. The microorganism count measuring apparatus according to the second embodiment is basically the same as the microorganism count measuring apparatus according to the first embodiment except that its light receiving unit measures scattered light from microorganisms. The description is omitted because it is described in the first embodiment.
[0045]
In the light receiving unit 7b of the second embodiment, the light receiving unit 7b is placed in a direction that forms an acute angle with the optical axis from the light source 7a. The light receiving unit 7b measures the amount of light scattered by the microorganism 10 from the light source 7a. As shown in FIG. 5, the amount of scattered light increases with the number of microorganisms, and reaches a peak when the number of microorganisms increases. Therefore, since it responds relatively sensitively even in a state where the number of microorganisms is small, it can be measured in a relatively short time rather than measuring the amount of reflected light.
[0046]
【The invention's effect】
According to the present invention, a maintenance-free system that can automatically measure the number of microorganisms quickly with a simple structure, high sensitivity, and any type of microorganisms, without the need for chemicals or special equipment. In addition, it is possible to provide a method for measuring the number of microorganisms capable of quickly measuring the number of microorganisms with high sensitivity, regardless of the sample containing any microorganism.
[Brief description of the drawings]
FIG. 1 is an overall configuration diagram of a microorganism count measuring apparatus according to Embodiment 1 of the present invention.
2A is a state diagram in which an attractive force is applied to an electric field concentrating portion with an electrode according to Embodiment 1 of the present invention. FIG.
(B) State diagram in which repulsive force from the electric field concentration portion is applied after the attractive force is applied to the electric field concentration portion with the electrode in the first embodiment of the present invention.
FIG. 3A is a diagram showing a dielectrophoretic force-frequency diagram in which a frequency band in which repulsive force is generated from an electric field concentration portion is narrow.
(B) Dielectrophoretic force-frequency diagram with a wide frequency band in which repulsive force is generated from the electric field concentration part
[Fig. 4] Relationship between the amount of reflected light and the number of microorganisms
[Figure 5] Relationship between the amount of scattered light and the number of microorganisms
FIG. 6 is an overall configuration diagram of a microorganism count measuring apparatus according to Embodiment 2 of the present invention.
[Explanation of symbols]
1 Measurement cell
1a electrode
2 Electrophoresis electrode
3 Solenoid valve
4 Sample piping
5 Electrophoresis power circuit
6 Frequency change means
7 Measurement section
7a Light source
7b Light receiver
8 Control means
9 Display means
10 Microorganisms

Claims

微生物含有の液体を内部に導入することができ、内壁面に円筒形状の電極が設けられた測定セルと、
前記測定セル内に設けられ前記微生物含有の液体に浸漬された円盤状の頭部をもつ板状の泳動電極と、
前記泳動電極と前記円筒形状の電極に接続され前記微生物を誘電泳動するため交流電圧を印加する泳動電源回路と、
前記交流電圧の周波数を変化させることができ、前記微生物に対して電界集中部からの斥力を生じさせることができる所定の周波数を選択する周波数変化手段と、
前記微生物の数を測定する測定部と、
前記周波数変化手段が選択した周波数の交流電圧が前記泳動電極に印加されたとき、前記測定部に微生物の数の測定を行わせる制御手段を備え、
前記泳動電極は、前記交流電圧の印加時にその頭部の円周方向端面に電界集中部が形成されるように配置され、
前記測定部は、前記微生物に対する電界集中部からの斥力によって、弱電界部分となる前記泳動電極の表面に集まった前記微生物の数を測定することを特徴とする微生物数測定装置。A liquid containing microorganisms can be introduced into the inside, and a measuring cell provided with a cylindrical electrode on the inner wall surface;
A plate-like electrophoresis electrode having a disk-like head provided in the measurement cell and immersed in the microorganism-containing liquid;
A migration power supply circuit for applying an AC voltage to dielectrophoresis said microorganism is connected to the electrodes of the cylindrical shape as the electrophoresis electrode,
Frequency changing means for selecting a predetermined frequency capable of changing the frequency of the alternating voltage and capable of causing repulsive force from the electric field concentration part on the microorganisms;
A measuring unit for measuring the number of microorganisms,
When the alternating voltage of the frequency selected by the frequency changing unit is applied to the electrophoresis electrode, the control unit includes a control unit that causes the measurement unit to measure the number of microorganisms,
The migration electrode is arranged such that an electric field concentration portion is formed on the circumferential end face of the head when the alternating voltage is applied,
The said measurement part measures the number of the said microorganisms collected on the surface of the said migration electrode used as a weak electric field part by the repulsive force from the electric field concentration part with respect to the said microorganisms , The microorganisms number measuring apparatus characterized by the above-mentioned .

微生物含有の液体を内部に導入することができ、内壁面に円筒形状の電気的に接地された電極が設けられた測定セルと、
前記測定セル内に設けられ前記微生物含有の液体に浸漬された円盤状の頭部をもつ板状の泳動電極と、
一出力端が前記泳動電極に接続されるとともに他出力端が接地され、前記微生物を誘電泳動するための交流電圧を印加する泳動電源回路と、
前記交流電圧の周波数を変化させることができ、前記微生物に対して電界集中部からの斥力を生じさせることができる所定の周波数を選択する周波数変化手段と、
前記微生物の数を測定する測定部と、
前記周波数変化手段が選択した周波数の交流電圧が前記泳動電極に印加されたとき、前記測定部に微生物の数の測定を行わせる制御手段を備え、
前記泳動電極は、前記交流電圧の印加時にその頭部の円周方向端面に電界集中部が形成されるように配置され、
前記測定部は、前記微生物に対する電界集中部からの斥力によって、弱電界部分となる前記泳動電極の表面に集まった前記微生物の数を測定することを特徴とする微生物数測定装置。A measuring cell in which a microorganism-containing liquid can be introduced, and a cylindrical electrically grounded electrode is provided on the inner wall ;
A plate-like electrophoresis electrode having a disk-like head provided in the measurement cell and immersed in the microorganism-containing liquid;
A migration power supply circuit to which an output terminal is the other output end is connected to the electrophoresis electrode is grounded, to apply an AC voltage for dielectrophoresis said microorganism,
Frequency changing means for selecting a predetermined frequency capable of changing the frequency of the alternating voltage and capable of causing repulsive force from the electric field concentration part on the microorganisms;
A measuring unit for measuring the number of microorganisms,
When the alternating voltage of the frequency selected by the frequency changing unit is applied to the electrophoresis electrode, the control unit includes a control unit that causes the measurement unit to measure the number of microorganisms,
The migration electrode is arranged such that an electric field concentration portion is formed on the circumferential end face of the head when the alternating voltage is applied,
The said measurement part measures the number of the said microorganisms collected on the surface of the said migration electrode used as a weak electric field part by the repulsive force from the electric field concentration part with respect to the said microorganisms , The microorganisms number measuring apparatus characterized by the above-mentioned .

前記測定部が光源と受光部を備え、前記光源から前記微生物に照射された光の反射強度により微生物の数を求めることを特徴とする請求項１または２記載の微生物数測定装置。 It said measuring unit comprises a light source and a light receiving portion, microbial count measuring apparatus according to claim 1, wherein the determination of the number of microorganisms by the reflection intensity of the irradiated light to said microorganisms from said light source.

前記周波数変化手段が前記電界集中部への引力を生じる所定の周波数を選択することができることを特徴とする請求項１〜３のいずれかに記載の微生物数測定装置。Microbial count measuring device according to any one of claims 1 to 3, characterized in that it is possible to said frequency changing means selects a predetermined frequency causing attraction to the electric field concentration.

内壁面に円筒形状の電極を備えた測定セル内に微生物含有の液体を導入し、該測定セル内の液体に円盤状の頭部をもつ板状の泳動電極を浸漬するとともに、該泳動電極と前記円筒形状の電極間に交流電圧を印加し、前記微生物を誘電泳動により収集して前記微生物の数を測定する微生物数測定方法であって、前記泳動電極は、前記交流電圧の印加時にその頭部の円周方向端面に電界集中部が形成されるように配置され、前記泳動電極と前記円筒形状の電極間に形成された前記電界集中部と前記微生物との間に前記電界集中部からの斥力を生じさせる周波数の交流電圧を印加し、弱電界部分となる前記泳動電極の表面に前記微生物を収集し、収集された微生物の数を測定して前記液体中の微生物数を算出することを特徴とする微生物数測定方法。A microorganism-containing liquid is introduced into a measurement cell having a cylindrical electrode on the inner wall surface, and a plate-like migration electrode having a disk-shaped head is immersed in the liquid in the measurement cell. the AC voltage is applied between the electrodes of the cylindrical shape, said microorganisms a microorganism number measuring method for measuring the number of microorganisms was collected by dielectrophoresis, the electrophoresis electrodes, the head upon application of the alternating voltage electric field concentration portion is arranged to be formed in the circumferential end parts, from the aforementioned electric field concentration between the electrophoresis electrode and the cylindrical electric field concentrated portions are formed between the electrodes of the shape and the microorganism that by applying an AC voltage having a frequency causing repulsion, and collecting the microorganism on the surface of the electrophoresis electrode to be a weak electric field portion, the number of collected microorganisms by measuring to calculate the number of microorganisms in the liquid Characteristic method for measuring the number of microorganisms

内壁面に円筒形状の電極を備えた測定セル内に微生物含有の液体を導入し、該測定セル内の液体に円盤状の頭部をもつ板状の泳動電極を浸漬するとともに、該泳動電極と前記円筒形状の電極間に交流電圧を印加し、前記微生物を誘電泳動により収集して前記微生物の数を測定する微生物数測定方法であって、前記泳動電極は、前記交流電圧の印加時にその頭部の円周方向端面に電界集中部が形成されるように配置され、前記泳動電極と前記円筒形状の電極間に形成された前記電界集中部と前記微生物との間に前記電界集中部への引力を生じさせる周波数の交流電圧を印加して、前記電界集中部分に前記微生物を収集し、さらに前記電界集中部からの斥力を生じさせる周波数の交流電圧を印加して、弱電界部分となる前記泳動電極の表面に微生物を収集し、収集された微生物の数を測定して前記液体中の微生物数を算出することを特徴とする微生物数測定方法。A microorganism-containing liquid is introduced into a measurement cell having a cylindrical electrode on the inner wall surface, and a plate-like migration electrode having a disk-shaped head is immersed in the liquid in the measurement cell. the AC voltage is applied between the electrodes of the cylindrical shape, said microorganisms a microorganism number measuring method for measuring the number of microorganisms was collected by dielectrophoresis, the electrophoresis electrodes, the head upon application of the alternating voltage electric field concentration portion is arranged to be formed in the circumferential end parts, to the aforementioned electric field concentration between the electrophoresis electrode and the cylindrical electric field concentrated portions are formed between the electrodes of the shape and the microorganism by applying an AC voltage of a frequency to cause an attractive force, the electric field concentration portion said microorganism were collected, and further applying an alternating voltage of a frequency to cause the repulsive force from the electric field concentration portion, the weak electric field portion wherein microorganisms on the surface of the electrophoresis electrode The collected microorganisms number measuring method characterized by measuring the number of collected microorganisms to calculate the number of microorganisms in said liquid.