JP3869687B2 - Microbial activity measuring apparatus and microbial activity measuring method - Google Patents

Description

ここで、複素誘電率Ｋは（数２）で表され、
【００１４】
【数２】

また、懸濁液の複素誘電率ε^＊ _ｍは（数３）で表され、
【００１５】
【数３】

ε_ｍ ：懸濁液の誘電率
ε^＊ _ｍ ：懸濁液の複素誘電率
ε^＊ _ｉ ：微生物の複素誘電率
σ 懸濁液の伝導率
ω ：電界の角周波数
また、
ａ ：球形近似したときの微生物の半径
Ｒｅ［Ｋ］ ：微生物と懸濁液の複素誘電率に依存するパラメータ
Ｅ ：電界強度
である。この（数１）（数２）（数３）から明らかなように、懸濁液と微生物の大きさが一定であれば、誘電泳動力Ｆ_ＤＥＰはパラメータＲｅ［Ｋ］に比例することがわかる。
【００１６】
図９は細胞質導電率をパラメータとするＲｅ［Ｋ］の周波数特性図である。図９において、誘電泳動に用いる電界の周波数fをパラメータとして、誘電泳動力を細胞質導電率σ_iの関数として表している。細胞質内部の溶液の導電率である。図９によると、周波数１０kHz〜１MHzで正の誘電泳動力が働き、それ以外では負の誘電泳動力が働くのがわかる。細胞質導電率σ_iと周波数ｆによっては負の誘電泳動力が働き、誘電泳動力Ｆ_ＤＥＰが微生物に作用しても、捕集できない場合があることが分かる。
【００１７】
従って、微生物（細胞質導電率σ_i）の種類や生死に応じて周波数ｆを選択すると、正の誘電泳動力を作用させて捕集したり、負の誘電泳動力を作用させて排除することが可能である。但し、実際の誘電泳動には懸濁液導電率等の影響も考慮しなければならない。
【００１８】
【発明が解決しようとする課題】
以上説明したように、従来から様々の分野で微生物の個体数と活性度を定量する必要があったが、コロニーカウント法に代わる適当な定量方法がなく、一般的にコロニーカウント法が用いられてきた。しかし、このコロニーカウント法は、コロニーが形成されるまでに1日から数日という非常に長い時間を必要とし、定量が行えるとはいえ、オフラインでしか使用できないものであった。
【００１９】
現在、食中毒等への対処や保険衛生の点から迅速性が強く求められ、また、酵母や有用な微生物を用いての有用物質の生産システムを高効率に制御したり、生産物の収率管理等を行うために、微生物の個体数と活性度をリアルタイムに定量することが求められている。しかし、上述したコロニーカウント法や、ＡＴＰ法は、定量自体にも時間がかかってリアルタイムの測定を実現するのは完全に無理である上に、測定に使用する試薬等も多く、当然専門家による高度で複雑な作業が必要で、とても一般の人間が測定を行えるようなものではなかった。
【００２０】
その点、本発明者が他の研究者らと一緒に提案したＤＥＰＩＭ法は、微生物の濃縮と菌濃度検出のいずれのプロセスも電気的に行うため、測定に時間がかからないという点で大きな特徴を有しており、検出に際して試薬を使用しないし、誘電泳動現象の経時変化を統計的にみることによって菌濃度の推定時間を短縮できるというきわめて優れた特徴を有している。
【００２１】
このように優れたＤＥＰＩＭ法であるが、微生物に関する（数１）の中の複素誘電率Ｋ、従って、Ｒｅ［Ｋ］は、Craneなど複数の研究者によって微生物の活性（生菌、死菌の区別）に強く影響を受けることが指摘されている。しかし、この活性を区別して定量化する方法は存在しない。ＤＥＰＩＭ法でリアルタイムに微生物活性を測定できるようになるためには、どうしても試料液中の微生物の生菌、死菌の区別を明確に付け、生菌だけを検出する手法の開発が必要である。
【００２２】
すなわち、食中毒や保険衛生の点から微生物の活性を検出する場合には、通常，検出と同時並行的に殺菌が行われているため死菌もリアルタイムに増えていくし、酵母や有用な微生物を用いての生産システムにおいても、リアルタイムに生菌、死菌の増減を把握しなければならない。
【００２３】
そこで、本発明はこのような問題を解決するために、リアルタイムに近い迅速性を備え、誰でも微生物活性を簡便且つ定量的に検出する微生物活性測定装置を提供することを目的とする。
【００２４】
また、本発明は、リアルタイムに近い迅速性を備え、誰でも微生物活性を簡便且つ定量的に検出する微生物活性の測定方法を提供することを目的とする。
【００２５】
【課題を解決するための手段】
上記の問題点を解決するため本発明の微生物活性測定装置は、微生物毎に懸濁液導電率，生菌だけのときのコンダクタンスと死菌だけのときのコンダクタンスの比をとったときに該死菌だけのコンダクタンスが１０％以下となる活性度測定最適周波数，検量線データを格納した活性度測定用テーブルが格納されたメモリ部とを備え、入力手段から試料液に含まれる微生物の種類と懸濁液導電率が入力されると、演算制御部が活性度測定用テーブルから活性度測定最適周波数を読み出し、誘電泳動用電源部が該活性度測定最適周波数の交流電圧を印加して微生物のうち生菌を濃縮し、測定部によって測定されたインピーダンスにより検量線データに基づいて電極間のコンダクタンス変化から微生物数を算出して出力することを特徴とする。
【００２６】
これにより、リアルタイムに近い迅速性を備え、誰でも微生物活性を簡便且つ定量的に検出することができる。
【００２７】
また、本発明の微生物活性の測定方法は、試料液に浸漬した一対の電極間に生菌だけのときのコンダクタンスと死菌だけのときのコンダクタンスの比をとったときに該死菌だけのコンダクタンスが１０％以下となる活性度測定最適周波数の交流電圧を印加し、該試料液に含有された特定の微生物中の生菌を誘電泳動して濃縮するとともに電極間のインピーダンスを測定し、測定したインピーダンスに基づいて電極間のコンダクタンス変化から微生物数を算出することを特徴とする。
【００２８】
これにより、リアルタイムに近い迅速性を備え、誰でも微生物活性を簡便且つ定量的に検出することができる。
【００２９】
【発明の実施の形態】
本発明の請求項１に記載の発明は、試料液を収容することができる測定チャンバーと、測定チャンバー内の試料液に浸漬され、不平等電界を発生して誘電泳動により該試料液に含有される微生物を濃縮するとともに、このときのインピーダンスを測定するための一対の電極と、電極間に交流電圧を印加する誘電泳動用電源部と、電極間のインピーダンスを測定することができる測定部と、誘電泳動用電源部と測定部の制御を行う演算制御部と、微生物毎に懸濁液導電率，生菌だけのときのコンダクタンスと死菌だけのときのコンダクタンスの比をとったときに該死菌だけのコンダクタンスが１０％以下となる活性度測定最適周波数，検量線データを格納した活性度測定用テーブルが格納されたメモリ部とを備え、入力手段から試料液に含まれる微生物の種類と懸濁液導電率が入力されると、演算制御部が活性度測定用テーブルから活性度測定最適周波数を読み出し、誘電泳動用電源部が該活性度測定最適周波数の交流電圧を印加して微生物のうち生菌を濃縮し、測定部によって測定されたインピーダンスにより検量線データに基づいて電極間のコンダクタンス変化から生菌の微生物数を算出して出力することを特徴とする微生物活性測定装置であるから、微生物名と懸濁液導電率を入力手段により入力すると、活性度測定用テーブルから活性度測定最適周波数を読み出し、この活性度測定最適周波数の交流電圧を印加して電極間に不平等電界を発生させ、これによって発生する誘電泳動力により微生物のうち生菌だけを誘電泳動で濃縮することができる。このとき、活性度測定最適周波数は、生菌に対しては正の誘電泳動力が作用し、死菌に対しては負の誘電泳動力が作用する、若しくは実用上（言い換えればほとんど）正の誘電泳動力が作用しないから、生菌と死菌の分離を実用上の精度を保ちつつ可能にするとともに、分離可能な周波数の中では正の誘電泳動力を大きくすることができる。これにより濃縮できるのは生菌だけとなる。この濃縮と同時にインピーダンス測定を行い、生菌が濃縮されて電極間のコンダクタンスが変化するのを検出し、コンダクタンスの変化が微生物数（生菌数）と比例していることを利用し、微生物のうち生菌の数を算出して出力することができる。活性度測定最適周波数を選択したときに、死菌が当初若干残留する理由は、測定開始時点に電極付近に存在した死菌に対しては、生菌に作用する大きな正の誘電泳動力は作用しないものの、負の誘電泳動力もしくは０近傍の誘電泳動力が作用し、これによって域外に排除されるのに時間がかかるからである。
【００３１】
本発明の請求項２に記載の発明は、演算制御部が、前記電極間のコンダクタンスの初期増加率から生菌の微生物数を算出して出力することを特徴とする請求項１記載の微生物活性測定装置であるから、誘電泳動により微生物を濃縮する際に、濃縮過程初期のコンダクタンスの増加率で試料液中の微生物濃度を推定することができ、リアルタイムと評価できるほどの迅速な測定が可能になる。
【００３２】
本発明の請求項３に記載の発明は、測定チャンバーには攪拌装置が設けられ、演算制御部が時計手段の行う計時に基づいて試料液の攪拌を行うことを特徴とする請求項１または２記載の微生物活性測定装置であるから、試料液を導入して誘電泳動するとき、微生物濃度を全体として均一化するとともに、より多くの微生物を電極近傍に導くことができる。
【００３３】
本発明の請求項４に記載の発明は、測定チャンバーには試料液の供給管と排出管が設けられるとともに、供給管に供給バルブ、排出管に排出バルブが設けられ、演算制御部が時計手段の行う計時に基づいて、供給バルブを開いて試料液を測定チャンバーに供給し、測定後排出バルブを開いて試料液を排出す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活性測定装置であるから、試料液を自動的に供給し、測定後自動的に排水することができる。
【００３４】
本発明の請求項５に記載の発明は、測定チャンバーには洗浄液の洗浄管が設けられるとともに、洗浄管に洗浄バルブが設けられ、演算制御部が時計手段の行う計時に基づいて、洗浄バルブを開いて洗浄液を測定チャンバーに供給し、試料液を排出した後チャンバー内を洗浄することを特徴とする請求項１〜４のいずれかに記載の微生物活性測定装置であるから、排水後洗浄液を測定チャンバーに導入し、自動的に測定チャンバー内を洗浄することができる。
【００３５】
本発明の請求項６に記載の発明は、試料液に浸漬した一対の電極間に生菌だけのときのコンダクタンスと死菌だけのときのコンダクタンスの比をとったときに該死菌だけのコンダクタンスが１０％以下となる活性度測定最適周波数の交流電圧を印加し、該試料液に含有された特定の微生物中の生菌を誘電泳動して濃縮するとともに電極間のインピーダンスを測定し、測定したインピーダンスに基づいて電極間のコンダクタンス変化から生菌の微生物数を算出することを特徴とする微生物活性の測定方法であるから、活性度測定最適周波数の交流電圧を印加して電極間に不平等電界を発生させ、これによって発生する誘電泳動力により微生物のうち生菌だけを誘電泳動で濃縮することができる。このとき、活性度測定最適周波数は、生菌に対しては正の誘電泳動力が作用し、死菌に対しては負の誘電泳動力が作用する、若しくは実用上（言い換えればほとんど）正の誘電泳動力が作用しないから、生菌と死菌の分離を実用上の精度を保ちつつ可能にするとともに、分離可能な周波数の中では正の誘電泳動力を大きくことができる。これにより濃縮できるのは生菌だけとなる。この濃縮と同時にインピーダンス測定を行い、生菌が濃縮されて電極間のコンダクタンスが変化するのを検出し、コンダクタンスの変化が微生物数（生菌数）と比例していることを利用し、微生物のうち生菌の数を算出して出力することができる。
【００３７】
本発明の請求項７に記載の発明は、電極間のコンダクタンスの初期増加率から微生物数を算出することを特徴とする請求項６記載の微生物活性の測定方法であるから、誘電泳動による濃縮過程初期のコンダクタンスの増加率で試料液中の微生物濃度を推定することができ、リアルタイムと評価できるほどの迅速な測定が可能になる。
【００３８】
（実施の形態１）
以下、本発明における実施の形態１における微生物活性測定装置及び微生物活性の測定方法について説明する。図１は本発明の実施の形態１における微生物活性測定装置の構成図、図２（ａ）は本発明の実施の形態１における微生物活性測定装置の活性度測定用テーブルの測定用制御データ図、図２（ｂ）は本発明の実施の形態１における微生物活性測定装置の活性度測定用テーブルの検量線図、図３は本発明の実施の形態１における微生物活性測定装置の誘電泳動力に及ぼす細胞質導電率と電界周波数の関係図、図４は本発明の実施の形態１における微生物活性測定装置の電界周波数１００ｋＨｚでの生菌と死菌の検出図、図５は本発明の実施の形態１における微生物活性測定装置の電界周波数１ＭＨｚでの生菌と死菌の検出図、図６は本発明の実施の形態１における微生物活性測定装置の生菌と死菌の混合懸濁液から生菌と死菌が分離されることを示す図、図７は本発明の実施の形態１における微生物活性測定装置の生菌数の加熱殺菌時間依存性を示す図、図８は図４は本発明の実施の形態１における微生物活性測定装置の加熱殺菌後の微生物活性測定図である。
【００３９】
図１において、１はＤＥＰＩＭ法 (Dielectrophoretic Impedance Measurement Method)で微生物中の生菌を測定するため試料液を収容する測定チャンバー、２は微生物活性を測定するためのチップ化された電極部、２ａ，２ｂは電極部２を構成する一対の電極である。電極２ａ，２ｂは、誘電泳動を行うために不平等電界を発生して、電極２ａ，２ｂ間にパールチェインと呼ばれる微生物の鎖を多数橋絡できる形状、例えばキャッスルウォール型電極、櫛歯型電極等の形状を備えておればよい。キャッスルウォール型電極は、両側に１ピッチ（例えば５０μｍ〜１００μｍ）おきに突出する多数の矩形電極片が形成された電極２ａ，２ｂを１ピッチずらして５μｍ〜１０μｍ離して配設したもので、電極２ａ，２ｂの各矩形電極片のエッジ間に電界が集中するものである。また、櫛歯型電極は、櫛のように歯（例えば３０μｍ〜１００μｍ幅）を形成された一対の電極が溝に入れ子状に挿入、組み合わされ、狭いギャップ（例えば５μｍ〜１０μｍ幅）で対向した電極であり、主として厚さ方向のエッジ間に不平等電界が形成され、パールチェインが多数形成されるものである。微生物の大きさでギャップやピッチは最も適当な値を選択するのが望ましい。
【００４０】
電極２ａ，２ｂはクロムや白金等の薄膜電極として構成し、ガラス基板やプラスチック基板にスパッタリングや蒸着、メッキ等で成膜し、フォトリソグラフィー等でエッチングして形成するのがよい。薄膜の厚さは５０ｎｍ〜２００ｎｍ程度のものが多数のパールチェインを形成する上で望ましい。なお、電極２ａ，２ｂの材質はクロムや白金に限らず、交流電圧を印加したとき電気分解が生じないイオン化傾向の小さい金属であればよい。
【００４１】
ところで、測定チャンバー１は、図１に示すように電極部２を別体として内部に収容するほかに、電極部２の基板を覆った構造とすることもできる。例えば、電極２ａ，２ｂの周囲にスペーサを配置して、このスペーサ上にガラス板やプラスチック板等を載せて接着すれば微小な測定チャンバー１をコンパクトな構造として作ることができる。これは試料液が少量である場合に有効であり、攪拌に代えて閉回路として流動させることで、微生物を電極２ａ，２ｂ付近に導くとともに微生物濃度を均一化するのが望ましい。
【００４２】
次に、３は電極２ａ，２ｂ間に誘電泳動を発生させるために交流電圧を印加する誘電泳動用電源部、４は電極２ａ，２ｂ間のインピーダンスを測定することができる測定部、５はマイクロプロセッサ等から構成され、作業域に制御用のプログラムや各種データをロードして機能し、少なくとも誘電泳動用電源部３及び測定部４を制御するとともに演算を行う演算制御部、６は時計手段である。誘電泳動用電源部３は、電極２ａ，２ｂ間に印加する交流電圧の電圧と周波数を演算制御部５によって制御される。本実施の形態１においては、１ｋＨｚ〜１０ＭＨｚの間で周波数を変えることができ、ピーク間電圧（以下、ｐｐと表す）１Ｖｐｐ〜２０Ｖｐｐを印加できるファンクションジェネレータが採用されている。なお、本明細書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三角波、方形波等の電圧を意味し、正負両サイドの電流の平均値が等しいものである。後述する活性度測定最適周波数は微生物の種類や懸濁液伝導率によって変化し、電圧は電極２ａ，２ｂ間で電気分解が発生せず、誘電泳動力が大きくなるような値が選択される。
【００４３】
測定部４には５００Ω程度の電流検出用の抵抗が設けられ、図１に示す電圧印加回路に直列に挿入されており、これを流れる電流を測定して電極２ａ，２ｂ間のコンダクタンス（抵抗成分の逆数）を算出している。すなわち、演算制御部５は、時計手段６の計時する、例えば１０ｓｅｃ間隔というタイミングで、誘電泳動用電源部３の周波数と電圧を制御し、電極２ａ，２ｂ間に活性度測定最適周波数の交流電圧を印加すると同時に、このとき測定部４に電圧印加回路を流れる電流の測定を行わせ、試料液中の微生物が濃縮されたことによる抵抗成分の測定を行い、逆数をとってコンダクタンスの算出をしている。時計手段６はこのサンプリングのタイミングだけでなく、実施の形態１における微生物活性測定装置の自動運転のためのスケジューリングのための時間管理を行う。
【００４４】
さて、７は演算制御部５にロードするプログラムや各種データを格納したメモリ部、７ａはメモリ部７の中に設けられ、微生物のうち生菌だけを捕集して濃縮するための測定用制御データと、コンダクタンスを示す測定部４の出力（以下、ＤＥＰＩＭ出力）から生菌数を換算するための検量線データ等を格納した活性度測定用テーブルである。測定用制御データは、微生物毎に、微生物を懸濁した試料液の懸濁液導電率と、生菌に正の誘電泳動力を作用し、死菌に負の誘電泳動力もしくは０近傍の誘電泳動力を作用させることが可能な活性度測定最適周波数がデータベース化されている。活性度測定最適周波数より大きい周波数を選択すると、生菌を濃縮するための正の誘電液動力が低下し、活性度測定最適周波数より小さい周波数を選択すると、生菌と死菌の分離が難しくなって測定精度が低下する。従って、活性度測定最適周波数を使用するのが最適であるが、これより大きい周波数を使用することも考えられる。しかし、測定に時間がかかるようになる上、測定部４との関係から好ましいものではない。
【００４５】
そこで、以下、活性度測定用テーブル７ａに格納されている測定用制御データや検量線データについて詳述する。図２（ａ）に示すように、測定用制御データには、少なくとも、微生物名，試料液の溶媒である懸濁液の懸濁液導電率，印加する交流電圧のピーク間電圧，活性度測定最適周波数が互いに関係付けられて格納されている。すなわち、測定用制御データとして格納されているのは、微生物の種類毎に関係付けられた懸濁液導電率と、このとき印加するピーク間電圧と、活性度測定最適周波数等である。図２（ａ）においては、懸濁液導電率は微生物毎に１種類の導電率しか格納されていないが、微生物毎に複数種類の導電率を格納し、それぞれにピーク間電圧と活性度測定最適周波数を関係付けるのが好適である。図２（ｂ）には、測定部４が検出したコンダクタンスを示すＤＥＰＩＭ出力と生菌数とを換算するための検量線が記載されている。この検量線データは、微生物の種類毎にＤＥＰＩＭ出力値と生菌数が１対１で対応付けられたテーブル状になっていてもよいが、検量線を１次関数として活性度測定用テーブル７ａに記憶しておくのもデータ量を小さくできて望ましい。
【００４６】
次に、活性度測定最適周波数について説明する。ＤＥＰＩＭ法による誘電泳動力を示す（数１）において、複素誘電率Ｋ、従って、Ｒｅ［Ｋ］は、複数の研究者によって微生物の活性（生菌、死菌の区別）に強く影響を受けることが指摘されている。Craneらの研究者は、加熱殺菌などによって微生物の細胞膜に含まれるタンパク質（酵素）が変性し細胞膜に欠陥が生じると、細胞膜内部の細胞質に含まれる各種イオンがこれら欠陥を通して外部に漏れ出すため、細胞質部分の導電率σ_iが時間と共に低下することを指摘している。
【００４７】
この細胞質内導電率σiが低下した場合、（数１）中のRｅ［Ｋ］すなわち誘電泳動力がどのように変化するかを理論的に求めた結果が図３である。図３においては、誘電泳動に用いる電界の周波数fをパラメータとして、誘電泳動力を細胞質導電率σiの関数として表現している。図３によると、周波数１００kHzでは誘電泳動力の細胞質導電率σiへの依存性は小さいが、周波数１MHzでは細胞質導電率σiの低下に伴い誘電泳動力が低下することがわかる。例えば細菌の失活によって細胞質導電率σiが初期値の１０^−１Ｓ／mから１０^−２Ｓ／mに低下した場合、周波数１００kHzでは誘電泳動力は５％しか低下しないが、１MHzでは７０％も低下している。
【００４８】
従って、周波数１MHzで細胞質導電率σ_iが低くなった微生物の集合（例えば、σ_iが２×１０^−３Ｓ／ｍ付近の大部分が死菌である集合に、生菌がわずか混じった集合）を誘電泳動した場合、細胞質導電率σ_iの低下に伴い正の誘電泳動力が作用する生菌はほとんど存在せず、負の誘電泳動力が作用する死菌がほとんど全部を占めることが分かる。しかし、これが周波数１００kHzの場合は、死菌に対しても正の誘電泳動力が作用し、周波数１MHzでは濃縮されなかった同じ微生物の集合が生菌死菌合わせて濃縮されることが分かる。これが、５００ｋＨｚの場合は、σ_iが２×１０^−３Ｓ／ｍ付近の大部分の死菌には誘電泳動力はほとんど作用せず、それ以上の細胞質導電率σ_iをもつ死菌に対してわずかながら正の誘電泳動力が作用している。
【００４９】
これらのことは、死菌に対して誘電泳動を行う周波数を高くしていくと、負の誘電泳動力かほとんど誘電泳動力が作用しない状態が招来され、生菌に対してだけ正の誘電泳動力が作用するようになることを示している。しかし、周波数の増加につれて誘電泳動力自体は減少傾向を示し、誘電泳動に時間を要するようになる上、１０ＭＨｚ以上の高周波数にすると、測定部４の能力から測定する精度が期待通りに得られなくなる。従って、生菌死菌を選択的に濃縮するのに活性度測定最適周波数が最も有効な周波数であることが分かる。
【００５０】
この活性度測定最適周波数を決定するには、次のような実験を行って決定するのがよい。すなわち、同じ微生物濃度で生菌だけの試料液と死菌だけの試料液をそれぞれ用意し、同じ周波数、電圧、懸濁液導電率で測定を行う。周波数を変化させて上昇させていくと、生菌だけのコンダクタンスを示すＤＥＰＩＭ信号が死菌のそれより相対的に大きくなっていく。この死菌に対する測定部４からのＤＥＰＩＭ信号が検出されないようになったときの周波数を活性度測定最適周波数とする。この状態においては、死菌は結果として電極２ａ，２ｂ近傍の測定領域から排除されているからである。図２（ａ）に示した活性度測定最適周波数はこのようにして決定されたものであり、微生物毎、懸濁液毎に実験を繰り返して精緻な活性度測定用テーブル７ａを作成しておくのが適当である。
【００５１】
ところで、以上説明したことから分かるとおり、この活性度測定最適周波数には近傍に最適とはいえないが許容される周波数の範囲が存在する。すなわち、死菌に対して実用上、ほとんど正の誘電泳動力が作用しない周波数の範囲である。死菌に対するＤＥＰＩＭ信号が検出されないまでも、生菌だけのときのＤＥＰＩＭ信号と死菌だけのときのそれとの比をとったとき１０％以下となる周波数であれば、測定精度を維持しつつ測定することが可能である。従って、この範囲も広義には活性度測定最適周波数（本発明の活性度測定最適周波数）に含めることができる。
【００５２】
そして、図９に記載された細胞質導電率をパラメータとするＲｅ［Ｋ］の周波数特性図を参照すると、細胞質導電率にもよるが１０ＭＨｚを超えると、１０ｋＨｚ以下と同様に、正の誘電泳動力が作用する状態から負の誘電泳動力が作用する状態に移行する。上述したように、１０ＭＨｚ以上の高周波領域では測定部４で精度が期待できなくなるため、本発明ではこれを避け、１０ＭＨｚ以下で周波数を上昇させながら活性度測定最適周波数を決定しているが、１０ＭＨｚ以上の高周波領域においても、生菌のＤＥＰＩＭ信号と死菌のそれとの比が１０％以下となる周波数があるのなら、ある程度の精度で微生物活性を測定する可能性はある。
【００５３】
続いて、図１において、８は微生物名や懸濁液導電率を入力するためのデータ入力部（実施の形態１における本発明の入力手段）、９はデータ入力のための表示を行ったり、演算制御部５が検量線データに基づいて算出した生菌数を表示するためのＬＣＤ等の表示部である。また、１０は微生物を含有した試料液を貯めた試料液槽、１１は試料液槽１０と測定チャンバー１を接続し、資料液槽１０内の試料液を測定チャンバー１に導く供給管である。１２は測定チャンバー１から測定後の試料液を排出するための排出管、１３は供給管１１に設けられた電磁弁等の供給バルブ、１４は排出管１２に設けられた排出バルブである。１５はスターラ等の撹拌装置であって、時計手段６の行う計時に基づいて演算制御部５が誘電泳動を行う前や誘電泳動時に試料液の攪拌を行うものである。これにより、試料液を供給管１１から導入して誘電泳動するとき、微生物濃度を全体として均一化するとともに、より多くの微生物を電極２ａ，２ｂ近傍に導くことができる。
【００５４】
また、測定チャンバー１を、電極２ａ，２ｂの周囲にスペーサを配置して、このスペーサ上にガラス板等を載せて接着した微小な測定チャンバー１の場合等には、攪拌装置１５に代えて排出管１２を試料液層１０に接続して閉じた状態で流動させることで微生物を電極２ａ，２ｂ付近に導くとともに、微生物濃度を均一化する方法と組み合わせで用いるのが望ましい。１６は洗浄液タンク、１７は洗浄管、１８は洗浄バルブである。演算制御部５が洗浄バルブ１８を開いて洗浄液を測定チャンバー１に供給し、試料液を排出した後測定チャンバー１内を洗浄する。時計手段６の時間管理で、排水後洗浄液を測定チャンバー１に洗浄液を導入し、自動的に測定チャンバー１内を洗浄して次の測定に備えることができる。本発明の微生物活性測定装置による測定にとって洗浄液で洗浄することは必須のことではないが、微生物含有の試料液を測定後直ちに洗浄したほうが望ましいので，本実施の形態１においては洗浄系が設けられている。
【００５５】
さらに、本実施の形態１の微生物活性測定装置は、測定セルフチェック機構を備えて、それほど知識がなくとも正しく測定できるようになっている。すなわち、本微生物活性測定装置が正しく準備されていなければ、測定を行っても無駄になってしまう。そこで、本微生物活性測定装置は測定開始直後にセルフチェックを行う。測定開始したばかりの状態では微生物が捕集されていない。この状態で電極２ａ，２ｂ間のインピーダンス測定を行ってＤＥＰＩＭ信号を検出すると、懸濁液の導電率と誘電率だけに依存した初期値を示す。もし、演算制御部５が、測定開始直後にＤＥＰＩＭ信号を検出して、懸濁液が示す初期値が通常値の範囲と大きく異なっていると判断すると、電極部２が破損して短絡されていたり汚染されているなどの可能性があり、アラームを鳴らし、または表示部９によって警告を表示して電極や装置の確認や交換を促す。懸濁液が示す初期値の通常値は、活性度測定用テーブル７ａに懸濁液毎に用意しておくか、格納されている検量線データを利用して判定すればよい。このようなセルフチェックを行うことによって、専門家でなくとも簡単に微生物活性の測定が確実に行える。
【００５６】
次に、本実施の形態１の微生物活性測定装置を動作させたときの動作手順と測定結果について説明する。図１において、図示しない電源スイッチを入力し、表示部９に表示されたガイダンスにより、データ入力部８から微生物の種類と、懸濁液導電率を入力する。演算制御部５はセルフチェックを行い、入力に基づいて活性度測定用テーブル７ａから印加する交流電圧と活性度測定最適周波数を読み出し、誘電泳動電源部３を制御して電極２ａ，２ｂに印加する。これとともに測定部４でインピーダンス測定を行う。
【００５７】
時計手段６の計時する所定時間（例えば１０ｓｅｃ）毎に、測定部４は微生物を濃縮したことによって変化するコンダクタンスを測定し、この結果に基づき演算制御部５は活性度測定用テーブル７ａから検量線データを読み出して生菌数を換算する。
【００５８】
次に、微生物として生菌と死菌の大腸菌を用い、懸濁液導電率が０．１ｍＳ／mの条件下で周波数１００ｋＨｚで微生物活性測定を行った場合の結果を説明する。死菌は加熱殺菌（８０℃，１５分）によって調製し、コロニーカウント法により失活を確認した。図４に示すように周波数１００ｋＨｚでは生菌、死菌共にほぼ同様のコンダクタンスの増加が認められる。このコンダクタンスの増加は、誘電泳動力により懸濁液中の大腸菌が電極ギャップ２ａ，２ｂ間に捕集された結果生じたものである。
【００５９】
そして、活性度測定最適周波数１ＭＨｚで同様の計測を行った結果を図５に示す。生菌では周波数１００ｋＨｚと同様のコンダクタンス増加が認められるが、死菌ではコンダクタンスはほぼ一定であった。顕微鏡を用いて電極近傍での誘電泳動現象を観測した結果、周波数１ＭＨｚでは死菌には誘電泳動力が作用せず、電極へは死菌が捕集されないことが確認された。このように、電界周波数を活性度測定最適周波数である１ＭＨｚにすることで生菌のみを検出できる。
【００６０】
次に、生菌と死菌が混濁された大腸菌懸濁試料液を対象に選択的微生物活性測定を行った例を図６に示す。生菌と死菌を混合した試料液のコンダクタンスを示すＤＥＰＩＭ出力は生菌のみの場合の出力とほぼ一致している。これにより、混合懸濁液から選択的に生菌のみを検出・定量することが可能であることが分かる。生菌の混合比を変化させると、それに対応して混合液のＤＥＰＩＭ出力が変化する。
【００６１】
加熱殺菌処理後の生菌数は、加熱時間に対し図７のように指数関数的に減少することが知られている。本実施の形態１の微生物活性測定装置により生菌数のみを選択的に検出することができるので、図６のような測定を加熱時間の経過と共に順次行えば、菌の活性（生菌の割合）を短時間で、ほぼリアルタイムといえる迅速さで検出することができる。上述の大腸菌懸濁試料液を対象として、ＤＥＰＩＭ法により生菌数の加熱殺菌時間依存性を測定した結果を図８に示す。図８には比較のため培養法により求めた生菌数変化も併せて示している。なお、図８のＤＥＰＩＭ出力は規格化したものである。同図に示すように、ＤＥＰＩＭ法で計測した生菌数は、培養法で決定したものとよく対応しており、加熱時間と共に指数関数的に生菌数が減少する様子を計測することができる。この場合、ＤＥＰＩＭ法の測定に要する時間は１０分以内であり、培養法に必要な時間（約２４時間）に比べて極めて短時間で生菌数変化を検出することができる。すなわち、インピーダンスの測定はリアルタイムで行うことができるので、ＤＥＰＩＭ法によって微生物の活性がほぼリアルタイムで測定することができる。
【００６２】
【発明の効果】
本発明の請求項１に記載の微生物活性測定装置は、入力手段から試料液に含まれる微生物の種類と懸濁液導電率が入力されると、演算制御部が活性度測定用テーブルから生菌だけのときのコンダクタンスと死菌だけのときのコンダクタンスの比をとったときに該死菌だけのコンダクタンスが１０％以下となる活性度測定最適周波数を読み出し、誘電泳動用電源部が活性度測定最適周波数の交流電圧を印加して微生物のうち生菌を濃縮し、測定部によって測定されたインピーダンスにより検量線データに基づいて電極間のコンダクタンス変化から微生物数を算出して出力するから、微生物名と懸濁液導電率を入力手段により入力すると、活性度測定用テーブルから活性度測定最適周波数を読み出し、この活性度測定最適周波数の交流電圧を印加して電極間に不平等電界を発生させ、これによって発生する誘電泳動力により微生物のうち生菌だけを誘電泳動で濃縮することができる。そして、活性度測定最適周波数は、生菌に対しては正の誘電泳動力が作用し、死菌に対しては負の誘電泳動力が作用する、若しくは実用上（言い換えればほとんど）正の誘電泳動力が作用しないから、生菌と死菌の分離を実用上の精度を保ちつつ可能にするとともに、分離可能な周波数の中で正の誘電泳動力を大きくにすることができる。これにより濃縮できるのは生菌だけとなる。この濃縮と同時にインピーダンス測定を行い、生菌が濃縮されて電極間のコンダクタンスが変化するのを検出し、コンダクタンスの変化が微生物数（生菌数）と比例していることを利用し、微生物のうち生菌の数を算出して出力することができる。リアルタイムの微生物活性の測定ができ、発酵プロセス制御において有用な情報が得られ、品質向上やコスト削減に役立つ。また、食品製造プロセス全般に対しても、殺菌効果を迅速に確認することができ、迅速かつ簡便な菌検出技術として確立することができる。
【００６４】
本発明の請求項２に記載の微生物活性測定装置は、電極間のコンダクタンスの初期増加率から微生物数を算出して出力するから、誘電泳動により微生物を濃縮する際に、濃縮過程初期のコンダクタンスの増加率で試料液中の微生物濃度を推定することができ、リアルタイムと評価できるほどの迅速な測定が可能になる。
【００６５】
本発明の請求項３に記載の微生物活性測定装置は、測定チャンバーには攪拌装置が設けられ、誘電泳動を行う前に試料液の攪拌を行うから、試料液を導入して誘電泳動するとき、微生物濃度を全体として均一化するとともに、より多くの微生物を電極近傍に導くことができる。
【００６６】
本発明の請求項４に記載の微生物活性測定装置は、供給バルブを開いて試料液を測定チャンバーに供給し、測定後排出バルブを開いて試料液を排出するから、試料液を自動的に供給し、測定後自動的に排水することができる。
【００６７】
本発明の請求項５に記載の微生物活性測定装置は、洗浄バルブを開いて洗浄液を測定チャンバーに供給し、試料液を排出した後チャンバー内を洗浄するから、排水後洗浄液を測定チャンバーに導入し、自動的に測定チャンバー内を洗浄することができる。
【００６８】
本発明の請求項６に記載の微生物活性の測定方法は、生菌だけのときのコンダクタンスと死菌だけのときのコンダクタンスの比をとったときに該死菌だけのコンダクタンスが１０％以下となる活性度測定最適周波数の交流電圧を印加し、試料液に含有された特定の微生物中の生菌を誘電泳動して濃縮するとともに電極間のインピーダンスを測定し、コンダクタンス変化から生菌の微生物数を算出するから、活性度測定最適周波数の交流電圧を印加して電極間に不平等電界を発生させ、これによって発生する誘電泳動力により微生物のうち生菌だけを誘電泳動で濃縮することができる。このとき、活性度測定最適周波数は、生菌に対しては正の誘電泳動力が作用し、死菌に対しては負の誘電泳動力が作用する、若しくは実用上（言い換えればほとんど）正の誘電泳動力が作用しないから、生菌と死菌の分離を実用上の精度を保ちつつ可能にするとともに、分離可能な周波数の中では正の誘電泳動力を大きくすることができる。これにより濃縮できるのは生菌だけとなる。この濃縮と同時にインピーダンス測定を行い、生菌が濃縮されて電極間のコンダクタンスが変化するのを検出し、コンダクタンスの変化が微生物数（生菌数）と比例していることを利用し、微生物のうち生菌の数を算出して出力することができる。リアルタイムの微生物活性の測定ができ、発酵プロセス制御において有用な情報が得られ、品質向上やコスト削減に役立つ。また、食品製造プロセス全般に対しても、殺菌効果を迅速に確認することができ、迅速かつ簡便な菌検出技術として確立することができる。
【００７０】
本発明の請求項７に記載の微生物活性の測定方法は、電極間のコンダクタンスの初期増加率から微生物数を算出するから、誘電泳動による濃縮過程初期のコンダクタンスの増加率で試料液中の微生物濃度を推定することができ、リアルタイムと評価できるほどの迅速な測定が可能になる。
【図面の簡単な説明】
【図１】本発明の実施の形態１における微生物活性測定装置の構成図
【図２】（ａ）本発明の実施の形態１における微生物活性測定装置の活性度測定用テーブルの測定用制御データ図（ｂ）本発明の実施の形態１における微生物活性測定装置の活性度測定用テーブルの検量線図
【図３】本発明の実施の形態１における微生物活性測定装置の誘電泳動力に及ぼす細胞質導電率と電界周波数の関係図
【図４】本発明の実施の形態１における微生物活性測定装置の電界周波数１００ｋＨｚでの生菌と死菌の検出図
【図５】本発明の実施の形態１における微生物活性測定装置の電界周波数１ＭＨｚでの生菌と死菌の検出図
【図６】本発明の実施の形態１における微生物活性測定装置の生菌と死菌の混合懸濁液から生菌と死菌が分離されることを示す図
【図７】本発明の実施の形態１における微生物活性測定装置の生菌数の加熱殺菌時間依存性を示す図
【図８】本発明の実施の形態１における微生物活性測定装置の加熱殺菌後の微生物活性測定図
【図９】細胞質導電率をパラメータとするＲｅ［Ｋ］の周波数特性図
【符号の説明】
１ 測定チャンバー
２ 電極部
２ａ，２ｂ 電極
３ 誘電泳動用電源部
４ 測定部
５ 演算制御部
６ 時計手段
７ メモリ部
７ａ 活性度測定用テーブル
８ データ入力部
９ 表示部
１０ 試料液槽
１１ 供給管
１２ 排出管
１３ 供給バルブ
１４ 排出バルブ
１５ 撹拌装置
１６ 洗浄液タンク
１７ 洗浄管
１８ 洗浄バルブ[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a microbial activity measuring apparatus that can perform near-real-time rapid measurement and anyone can easily and quantitatively detect microbial activity, and a microbial activity measuring method used at that time.
[0002]
[Prior art]
In recent years, food poisoning incidents caused by microorganisms, especially malignant bacteria, have become one of the social problems, and such incidents are reported in summer as usual. In many cases of food poisoning, the cause is unknown, but more than 80% of cases are caused by bacteria, suggesting that bacterial testing in food management is extremely important.
[0003]
And in the present situation where the plantation of the food industry has progressed, it can cause serious social damage due to bacterial contamination occurring in one factory. For this reason, food companies carry out meticulous hygiene management and test bacteria for food production. In the bacteria test, the number of bacteria is generally counted by a colony counting method described later.
[0004]
By the way, not only the above-mentioned malignant bacteria but also many useful microorganisms are included in the microorganisms. Traditionally, brewing techniques that have been nurtured for more than a thousand years cannot fulfill their purpose without useful bacteria such as yeast for fermentation.For example, lactic acid bacteria are used in the production of dairy products, and recently developed antibiotics are produced. It is well known that molds and radiation bacteria are indispensable.
[0005]
In the process of producing useful substances using these useful microorganisms, as in other industrial processes, many physical state quantities and chemical state quantities are measured and used to improve the yield of the target product. Yes. Above all, the number and activity of microorganisms in the reactor (hereinafter also referred to as the number of viable bacteria) are particularly important because they are important parameters that directly indicate the reaction. Has been.
[0006]
As can be seen from the above explanation, in the present specification, microorganisms are generally called bacteria, fungi, actinomycetes, rickettsia, mycoplasma, viruses, etc., in addition to yeasts and molds used in such processes. Contains all microorganisms that are subject to microbiology.
[0007]
In order to quantify the number and activity of such microorganisms, a colony counting method has been generally used. That is, in this method, a suspension sample containing microorganisms is sprayed on a medium, and the number of viable bacteria is quantified from the number of colonies (colonies) formed as the microorganisms grow. However, this colony counting method requires a surprisingly long time of 1 to several days until colonies are formed, and although it can be quantified, it can only be used offline. This makes it impossible to control the process by grasping the state in the reactor. In addition, high-level knowledge and complicated manual operations such as sampling from the test object, concentration and dilution, planting in the culture medium, and counting the number of colonies (CFU) are necessary. For these reasons, it could not be said that there was no increase in running costs necessary for the inspection and no human error.
[0008]
As another method, there is an ATP (Adenosine Tri Phosphate) method using luminescence. Since cells such as animals, plants, and bacteria always contain ATP (adenosine triphosphate), the ATP contained in the bacteria in the test sample is extracted with an ATP extraction reagent, and the extracted ATP is then added to the firefly. The amount of ATP is detected by measuring the amount of luminescence with the enzyme (luciferase). Since the ATP content in the microorganism is a certain numerical value, the number of bacteria contained in the sample is estimated by detecting the amount of ATP estimated from the luminescence intensity.
[0009]
However, extraction of ATP with an ATP extraction reagent, enzyme action, measurement of luminescence, estimation of ATP, etc. require sophisticated and complicated work by experts, take time, and can be measured in real time It wasn't.
[0010]
Therefore, the present inventor, for the purpose of quantifying the number of microorganisms without using the colony counting method, measures the number of microorganisms together with other inventors by combining dielectrophoresis and electrical impedance (DEPIM method ( Dielectrophoretic Impedance Measurement Method)) was proposed.
[0011]
This DEPIM method is characterized in that both the microbial concentration process and the microbial concentration detection process are electrically performed. The concentration process concentrates bacteria on the integrated microelectrode by a dielectrophoresis phenomenon (a phenomenon in which an electric force acts on dielectric particles polarized in an electric field and moves in a certain direction). Quantitative estimation of bacterial concentration from changes in interelectrode impedance during concentration (Yanami, et al .: “Measurement of suspension concentration of Escherichia coli by dielectrophoretic impedance measurement (1): Principle and outline of the device”), IEEJ Proceedings '99, pp.337-340 (1999); Tomita, et al .: “Measurement of Suspension Concentration of Escherichia coli by Dielectrophoresis Impedance Measurement (2)-Estimated Model of Suspension Concentration”, Collection '99, pp.341-344 (1999)).
[0012]
In order to detect microorganisms by this DEPIM method, it is necessary to collect the microorganisms on the microelectrode by dielectrophoretic force. In other words, the number of bacteria collected on the microelectrode is reduced under conditions where the dielectrophoretic force does not sufficiently act, so that the detection signal is reduced. Dielectrophoretic force F acting on microorganisms_DEPIn terms of complex numbers, is theoretically given by (Equation 1) below.
[0013]
[Expression 1]

Here, the complex dielectric constant K is expressed by (Equation 2),
[0014]
[Expression 2]

Also, the complex permittivity ε of the suspension^* _mIs represented by (Equation 3),
[0015]
[Equation 3]

ε_m  : Dielectric constant of suspension
ε^* _m  : Complex permittivity of suspension
ε^* _i  : Complex permittivity of microorganisms
σ Suspension conductivity
ω: Angular frequency of electric field
Also,
a: Radius of microorganism when approximated by a sphere
Re [K]: a parameter that depends on the complex permittivity of the microorganism and the suspension
E: Electric field strength
It is. As is clear from (Equation 1), (Equation 2), and (Equation 3), if the size of the suspension and the microorganism is constant, the dielectrophoretic force F_DEPIs proportional to the parameter Re [K].
[0016]
FIG. 9 is a frequency characteristic diagram of Re [K] using cytoplasmic conductivity as a parameter. In FIG. 9, the frequency f of the electric field used for dielectrophoresis is used as a parameter, and the dielectrophoretic force is expressed as cytoplasmic conductivity σ._iIt is expressed as a function of It is the conductivity of the solution inside the cytoplasm. According to FIG. 9, it can be seen that a positive dielectrophoretic force works at a frequency of 10 kHz to 1 MHz, and a negative dielectrophoretic force works otherwise. Cytoplasmic conductivity σ_iDepending on the frequency f, the negative dielectrophoretic force works, and the dielectrophoretic force F_DEPIt can be seen that there are cases in which even if it acts on microorganisms, it cannot be collected.
[0017]
Therefore, microorganisms (cytoplasmic conductivity σ_iWhen the frequency f is selected according to the type and life and death, it can be collected by applying a positive dielectrophoretic force or eliminated by applying a negative dielectrophoretic force. However, in actual dielectrophoresis, the influence of suspension conductivity and the like must be taken into consideration.
[0018]
[Problems to be solved by the invention]
As described above, it has been necessary to quantify the number and activity of microorganisms in various fields, but there is no appropriate quantification method to replace the colony counting method, and the colony counting method has generally been used. It was. However, this colony counting method requires a very long time of 1 to several days until a colony is formed, and although it can be quantified, it can only be used offline.
[0019]
Currently, there is a strong demand for speed in dealing with food poisoning and insurance hygiene. In addition, the production system for useful substances using yeast and useful microorganisms is controlled with high efficiency, and yield control of products is performed. Therefore, it is required to quantify the number and activity of microorganisms in real time. However, the above-described colony counting method and ATP method take time for quantification itself and cannot completely realize real-time measurement, and there are many reagents used for the measurement. It was an advanced and complex task, and it wasn't possible for ordinary people to make measurements.
[0020]
In that respect, the DEPIM method proposed by the inventor together with other researchers has a great feature in that it does not take a long time to measure because both processes of microbial concentration and microbial concentration detection are electrically performed. In addition, it has an extremely excellent feature that the estimation time of the bacteria concentration can be shortened by using no reagent for detection and statistically examining the time-dependent change of the dielectrophoresis phenomenon.
[0021]
Although this is an excellent DEPIM method, the complex dielectric constant K in (Equation 1) relating to microorganisms, and therefore Re [K], is determined by the microorganisms (such as Crane) as the activity of microorganisms (live and dead). It is pointed out that it is strongly influenced by However, there is no method for distinguishing and quantifying this activity. In order to be able to measure microbial activity in real time using the DEPIM method, it is necessary to clearly distinguish between live and dead microorganisms in a sample solution and to develop a technique for detecting only live bacteria.
[0022]
That is, when detecting the activity of microorganisms from the viewpoint of food poisoning and health insurance, sterilization is usually performed in parallel with detection, and dead bacteria increase in real time. Even in the production system used, it is necessary to grasp the increase and decrease of live and dead bacteria in real time.
[0023]
Therefore, in order to solve such a problem, an object of the present invention is to provide a microbial activity measuring apparatus that has near-real-time speediness and allows anyone to detect microbial activity simply and quantitatively.
[0024]
Another object of the present invention is to provide a method for measuring microbial activity, which has rapidity close to real time and allows anyone to detect microbial activity simply and quantitatively.
[0025]
[Means for Solving the Problems]
  In order to solve the above problems, the microbial activity measuring apparatus of the present invention has a suspension conductivity for each microorganism., When the ratio of conductance with only live bacteria and conductance with only dead bacteria is taken, the conductance of only dead bacteria becomes 10% or lessAnd a memory unit storing an activity measurement optimum frequency and an activity measurement table storing calibration curve data, and when the type of microorganism contained in the sample solution and the suspension conductivity are input from the input means, The calculation control unit reads the optimum frequency for activity measurement from the activity measurement table, and the power supply unit for dielectrophoresis applies an alternating voltage of the optimum frequency for activity measurement to concentrate viable bacteria among the microorganisms, and measures by the measurement unit Based on the calibration curve data, the number of microorganisms is calculated from the change in conductance between the electrodes based on the measured impedance and output.
[0026]
Thereby, anyone can detect microbial activity simply and quantitatively with near real time rapidity.
[0027]
  In addition, the method for measuring microbial activity of the present invention comprises a pair of electrodes immersed in a sample solution.When the ratio of the conductance for only living bacteria and the conductance for only dead bacteria is taken, the conductance of only the dead bacteria becomes 10% or less.Apply an alternating voltage of the optimal frequency for activity measurement, dielectrophoresis and concentrate live bacteria in a specific microorganism contained in the sample solution, measure the impedance between the electrodes, and measure the impedance between the electrodes based on the measured impedance The number of microorganisms is calculated from the change in conductance.
[0028]
Thereby, anyone can detect microbial activity simply and quantitatively with near real time rapidity.
[0029]
DETAILED DESCRIPTION OF THE INVENTION
  The invention according to claim 1 of the present invention is a measurement chamber that can store a sample solution, and is immersed in the sample solution in the measurement chamber, generates an unequal electric field, and is contained in the sample solution by dielectrophoresis. A pair of electrodes for measuring the impedance at this time, a power source unit for dielectrophoresis that applies an alternating voltage between the electrodes, a measurement unit that can measure the impedance between the electrodes, Computational control unit that controls the dielectrophoresis power supply unit and measurement unit, and suspension conductivity for each microorganism, When the ratio of conductance with only live bacteria and conductance with only dead bacteria is taken, the conductance of only dead bacteria becomes 10% or lessAnd a memory unit storing an activity measurement optimum frequency and an activity measurement table storing calibration curve data, and when the type of microorganism contained in the sample solution and the suspension conductivity are input from the input means, The calculation control unit reads the optimum frequency for activity measurement from the activity measurement table, and the power supply unit for dielectrophoresis applies an alternating voltage of the optimum frequency for activity measurement to concentrate viable bacteria among the microorganisms, and measures by the measurement unit This is a microbial activity measuring device that calculates and outputs the number of viable microorganisms from changes in conductance between electrodes based on calibration curve data based on the measured impedance. When input by means, the optimum frequency for activity measurement is read from the activity measurement table, and an unequal electric field is generated between the electrodes by applying an AC voltage of this activity measurement optimum frequency. Thereby, only the live cells of the microorganism can be concentrated by dielectrophoresis by dielectrophoretic force generated thereby.At this time, the optimal frequency for measuring the activity is a positive dielectrophoretic force acting on live bacteria, and a negative dielectrophoretic force acting on dead bacteria, or practically (in other words, almost) positive. Since the dielectrophoretic force does not act, it is possible to separate live and dead bacteria while maintaining practical accuracy, and the positive dielectrophoretic force can be increased within the separable frequency. This can concentrateOnly live bacteria. Simultaneously with this concentration, impedance measurement is performed to detect changes in the conductance between the electrodes due to the concentration of viable bacteria. By utilizing the fact that the change in conductance is proportional to the number of microorganisms (viable count), The number of viable bacteria can be calculated and output.When the optimal frequency for activity measurement is selected, the reason why some dead bacteria remain at the beginning is that a large positive dielectrophoretic force acting on live bacteria acts on dead bacteria that existed near the electrode at the start of measurement. Although it does not, negative dielectrophoretic force or dielectrophoretic force in the vicinity of 0 acts, and it takes time to be excluded out of the region.
[0031]
  Claims of the invention2According to the invention described in (2), the arithmetic and control unit determines whether the conductance between the electrodes is initially increased.ViableThe number of microorganisms is calculated and output.1Since the microorganism activity measuring apparatus is described, when concentrating microorganisms by dielectrophoresis, it is possible to estimate the microorganism concentration in the sample liquid with the increase rate of conductance at the initial stage of the concentration process, so that it can be evaluated rapidly in real time. Measurement becomes possible.
[0032]
  Claims of the invention3In the invention described in item 1, the stirring chamber is provided in the measurement chamber, and the calculation control unit stirs the sample liquid based on the time measured by the clock means.Or 2Since the microbial activity measuring apparatus is described, when the sample solution is introduced for dielectrophoresis, the microbial concentration can be made uniform as a whole and more microorganisms can be led to the vicinity of the electrode.
[0033]
  Claims of the invention4According to the invention, the measurement chamber is provided with a supply pipe and a discharge pipe for the sample solution, a supply valve is provided in the supply pipe, and a discharge valve is provided in the discharge pipe. And opening the supply valve to supply the sample liquid to the measurement chamber, and opening the discharge valve after the measurement to discharge the sample liquid.1-3Therefore, the sample liquid can be automatically supplied and drained automatically after the measurement.
[0034]
  Claims of the invention5The measurement chamber is provided with a cleaning pipe for cleaning liquid, and a cleaning valve is provided for the cleaning pipe. Based on the time measured by the clock control means, the arithmetic control unit opens the cleaning valve and supplies the cleaning liquid to the measurement chamber. The inside of the chamber is washed after the sample liquid is supplied and discharged.1-4Since the microbial activity measuring apparatus according to any one of the above, the post-drainage cleaning liquid can be introduced into the measuring chamber and the inside of the measuring chamber can be automatically cleaned.
[0035]
  Claims of the invention6The invention described in is between a pair of electrodes immersed in a sample solution.When the ratio of the conductance for only living bacteria and the conductance for only dead bacteria is taken, the conductance of only the dead bacteria becomes 10% or less.Apply an alternating voltage of the optimal frequency for activity measurement, dielectrophoresis and concentrate live bacteria in a specific microorganism contained in the sample solution, measure the impedance between the electrodes, and measure the impedance between the electrodes based on the measured impedance From the conductance change ofViableSince it is a method for measuring microbial activity characterized by calculating the number of microorganisms, an inequality electric field is generated between the electrodes by applying an alternating voltage at the optimum frequency for activity measurement, and the microorganisms are generated by the dielectrophoretic force generated thereby. Of these, only viable bacteria can be concentrated by dielectrophoresis.At this time, the optimal frequency for measuring the activity is a positive dielectrophoretic force acting on live bacteria, and a negative dielectrophoretic force acting on dead bacteria, or practically (in other words, almost) positive. Since the dielectrophoretic force does not act, it is possible to separate live and dead bacteria while maintaining practical accuracy, and the positive dielectrophoretic force can be increased within the separable frequency. This can concentrateOnly live bacteria. Simultaneously with this concentration, impedance measurement is performed to detect changes in the conductance between the electrodes due to the concentration of viable bacteria. By utilizing the fact that the change in conductance is proportional to the number of microorganisms (viable count), The number of viable bacteria can be calculated and output.
[0037]
  Claims of the invention7The invention according to claim 2, wherein the number of microorganisms is calculated from an initial increase rate of conductance between the electrodes.6Since the microbial activity measuring method is described, the microbial concentration in the sample liquid can be estimated by the conductance increase rate at the initial stage of the concentration process by dielectrophoresis, and the measurement can be performed rapidly enough to evaluate in real time.
[0038]
(Embodiment 1)
Hereinafter, the microbial activity measuring apparatus and the microbial activity measuring method according to Embodiment 1 of the present invention will be described. FIG. 1 is a configuration diagram of a microbial activity measuring apparatus according to Embodiment 1 of the present invention, FIG. 2A is a control data diagram for measurement of an activity measuring table of the microbial activity measuring apparatus according to Embodiment 1 of the present invention, 2B is a calibration diagram of the activity measurement table of the microorganism activity measuring apparatus according to the first embodiment of the present invention, and FIG. 3 affects the dielectrophoretic force of the microorganism activity measuring apparatus according to the first embodiment of the present invention. FIG. 4 is a relationship diagram between the cytoplasmic conductivity and the electric field frequency, FIG. 4 is a detection diagram of viable and dead bacteria at the electric field frequency of 100 kHz of the microbial activity measuring apparatus according to the first embodiment of the present invention, and FIG. 5 is the first embodiment of the present invention. FIG. 6 is a detection diagram of viable and dead bacteria at the electric field frequency of 1 MHz of the microbial activity measuring apparatus in FIG. 6, and FIG. 6 shows viable bacteria and dead bacteria from the mixed suspension of viable and dead bacteria in the microbial activity measuring apparatus in Embodiment 1 of the present invention. That dead bacteria are isolated FIG. 7 is a diagram showing the heat sterilization time dependence of the viable cell count of the microbial activity measuring apparatus according to the first embodiment of the present invention. FIG. 8 is a microbial activity measuring apparatus according to the first embodiment of the present invention. It is a microbe activity measurement figure after heat-sterilization of.
[0039]
In FIG. 1, 1 is a measuring chamber for storing a sample solution for measuring viable bacteria in microorganisms by the DEPIM method (Dielectrophoretic Impedance Measurement Method), 2 is a chip electrode part for measuring microbial activity, 2a, Reference numeral 2 b denotes a pair of electrodes constituting the electrode unit 2. The electrodes 2a and 2b generate a non-uniform electric field in order to perform dielectrophoresis, and have a shape capable of bridging a large number of microbial chains called pearl chains between the electrodes 2a and 2b, such as castle wall electrodes and comb-shaped electrodes. Or the like. The castle wall type electrode is formed by disposing electrodes 2a and 2b each having a plurality of rectangular electrode pieces protruding at intervals of one pitch (for example, 50 μm to 100 μm) on both sides, with a pitch of 5 μm to 10 μm. The electric field concentrates between the edges of the rectangular electrode pieces 2a and 2b. In addition, the comb-shaped electrode has a pair of electrodes in which teeth (for example, 30 μm to 100 μm width) formed like a comb are nested and inserted into the groove, and is opposed by a narrow gap (for example, 5 μm to 10 μm width). It is an electrode, and an unequal electric field is mainly formed between edges in the thickness direction, and a number of pearl chains are formed. It is desirable to select the most appropriate value for the gap and pitch depending on the size of the microorganism.
[0040]
The electrodes 2a and 2b are preferably formed as thin film electrodes such as chromium and platinum, formed on a glass substrate or plastic substrate by sputtering, vapor deposition, plating or the like, and etched by photolithography or the like. A thin film having a thickness of about 50 nm to 200 nm is desirable for forming a large number of pearl chains. The material of the electrodes 2a and 2b is not limited to chromium or platinum, but may be any metal that does not cause electrolysis when an AC voltage is applied and has a low ionization tendency.
[0041]
By the way, as shown in FIG. 1, the measurement chamber 1 can be configured to cover the substrate of the electrode unit 2 in addition to accommodating the electrode unit 2 as a separate body. For example, if a spacer is arranged around the electrodes 2a and 2b and a glass plate, a plastic plate or the like is placed on the spacer and bonded, the minute measurement chamber 1 can be made as a compact structure. This is effective when the amount of the sample solution is small, and it is desirable to guide the microorganisms to the vicinity of the electrodes 2a and 2b and make the microorganism concentration uniform by flowing as a closed circuit instead of stirring.
[0042]
Next, 3 is a power supply unit for dielectrophoresis that applies an alternating voltage to generate dielectrophoresis between the electrodes 2a and 2b, 4 is a measurement unit that can measure the impedance between the electrodes 2a and 2b, and 5 is a micro An arithmetic control unit comprising a processor and the like, functions by loading a control program and various data into the work area, controls at least the dielectrophoresis power supply unit 3 and the measurement unit 4, and 6 is a clock means. is there. The power source unit 3 for dielectrophoresis is controlled by the arithmetic control unit 5 with respect to the voltage and frequency of the alternating voltage applied between the electrodes 2a and 2b. In the first embodiment, a function generator that can change the frequency between 1 kHz and 10 MHz and can apply a peak-to-peak voltage (hereinafter referred to as pp) of 1 Vpp to 20 Vpp is employed. In this specification, the AC voltage means a voltage such as a sine wave, a triangular wave, a square wave, etc. that changes the flow direction at a substantially constant cycle, and the average value of the currents on both the positive and negative sides is equal. is there. The optimum frequency for measuring the activity described later varies depending on the type of microorganism and the conductivity of the suspension, and the voltage is selected such that the electrophoretic force does not occur between the electrodes 2a and 2b and the dielectrophoretic force is increased.
[0043]
The measurement unit 4 is provided with a resistance for current detection of about 500Ω and is inserted in series in the voltage application circuit shown in FIG. 1, and the current flowing through this is measured to determine the conductance (resistance component between the electrodes 2a and 2b. The reciprocal of) is calculated. That is, the arithmetic control unit 5 controls the frequency and voltage of the power source unit 3 for dielectrophoresis at a timing of, for example, 10 sec, which is measured by the clock means 6, and an AC voltage having an optimum frequency for activity measurement between the electrodes 2a and 2b. At the same time, the measurement unit 4 measures the current flowing through the voltage application circuit, measures the resistance component due to the concentration of microorganisms in the sample solution, and calculates the conductance by taking the reciprocal. ing. The clock means 6 performs not only the sampling timing but also time management for scheduling for the automatic operation of the microorganism activity measuring apparatus according to the first embodiment.
[0044]
Now, 7 is a memory unit storing a program to be loaded into the arithmetic control unit 5 and various data, 7a is provided in the memory unit 7, and is a measurement control for collecting and concentrating only living bacteria among microorganisms. It is an activity measurement table that stores data and calibration curve data for converting the number of viable bacteria from the output of the measurement unit 4 indicating conductance (hereinafter referred to as DEPIM output). The control data for measurement includes, for each microorganism, the suspension conductivity of the sample solution in which the microorganism is suspended, the positive dielectrophoretic force acting on the living bacteria, and the negative dielectrophoretic force on the dead bacteria or a dielectric near zero. A database of optimum frequencies for activity measurement capable of applying the electrophoretic force. Selecting a frequency greater than the optimal frequency for activity measurement reduces the positive dielectric fluid power for concentrating viable bacteria, and selecting a frequency lower than the optimal frequency for activity measurement makes it difficult to separate live and dead bacteria. Measurement accuracy decreases. Therefore, it is optimal to use the optimum frequency for activity measurement, but it is also conceivable to use a frequency larger than this. However, the measurement takes time and is not preferable from the relationship with the measurement unit 4.
[0045]
Therefore, hereinafter, the measurement control data and calibration curve data stored in the activity measurement table 7a will be described in detail. As shown in FIG. 2A, the measurement control data includes at least the name of the microorganism, the suspension conductivity of the suspension that is the solvent of the sample solution, the peak-to-peak voltage of the applied AC voltage, and the activity measurement. Optimal frequencies are stored in relation to each other. That is, what is stored as measurement control data is the suspension conductivity associated with each type of microorganism, the peak-to-peak voltage applied at this time, the activity measurement optimum frequency, and the like. In FIG. 2 (a), only one type of conductivity is stored for each microorganism in the suspension conductivity. However, a plurality of types of conductivity are stored for each microorganism, and the peak-to-peak voltage and the activity are measured for each type. It is preferred to relate the optimum frequency. FIG. 2B shows a calibration curve for converting the DEPIM output indicating the conductance detected by the measurement unit 4 and the viable count. The calibration curve data may be in the form of a table in which the DEPIM output value and the number of viable bacteria are correlated one to one for each type of microorganism, but the activity measurement table 7a with the calibration curve as a linear function. It is also desirable to store the data in the memory because the data amount can be reduced.
[0046]
Next, the optimum frequency for activity measurement will be described. In (Equation 1) showing the dielectrophoretic force by the DEPIM method, the complex dielectric constant K, and hence Re [K], is strongly influenced by the activity of microorganisms (distinguishing between live and dead bacteria) by multiple researchers. Has been pointed out. Researchers such as Crane et al. Say that when proteins (enzymes) contained in the cell membrane of microorganisms are denatured due to heat sterilization, etc., and defects occur in the cell membrane, various ions contained in the cytoplasm inside the cell membrane leak out through these defects, Conductivity σ of cytoplasm_iPoint out that it decreases with time.
[0047]
  FIG. 3 shows the result of theoretical determination of how the Re [K] in (Equation 1), that is, the dielectrophoretic force, changes when the cytoplasmic conductivity σi decreases. In FIG. 3, the dielectrophoretic force is expressed as a function of the cytoplasmic conductivity σi with the frequency f of the electric field used for dielectrophoresis as a parameter. According to FIG. 3, the dependence of the dielectrophoretic force on the cytoplasmic conductivity σi is small at a frequency of 100 kHz, but the dielectrophoretic force decreases with a decrease in the cytoplasmic conductivity σi at a frequency of 1 MHz. For example, the cytoplasmic conductivity σi is an initial value of 10 due to bacterial inactivation.^-1S/m to 10^-2S/When the frequency drops to m, the dielectrophoretic force decreases only by 5% at a frequency of 100 kHz, but decreases by 70% at 1 MHz.
[0048]
Therefore, cytoplasmic conductivity σ at a frequency of 1 MHz_iA collection of microorganisms with low (for example, σ_iIs 2 × 10^-3In the case of dielectrophoresis of a collection in which most of the vicinity of S / m is dead bacteria and a collection of living bacteria is mixed, cytoplasmic conductivity σ_iIt can be seen that there are few viable bacteria to which a positive dielectrophoretic force acts with the decrease in the number, and that dead bacteria to which a negative dielectrophoretic force acts occupy almost all. However, when this is a frequency of 100 kHz, a positive dielectrophoretic force acts on dead bacteria, and it can be seen that the same set of microorganisms that were not concentrated at a frequency of 1 MHz are concentrated together with the dead bacteria. If this is 500 kHz, σ_iIs 2 × 10^-3Dielectrophoretic force hardly acts on most dead bacteria near S / m, and the cytoplasmic conductivity σ beyond that_iA slightly positive dielectrophoretic force acts on dead bacteria with.
[0049]
These facts indicate that when the frequency of performing dielectrophoresis on dead bacteria is increased, negative dielectrophoretic force or almost no dielectrophoretic force acts, and positive dielectrophoresis occurs only on live bacteria. It shows that force comes to act. However, as the frequency increases, the dielectrophoretic force itself tends to decrease, and the time required for the dielectrophoresis becomes longer. In addition, when the frequency is higher than 10 MHz, the measurement accuracy can be obtained as expected from the capability of the measurement unit 4. Disappear. Therefore, it can be seen that the optimum frequency for activity measurement is the most effective frequency for selectively concentrating viable dead bacteria.
[0050]
In order to determine the optimum frequency for activity measurement, it is preferable to determine by performing the following experiment. That is, a sample solution containing only live bacteria and a sample solution containing only dead bacteria are prepared at the same microorganism concentration, and measurement is performed at the same frequency, voltage, and suspension conductivity. As the frequency is increased, the DEPIM signal indicating the conductance of only live bacteria becomes relatively larger than that of dead bacteria. The frequency when the DEPIM signal from the measurement unit 4 for the dead bacteria is not detected is set as the optimum activity measurement frequency. This is because, in this state, dead bacteria are excluded from the measurement region in the vicinity of the electrodes 2a and 2b as a result. The optimum frequency for activity measurement shown in FIG. 2A is determined in this way, and the experiment is repeated for each microorganism and each suspension to create a precise activity measurement table 7a. Is appropriate.
[0051]
  By the way, as can be seen from the above description, there is a range of allowable frequencies that are not optimal in the vicinity of the optimal frequency for activity measurement. That is, it is a frequency range where practically no positive dielectrophoretic force acts on dead bacteria. Even if no DEPIM signal for dead bacteria is detected, if the frequency is 10% or less when the ratio of the DEPIM signal for live bacteria to that for dead bacteria is taken, measurement is performed while maintaining measurement accuracy. Is possible. Therefore, this range is also broadly defined as the optimum frequency for activity measurement.(Optimum frequency for activity measurement of the present invention)Can be included.
[0052]
Then, referring to the frequency characteristic diagram of Re [K] with the cytoplasmic conductivity as a parameter shown in FIG. 9, although it depends on the cytoplasmic conductivity, when it exceeds 10 MHz, the positive dielectrophoretic force is similar to 10 kHz or less. Shifts from a state in which the negative action acts to a state in which a negative dielectrophoretic force acts. As described above, in the high frequency region of 10 MHz or higher, the measurement unit 4 cannot expect accuracy. Therefore, in the present invention, this is avoided, and the optimum activity measurement frequency is determined while increasing the frequency below 10 MHz. Even in the above high-frequency region, if there is a frequency at which the ratio of the DEPIM signal of viable bacteria to that of dead bacteria is 10% or less, there is a possibility of measuring the microbial activity with a certain degree of accuracy.
[0053]
Subsequently, in FIG. 1, 8 is a data input unit (input means of the present invention in Embodiment 1) for inputting the name of microorganism and suspension conductivity, 9 is a display for data input, It is a display unit such as an LCD for displaying the viable cell count calculated by the arithmetic control unit 5 based on the calibration curve data. Reference numeral 10 denotes a sample solution tank that stores a sample solution containing microorganisms, and 11 denotes a supply pipe that connects the sample solution tank 10 and the measurement chamber 1 and guides the sample solution in the sample solution tank 10 to the measurement chamber 1. Reference numeral 12 denotes a discharge pipe for discharging the measured sample solution from the measurement chamber 1, 13 denotes a supply valve such as an electromagnetic valve provided in the supply pipe 11, and 14 denotes a discharge valve provided in the discharge pipe 12. Reference numeral 15 denotes a stirrer such as a stirrer, which stirs the sample liquid before or during the dielectrophoresis by the arithmetic control unit 5 based on the time measured by the clock means 6. As a result, when the sample solution is introduced from the supply pipe 11 and subjected to dielectrophoresis, the concentration of microorganisms can be made uniform as a whole, and more microorganisms can be led to the vicinity of the electrodes 2a and 2b.
[0054]
  Further, in the case of a minute measurement chamber 1 in which a spacer is arranged around the electrodes 2a and 2b and a glass plate or the like is placed on and adhered to the measurement chamber 1, the measurement chamber 1 is discharged instead of the stirring device 15. Connect the tube 12 to the sample liquid layer 10In the closed stateIt is desirable to use it in combination with a method in which the microorganisms are guided to the vicinity of the electrodes 2a and 2b by flowing, and the concentration of the microorganisms is made uniform. Reference numeral 16 is a cleaning liquid tank, 17 is a cleaning pipe, and 18 is a cleaning valve. The arithmetic control unit 5 opens the cleaning valve 18 to supply the cleaning liquid to the measurement chamber 1, and after discharging the sample liquid, the inside of the measurement chamber 1 is cleaned. With the time management of the clock means 6, the cleaning liquid after draining can be introduced into the measurement chamber 1, and the inside of the measurement chamber 1 can be automatically cleaned to prepare for the next measurement. Although it is not indispensable to wash with a washing liquid for the measurement by the microbial activity measuring apparatus of the present invention, it is desirable to wash the microorganism-containing sample liquid immediately after the measurement, so in this Embodiment 1, a washing system is provided. ing.
[0055]
Furthermore, the microorganism activity measuring apparatus according to the first embodiment is provided with a measurement self-checking mechanism so that it can correctly measure without much knowledge. That is, if the microbial activity measuring device is not properly prepared, it will be useless even if measurement is performed. Therefore, the present microbial activity measuring apparatus performs a self-check immediately after the start of measurement. Microorganisms are not collected in the state where measurement has just started. When the DEPIM signal is detected by measuring the impedance between the electrodes 2a and 2b in this state, the initial value depends only on the conductivity and dielectric constant of the suspension. If the calculation control unit 5 detects the DEPIM signal immediately after the start of measurement and determines that the initial value indicated by the suspension is significantly different from the normal value range, the electrode unit 2 is damaged and short-circuited. The alarm is sounded or a warning is displayed on the display unit 9 to prompt the user to check or replace the electrode or the device. The normal value of the initial value indicated by the suspension may be determined for each suspension in the activity measurement table 7a or may be determined using the stored calibration curve data. By performing such a self-check, microbial activity can be easily and reliably measured without being an expert.
[0056]
Next, an operation procedure and a measurement result when the microorganism activity measuring apparatus according to the first embodiment is operated will be described. In FIG. 1, a power switch (not shown) is input, and the type of microorganism and the suspension conductivity are input from the data input unit 8 according to the guidance displayed on the display unit 9. The arithmetic control unit 5 performs a self-check, reads an AC voltage and an optimum activity measurement frequency applied from the activity measurement table 7a based on the input, controls the dielectrophoresis power supply unit 3, and applies them to the electrodes 2a and 2b. . At the same time, the measurement unit 4 performs impedance measurement.
[0057]
Every predetermined time (for example, 10 sec) timed by the clock means 6, the measuring unit 4 measures the conductance that changes as the microorganisms are concentrated, and based on this result, the arithmetic control unit 5 uses the calibration curve from the activity measurement table 7a. Read the data and convert the number of viable bacteria.
[0058]
  Next, the results when microbial activity is measured at a frequency of 100 kHz under the condition of suspension conductivity of 0.1 mS / m using live and dead Escherichia coli as microorganisms will be described. Dead bacteria were prepared by heat sterilization (80 ° C., 15 minutes), and inactivation was confirmed by a colony counting method. Figure4As shown in FIG. 4, at the frequency of 100 kHz, almost the same increase in conductance is observed for both live and dead bacteria. This increase in conductance is a result of E. coli in the suspension being collected between the electrode gaps 2a and 2b by the dielectrophoretic force.
[0059]
  And the result of having performed the same measurement at the optimum frequency of activity measurement 1MHz5Shown in In live bacteria, an increase in conductance similar to that at a frequency of 100 kHz was observed, but in dead bacteria, the conductance was almost constant. As a result of observing the dielectrophoresis phenomenon in the vicinity of the electrode using a microscope, it was confirmed that the dielectrophoretic force did not act on the dead bacteria at the frequency of 1 MHz, and the dead bacteria were not collected on the electrode. Thus, only viable bacteria can be detected by setting the electric field frequency to 1 MHz which is the optimum frequency for activity measurement.
[0060]
Next, FIG. 6 shows an example in which selective microbial activity measurement was performed on an E. coli suspension sample solution in which live and dead bacteria were turbid. The DEPIM output indicating the conductance of the sample liquid in which live bacteria and dead bacteria are mixed is almost identical to the output in the case of only living bacteria. Thereby, it turns out that only a living microbe can be selectively detected and quantified from mixed suspension. When the mixing ratio of viable bacteria is changed, the DEPIM output of the mixed solution changes correspondingly.
[0061]
It is known that the viable cell count after the heat sterilization treatment decreases exponentially with respect to the heating time as shown in FIG. Since only the viable cell count can be selectively detected by the microbial activity measuring apparatus of the first embodiment, if the measurement as shown in FIG. ) Can be detected in a short period of time and almost as quickly as real time. FIG. 8 shows the results of measuring the number of viable bacteria depending on the heat sterilization time by the DEPIM method for the above-mentioned E. coli suspension sample solution. FIG. 8 also shows changes in the number of viable bacteria determined by the culture method for comparison. Note that the DEPIM output in FIG. 8 is standardized. As shown in the figure, the viable cell count measured by the DEPIM method corresponds well with that determined by the culture method, and it can be measured how the viable cell count decreases exponentially with the heating time. . In this case, the time required for the measurement by the DEPIM method is within 10 minutes, and a change in the viable cell count can be detected in a very short time compared to the time required for the culture method (about 24 hours). That is, since the impedance can be measured in real time, the activity of the microorganism can be measured almost in real time by the DEPIM method.
[0062]
【The invention's effect】
  In the microorganism activity measuring apparatus according to claim 1 of the present invention, when the type of microorganism contained in the sample solution and the suspension conductivity are input from the input means, the arithmetic control unit reads from the activity measuring table.When the ratio of the conductance for only living bacteria and the conductance for only dead bacteria is taken, the conductance of only the dead bacteria becomes 10% or less.Reads the optimum frequency for activity measurement, the power supply unit for dielectrophoresis applies an AC voltage of the optimum frequency for activity measurement to concentrate viable bacteria among the microorganisms, and the electrode based on the calibration curve data by the impedance measured by the measurement unit Since the number of microorganisms is calculated from the change in conductance between them and output, when the microorganism name and suspension conductivity are input by the input means, the optimum activity measurement frequency is read from the activity measurement table, and this activity measurement optimum frequency is read. In this way, an inequality electric field is generated between the electrodes, and only the living bacteria among the microorganisms can be concentrated by dielectrophoresis by the dielectrophoretic force generated thereby.The optimum frequency for measuring the activity is a positive dielectrophoretic force acting on live bacteria and a negative dielectrophoretic force acting on dead bacteria, or practically (in other words, a positive dielectric). Since the electrophoretic force does not act, it is possible to separate live and dead bacteria while maintaining practical accuracy and to increase the positive dielectrophoretic force within the separable frequency. This can concentrateOnly live bacteria. Simultaneously with this concentration, impedance measurement is performed to detect changes in the conductance between the electrodes due to the concentration of viable bacteria. By utilizing the fact that the change in conductance is proportional to the number of microorganisms (viable count), The number of viable bacteria can be calculated and output. Real-time microbial activity can be measured, useful information for fermentation process control can be obtained, and it is useful for quality improvement and cost reduction. Moreover, the bactericidal effect can be confirmed quickly for the whole food production process, and it can be established as a quick and simple bacteria detection technique.
[0064]
  Claims of the invention2Since the microbial activity measuring apparatus described in (1) calculates and outputs the number of microorganisms from the initial increase rate of conductance between the electrodes, when concentrating the microorganisms by dielectrophoresis, the conductance increase rate at the initial stage of the concentration process is increased in the sample solution. The microbial concentration can be estimated, and the measurement can be performed rapidly enough to evaluate in real time.
[0065]
  Claims of the invention3In the microbial activity measuring apparatus described in 1, the stirring chamber is provided in the measurement chamber, and the sample liquid is stirred before dielectrophoresis. Therefore, when introducing the sample liquid and performing dielectrophoresis, the microbial concentration is uniform throughout. And more microorganisms can be led to the vicinity of the electrode.
[0066]
  Claims of the invention4The microbial activity measuring device described in 1) opens the supply valve to supply the sample liquid to the measurement chamber, and after the measurement opens the discharge valve to discharge the sample liquid. Therefore, the sample liquid is automatically supplied and automatically measured after the measurement. Can be drained.
[0067]
  Claims of the invention5The microbial activity measuring device described in 1) opens the cleaning valve, supplies the cleaning liquid to the measuring chamber, discharges the sample liquid, and then cleans the inside of the chamber. After draining, the cleaning liquid is introduced into the measuring chamber and automatically The inside can be cleaned.
[0068]
  The method for measuring microbial activity according to claim 6 of the present invention comprises:When the ratio of the conductance for only living bacteria and the conductance for only dead bacteria is taken, the conductance of only the dead bacteria becomes 10% or less.The AC voltage of the optimal frequency of the activity measurement is applied, and viable bacteria in a specific microorganism contained in the sample solution are dielectrophoretically concentrated and the impedance between the electrodes is measured.ViableSince the number of microorganisms is calculated, an AC voltage of the optimal frequency for activity measurement is applied to generate an unequal electric field between the electrodes,By virtue of the dielectrophoretic force generated thereby, only viable bacteria among the microorganisms can be concentrated by dielectrophoresis. At this time, the optimal frequency for measuring the activity is a positive dielectrophoretic force acting on live bacteria, and a negative dielectrophoretic force acting on dead bacteria, or practically (in other words, almost) positive. Since the dielectrophoretic force does not act, it is possible to separate live and dead bacteria while maintaining practical accuracy, and the positive dielectrophoretic force can be increased within the separable frequency. This can concentrateOnly live bacteria. Simultaneously with this concentration, impedance measurement is performed to detect changes in the conductance between the electrodes due to the concentration of viable bacteria. By utilizing the fact that the change in conductance is proportional to the number of microorganisms (viable count), The number of viable bacteria can be calculated and output. Real-time microbial activity can be measured, useful information for fermentation process control can be obtained, and it is useful for quality improvement and cost reduction. Moreover, the bactericidal effect can be confirmed quickly for the whole food production process, and it can be established as a quick and simple bacteria detection technique.
[0070]
  Claims of the invention7Since the method for measuring microbial activity described in (1) calculates the number of microorganisms from the initial increase rate of conductance between electrodes, the concentration of microorganisms in the sample solution can be estimated from the increase rate of conductance at the beginning of the concentration process by dielectrophoresis. Real-time and fast enough to be evaluated.
[Brief description of the drawings]
FIG. 1 is a configuration diagram of a microbial activity measuring apparatus according to a first embodiment of the present invention.
FIG. 2A is a control data diagram for measurement in the activity measurement table of the microorganism activity measuring apparatus according to the first embodiment of the present invention. FIG. 2B is a diagram showing the activity measurement of the microorganism activity measuring apparatus according to the first embodiment of the present invention. Calibration curve for table
FIG. 3 is a relationship diagram of cytoplasmic conductivity and electric field frequency on the dielectrophoretic force of the microorganism activity measuring apparatus according to the first embodiment of the present invention.
FIG. 4 is a detection diagram of viable and dead bacteria at an electric field frequency of 100 kHz of the microbial activity measuring apparatus according to the first embodiment of the present invention.
FIG. 5 is a detection diagram of viable and dead bacteria at an electric field frequency of 1 MHz of the microorganism activity measuring apparatus according to Embodiment 1 of the present invention.
FIG. 6 is a diagram showing that live bacteria and dead bacteria are separated from a mixed suspension of live bacteria and dead bacteria in the microorganism activity measuring apparatus according to Embodiment 1 of the present invention.
FIG. 7 is a diagram showing the heat sterilization time dependence of the viable cell count of the microbial activity measuring apparatus according to Embodiment 1 of the present invention.
FIG. 8 is a microbial activity measurement diagram after heat sterilization of the microbial activity measuring apparatus according to Embodiment 1 of the present invention.
FIG. 9 is a frequency characteristic diagram of Re [K] with the cytoplasmic conductivity as a parameter.
[Explanation of symbols]
1 Measurement chamber
2 electrodes
2a, 2b electrode
3 Power supply for dielectrophoresis
4 Measurement section
5 Operation control unit
6 Clock means
7 Memory part
7a Activity measurement table
8 Data input part
9 Display
10 Sample solution tank
11 Supply pipe
12 Discharge pipe
13 Supply valve
14 Discharge valve
15 Stirrer
16 Cleaning liquid tank
17 Washing tube
18 Cleaning valve

Claims (7)

A measurement chamber capable of containing a sample solution;
A pair of electrodes that are immersed in the sample solution in the measurement chamber, generate an unequal electric field, concentrate microorganisms contained in the sample solution by dielectrophoresis, and measure impedance at this time;
A dielectrophoresis power supply for applying an alternating voltage between the electrodes;
A measurement unit capable of measuring impedance between the electrodes;
A calculation control unit for controlling the power supply unit for dielectrophoresis and the measurement unit;
The optimum activity measurement frequency and calibration curve for which the conductance of only the dead bacteria is 10% or less when the ratio of the conductance of only the dead bacteria and the conductance of the suspension is determined for each microorganism. A memory unit storing an activity measurement table storing data;
When the type of microorganism and suspension conductivity contained in the sample solution are input from the input means, the calculation control unit reads the optimum activity measurement frequency from the activity measurement table, and the power source unit for dielectrophoresis Applies an alternating voltage of the optimal frequency for measuring the activity to concentrate viable bacteria among the microorganisms, and from the change in conductance between the electrodes based on the calibration curve data according to the impedance measured by the measurement unit , A microorganism activity measuring apparatus characterized by calculating and outputting the number of microorganisms.   The microbial activity measuring apparatus according to claim 1, wherein the arithmetic control unit calculates and outputs the number of microorganisms from an initial increase rate of conductance between the electrodes. The microbial activity measuring apparatus according to claim 1 or 2 , wherein a stirring device is provided in the measurement chamber, and the calculation control unit stirs the sample liquid based on the time measured by the clock means. The measurement chamber is provided with a supply pipe and a discharge pipe for the sample liquid, a supply valve is provided in the supply pipe, a discharge valve is provided in the discharge pipe, and the arithmetic control unit is based on the time measured by the clock means, The microbial activity measuring apparatus according to any one of claims 1 to 3 , wherein a sample solution is supplied to the measurement chamber by opening a supply valve, and the sample valve is discharged after the measurement by opening the discharge valve. The measurement chamber is provided with a cleaning liquid cleaning pipe, and the cleaning pipe is provided with a cleaning valve. Based on the time measured by the clock controller, the arithmetic control unit opens the cleaning valve to supply the cleaning liquid to the measurement chamber. The microbial activity measuring apparatus according to any one of claims 1 to 4 , wherein the inside of the chamber is washed after being supplied and the sample liquid is discharged. When the ratio of the conductance for only living bacteria and the conductance for only dead bacteria is taken between a pair of electrodes immersed in the sample solution, the AC of the optimal frequency for activity measurement is such that the conductance of only the dead bacteria is 10% or less. applying a voltage, the viable bacteria in the particular microorganism contained in the sample solution by measuring the impedance between the electrodes as well as concentrated dielectrophoresis, based on the measured impedance live from change in conductance between the electrodes A method for measuring microbial activity, comprising calculating the number of microorganisms of a bacterium . The method for measuring microbial activity according to claim 6, wherein the number of viable microorganisms is calculated from an initial increase rate of conductance between the electrodes.

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