JPH045440B2 - - Google Patents
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- Publication number
- JPH045440B2 JPH045440B2 JP58052772A JP5277283A JPH045440B2 JP H045440 B2 JPH045440 B2 JP H045440B2 JP 58052772 A JP58052772 A JP 58052772A JP 5277283 A JP5277283 A JP 5277283A JP H045440 B2 JPH045440 B2 JP H045440B2
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Links
- 238000000034 method Methods 0.000 claims description 22
- 239000006285 cell suspension Substances 0.000 claims description 11
- 239000000725 suspension Substances 0.000 claims description 7
- MWVTWFVJZLCBMC-UHFFFAOYSA-N 4,4'-bipyridine Chemical compound C1=NC=CC(C=2C=CN=CC=2)=C1 MWVTWFVJZLCBMC-UHFFFAOYSA-N 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 description 51
- 241000894006 Bacteria Species 0.000 description 14
- 238000005259 measurement Methods 0.000 description 13
- 244000005700 microbiome Species 0.000 description 10
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 6
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 6
- 235000014469 Bacillus subtilis Nutrition 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- 244000063299 Bacillus subtilis Species 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000002484 cyclic voltammetry Methods 0.000 description 3
- 238000001736 differential pulse polarography Methods 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 238000004832 voltammetry Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000186840 Lactobacillus fermentum Species 0.000 description 2
- 241000192130 Leuconostoc mesenteroides Species 0.000 description 2
- VZUNGTLZRAYYDE-UHFFFAOYSA-N N-methyl-N'-nitro-N-nitrosoguanidine Chemical compound O=NN(C)C(=N)N[N+]([O-])=O VZUNGTLZRAYYDE-UHFFFAOYSA-N 0.000 description 2
- 238000000086 alternating current polarography Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000003411 electrode reaction Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229940012969 lactobacillus fermentum Drugs 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 229910052709 silver Inorganic materials 0.000 description 2
- 239000004332 silver Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000000067 square-wave polarography Methods 0.000 description 2
- 238000010408 sweeping Methods 0.000 description 2
- 238000001075 voltammogram Methods 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 238000010953 Ames test Methods 0.000 description 1
- 231100000039 Ames test Toxicity 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 241000227653 Lycopersicon Species 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000001903 differential pulse voltammetry Methods 0.000 description 1
- 238000001318 differential pulse voltammogram Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
- 229910002804 graphite Inorganic materials 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 235000015193 tomato juice Nutrition 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本発明は各種生細胞の電気化学的活性測定方法
に係わり、より詳しくは微生物、動植物等の生細
胞懸濁液に於ける細胞数（生菌数）やその種類の
新規な測定乃至識別方法に関する。
微生物、動物、植物の細胞数の測定は極めて重
要である。従来、細胞数は濁度法や顕微鏡による
直接計数により測定されてきた。しかしこれらの
方法では、生細胞と死細胞の区別は困難であつ
た。また、コロニー計数法は生細胞数のみを計測
することができるが、測定に非常に時間がかかり
結果がでるまで１日以上要する。同じく、微生物
等の種類の分類学的識別乃至同定は臨床検査分野
を始めとして広汎な各種産業分野等に於いて極め
て重要な位置を占めているものであるが、その実
施は所謂選択培地を使用したコロニー計数法、顕
微鏡直接観察法等に依るものであるため著るしく
煩雑且つ長時間を要するものとならざるを得なか
つた。
他方、本発明者らは先に生細胞が電極に直接接
触すると電流が得られる現象を発見し、この現象
を利用した電気化学的菌数計測法を提案した
（Anal.Chim.Acta、98、25（1978）；Appl.
Environ.Microbiol.、37、117（1979）及びEur.J.
Appl.Microbiol.Biotechnol.、10、125（1980）も
のであるが、精度等の点で不充分なものであるの
みならず細胞の種類、菌学的性質等の識別に関し
ては全然未解明であつた。
上記に鑑み本発明者らは更に鋭意研究の結果、
細胞懸濁液に電流増強剤として４，4′−ビピリジ
ンの存在下所謂サイクリツクボルタメトリを始め
として微分パルスポーラログラフ、位相差弁交流
ポーラログラフ、矩形波ポーラログラフ等々の各
種ボルタメトリの手法に準じて走査電位を印加し
生起電流値を測定すれば極めて高精度で細胞数が
計測され得るのみならずピーク電位値等の異同に
より同時に細胞種類判別の情報も与えられること
を知見し、本発明に到達したものである。特に、
細胞懸濁液に微分パルスポーラログラフ、位相弁
別交流ポーラログラフ或いは矩形波ポーラログラ
フ等々の手法に準じて漸増直流走査電位とこれに
重畳された適切な微小電位とを印加し４，4′−ビ
ピリジンにより増強される生起微分電流値を測定
すれば極めて高精度に細胞の種類が識別され得る
ことが知見された。
すなわち、上記各方法により得られる電流値の
極値を与える電位値（ピーク電位値）は細胞の種
類に応じて相互に相違するのでこれにより各細胞
（微生物）相互の弁別、同定も又極めて明瞭に達
成され得るものとなる。
以下、本発明の構成等につきより詳細に分説す
る。
測定対象
細菌類、放線菌、カビ類、微細藻類、酵母類等
の各種微生物、赤血球、白血球、腫瘍細胞及び培
養動植物細胞等々の各種動植物細胞など、生細胞
であれば殆んど全ての微細生物が測定対象となり
得る。
ここに於いて、本発明方法に依ればこれら各種
微細生物の細胞濃度のみならずその菌学的弁別、
同定を始めとして、例えばグラム陰陽性菌の分
別、所謂Amesテストに於ける変異復帰又は非復
帰菌株の弁別等々、適切な測定条件を設定すれば
極めて広範囲に亘る識別が可能となるものであ
る。
測定諸条件及び適用分野
装置としては通常の各種ボルタメトリ用装置が
使用され得るものであるが、サイクリツクボルタ
メトリ用装置の１例につき模式説明図を示せば第
１図の通りである。すなわち、例示の装置は作用
極１、対極２及びSSCE等の参照極３を具備する
電解セル４、ポテンシオスタツト５、線型走査乃
至掃引電源６及びXY記録計乃至シンクロスコー
プ７より構成されている。ここに於いて、電極と
しては通常の白金、金、銀、炭素等々の電極及び
これらを高分子等で被覆等の各種修飾電極が使用
され得、又、対極２の電位が安定不変である場合
は参照電極３を欠く通常のポーラログラフと同等
の回路構成で足りる。測定は電解セル４に微生物
培養液等の生細胞懸濁液を注入し、電極間に周期
的走査（掃引）電位を印加して生起電流を測定す
ることによりなされるが、通常、その電位走査と
しては時間に比例して電位を変化させる所謂線型
走査（linear Sweep）が好適に採用される。
このようにして得られる電流−電位曲線
（Voltammogram）は、後に詳述する通り細胞濃
度に比例する極大電流値（ピーク電流）を与える
のみならず細胞の種類により相違するピーク電位
値等その曲線形状の特異性により細菌等その曲線
形状の特異性により細菌等の微生物の同定に充分
な情報をも与えるものとなる。すなわち、従来技
術と対比するとき本発明方法に依れば、応答時間
ひいては測定時間の著るしい短縮、走査電位によ
るため電極反応に於ける撹乱的諸要因が排除され
得るのでより精確な測定が可能となること並びに
細胞濃度（数）のみならずその種類の同定も同時
になされ得ること等々の実用上多大の利点が得ら
れるものである。従つて、本発明は発酵プロセス
のリアル・タイム制御用センサ、水質の微生物汚
染度の測定、赤血球や白血球数の測定等々、広汎
な各分野に於けるセンサ手段として極めて有用な
ものと云い得る。
他方、細胞識別に特に有用な微分パルスポーラ
ログラフ用装置の１例につき模式説明図を示せば
第２図の通りである。すなわち、例示の装置は
BPG（Basal Plane Pyrolytic Graphite）、HPG
（高純度分光分析用カーボン）等の炭素、白金、
金、銀等々より成る作用電極１１及び対極１２と
SSCE（飽和塩化ナトリウムカロメロ電極）等の
参照電極１３を具備するセル１４、パルスシーケ
ンサ１５、ポテンシヤルプログラマ１６、ポテン
シオスタツト１７、ドロツプノツカ１８、ｉ／Ｅ
コンバータ１９、サンプルホルド（τ）１１０及
び同（τ′）１１１、デイフアレンスアンプリフイ
ア１１２及びレコーダ１１３より構成されてい
る。
この場合、測定はセル１４に微生物培養液等の
生細胞懸濁液を注入し、電極間に微小電位の重畳
された漸増走査（掃引）電位を印加して生起電流
を測定することによりなされるが、通常、その電
位走査としては時間に比例して電位を変化させる
所謂線型走査（Linear Sweep）が好適に採用さ
れる。
走査電位に重畳される微小電位としては、前述
の通り微分電流値を与え得る適切な波形及び周期
を有するものが適宜選択使用され得る。
このようにして得られる電流−電位曲線
（Voltammogram）は細胞の種類によつて相違す
る明瞭なピーク電位値を与えこれによりそれらの
識別を可能とするのみならずそのピーク波形の解
析により細菌等の微生物の電気化学的活性に関す
る情報をも与えるものとなる。すなわち、従来技
術と対比するとき本発明方法に依れば各種細菌等
の識別、同定が極めて短時間且つ容易に達成され
るものである。
ここで、本発明方法に於ける細胞−電極間の電
子伝達の重要な賦活剤である４，4′−ビピリジン
（4.4′−bipyridine：BP）の使用条件につき要約
して示せば次の通りである。
すなわち、本発明に於いてBPは懸濁液中に直
接添加されて或いはニトロセルロース膜等に固定
されて電極に装着されることにより細胞−電極間
反応に関与するものとなる。尚、細胞懸濁液中に
於けるBPの濃度は対象細胞の種類によつて変動
するものであるが、一般には数mM〜100mM程
度である。
このようにしてBP共存下でボルタメトリを行
なうことにより、ピーク電流値の1.5〜2.5倍程度
の増強及び波形の鮮明化、微分電流値にあつては
ピークの先鋭化がもたらされるものとなる。
実施例 １
Saccharomyces cerevisiae（パン酵母；ATCC
7754）をペプトン２％、グルコース２％、酵母エ
キス１％からなる培地（PH 6.0）中で、８時間
好気培養した。これを集菌後、0.1Mリン酸緩衝
液で洗浄し、任意の濃度の細胞懸濁液（PH 7.0）
を調製した。この細胞懸濁液を15mlＨ型セルに入
れ、空気を10分間導入した後、直径６mmBPG電
極を挿入し、走査速度10mV／secでサイクリツ
クボルタメトリ法（第１図参照）により電気化学
的挙動を調べた。対極としては白金線電極、参照
電極としてはSSCE電極を用いた。
生細胞懸濁液（1.5×108個／ml）に電極を挿入
した時の、各走査電位におけるサイクリツクボル
タグラムを第３図に示す（図中、ｉは電流値
（μA）；Ｅは電位（Ｖ）；ａ、ｂは夫々50mMBP
無添加、添加の場合の波形である）。第３図から
明らかなように、0Vから1V（vs.SSCE）において
電位走査を行うと、BP添加の場合特に鮮明に
0.7V付近に増幅された電流のピークが得られた。
このピークはリン酸緩衝液のみのときや、オート
クレープで110℃、10分間減菌した細胞懸濁液で
は得られなかつた。また、BPG電極表面を透析
膜で覆つて、電極と細胞が直接接することができ
ないような状態でもピークが得られなかつた。以
上のことから、このピーク電流は生細胞が電極に
接触するとき得られることが明らかになつた。
ここで、50mMBP共存下で上記と同様にして
測定した細胞濃度Coとピーク電流値ipの関係を
第４図に示す。ipとCoの間には直線関係が成り
立つことが確かめられた。したがつて、ipの値よ
り酵母の細胞濃度を測定できることが明らかにな
つた。測定できる細胞濃度の範囲は106cells／ml
から109cells／mlで実際に発酵で測定の必要な範
囲を充分満たしているので、この方法により発酵
槽中の菌数の測定も可能であることが明らかにな
つた。
更に、第５図は細胞濃度1.5×108cells／mlの時
のBP濃度とピーク電流値ipの関係を示すもので
ある。
尚、Bacillus subtilis IFO 3009、
Lactobacillus fermentum IFO 3071、
Leuconostoc mesenteroides IFO 3832、
Escherichia coli等の場合も、BP共存により濃
度に比例するピーク電流値が1.5〜2.5倍増幅され
ると共にピーク波形の先鋭化が確認された。
実施例 ２
パン酵母（Saccharomyces cerevisiae）及び
枯草菌（Bacillus subtilis；IFO 3009）を下記第
１乃至第２表の組成の各培地（PH 7.0）で37℃、
常法により通気培養し、前記例各電極を培養槽に
挿入して10mMBP共存下リアル・タイムで生菌
数とピーク電流値ipとを追跡した（生菌数はコロ
ニー計数法で別途測定）。
結果を第３乃至４表に要約して示す。
これらから明らかなように、両者の極めて良好
な相関が認められた。
第１表
グルコース 40ｇ
ペプトン 10ｇ
イーストエキストラクト ５ｇ
KH2PO4 ５ｇ
MgSO4 ２ｇ
（精製水を加えて１）
第２表
グルコース 10ｇ
ペプトン 10ｇ
同エキス ５ｇ
NaCl ５ｇ
（精製水を加えて１）
The present invention relates to a method for measuring the electrochemical activity of various living cells, and more specifically to a novel method for measuring and identifying the number of cells (viable cell count) and their types in a suspension of living cells of microorganisms, animals, plants, etc. . Measuring the number of microbial, animal, and plant cells is extremely important. Conventionally, cell number has been measured by turbidity method or direct counting using a microscope. However, with these methods, it has been difficult to distinguish between live cells and dead cells. In addition, although the colony counting method can only measure the number of living cells, the measurement takes a very long time and takes more than one day to obtain the results. Similarly, taxonomic identification or identification of types of microorganisms, etc., plays an extremely important role in a wide variety of industrial fields, including the field of clinical testing, but this is carried out using so-called selective media. This method is extremely complicated and takes a long time because it relies on the colony counting method and the direct observation method using a microscope. On the other hand, the present inventors previously discovered a phenomenon in which an electric current is obtained when living cells come into direct contact with an electrode, and proposed an electrochemical bacterial counting method that utilizes this phenomenon (Anal.Chim.Acta, 98 , 25 (1978); Appl.
Environ.Microbiol., 37 , 117 (1979) and Eur.J.
Appl.Microbiol.Biotechnol., 10 , 125 (1980), but not only is it insufficient in terms of accuracy, but the identification of cell types, mycological properties, etc. is completely unknown. Ta. In view of the above, the present inventors further conducted extensive research and found that
In the presence of 4,4'-bipyridine as a current enhancer in the cell suspension, scanning potentials were measured according to various voltammetry techniques such as so-called cyclic voltammetry, differential pulse polarography, phase difference valve alternating current polarography, and square wave polarography. The present invention was achieved based on the discovery that not only can the number of cells be counted with extremely high accuracy by applying the voltage and measuring the generated current value, but also that information for distinguishing cell types can be provided based on differences in peak potential values, etc. It is. especially,
A gradually increasing DC scanning potential and an appropriate micropotential superimposed thereon are applied to the cell suspension according to methods such as differential pulse polarography, phase discrimination AC polarography, or square wave polarography, and the voltage is enhanced by 4,4'-bipyridine. It has been found that cell types can be identified with extremely high accuracy by measuring the differential current value generated by the cell. In other words, since the potential value (peak potential value) that gives the extreme value of the current value obtained by each of the above methods differs depending on the type of cell, it is also possible to distinguish and identify each cell (microorganism) from each other very clearly. can be achieved. Hereinafter, the structure of the present invention will be explained in more detail. Measurement targets: Almost all living microorganisms, including various microorganisms such as bacteria, actinomycetes, molds, microalgae, and yeast, as well as various animal and plant cells such as red blood cells, white blood cells, tumor cells, and cultured animal and plant cells. can be the measurement target. Here, according to the method of the present invention, not only the cell concentration of these various microorganisms but also their mycological discrimination,
In addition to identification, it is possible to differentiate over a very wide range by setting appropriate measurement conditions, such as the separation of Gram-negative bacteria and the discrimination of mutation-reverting and non-reverting bacterial strains in the so-called Ames test. Measurement Conditions and Field of Application Various conventional voltammetry devices can be used as the device, and FIG. 1 is a schematic illustration of an example of a cyclic voltammetry device. That is, the illustrated apparatus comprises an electrolytic cell 4 having a working electrode 1, a counter electrode 2, and a reference electrode 3 such as SSCE, a potentiostat 5, a linear scanning or sweeping power supply 6, and an XY recorder or synchroscope 7. . Here, as the electrode, ordinary platinum, gold, silver, carbon, etc. electrodes and various modified electrodes such as those coated with polymers etc. can be used, and if the potential of the counter electrode 2 is stable and unchanged. A circuit configuration equivalent to that of a normal polarograph lacking the reference electrode 3 is sufficient. Measurement is performed by injecting a living cell suspension such as a microbial culture solution into the electrolytic cell 4 and measuring the generated current by applying a periodic scanning (sweeping) potential between the electrodes. As such, a so-called linear sweep in which the potential is changed in proportion to time is preferably employed. The current-potential curve (Voltammogram) obtained in this way not only gives a maximum current value (peak current) proportional to the cell concentration, but also has a curve shape such as a peak potential value that differs depending on the type of cell. The specificity of the curve shape provides sufficient information for the identification of microorganisms such as bacteria. That is, when compared with the prior art, the method of the present invention significantly shortens the response time and measurement time, and because it uses a scanning potential, disturbing factors in the electrode reaction can be eliminated, resulting in more accurate measurements. This provides many practical advantages, such as being able to identify not only the cell concentration (number) but also the type of cells at the same time. Therefore, the present invention can be said to be extremely useful as a sensor means in a wide variety of fields, such as a sensor for real-time control of fermentation processes, measurement of the degree of microbial contamination of water, and measurement of red blood cell and white blood cell counts. On the other hand, a schematic illustration of an example of a differential pulse polarography device particularly useful for cell identification is shown in FIG. 2. That is, the exemplary device
BPG (Basal Plane Pyrolytic Graphite), HPG
Carbon such as (high purity carbon for spectroscopic analysis), platinum,
A working electrode 11 and a counter electrode 12 made of gold, silver, etc.
A cell 14 equipped with a reference electrode 13 such as SSCE (saturated sodium chloride Calomelo electrode), pulse sequencer 15, potentiometer programmer 16, potentiostat 17, drop-knocker 18, i/E
It is composed of a converter 19, a sample hold (τ) 110 and a sample hold (τ') 111, a difference amplifier amplifier 112, and a recorder 113. In this case, the measurement is performed by injecting a living cell suspension such as a microbial culture solution into the cell 14 and measuring the generated current by applying a gradually increasing scanning (sweep) potential with a superimposed micropotential between the electrodes. However, as the potential scanning, so-called linear scanning (Linear Sweep) in which the potential is changed in proportion to time is usually suitably employed. As the minute potential to be superimposed on the scanning potential, one having an appropriate waveform and period capable of giving a differential current value can be appropriately selected and used as described above. The current-potential curve (Voltammogram) obtained in this way gives clear peak potential values that differ depending on the type of cell, and this not only makes it possible to identify them, but also allows analysis of the peak waveform to identify bacteria, etc. It also provides information regarding the electrochemical activity of microorganisms. That is, when compared with the prior art, the method of the present invention allows the identification and identification of various types of bacteria to be achieved in an extremely short time and easily. Here, the conditions for using 4,4'-bipyridine (BP), which is an important activator of electron transfer between cells and electrodes in the method of the present invention, are summarized as follows. be. That is, in the present invention, BP participates in the cell-electrode reaction by being added directly to the suspension or fixed to a nitrocellulose membrane or the like and attached to the electrode. Note that the concentration of BP in the cell suspension varies depending on the type of target cells, but is generally about several mM to 100 mM. By performing voltammetry in the presence of BP in this manner, the peak current value is enhanced by about 1.5 to 2.5 times, the waveform becomes clearer, and the peak becomes sharper in the case of the differential current value. Example 1 Saccharomyces cerevisiae (baker's yeast; ATCC
7754) was aerobically cultured for 8 hours in a medium (PH 6.0) consisting of 2% peptone, 2% glucose, and 1% yeast extract. After collecting the cells, wash with 0.1M phosphate buffer to obtain a cell suspension of any concentration (PH 7.0).
was prepared. This cell suspension was placed in a 15 ml H-type cell, and after introducing air for 10 minutes, a 6 mm diameter BPG electrode was inserted and the electrochemical behavior was measured by cyclic voltammetry (see Figure 1) at a scanning speed of 10 mV/sec. Examined. A platinum wire electrode was used as the counter electrode, and an SSCE electrode was used as the reference electrode. Figure 3 shows the cyclic voltamgram at each scanning potential when the electrode is inserted into a living cell suspension (1.5 x 10 8 cells/ml) (in the figure, i is the current value (μA); E is the Potential (V); a and b are each 50mMBP
The waveforms are for cases with and without additives). As is clear from Figure 3, when the potential is scanned from 0V to 1V (vs. SSCE), it becomes clear especially in the case of BP addition.
A peak of the amplified current was obtained around 0.7V.
This peak could not be obtained with phosphate buffer alone or with cell suspensions sterilized in an autoclave at 110°C for 10 minutes. Furthermore, no peak could be obtained even when the BPG electrode surface was covered with a dialysis membrane so that the electrode and cells could not come into direct contact. From the above, it has become clear that this peak current is obtained when living cells come into contact with the electrode. Here, FIG. 4 shows the relationship between the cell concentration Co and the peak current value ip measured in the same manner as above in the coexistence of 50mMBP. It was confirmed that a linear relationship exists between ip and Co. Therefore, it has become clear that yeast cell concentration can be measured from the ip value. The measurable cell concentration range is 10 6 cells/ml
Since 10 9 cells/ml actually satisfies the range required for measurement in fermentation, it has become clear that this method can also be used to measure the number of bacteria in a fermenter. Furthermore, FIG. 5 shows the relationship between the BP concentration and the peak current value ip when the cell concentration is 1.5×10 8 cells/ml. Furthermore, Bacillus subtilis IFO 3009,
Lactobacillus fermentum IFO 3071,
Leuconostoc mesenteroides IFO 3832,
In the case of Escherichia coli, etc., it was confirmed that the peak current value proportional to the concentration was amplified by 1.5 to 2.5 times due to the coexistence of BP, and the peak waveform became sharper. Example 2 Baker's yeast (Saccharomyces cerevisiae) and Bacillus subtilis (IFO 3009) were grown at 37°C in each medium (PH 7.0) having the composition shown in Tables 1 and 2 below.
Aerated culture was carried out using a conventional method, and each electrode in the above example was inserted into a culture tank, and the number of viable bacteria and peak current value ip were tracked in real time in the coexistence of 10mMBP (the number of viable bacteria was measured separately by colony counting method). The results are summarized in Tables 3 and 4. As is clear from these results, an extremely good correlation between the two was observed. Table 1 Glucose 40g Peptone 10g Yeast Extract 5g KH 2 PO 4 5g MgSO 4 2g (Add purified water 1) Table 2 Glucose 10g Peptone 10g Extract 5g NaCl 5g (Add purified water 1)
【表】【table】
【表】
実施例 ３
Bacillus subtilis IFO 3009をNutrient Broth
（牛肉エキス１％、ペプトン１％）中で培養し、
対数期に集菌し0.1Mリン酸緩衝液（PH 7.0）で
洗浄後、同緩衝液に懸濁し細胞懸濁液（1.2×
109cellscm-3）を調製した。
同様にしてSaccharomyces cerevisiae（YPD
培地：酵母エキス１％、ポリペプトン２％、グル
コース２％）、Lactobacillus fermentum IFO
3071（Nutrient Broth）、Leuconostoc
mesenteroides IFO 3832（Tomato Juice
Broth：トリブトン１％、酵母エキス１％、トマ
トジユース20％）及びEscherichia coli
（Nutrient Broth）を夫々培養し、対数期に集菌
し前記緩衝液に懸濁して生菌数濃度1.03×109、
5.0×108、1.3×109及び1.0×1010cellscm-3の各懸
濁液を調製した。
次にこれら生細胞各懸濁液を15mlＨ型セルに入
れ、作用電極BPG、対極白金線及び参照極SSCE
を用いて50mMBP共存下、走査電位０〜1.0V
（vs.SSCE）、サンプリング・タイム20ms、変調
電圧50mV及び100mV、電位単掃引0.5mV／ｓ、
測定温度25℃の条件下、デイフアレンシヤル・パ
ルスボルタメトリを実施した。
尚、装置は扶桑製作所製“ポーラログラフ312
型”を使用した。こうして得られた微分電流のピ
ークは極めて先鋭明瞭であり且つ各細菌毎に相違
するピーク電位値を与えるものであつた。
各細菌のピーク電位置を下記第５表に要約して
示す。
同表からも明らかなように、本発明方法に依れ
ば細菌相互の識別が極めて明瞭且つ容易になされ
得るものであり、特にグラム陰性菌であるE.coli
とその余の各グラム陽性菌とが明瞭に区別され得
ることは注目に値するものである。
第５表 微生物 ピーク電位（Ｖ vs.SSCE）
B.subtilis 0.68
S.cerevisiae 0.74
Lact.fermentum 0.75
Leuco.mesenteroides 0.80
E.coli 0.85
実施例 ５
Salmonella typhimurium TA98およびTA100
をそれぞれHistidine Bioassay Medium
（TAKARA KOHSAN CO.、LTD）で、ヒス
チジン（0.075mM）を加えたもの、ヒスチジン
（0.075mM）とMNNG（Ｎ−メチル−N′ニトロ−
Ｎ−ニトロソグアニジン）（0.07μｇ／ml）を加え
たもの、さらにHistidine Bioassay Mediumの
みのものそれぞれで培養し、TA98、TA100それ
ぞれについて変異していないもの、変異したもの
を得た。
次いでこれら微生物の50mMBP、0.1Mリン酸
緩衝液（PH 7.0）懸濁液（2.1×109cellscm-3）を
調製し、前記例と同様にデイフアレンシヤル・パ
ルス・ボルタモグラムを測定した。
各菌株のピーク電位値を下記第６表に要約して
示す。[Table] Example 3 Add Bacillus subtilis IFO 3009 to Nutrient Broth
(beef extract 1%, peptone 1%),
Collect cells in the logarithmic phase, wash with 0.1M phosphate buffer (PH 7.0), suspend in the same buffer, and make a cell suspension (1.2
109 cellscm -3 ) were prepared. Similarly, Saccharomyces cerevisiae (YPD
Medium: yeast extract 1%, polypeptone 2%, glucose 2%), Lactobacillus fermentum IFO
3071 (Nutrient Broth), Leuconostoc
mesenteroides IFO 3832 (Tomato Juice
Broth: Tributone 1%, yeast extract 1%, tomato youth 20%) and Escherichia coli
(Nutrient Broth) were cultured respectively, and the bacteria were collected in the logarithmic phase and suspended in the above buffer solution to a viable cell count concentration of 1.03×10 9 .
Suspensions of 5.0×10 8 , 1.3×10 9 and 1.0×10 10 cells cm −3 were prepared. Next, each suspension of these living cells was placed in a 15 ml H-type cell, and the working electrode BPG, the counter electrode platinum wire, and the reference electrode SSCE were placed in a 15 ml H-type cell.
using a scanning potential of 0 to 1.0 V in the coexistence of 50mMBP.
(vs.SSCE), sampling time 20ms, modulation voltage 50mV and 100mV, potential single sweep 0.5mV/s,
Differential pulse voltammetry was performed at a measurement temperature of 25°C. The device is “Polarograph 312” manufactured by Fuso Seisakusho.
The peak of the differential current thus obtained was extremely sharp and clear, and gave a different peak potential value for each bacterium. The peak potential positions of each bacterium are summarized in Table 5 below. As is clear from the same table, according to the method of the present invention, bacteria can be distinguished from each other very clearly and easily, and in particular, E. coli, which is a Gram-negative bacterium, can be distinguished from each other very clearly and easily.
It is noteworthy that the bacteria can be clearly distinguished from the rest of the Gram-positive bacteria. Table 5 Microorganisms Peak potential (V vs. SSCE) B.subtilis 0.68 S.cerevisiae 0.74 Lact.fermentum 0.75 Leuco.mesenteroides 0.80 E.coli 0.85 Example 5 Salmonella typhimurium TA98 and TA100
Histidine Bioassay Medium
(TAKARA KOHSAN CO., LTD) with histidine (0.075mM), histidine (0.075mM) and MNNG (N-methyl-N'nitro-
N-nitrosoguanidine) (0.07 μg/ml) and Histidine Bioassay Medium alone were used to obtain unmutated and mutated samples for TA98 and TA100, respectively. Next, suspensions (2.1×10 9 cellscm −3 ) of these microorganisms in 50 mMBP and 0.1 M phosphate buffer (PH 7.0) were prepared, and differential pulse voltammograms were measured in the same manner as in the previous example. The peak potential values of each strain are summarized in Table 6 below.
添付第１乃至２図は本発明実施例で使用の装置
の模式説明図、第３乃至５図は同実験説明図であ
る。
４……電解セル、５……ポテンシオスタツト、
６……リニア・スイーブ電源、７……XY記録計
乃至シンクロスコープ、１１……作用電極、１２
……対極、１３……参照電極、１４……セル、１
５……パルスシーケンサ、１７……ポテンシオス
タツト、１１２……デイフアレンスアンプリフイ
ア及び１１３……レコーダ。
Attached Figures 1 to 2 are schematic explanatory diagrams of the apparatus used in the examples of the present invention, and Figures 3 to 5 are explanatory diagrams of the same experiment. 4... Electrolytic cell, 5... Potentiostat,
6... Linear sweep power supply, 7... XY recorder or synchroscope, 11... Working electrode, 12
... Counter electrode, 13 ... Reference electrode, 14 ... Cell, 1
5... Pulse sequencer, 17... Potentiostat, 112... Differential amplifier amplifier, and 113... Recorder.
Claims (1)
し、４，4′−ビピリジンの共存下前記電極間に走
査電位を印加して生起する電流値を測定すること
を特徴とする細胞の電気化学的活性測定方法。 ２ 前記懸濁液中の細胞数を測定すべく前記電流
値のピーク値を計測することを特徴とする特許請
求の範囲第１項に記載の前記方法。 ３ 前記懸濁液中の細胞の種類を識別すべく前記
電流値と前記走査電位との対応曲線を測定するこ
とを特徴とする特許請求の範囲第１乃至２項に記
載の前記方法。[Claims] 1. At least one pair of electrodes is inserted into a cell suspension, and a scanning potential is applied between the electrodes in the coexistence of 4,4'-bipyridine to measure the generated current value. A method for measuring electrochemical activity of cells. 2. The method according to claim 1, characterized in that the peak value of the current value is measured in order to measure the number of cells in the suspension. 3. The method according to any one of claims 1 to 2, characterized in that a correspondence curve between the current value and the scanning potential is measured in order to identify the type of cells in the suspension.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP58052772A JPS59179097A (en) | 1983-03-30 | 1983-03-30 | Electrochemical measurement of cell activity |
| US06/546,101 US4528270A (en) | 1982-11-02 | 1983-10-27 | Electrochemical method for detection and classification of microbial cell |
| DE19833339408 DE3339408A1 (en) | 1982-11-02 | 1983-10-29 | METHOD FOR ELECTROCHEMICALLY DETECTING AND CLASSIFYING MICROBIAL CELLS |
| GB08329095A GB2131954B (en) | 1982-11-02 | 1983-11-01 | Electrochemical method for detection and classification of microbial cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP58052772A JPS59179097A (en) | 1983-03-30 | 1983-03-30 | Electrochemical measurement of cell activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS59179097A JPS59179097A (en) | 1984-10-11 |
| JPH045440B2 true JPH045440B2 (en) | 1992-01-31 |
Family
ID=12924154
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP58052772A Granted JPS59179097A (en) | 1982-11-02 | 1983-03-30 | Electrochemical measurement of cell activity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS59179097A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07184686A (en) * | 1993-12-28 | 1995-07-25 | Nec Corp | Method for measuring cell activity |
| GB201020619D0 (en) * | 2010-12-06 | 2011-01-19 | Syngenta Ltd | Pathogen sensor |
-
1983
- 1983-03-30 JP JP58052772A patent/JPS59179097A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS59179097A (en) | 1984-10-11 |
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