JP2014097027A - Viable cell count measuring method and viable cell count measuring kit - Google Patents
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本発明は、生菌数を電気化学的に計測し得る生菌数計測方法及び生菌数計測キットに関するものであり、特に、極めて簡易な構成でありながら迅速に生菌数を計測し得る生菌数計測方法及び生菌数計測キットに関する。 The present invention relates to a viable cell count method and a viable cell count kit capable of electrochemically measuring the viable cell count, and in particular, a viable cell count capable of quickly measuring the viable cell count with an extremely simple configuration. The present invention relates to a method for measuring the number of bacteria and a kit for measuring the number of viable bacteria.
例えば食品分野においては、大腸菌等、各種細菌による汚染の防止が大きな課題となっている。細菌による汚染は、食中毒等の原因にもなることから、これを極力排除する必要がある。そのためには、細菌による汚染度合い（食品等に付着している生菌の数）を正確に把握する必要があり、できる限り簡易に、且つ短時間に生菌の数を計測し得ることが望まれる。 For example, in the food field, prevention of contamination by various bacteria such as Escherichia coli is a major issue. Contamination with bacteria can cause food poisoning and the like, so it is necessary to eliminate it as much as possible. For that purpose, it is necessary to accurately grasp the degree of contamination by bacteria (the number of viable bacteria adhering to food, etc.), and it is desirable to be able to measure the number of viable bacteria as easily and in a short time as possible. It is.
このような状況から、各方面で生菌数の計測方法に関する検討が進められ、様々な方式の生菌数計測方法、生菌数計測装置が提案されている（特許文献１〜３等を参照）。 Under such circumstances, studies on the method for measuring the number of viable bacteria are being promoted in various directions, and various methods for measuring the number of viable bacteria and a viable cell count measuring apparatus have been proposed (see Patent Documents 1 to 3, etc.) ).
例えば特許文献１には、酵素電極の先端部に被検菌体を固定したメンブランフィルターを装着し、メンブランフィルターを酵素電極の先端部と共に透析膜で被覆して生菌数の計測を行う生菌数計測用センサが開示されている。 For example, in Patent Document 1, a membrane filter in which a test cell is fixed to the tip of an enzyme electrode is attached, and the membrane filter is covered with a dialysis membrane together with the tip of the enzyme electrode to measure the number of viable bacteria. A number measuring sensor is disclosed.
特許文献２には、微生物を含有する試料溶液中の溶存酸素の減少量を酸素電極を用いて測定することにより試料中の生菌数を測定する方法が開示されており、溶存酸素の減少量の測定を試料溶液を容器内に密閉した状態で行うことが記載されている。 Patent Document 2 discloses a method for measuring the number of viable bacteria in a sample by measuring the amount of decrease in dissolved oxygen in a sample solution containing microorganisms using an oxygen electrode. It is described that the measurement is performed with the sample solution sealed in a container.
特許文献３には、濾過膜に試料液を通水することによって試料液中の細菌を濾過膜上に捕集し、該濾過膜を溶菌剤及び酵素反応基質に接触させ、ターゲット細菌中酵素の酵素反応基質に対する酵素活性値を求めることによって、試料液中の細菌数を定量する細菌の検出方法が開示されている。 In Patent Document 3, bacteria in a sample solution are collected on the filtration membrane by passing the sample solution through the filtration membrane, the filtration membrane is brought into contact with a lysing agent and an enzyme reaction substrate, and the enzyme in the target bacteria is detected. A method for detecting bacteria is disclosed in which the number of bacteria in a sample solution is quantified by determining an enzyme activity value for an enzyme reaction substrate.
しかしながら、特許文献１記載の生菌数計測センサの場合、酵素電極とメンブランフィルターの間にテフロン（登録商標）膜を挟み、透析膜で外側を包み、２種類の溶液に浸して電流値を測定し、その差から生菌数を計測しており、輪ゴムで留める等、メンブランフィルターの酵素電極への装着が煩雑である。また、計測自体も、２種類の溶液（リン酸緩衝液と培養液）についてそれぞれ計測を行う必要があり、煩雑である。 However, in the case of the viable cell count sensor described in Patent Document 1, a Teflon (registered trademark) membrane is sandwiched between the enzyme electrode and the membrane filter, the outside is wrapped with a dialysis membrane, and the current value is measured by immersing in two types of solutions. However, the viable count is measured from the difference, and it is complicated to attach the membrane filter to the enzyme electrode, such as fastening with a rubber band. Further, the measurement itself is complicated because it is necessary to measure each of the two types of solutions (phosphate buffer solution and culture solution).
一方、特許文献２記載の方法の場合、メンブランによる濃縮を行っていないため、感度が不十分である他、酸素電極を用いて溶存酸素の減少量を計測しているため、装置構成が煩雑で、現場での簡易検査等には向いていない。また、特許文献３記載の方法は、酵素反応を利用したものであるので、試薬の数も多く、やはり簡易検査には向いていない。 On the other hand, in the case of the method described in Patent Document 2, since the concentration by the membrane is not performed, the sensitivity is insufficient and the amount of dissolved oxygen is measured using an oxygen electrode, so that the apparatus configuration is complicated. It is not suitable for on-site simple inspections. In addition, since the method described in Patent Document 3 uses an enzyme reaction, the number of reagents is large and it is not suitable for simple inspection.
本発明は、以上のような従来技術の抱える課題を解消することを目的とするものであり、簡易な構成でありながら迅速に生菌数を正確に計測することができ、取り扱いや操作が容易で、あらゆる状況において、生菌数の計測を行うことが可能な生菌数測定方法及び生菌数測定キットを提供することを目的とする。 The object of the present invention is to solve the above-mentioned problems of the prior art, and it is possible to measure the number of viable bacteria quickly and accurately with a simple configuration, and easy to handle and operate. Thus, an object of the present invention is to provide a viable cell count measurement method and a viable cell count measurement kit capable of measuring the viable cell count in any situation.
前述の目的を達成するために、本発明の生菌数測定方法は、検査対象液を細菌捕集可能なメンブランで濾過し、前記濾過に使用したメンブランを酸素透過性のない袋状容器内に収容するとともに、印刷電極をメンブランに接触するように袋状容器内に挿入し、袋状容器内の酸素減少量を電気化学的に測定し生菌数を計測することを特徴とする。 In order to achieve the above-mentioned object, the method for measuring the viable cell count of the present invention is to filter the liquid to be examined with a membrane capable of collecting bacteria, and place the membrane used for the filtration in a bag-like container having no oxygen permeability. In addition, the printed electrode is inserted into a bag-like container so as to be in contact with the membrane, and the oxygen reduction amount in the bag-like container is electrochemically measured to count the number of viable bacteria.
また、本発明の生菌数測定キットは、検査対象液を濾過する細菌捕集可能なメンブランと、前記濾過に使用したメンブランを収容する酸素透過性のない袋状容器と、前記メンブランに接触するように袋状容器内に挿入される印刷電極とを備え、袋状容器内の酸素減少量を電気化学的に測定することにより生菌数を計測することを特徴とする。 The viable cell count kit of the present invention is in contact with the membrane, a membrane-capable membrane for filtering the liquid to be tested, a non-oxygen permeable bag-like container containing the membrane used for the filtration, and the membrane. And a printed electrode inserted into the bag-like container, and the number of viable bacteria is measured by electrochemically measuring the amount of oxygen reduction in the bag-like container.
本発明によれば、メンブランによる濃縮を採用しているので、生菌数を感度良く短時間に測定することが可能である。また、酵素電極や酸素電極は必要なく、極めて簡易な印刷電極を用いており、他に必要なのは袋状容器のみであり、非常に簡便な測定方法、測定キットを実現することが可能である。さらに、本発明によれば、輪ゴムによるメンブランの装着や、電極移動等も不要であり、測定操作や取り扱いも容易である。 According to the present invention, since concentration by a membrane is employed, it is possible to measure the viable cell count with high sensitivity in a short time. In addition, an enzyme electrode and an oxygen electrode are not required, and a very simple printed electrode is used. In addition, only a bag-like container is required, and a very simple measurement method and measurement kit can be realized. Furthermore, according to the present invention, it is not necessary to attach a membrane with a rubber band, move an electrode, or the like, and the measurement operation and handling are easy.
以下、本発明を適用した生菌数測定方法及び生菌数測定キットの実施形態について、図面を参照して詳細に説明する。 Hereinafter, embodiments of a viable cell count measurement method and a viable cell count measurement kit to which the present invention is applied will be described in detail with reference to the drawings.
本発明の生菌数測定方法及び生菌数測定キットは、例えば食品等に存在する生菌数を測定するものである。その測定原理は、生菌による酸素消費量を指標とするもので、検査対象サンプルを洗浄した液体をメンブランで濾過した後、前記濾過に使用したメンブランを酸素透過性のない袋状容器内に収容するとともに、印刷電極をメンブランに接触するように袋状容器内に挿入し、袋状容器内の酸素減少量を電気化学的に測定するという、極めて簡便なものである。 The viable cell count measurement method and the viable cell count measurement kit of the present invention measure the viable cell count present in, for example, food. The measurement principle is based on the consumption of oxygen by viable bacteria, and after filtering the liquid in which the sample to be examined is washed with a membrane, the membrane used for the filtration is stored in a bag-like container that is not permeable to oxygen. At the same time, the printing electrode is inserted into the bag-like container so as to be in contact with the membrane, and the oxygen reduction amount in the bag-like container is measured electrochemically.
図１は、本発明による生菌数の測定の様子を示すものである。測定に際しては、先ず、検査対象液を細菌捕集可能なメンブランで濾過する。これにより、検査対象液に含まれる生菌がメンブランに濃縮され固定される。検査対象液としては、被検対象サンプル（例えば食品等）が液体でない場合には、その表面を細菌を含まない液体（水や培地等）で洗浄した洗浄液を用いるか、あるいは綿棒で表面をぬぐい、その綿棒を洗浄した洗浄液を用いる。検査対象サンプルが液体である場合には、液体サンプルをそのままの状態で検査対象液として用いる。いずれの場合にも、これらを前処理カラムで濾過して用いることも可能である。 FIG. 1 shows how the number of viable bacteria is measured according to the present invention. In the measurement, first, the test solution is filtered through a membrane capable of collecting bacteria. As a result, viable bacteria contained in the test solution are concentrated and fixed on the membrane. If the sample to be tested (for example, food) is not liquid, use a cleaning solution whose surface is washed with a liquid that does not contain bacteria (water, medium, etc.), or wipe the surface with a cotton swab. Use a cleaning solution for cleaning the swab. When the inspection target sample is a liquid, the liquid sample is used as it is as the inspection target liquid. In any case, these can be used after being filtered through a pretreatment column.
次いで、図１に示すように、濾過後のメンブラン１を酸素透過性のない袋状容器２に入れる。袋状容器２は、例えば、２枚重ねにしたプラスチックフィルムの２辺をヒートシールしたものであり、市販のものを使用することも可能である。 Next, as shown in FIG. 1, the membrane 1 after filtration is put into a bag-like container 2 having no oxygen permeability. The bag-like container 2 is, for example, one obtained by heat-sealing two sides of a two-layer plastic film, and a commercially available one can also be used.
袋状容器２に菌を固定したメンブラン１を入れた後、メンブラン１に接触するように平板な印刷電極３を袋状容器２内に挿入する。印刷電極３は、例えばカーボン印刷電極等、所定の電極パターン（例えば、作用極、参照極、及び対極）が印刷された簡単な構造のものを使用することができる。 After the membrane 1 in which bacteria are fixed is put into the bag-like container 2, a flat printed electrode 3 is inserted into the bag-like container 2 so as to come into contact with the membrane 1. The printed electrode 3 may have a simple structure on which a predetermined electrode pattern (for example, a working electrode, a reference electrode, and a counter electrode) is printed, such as a carbon printed electrode.
この状態で酸素減少量をサイクリックボルタンメトリー（cyclic voltammetry）を用いて電気化学的に測定する。例えば、培養液を少量入れた状態で、電圧を変化させ、酸素濃度を反映する０．８Ｖ付近のピークの減少を測定する。電圧変化は、例えば５０ｍＶ／秒で行う。この減少の度合いを、予め作成しておいた検量線と照らし合わせることで、生菌数を計測することができる。なお、電気化学的測定としては、前記のようにピーク電流値を指標とするのが良いが、ピークの現れる範囲を積分して指標とすることも可能である。 In this state, the amount of oxygen decrease is measured electrochemically using cyclic voltammetry. For example, in a state where a small amount of culture solution is added, the voltage is changed, and the decrease in the peak around 0.8 V reflecting the oxygen concentration is measured. The voltage change is performed at 50 mV / second, for example. The number of viable bacteria can be measured by comparing the degree of this decrease with a calibration curve prepared in advance. For electrochemical measurement, the peak current value is preferably used as an index as described above, but it is also possible to integrate the range in which the peak appears as an index.
一般的な生菌数測定方法では、２４時間以上かけて培養を行い、それから測定を行うため、測定に長時間を要することになる。本発明の測定方法では、メンブラン１に濃縮された菌がごく少量の培地中の酸素を消費するので、酸素減少量が検出し易く、２０分〜６０分程度で測定を完了することが可能である。生菌数が少ない場合、従来法では測定が難しいが、本発明方法を採用した場合、例えば１０３個／ｍｌ程度の生菌数であっても、１時間〜２時間程度で測定が可能である。 In a general method for measuring the number of viable bacteria, culturing is performed for 24 hours or more, and then measurement is performed. Therefore, the measurement takes a long time. In the measurement method of the present invention, since the bacteria concentrated in the membrane 1 consume a very small amount of oxygen in the medium, the amount of oxygen decrease is easy to detect, and the measurement can be completed in about 20 to 60 minutes. is there. When the number of viable bacteria is small, it is difficult to measure by the conventional method, but when the method of the present invention is adopted, even if the number of viable bacteria is, for example, about 10 3 cells / ml, it can be measured in about 1 to 2 hours. is there.
前述の通り、本発明方法を採用することで、簡単な操作で、迅速且つ高感度に被検対象サンプルの生菌数を計測することが可能である。また、係る測定を行うための生菌数測定キットについても、メンブランと袋状容器、印刷電極、さらには必要に応じてシリンジやメンブランホルダ等をセットにして提供すればよく、極めて簡易な構成でありながら、高性能な生菌数測定キットを提供することが可能である。 As described above, by employing the method of the present invention, it is possible to measure the number of viable bacteria in a sample to be examined quickly and with high sensitivity by a simple operation. In addition, a viable cell count kit for performing such measurement may be provided as a set of a membrane, a bag-like container, a printed electrode, and a syringe, a membrane holder, etc. as necessary. Nevertheless, it is possible to provide a high-performance viable count kit.
以上、本発明を適用した実施形態についてを説明してきたが、本発明が前述の実施形態に限られるものでないことは言うまでもなく、本発明の要旨を逸脱しない範囲において、多様な変更または改良を加えることが可能である。 Although the embodiment to which the present invention is applied has been described above, it goes without saying that the present invention is not limited to the above-described embodiment, and various modifications or improvements are made without departing from the gist of the present invention. It is possible.
次に、本発明を適用した具体的な実施例について、実験結果を基に説明する。 Next, specific examples to which the present invention is applied will be described based on experimental results.
浸漬法による測定（比較例）
生菌（Ｎ−３株）を１×１０３個／ｍＬ〜１×１０８個／ｍＬ含む液体をエッペンチューブに入れ、印刷電極を挿入し、電圧を変化させながら電流値の変化を測定した。測定開始時及び３０分後のピーク高をプロットしたものを図２に示す。
Measurement by immersion method (comparative example)
A liquid containing 1 × 10 3 cells / mL to 1 × 10 8 cells / mL of viable bacteria (N-3 strain) was placed in an Eppendorf tube, a printed electrode was inserted, and the change in current value was measured while changing the voltage. . A plot of the peak height at the start of measurement and after 30 minutes is shown in FIG.
図２から明らかなように、いずれの濃度のサンプルにおいても、測定開始時に比べて３０分後のピーク高は減少していた。平均値では、菌濃度が高くなるにしたがってピークの大きさは小さくなっているが、例えば１×１０５個／ｍＬのサンプルと１×１０６個／ｍＬのサンプルでは、有意な差は認められなかった。 As is apparent from FIG. 2, the peak height after 30 minutes was decreased in the samples of any concentration compared to the measurement start time. In the average value, the peak size decreases as the bacterial concentration increases. However, for example, a significant difference is observed between a sample of 1 × 10 5 cells / mL and a sample of 1 × 10 6 cells / mL. There was no.
メンブランを用いた濃縮法（実施例）
１×１０４個／ｍＬ〜１×１０６個／ｍＬの生菌（Ｎ−３株）を含む培養液５０ｍＬをメンブランで濾過して生菌をメンブランに固定し、この生菌を固定したメンブランをビニールパウチに入れた。さらに、ビニールパウチ（袋状容器）内に１００μＬの培養液を入れ、印刷電極をメンブランに密着するようにビニールパウチ内に挿入した。
Concentration method using membrane (Example)
A culture solution containing 1 × 10 4 cells / mL to 1 × 10 6 cells / mL viable bacteria (N-3 strain) was filtered through a membrane to fix the live cells to the membrane, and the membrane to which the live cells were fixed Was put in a vinyl pouch. Further, 100 μL of the culture solution was placed in a vinyl pouch (bag-like container), and the printed electrode was inserted into the vinyl pouch so as to be in close contact with the membrane.
この状態で、比較例と同様、印刷電極により電圧を変化させながら電流値の変化を測定した。１×１０４個／ｍＬの生菌（Ｎ−３株）を含む培養液５０ｍＬを濾過したメンブランについての測定結果を図３に示す。いずれの濃度においても、浸漬法（比較例）と同様、時間とともに減少する酸素ピークが確認された。 In this state, as in the comparative example, the change in the current value was measured while changing the voltage with the printed electrode. The measurement result about the membrane which filtered 50 mL of culture solution containing 1 * 10 < 4 > piece / mL viable bacteria (N-3 strain | stump | stock) is shown in FIG. At any concentration, as in the immersion method (Comparative Example), an oxygen peak decreasing with time was confirmed.
１×１０６個／ｍＬの生菌（Ｎ−３株）を含む培養液５０ｍＬを濾過したメンブランについては、測定開始から１０分でもピークはほとんど認められないくらいに減少しており、浸漬法では測定できない範囲においても、本発明方法であれば測定が可能であると考えられる。 The membrane obtained by filtering 50 mL of the culture solution containing 1 × 10 6 cells / mL viable bacteria (N-3 strain) has decreased to such a degree that almost no peak is observed even after 10 minutes from the start of measurement. Even in a range where measurement is not possible, the method of the present invention is considered to allow measurement.
図４は、測定開始３０分後のピーク電流値を指標とした検量線である。１×１０４個／ｍＬ、１×１０５個／ｍＬ、１×１０６個／ｍＬで、それぞれ有意な差が認められた。培養液の量を１００ｍＬとすると、１×１０４個／ｍＬと１×１０３個／ｍＬでも差が認められた。図５は、培養液を５００ｍｌに増やした場合の検量線である。１×１０３個／ｍＬと１×１０４個／ｍＬの差は、さらに顕著なものとなった。 FIG. 4 is a calibration curve using the peak current value 30 minutes after the start of measurement as an index. Significant differences were observed at 1 × 10 4 cells / mL, 1 × 10 5 cells / mL, and 1 × 10 6 cells / mL. When the amount of the culture solution was 100 mL, a difference was observed even at 1 × 10 4 cells / mL and 1 × 10 3 cells / mL. FIG. 5 is a calibration curve when the culture solution is increased to 500 ml. The difference between 1 × 10 3 cells / mL and 1 × 10 4 cells / mL was even more remarkable.
図３や図４に示す検量線は、生菌の培養液中の濃度に対して作成したものである。そこで、メンブレン上の生菌数とピーク電流値の関係を検討したところ、図６に示す関係となった。図６から明らかなように、濾過した液量に関わらず、シグモイド曲線上に載っていることから、例えば生菌数センサにプリセットする検量線として使用できるものと考えられる。
The calibration curves shown in FIG. 3 and FIG. 4 are prepared with respect to the concentration of viable bacteria in the culture solution. Then, when the relationship between the number of viable bacteria on the membrane and the peak current value was examined, the relationship shown in FIG. 6 was obtained. As apparent from FIG. 6, since it is on the sigmoid curve regardless of the amount of filtered liquid, it can be considered that it can be used, for example, as a calibration curve preset in a viable cell count sensor.
Claims (5)
前記濾過に使用したメンブランを酸素透過性のない袋状容器内に収容するとともに、印刷電極をメンブランに接触するように袋状容器内に挿入し、
袋状容器内の酸素減少量を電気化学的に測定し生菌数を計測することを特徴とする生菌数測定方法。 Filter the fluid to be tested through a membrane that can collect bacteria,
The membrane used for the filtration is housed in a bag-like container having no oxygen permeability, and the printed electrode is inserted into the bag-like container so as to contact the membrane,
A method for measuring the number of viable bacteria, characterized in that the amount of oxygen reduction in a bag-like container is electrochemically measured to count the number of viable bacteria.
前記濾過に使用したメンブランを収容する酸素透過性のない袋状容器と、
前記メンブランに接触するように袋状容器内に挿入される印刷電極とを備え、
袋状容器内の酸素減少量を電気化学的に測定することにより生菌数を計測することを特徴とする生菌数測定キット。 A membrane capable of collecting bacteria that filters the liquid to be tested;
A non-oxygen permeable bag-like container containing the membrane used for the filtration;
A printed electrode inserted into the bag-like container so as to contact the membrane,
A viable cell count kit characterized in that the viable cell count is measured by electrochemically measuring an oxygen reduction amount in a bag-like container.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020235403A1 (en) * | 2019-05-20 | 2020-11-26 | 国立研究開発法人物質・材料研究機構 | Detection device and data collection method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020235403A1 (en) * | 2019-05-20 | 2020-11-26 | 国立研究開発法人物質・材料研究機構 | Detection device and data collection method |
| JPWO2020235403A1 (en) * | 2019-05-20 | 2020-11-26 | ||
| JP7136508B2 (en) | 2019-05-20 | 2022-09-13 | 国立研究開発法人物質・材料研究機構 | Detection device and data collection method |
| EP3973915A4 (en) * | 2019-05-20 | 2023-05-17 | National Institute for Materials Science | Detection device and data collection method |
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