JP2735901B2 - Method for measuring the number of living cells, dead cells, and particles other than microorganism cells of microorganisms - Google Patents
Method for measuring the number of living cells, dead cells, and particles other than microorganism cells of microorganismsInfo
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- JP2735901B2 JP2735901B2 JP1269130A JP26913089A JP2735901B2 JP 2735901 B2 JP2735901 B2 JP 2735901B2 JP 1269130 A JP1269130 A JP 1269130A JP 26913089 A JP26913089 A JP 26913089A JP 2735901 B2 JP2735901 B2 JP 2735901B2
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Description
【発明の詳細な説明】 本発明は微生物細胞とその他の粒子とを含む試料に光
を照射し、上記試料中の全体の粒子から、微生物の生細
胞、死細胞および微生物以外の粒子の数を測定する方法
に関し、特に食品製造プラント、医薬品製造プラントに
おける製品の品質管理や殺菌装置の性能把握等、半導体
製造に使用される超純水製造装置のチェック等に有利に
適用される方法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention irradiates a sample containing microbial cells and other particles with light, and counts the number of living cells, dead cells, and non-microbial particles of the microorganism from the total particles in the sample. The present invention relates to a method for measuring, particularly to a method advantageously applied to checking of ultrapure water production equipment used for semiconductor production, such as quality control of products in food production plants and pharmaceutical production plants, and grasp of performance of sterilization equipment.
食品、医薬品製造分野、発酵工業等においては、原料
や製品の品質管理、殺菌装置の性能確認のため微生物検
査、測定が不可欠である。また半導体製造に使用される
超純水では、微生物細胞も含め、無機粒子の検査も重要
なチェック項目となっている。In the food and pharmaceutical manufacturing fields, the fermentation industry, and the like, microbial testing and measurement are indispensable for quality control of raw materials and products and performance confirmation of sterilizers. In ultrapure water used for semiconductor production, inspection of inorganic particles including microbial cells is also an important check item.
従来の微生物検査・測定方法には、顕微鏡法、寒天培
養法、バイオルミネッセンス法などがあるが、いずれの
方法も微生物細胞の生死までは判別できず、殺菌された
かどうかの評価はできないという欠点がある。すなわ
ち、寒天培養法は生細胞数の測定方法として最も広く用
いられている方法であるが、結果を得るまでに数十時間
以上と長い時間を要するため、殺菌管理に支障をきたす
場合が多い。バイオルミネッセンス法中で最も代表的な
ATP（アデノシン三リン酸）法は寒天培養法に比較し、
測定時間がいくぶん短縮される利点はあるが、生細胞数
が103〜104個/ml以上と比較的高濃度領域にしか適用で
きないという欠点がある。また前述の三者の方法では微
生物細胞とそれ以外の粒子の識別はできない。Conventional methods for testing and measuring microorganisms include microscopy, agar culture, and bioluminescence.However, none of these methods can determine the viability of microbial cells and cannot evaluate whether they have been killed. is there. That is, the agar culture method is the most widely used method for measuring the number of viable cells, but it takes a long time of several tens of hours or more to obtain a result, which often hinders sterilization management. The most typical bioluminescence method
ATP (adenosine triphosphate) method is compared with agar culture method,
Although there is an advantage that the measurement time is somewhat shortened, there is a disadvantage that the number of living cells is 10 3 to 10 4 cells / ml or more and can be applied only to a relatively high concentration region. In addition, the above three methods cannot distinguish between microbial cells and other particles.
従来、粒子計測方法としてはパーティクルカウンタが
実用化されている。この方法はセル中を通過する試料に
光を照射し、粒子の散乱光を計測することにより粒子数
を求める方法であるが、微生物細胞とそれ以外の粒子を
識別することはできないため、殺菌管理等には使用でき
ない。Conventionally, a particle counter has been put to practical use as a particle measurement method. This method irradiates a sample passing through the cell with light and measures the scattered light of the particles to determine the number of particles.However, since it is not possible to distinguish microbial cells from other particles, sterilization control is performed. Cannot be used for etc.
また、微生物細胞の生死を判別する方法として従来ア
クリジンオレンジという蛍光色素を使用すると、生細胞
は緑色に、死細胞は橙赤色に蛍光発色することから、こ
の差でもって生死判別が試みられたことがあるが、試料
の状態や試験条件のわずかな差により判別が不十分にな
り、信頼性に乏しいことから現在ではほとんど使われて
いない。In addition, when a fluorescent dye called acridine orange was used as a conventional method for determining the viability of microbial cells, viable cells glowed green and dead cells glowed orange red. However, discrimination is insufficient due to slight differences in the condition of the sample and test conditions, and the reliability is poor.
以上のように従来技術には、試料中の微生物細胞の生
−死、微生物細胞以外の粒子の識別−計測を迅速かつ、
同時に、低濃度の試料に対しても行えるようなものはな
かった。As described above, in the prior art, the life-death of microbial cells in a sample, identification of particles other than microbial cells
At the same time, there was nothing that could be performed on low concentration samples.
従来技術では、微生物細胞の生−死の識別ができな
い。生細胞を測定するには長時間必要（寒天培養
法）。高濃度の微生物細胞しか測定できない（バイオ
ルミネッセンス法）。微生物細胞とそれ以外の粒子と
の識別はできない（パーティクルカウンター）。などの
課題があり、原料や製品の品質管理、殺菌管理に支障を
きたすと共に使用範囲が限定されていた。In the prior art, it is not possible to distinguish between the life and death of microbial cells. It takes a long time to measure live cells (agar culture method). Only high concentrations of microbial cells can be measured (bioluminescence method). Microbial cells cannot be distinguished from other particles (particle counter). However, there is a problem in quality control and sterilization control of raw materials and products, and the use range is limited.
本発明はこれらの課題を解決し、微生物細胞とそれ以
外の粒子の識別、微生物細胞の生−死の識別を同時にか
つ短時間で、そして細胞濃度が１個/ml以下の低濃度で
も高感度に測定できる方法を提供しようとするものであ
る。The present invention solves these problems, distinguishes microbial cells from other particles, distinguishes the life-death of microbial cells simultaneously and in a short time, and has high sensitivity even at a low cell concentration of 1 cell / ml or less. It is intended to provide a method that can be used for measurement.
本発明者らは生細胞、死細胞、微生物細胞以外の粒子
を短時間で高感度に識別、計測する方法について実験・
検討を重ねた結果、 生細胞だけを識別する方法として、生細胞中に含ま
れる酵素又は補酵素と反応し蛍光を発する物質を作用さ
せ生細胞を光の点として計測する。The present inventors have experimented with a method for quickly and highly sensitively identifying and measuring particles other than living cells, dead cells, and microbial cells.
As a result of repeated studies, as a method of identifying only living cells, a substance that reacts with an enzyme or coenzyme contained in the living cells and emits fluorescence is acted on, and the living cells are measured as light points.
死細胞だけを識別する方法として、細胞内に存在す
る核酸に作用する蛍光物質を試料に作用させ、光の点と
して計測する。As a method of identifying only dead cells, a fluorescent substance that acts on nucleic acids present in cells is made to act on a sample, and measurement is performed as light spots.
微生物以外の粒子の計測は、微生物細胞を含む全粒
子数を散乱光で計測し、，で測定した生細胞数、死
細胞数を差し引いて求める。The measurement of particles other than microorganisms is obtained by measuring the total number of particles including microbial cells using scattered light, and subtracting the number of live cells and the number of dead cells measured by,.
という，，を組み合せた手段を用いることによ
り、生細胞数、死細胞数、微生物細胞以外の粒子が各々
識別・計測できることを確認した。It has been confirmed that the use of a combination of the above-mentioned methods enables the identification and measurement of live cells, dead cells, and particles other than microbial cells.
本発明は上記知見によって完成されたものであって、
微生物細胞とその他の粒子とを含む試料に光を照射し、
上記試料中の全体の粒子から生じる個々の散乱光を光の
点として検出して全体の粒子数を計測する工程と、上記
試料中の生きている微生物細胞の酵素あるいは補酵素と
反応して蛍光物質を生成する試薬を添加し、生成する蛍
光物質を励起させる光を照射して微生物細胞から生じる
蛍光を光の点として検出して生きている微生物細胞の数
を計測する工程と、上記試料中の死んでいる微生物細胞
の核酸に作用する蛍光物質を添加し、該蛍光物質を励起
させる光を照射して微生物細胞から生じる蛍光を光の点
として検出して死んでいる微生物細胞の数を計測する工
程とを有することを特徴とする微生物の生細胞、死細胞
および微生物細胞以外の粒子の数の測定方法である。The present invention has been completed based on the above findings,
Irradiating a sample containing microbial cells and other particles with light,
A step of detecting individual scattered light generated from all the particles in the sample as light points to count the total number of particles, and reacting with an enzyme or coenzyme of a living microbial cell in the sample to obtain a fluorescence. Adding a reagent that generates a substance, irradiating light that excites the fluorescent substance to be generated, detecting fluorescence generated from the microbial cells as light points, and counting the number of living microbial cells; and Add a fluorescent substance that acts on the nucleic acids of dead microbial cells, irradiate light that excites the fluorescent substance, detect the fluorescence generated from the microbial cells as light spots, and count the number of dead microbial cells And measuring the number of particles other than living cells, dead cells, and microbial cells of the microorganism.
本発明において、生細胞の識別に用いる物質は、細胞
中に含まれる酵素又は補酵素と反応して蛍光を発するも
ので、具体的にはフルオレセイン二酢酸、ホモバニリン
酸、Ｏ−フタルアルデヒド、ニコチンアミドアデニンジ
ヌクレチドなどが使用できるが、中でもスルオレセイン
二酢酸は生細胞に作用して特に強い蛍光を発するので特
に好ましい。In the present invention, the substance used for identifying living cells emits fluorescence when reacted with an enzyme or coenzyme contained in the cells, and specifically, fluorescein diacetate, homovanillic acid, O-phthalaldehyde, nicotinamide Adenine dinucleotide and the like can be used, and among them, sulorescein diacetate is particularly preferable because it acts on living cells and emits particularly strong fluorescence.
また死細胞の識別に用いた物質は、DAPI（４′,6−ジ
アミジノ２−フェニルインドール）、ローダミン、臭化
エチジューム、臭化プロピジュームであり、これらのい
ずれも死菌に対し、強い蛍光を発するので死菌を高感度
に検知・計測することができる。The substances used to identify dead cells are DAPI (4 ', 6-diamidino-2-phenylindole), rhodamine, ethidium bromide and propidium bromide, all of which have strong fluorescence against dead bacteria. Since it is emitted, dead bacteria can be detected and measured with high sensitivity.
生細胞の識別に用いた例えばフルオレセン二酢酸は生
細胞に作用させると細胞内に浸透して細胞内に存在する
酵素、エラテラーゼの作用により加水分解されフルオレ
セインを生成する。このフルオレセインは450〜490nmの
波長を有する励起光を照射してやることにより510〜520
nmをピーク波長とする蛍光を発するため、細胞が発光し
て見える。For example, fluorescein diacetate used to identify living cells, when applied to living cells, penetrates into the cells and is hydrolyzed by the action of an enzyme, elastase, present in the cells to produce fluorescein. This fluorescein is irradiated with an excitation light having a wavelength of 450 to 490 nm to produce 510 to 520.
The cells emit light because they emit fluorescence having a peak wavelength of nm.
また、死細胞を識別する場合、例えばDAPIを作用させ
ると、死細胞は生細胞に比較して細胞壁、細胞幕が弱く
なって損傷を受けているため、DAPIが細胞内に入りやす
くなって核酸と結びつきやすくなる。核酸と結びついた
DAPIは330〜380nmの励起光を照射することにより、430
〜440nmをピーク波長とする蛍光を発するため死細胞が
明るく発色して見える。In the case of identifying dead cells, for example, when DAPI is acted on, the dead cells are damaged due to weakened cell walls and cell curtains compared to live cells, so that DAPI can easily enter the cells and nucleic acids It becomes easy to connect with. Associated with nucleic acids
DAPI emits 330 to 380 nm excitation light to generate 430
Dead cells appear brightly colored because they emit fluorescence with a peak wavelength of ~ 440 nm.
また微生物細胞を含む全粒子数を計測する場合、例え
ば生菌測定に用いた450〜490nmの励起光を利用し、これ
を照射してやれば励起光と同一波長域の450〜490nmの散
乱光が各粒子から発するので、これを検知・計測すれば
全粒子数が求まる。When counting the total number of particles including microbial cells, for example, 450-490 nm excitation light used for live bacteria measurement is used, and if irradiated, 450-490 nm scattered light in the same wavelength range as the excitation light will be emitted. Since it is emitted from particles, if this is detected and measured, the total number of particles can be obtained.
本発明の一実施例を第１図によって説明する。第１図
において、１は試料に含まれる粒子に光を照射するため
の光源で、実施例では100Wの水銀灯を用いているが、蛍
光色素を励起する波長を有するものであればキセノンラ
ンプ、レーザー光でも構わない。１はハーフミラーで光
源１からの光を２軸方向に分割し、一方はセル24、他方
は全反射ミラー３を介してセル22に照射する。One embodiment of the present invention will be described with reference to FIG. In FIG. 1, reference numeral 1 denotes a light source for irradiating the particles contained in the sample with light. In this embodiment, a 100 W mercury lamp is used. However, a xenon lamp or a laser having a wavelength for exciting a fluorescent dye is used. It can be light. Reference numeral 1 denotes a half mirror that divides the light from the light source 1 in two axial directions.
22は生細胞と全粒子数の計数用セル、24は死細胞計測
用セルである。４は死細胞に作用させた色素を励起する
に必要な波長を通過させるフィルター、５は集光レンズ
である。また６は生細胞に作用させた色素を励起するに
必要な波長を通過させるフィルター、７は集光レンズで
ある。Reference numeral 22 denotes a cell for counting the number of living cells and the total number of particles, and reference numeral 24 denotes a cell for counting dead cells. Reference numeral 4 denotes a filter for passing a wavelength necessary to excite the dye applied to the dead cells, and reference numeral 5 denotes a condenser lens. Reference numeral 6 denotes a filter for passing a wavelength necessary to excite the dye applied to living cells, and reference numeral 7 denotes a condenser lens.
微生物の生細胞、死細胞、それ以外の粒子を含む試料
はラインP1を経由して反応槽19に入るが、その時ライン
P2より、生菌検知物質を所定濃度で注入してやる。20は
温度ヒーター付きマグネチックスターラ、21は回転子で
試料を所定温度で撹拌混合するためのものである。反応
槽19にて所定時間撹拌反応を行うことにより、生細胞内
では蛍光物質が生成するようになる。この試料をライン
P3を経由して、セル22内を通過させる。セル22には蛍光
物質を励起するに必要な波長の光が照射されており、試
料が通過すると各粒子は散乱光を発する。各粒子の中で
生細胞は散乱光以外に蛍光も発する。Live cells of microorganism, dead cells, although the sample enters the reaction vessel 19 via line P 1 containing other particles, then the line
Than P 2, it'll live bacteria detection substance is injected at a predetermined concentration. Reference numeral 20 denotes a magnetic stirrer with a temperature heater, and reference numeral 21 denotes a rotor for stirring and mixing the sample at a predetermined temperature. By performing the stirring reaction in the reaction tank 19 for a predetermined time, a fluorescent substance is generated in the living cells. Line this sample
Via the P 3, passing the cell 22. The cell 22 is irradiated with light having a wavelength necessary to excite the fluorescent substance, and when the sample passes, each particle emits scattered light. Living cells emit fluorescence in addition to scattered light in each particle.
８は散乱光をカットするための蛍光フィルタで生細胞
の発する蛍光を通過させレンズ９を介して受光器10に受
け、同受光器10の出力をパルスカウンタ11により生細胞
から発する蛍光を光の点としてパルスカウントすること
により生細胞数を計測する。Reference numeral 8 denotes a fluorescent filter for cutting scattered light, which passes the fluorescence emitted from the living cells through a lens 9 to a light receiver 10, and outputs the output of the light receiver 10 by a pulse counter 11 to convert the fluorescence emitted from the living cells into light. The number of living cells is counted by pulse counting as points.
また、12はセル内を通過する粒子に励起光を照射した
時、各粒子から発する散乱光を集光するレンズで、13は
散乱光を受光するための受光器、14はパルスカウンタ
で、全粒子数を光の点として計測する。Reference numeral 12 denotes a lens that collects scattered light emitted from each particle when the particles passing through the cell are irradiated with excitation light, 13 denotes a light receiver for receiving the scattered light, and 14 denotes a pulse counter. The number of particles is measured as a point of light.
セル22を出た試料はラインP4を介して反応槽23に入る
が、この時、ラインP5より死菌検知物質を所定濃度で添
加し反応槽23で所定時間反応させた後、ラインP6を介し
てセル24に入る。セル24には死細胞に作用させた色素を
励起するに必要な波長の光を照射しており、このセル内
を通過する粒子は散乱光および死細胞は散乱光以外に蛍
光を発する。15は散乱光をカットする蛍光フィルタで死
細胞の発する蛍光だけを通過させる。レンズ16を介して
受光器17により蛍光を受光し、パルスカウンタ18により
死細胞の発する蛍光を光の点としてパルスカウントし死
細胞数を求める。After the sample exiting the cell 22 is entering the reaction vessel 23 via line P 4, which this time, the reaction vessel 23 from the line P 5 was added killed detection substance at a predetermined concentration for a predetermined time response, line P Enter cell 24 via 6 . The cell 24 is irradiated with light having a wavelength necessary to excite the dye that has acted on the dead cells. Particles passing through this cell emit scattered light and dead cells emit fluorescence in addition to scattered light. Reference numeral 15 denotes a fluorescent filter that cuts scattered light and allows only the fluorescence emitted from dead cells to pass. Fluorescent light is received by a light receiver 17 via a lens 16, and pulse counting is performed by a pulse counter 18 using the fluorescent light emitted from dead cells as light points to determine the number of dead cells.
なお、生菌検知物質としてフルオレセイン二酢酸を用
いた場合、反応槽19での反応時間は10分以内でありまた
死菌検知物質としてDAPIを用いた場合、反応槽23での反
応時間は１分以内であり、本発明方法では約10分という
従来の寒天培養法の数十時間の測定時間に比較して格段
に短くてすみ、またセル内を通過する個々の粒子を１
個、１個、光の点のパルスとしてカウントする方式であ
るため、１個/ml以下という極めて低い細胞濃度でも測
定を可能とする。When fluorescein diacetate is used as a viable cell detection substance, the reaction time in the reaction tank 19 is within 10 minutes, and when DAPI is used as a dead cell detection substance, the reaction time in the reaction tank 23 is 1 minute. And the method of the present invention requires much less time than the conventional agar culture method of about 10 minutes, which is several tens of hours.
Since this method counts individual pulses as light pulses, light can be measured even at an extremely low cell concentration of 1 / ml or less.
本発明においては、生細胞および死細胞の発する蛍光
量の強さ、およびこの蛍光を検知するために必要な受光
器の選定が最も大きな技術的ポイントである。そこで本
発明者等はこれらの点について検討するため対象試料と
して酵母を用い光学的、電気的に生細胞、死細胞からの
蛍光量の測定を行った結果、生細胞１個当りの蛍光発光
量はフルオレセイン二酢酸を50μg/ml作用させた時、30
〜80pW、そして死菌１個当りの蛍光発光量はDAPIを１μ
g/ml作用させた時、約100〜200pWという数値を得た。そ
こで、この光エネルギーを検知する能力を有する受光器
を選定し試験に使用した。In the present invention, the most important technical points are the intensity of the amount of fluorescence emitted from living cells and dead cells, and the selection of a light receiver necessary for detecting the fluorescence. In order to examine these points, the present inventors optically and electrically measured the amount of fluorescence from living cells and dead cells using yeast as a target sample. As a result, the amount of fluorescence emitted per living cell was measured. Is 50 μg / ml fluorescein diacetate, 30
~ 80pW, and the amount of fluorescence per killed cell is 1μ for DAPI.
When g / ml was applied, a value of about 100 to 200 pW was obtained. Therefore, a photodetector having the ability to detect this light energy was selected and used for the test.
なお、この実施例では、セル22により生菌と全粒子数
を、セル24にて死菌数を計測する例を示したが、セル22
で死菌数をセル24にて生菌数を計測してもよいし、散乱
光の測定は22,24のどちらのセルを行ってもかまわな
い。In this embodiment, an example is shown in which the number of live bacteria and the total number of particles is measured by the cell 22, and the number of dead bacteria is measured by the cell 24.
Then, the number of dead bacteria may be measured in the cell 24 and the number of viable bacteria may be measured in the cell 24, and the measurement of the scattered light may be performed in either the cell 22 or the cell 24.
次に本装置を用いた試験実施例を示す。 Next, a test example using the present apparatus will be described.
（１）試料溶液 48時間培養したBaker′s Yeast（酵母）を遠心分離器
により遠心濃縮（3000rpm、５分間）して細胞を回収
し、pH7.0、1/15Mリン酸バッファー液で洗浄したものを
生細胞とした。(1) Sample solution Baker's Yeast (yeast) cultured for 48 hours was centrifugally concentrated (3000 rpm, 5 minutes) with a centrifuge to collect cells, and washed with a pH 7.0, 1/15 M phosphate buffer solution. Those were defined as living cells.
また死細胞はこれを121℃５分間加熱処理したものを
使用した。微生物細胞以外の粒子としてはポリスチレン
ラテックス標準粒子を使用し生細胞：死細胞：ポリスチ
レンラテックス標準粒子＝1:10:10の割合で混合し、総
粒子数として約100個/mlの濃度になるように調整した。The dead cells used were heat-treated at 121 ° C. for 5 minutes. Polystyrene latex standard particles are used as particles other than microbial cells, and live cells: dead cells: polystyrene latex standard particles are mixed at a ratio of 1:10:10 so that the total number of particles becomes about 100 particles / ml. Was adjusted.
（２）フルオレセイン二酢酸溶液 アセトンにフルオレセイン二酢酸を溶解して1mg/mlの
濃度に調整したものを試料に対し50μg/mlの濃度になる
ように添加した。(2) Fluorescein diacetate solution Fluorescein diacetate dissolved in acetone and adjusted to a concentration of 1 mg / ml was added to the sample to a concentration of 50 μg / ml.
（３）DAPI溶液 純水にDAPIを100μg/mlの濃度になるように調整し試
料に対し１μg/mlの濃度で添加した。(3) DAPI solution DAPI was adjusted to a concentration of 100 μg / ml in pure water and added to the sample at a concentration of 1 μg / ml.
（４）試料処理量、反応条件 試料を10mg/minの処理速度で内径1mmφのガラス製セ
ル内を通過させたが、この時、反応槽19では37℃、５分
間、また反応槽23では室温で１分間試料と色素との反応
を行わせた。(4) Sample throughput and reaction conditions The sample was passed through a glass cell with an inner diameter of 1 mm at a processing rate of 10 mg / min. To react the sample with the dye for 1 minute.
（５）測定条件 照射光として100W水銀ランプを使用し、励起フィルタ
６には450〜490nmの波長域を通過するもの、また蛍光フ
ィルタ８は510nm以上の波長域を通過するものを使用し
た。また死菌を励起させるためのフィルタ４には、330
〜380nmの波長域を通過するもの、蛍光フィルタ15には4
20nm以上の波長域を通過するものを用いた。(5) Measurement conditions A 100 W mercury lamp was used as the irradiation light, and the excitation filter 6 used passed through a wavelength range of 450 to 490 nm, and the fluorescent filter 8 used passed through a wavelength range of 510 nm or more. In addition, the filter 4 for exciting dead bacteria has 330
~ 380nm wavelength band, 4 for fluorescent filter 15
Those that pass a wavelength range of 20 nm or more were used.
（６）測定結果 パルスカウンタにてカウントされた各々の粒子数を第
１表に示す。この値は試料10ml当りの粒子カウント数で
ある。この結果より生菌：死菌：微生物細胞以外の粒子
数（全粒子数−生菌数−死菌数）＝52:508:480となり初
期設定値の1:10:10にほぼ近い数値を得ることができ
た。(6) Measurement results Table 1 shows the number of each particle counted by the pulse counter. This value is the number of particles counted per 10 ml of the sample. From this result, live bacteria: dead bacteria: the number of particles other than microbial cells (the total number of particles-the number of live bacteria-the number of dead bacteria) = 52: 508: 480, which is a value close to the initial set value of 1:10:10. I was able to.
〔発明の効果〕 本発明の効果として （１） 従来法では不可能であった生菌、死菌、微生物
細胞以外の粒子数をそれぞれ識別して計測できる。 [Effects of the Invention] The effects of the present invention are as follows: (1) The number of particles other than live bacteria, dead bacteria, and microbial cells, which was impossible with the conventional method, can be identified and measured.
（２） 従来寒天培養法では数十時間必要であった微生
物細胞の計測が本発明方法により10分以内で測定でき
る。(2) The measurement of microbial cells, which conventionally required several tens of hours in the agar culture method, can be measured within 10 minutes by the method of the present invention.
（３） また、各細胞、粒子を光の点としてパルスカウ
ントすることにより低い細胞濃度の試料でも測定でき
る。(3) In addition, it is possible to measure even a sample having a low cell concentration by pulse counting each cell or particle as a light point.
となり、殺菌装置の性能把握、原料、製品の品質管理が
高精度かつ迅速に行えるようになった。As a result, the performance of the sterilizer and the quality control of raw materials and products can be accurately and quickly performed.
第１図は本発明の実施例を説明するための説明図であ
る。FIG. 1 is an explanatory diagram for explaining an embodiment of the present invention.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 土井 崇史 神奈川県横浜市金沢区幸浦１丁目８番地 １ 三菱重工業株式会社基盤技術研究所 内 (56)参考文献 国際公開89／8714（ＷＯ，Ａ１) ────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Takashi Doi 1-8-1 Koura, Kanazawa-ku, Yokohama-shi, Kanagawa 1 Mitsubishi Heavy Industries, Ltd. Basic Technology Research Laboratory (56) References International Publication 89/8714 (WO, A1)
Claims (1)
光を照射し、上記試料中の全体の粒子から生じる個々の
散乱光を光の点として検出して全体の粒子数を計測する
工程と、上記試料中の生きている微生物細胞の酵素ある
いは補酵素と反応して蛍光物質を生成する試薬を添加
し、生成する蛍光物質を励起させる光を照射して微生物
細胞から生じる蛍光を光の点として検出して生きている
微生物細胞の数を計測する工程と、上記試料中の死んで
いる微生物細胞の核酸に作用する蛍光物質を添加し、該
蛍光物質を励起させる光を照射して微生物細胞から生じ
る蛍光を光の点として検出して死んでいる微生物細胞の
数を計測する工程とを有することを特徴とする微生物の
生細胞、死細胞および微生物細胞以外の粒子の数の測定
方法。1. A step of irradiating a sample containing microbial cells and other particles with light, detecting individual scattered light generated from all the particles in the sample as light points, and counting the total number of particles. And a reagent that generates a fluorescent substance by reacting with the enzyme or coenzyme of living microbial cells in the sample, and irradiates light that excites the generated fluorescent substance to generate fluorescence generated from the microbial cells. Measuring the number of living microbial cells by detecting them as spots, adding a fluorescent substance that acts on the nucleic acid of the dead microbial cells in the sample, and irradiating light that excites the fluorescent substance to the microorganism. Measuring the number of dead microbial cells by detecting the fluorescence generated from the cells as light spots, and measuring the number of living cells, dead cells, and particles other than the microbial cells of the microorganism.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1269130A JP2735901B2 (en) | 1989-10-18 | 1989-10-18 | Method for measuring the number of living cells, dead cells, and particles other than microorganism cells of microorganisms |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1269130A JP2735901B2 (en) | 1989-10-18 | 1989-10-18 | Method for measuring the number of living cells, dead cells, and particles other than microorganism cells of microorganisms |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03131743A JPH03131743A (en) | 1991-06-05 |
| JP2735901B2 true JP2735901B2 (en) | 1998-04-02 |
Family
ID=17468110
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1269130A Expired - Fee Related JP2735901B2 (en) | 1989-10-18 | 1989-10-18 | Method for measuring the number of living cells, dead cells, and particles other than microorganism cells of microorganisms |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2735901B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003063078A1 (en) * | 2002-01-21 | 2003-07-31 | Matsushita Ecology Systems Co., Ltd. | Imaging device |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8071051B2 (en) * | 2004-05-14 | 2011-12-06 | Honeywell International Inc. | Portable sample analyzer cartridge |
| KR100616465B1 (en) * | 2001-03-28 | 2006-08-29 | 한국원자력연구소 | Methods for the measurement of radiation dose of radio-sensitive organs using apoptotic fragment assay |
| WO2016013394A1 (en) * | 2014-07-22 | 2016-01-28 | 株式会社日立ハイテクノロジーズ | Cell-number-concentration adjustment device, and automatic subculture system using same |
| CN111044435B (en) * | 2019-12-16 | 2021-01-19 | 中国科学院昆明动物研究所 | Multi-index detection light path system for flow cytometer |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2628531B1 (en) * | 1988-03-08 | 1992-10-02 | Chemunex Sa | METHOD FOR SEARCHING, SPECIFIC DETECTION AND NUMBERING OF MICRO-ORGANISMS, INSTALLATION FOR IMPLEMENTING SAID METHOD |
-
1989
- 1989-10-18 JP JP1269130A patent/JP2735901B2/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003063078A1 (en) * | 2002-01-21 | 2003-07-31 | Matsushita Ecology Systems Co., Ltd. | Imaging device |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH03131743A (en) | 1991-06-05 |
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