JPH04287681A - Automatic and continuous measuring device of number of viable microorganism in water - Google Patents
Automatic and continuous measuring device of number of viable microorganism in waterInfo
- Publication number
- JPH04287681A JPH04287681A JP15081191A JP15081191A JPH04287681A JP H04287681 A JPH04287681 A JP H04287681A JP 15081191 A JP15081191 A JP 15081191A JP 15081191 A JP15081191 A JP 15081191A JP H04287681 A JPH04287681 A JP H04287681A
- Authority
- JP
- Japan
- Prior art keywords
- water
- reagent
- tested
- automatically
- test water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 134
- 244000005700 microbiome Species 0.000 title abstract description 10
- 238000012360 testing method Methods 0.000 claims abstract description 58
- 238000006243 chemical reaction Methods 0.000 claims abstract description 47
- 238000003860 storage Methods 0.000 claims abstract description 29
- 239000003153 chemical reaction reagent Substances 0.000 claims description 107
- 241000894006 Bacteria Species 0.000 claims description 27
- 238000005259 measurement Methods 0.000 claims description 24
- 238000012546 transfer Methods 0.000 claims description 18
- 238000005375 photometry Methods 0.000 claims description 6
- 238000007599 discharging Methods 0.000 claims description 4
- 244000013123 dwarf bean Species 0.000 claims 1
- 235000021331 green beans Nutrition 0.000 claims 1
- 238000005406 washing Methods 0.000 claims 1
- 108060001084 Luciferase Proteins 0.000 abstract description 10
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 abstract description 9
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 abstract description 9
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 abstract description 9
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 abstract description 9
- 239000008235 industrial water Substances 0.000 abstract description 9
- 239000005089 Luciferase Substances 0.000 abstract description 7
- 108090000790 Enzymes Proteins 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 abstract 1
- 230000001939 inductive effect Effects 0.000 abstract 1
- 238000005070 sampling Methods 0.000 description 24
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 23
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 23
- 239000008223 sterile water Substances 0.000 description 18
- 238000004140 cleaning Methods 0.000 description 16
- 238000000034 method Methods 0.000 description 16
- 238000012136 culture method Methods 0.000 description 8
- 239000010842 industrial wastewater Substances 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 238000004020 luminiscence type Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 229910000831 Steel Inorganic materials 0.000 description 3
- 239000000498 cooling water Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002736 nonionic surfactant Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000010959 steel Substances 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 2
- 239000006177 biological buffer Substances 0.000 description 2
- 238000005415 bioluminescence Methods 0.000 description 2
- 230000029918 bioluminescence Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000001678 irradiating effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 150000002894 organic compounds Chemical class 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000002351 wastewater Substances 0.000 description 2
- KDKGWGUUUVROTO-UHFFFAOYSA-N 1-hydroxypiperazine Chemical compound ON1CCNCC1 KDKGWGUUUVROTO-UHFFFAOYSA-N 0.000 description 1
- 230000002407 ATP formation Effects 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- 241000270295 Serpentes Species 0.000 description 1
- 239000004809 Teflon Substances 0.000 description 1
- 229920006362 Teflon® Polymers 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 241000238583 Vargula hilgendorfii Species 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N adenyl group Chemical group N1=CN=C2N=CNC2=C1N GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000012840 feeding operation Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- LIYGYAHYXQDGEP-UHFFFAOYSA-N firefly oxyluciferin Natural products Oc1csc(n1)-c1nc2ccc(O)cc2s1 LIYGYAHYXQDGEP-UHFFFAOYSA-N 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 230000009931 harmful effect Effects 0.000 description 1
- 230000020169 heat generation Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 150000002605 large molecules Chemical class 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- JJVOROULKOMTKG-UHFFFAOYSA-N oxidized Photinus luciferin Chemical compound S1C2=CC(O)=CC=C2N=C1C1=NC(=O)CS1 JJVOROULKOMTKG-UHFFFAOYSA-N 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 229920000151 polyglycol Polymers 0.000 description 1
- 239000010695 polyglycol Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【０００１】0001
【産業上の利用分野】本発明は水中の生菌数を自動的且
つ連続的にリアルタイムで測定する装置に関する。本発
明は、例えば、製紙工場の抄紙白水、或は各種化学工場
及び製鉄工場等各種工場或は発電所等の循環式冷却水そ
の他各種工業用水および排水の処理・管理に利用される
。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for automatically and continuously measuring the number of viable bacteria in water in real time. The present invention is utilized, for example, for the treatment and management of papermaking white water in paper mills, circulating cooling water in various factories such as chemical factories and steel factories, power plants, and various other industrial water and wastewater.
【０００２】0002
【従来技術】製紙工場の抄紙白水、各種化学工場及び製
鉄工場等各種工場及び発電所等の循環式冷却水その他各
種工業用水および排水等（以下これらを包括して“各種
工業用水および産業排水等”と略記する場合がある）は
微生物が発生し増殖し易い条件にあるか或は工業用水の
様に微生物が存在している表流水を使用している場合が
ある。これらの微生物は直接又は間接に各種の弊害の原
因となる。[Prior Art] Papermaking white water of paper mills, circulating cooling water of various factories such as various chemical factories and steel factories, power plants, etc., and various industrial water and wastewater (hereinafter collectively referred to as "various industrial water and industrial wastewater, etc.") (sometimes abbreviated as ")" may be used in conditions where microorganisms are likely to occur and multiply, or in which surface water, such as industrial water, contains microorganisms. These microorganisms directly or indirectly cause various harmful effects.
【０００３】即ち、詳述するならば、製紙工場において
抄紙工程の白水中には原料由来や添加された各種の有機
化合物が溶存しているので、これを栄養源として微生物
が増殖し、スライム障害を惹起す。又、石油化学工場、
製鉄工場、発電所等での循環式冷却水では冷却塔を用い
た淡水の循環使用を行なっているので、水の濃縮がおこ
り栄養塩類が濃縮され、これを利用して水中に微生物が
繁殖して冷却効率の低下などの障害を惹起す。Specifically, in paper mills, various organic compounds derived from raw materials or added are dissolved in the white water of the papermaking process, and microorganisms multiply using this as a nutrient source, resulting in slime damage. cause Also, petrochemical factories,
In circulating cooling water at steel factories, power plants, etc., fresh water is recycled using cooling towers, which causes the water to become concentrated and nutrients to be concentrated, which can be used to propagate microorganisms in the water. This may cause problems such as a decrease in cooling efficiency.
【０００４】従って、各種工業用水及び産業排水等の微
生物管理、即ちより具体的に云うならば、前記用水およ
び排水等に存在する生菌数を測定することによって然る
べき後続手段を講じて細菌の増殖を抑制することは製造
管理、工場管理、環境管理等から重要なことである。更
に、排水に関しては排水中の菌数が法的に規制されてい
る。即ち、工場排水では大腸菌群数は３，０００個／ｍ
ｌ以下と法規制されている。[0004] Therefore, microbial control of various industrial water and industrial wastewater, etc., more specifically, by measuring the number of viable bacteria present in the above-mentioned industrial water and wastewater, etc., appropriate subsequent measures are taken to control the growth of bacteria. Controlling this is important from the standpoint of manufacturing management, factory management, environmental management, etc. Furthermore, regarding wastewater, the number of bacteria in wastewater is legally regulated. In other words, the number of coliform bacteria in industrial wastewater is 3,000/m
It is regulated by law to be less than l.
【０００５】従来、水中の生菌数の測定は通常、培養法
が用いられている。尚、培養法による測定は、１つの集
落が１個の生きた菌がもとになって形成されるという前
提のもとに行う方法である。従って、正確には、この方
法で知られるのは菌数というより、集落形成単位である
。培養法にはイ．平板塗布法、ロ．混和培養法及びハ．
メンブランフィルター法があるが、その１つの混和培養
法が広く採用されている。この方法は、試料水を無菌操
作により寒天平板上に植種し、３７℃で２４時間ないし
４８時間培養後、発生した細菌集落数を測定することに
よって水中に存在していた生菌数を測定するものである
。これら従来法の欠点の１つは生菌数は培養後つまり、
１乃至２日後でないとわからないので判明した時点では
生菌数を測定しようとした水はすでに系内に存在しない
ので、現状把握が出来ないということである。即ち、リ
アルタイム性（即時性、同時性）に欠けるということで
ある。[0005] Conventionally, the number of viable bacteria in water has usually been measured using a culture method. The culture method is a method based on the premise that one colony is formed from one living bacterium. Therefore, to be more precise, what is known with this method is the colony forming unit rather than the number of bacteria. The culture method includes a. Flat plate coating method, b. Mixed culture method and c.
There are membrane filter methods, one of which is the mixed culture method, which is widely used. In this method, sample water is inoculated onto an agar plate using aseptic operation, and after culturing at 37°C for 24 to 48 hours, the number of viable bacteria present in the water is determined by measuring the number of bacterial colonies that have formed. It is something to do. One of the drawbacks of these conventional methods is that the number of viable bacteria is low after culturing.
This is not known until one or two days later, and by the time it is known, the water in which the number of viable bacteria was to be measured is no longer present in the system, so the current situation cannot be ascertained. In other words, it lacks real-time properties (immediacy, simultaneity).
【０００６】所で、近年細菌の菌体に含まれるアデノシ
ントリホスフェート（ＡＴＰ）を化学発光測定装置を用
いて瞬時に測定する方法が開発された。然しながら、こ
の方法を水中の生菌数の測定に応用してもリアルタイム
で結果が得られる訳ではない。即ち、被試験水の採取、
サンプルの調製、測定操作等に人手を要し、最終結果を
得るのに長時間を必要とする。In recent years, a method has been developed for instantly measuring adenosine triphosphate (ATP) contained in bacterial cells using a chemiluminescent measuring device. However, even if this method is applied to measuring the number of viable bacteria in water, results cannot be obtained in real time. That is, sampling of the water to be tested;
Sample preparation, measurement operations, etc. require manpower, and it takes a long time to obtain the final results.
【０００７】上述した様に、水中の生菌数を自動的且つ
連続的にリアルタイムで測定する方法及び装置は従来な
かった。[0007] As mentioned above, there has been no method or device for automatically and continuously measuring the number of viable bacteria in water in real time.
【０００８】[0008]
【発明が解決しようとする課題】本発明が解決しようと
する課題は、水中、なかんずく各種産業排水及び工業用
水等の生菌数を自動的且つ連続的にリアルタイムで測定
する装置を開発することである。[Problem to be solved by the invention] The problem to be solved by the present invention is to develop a device that automatically and continuously measures the number of viable bacteria in water, especially in various industrial wastewater and industrial water, in real time. be.
【０００９】発明が解決しようとする別の課題は、いわ
ゆる各種工業用水及び産業排水等の生菌数を自動的且つ
連続的にリアルタイムで測定し、その結果に依り前記各
種工業用水及び産業排水等の微生物管理、広義には水質
管理を自動的に行うシステムを確立することである。Another problem to be solved by the invention is to automatically and continuously measure the number of viable bacteria in various types of industrial water, industrial wastewater, etc. in real time, and based on the results, to The goal is to establish a system that automatically controls microorganisms, or in a broader sense, water quality control.
【００１０】発明が解決しようとする更なる課題は以下
逐次明らかにされる。[0010] Further problems to be solved by the invention will be successively clarified below.
【００１１】[0011]
【課題を解決するための手段】課題を解決するための基
本的な手段は、被試験水を収容する装置から被試験水を
、被試験水を一時的に貯溜する一時貯溜槽に自動的且つ
連続的に移送、還流させながら所定量ずつ反応管へ分注
した後一定のインターバルで第１試薬ｐＨ調整液、第２
試薬ＡＴＰ放出液及びルシフェリン、発光酵素ルシフェ
ラーゼ及びＭｇ２＋から成る第３試薬を分注し、被試験
水中に存在する生菌数が保有するアデノシントリフォス
フェートと第３試薬を反応管内で反応させた際に発生す
る光子の量を測定することである。[Means for solving the problem] The basic means for solving the problem is to automatically transfer the test water from the device containing the test water to a temporary storage tank that temporarily stores the test water. After dispensing a predetermined amount into the reaction tube while continuously transferring and refluxing, the first reagent pH adjustment solution and the second
When the third reagent consisting of the reagent ATP release liquid and luciferin, the luminescent enzyme luciferase, and Mg2+ is dispensed, and the third reagent is reacted with the adenosine triphosphate possessed by the number of viable bacteria present in the test water in the reaction tube. The method is to measure the amount of photons that are generated.
【００１２】本発明で使用する各試薬について解説する
。[0012] Each reagent used in the present invention will be explained.
【００１３】第１試薬は生物学的緩衝剤で被試験水をｐ
Ｋａ＝７〜７．８に調整する作用をする。ルシフエリン
及びルシフェラーゼとの相溶性等を考慮するとＮ−２−
ヒドロキシルピペラジンＮ′−２−エタンスルフォン酸
が最も好ましい。これはルーマックインターナショナル
社より“ＨＥＰＥＳ▲Ｒ▼”という商標名で販売されて
いるものが容易に人手出来るが勿論均等物が使用出来る
。[0013] The first reagent is a biological buffer that purifies the water to be tested.
It functions to adjust Ka to 7 to 7.8. Considering the compatibility with luciferin and luciferase, N-2-
Most preferred is hydroxylpiperazine N'-2-ethanesulfonic acid. This can be easily done by hand using a product sold under the trade name "HEPES▲R▼" by Lumac International Co., Ltd., but of course equivalent products can also be used.
【００１４】第２試薬は細菌の細胞膜の透過性を高めそ
れによってＡＴＰのような低分子量の分子を細胞外に放
出させ、一方アルカリフォスフアターゼのような大きな
分子を細胞内に閉じ込めておく作用をする化学抽出用試
薬であって、沸騰したトリス緩衝液や“Ｔｒｉｔｏｎｘ
−１００”のような体細胞用抽出用試薬でもよい。然し
ながら、測定精度や速度を高めるには界面活性剤、それ
も特定の非イオン性界面活性剤が有効であることがわか
った。即ち、このような非イオン界面活性剤は化１The second reagent has the effect of increasing the permeability of the bacterial cell membrane, thereby allowing low molecular weight molecules such as ATP to be released outside the cell, while keeping large molecules such as alkaline phosphatase confined within the cell. A chemical extraction reagent that can be used in boiling Tris buffer or
-100" may be used. However, it has been found that surfactants, especially specific nonionic surfactants, are effective in increasing measurement accuracy and speed. Namely, Such a nonionic surfactant has chemical formula 1
【０
０１５】0
015]
【化１】
を有するエトキシル化アルキルフェノール類およびオク
チルフェノール類ならびに化２Ethoxylated alkylphenols and octylphenols having formula 1 and formula 2
【００１６】[0016]
【化２】
を有する脂肪酸ポリグリコールエーテル類が好ましい。
第２試薬は“ＡＴＰ放出剤”あるいは“ＡＴＰ抽出剤”
等と呼称してもよい。Fatty acid polyglycol ethers having the following formula are preferred. The second reagent is an “ATP release agent” or “ATP extractant”
etc. may also be called.
【００１７】第３試薬は発光素ルシフェリン、発光酵素
ルシフェラーゼ及びＭｇ２＋を配合した試薬である。The third reagent is a reagent containing a luminescent element luciferin, a luminescent enzyme luciferase, and Mg2+.
【００１８】ここで、本発明を開発する基礎になった理
論的背景を説明する。The theoretical background on which the present invention was developed will now be explained.
【００１９】総ての細菌は、有機化合物の分解によって
、それに含まれる化学的エネルギーを取り出して、アデ
ノシントリフォスフェート（以下“ＡＴＰ”と略記する
）の形で捕捉する過程、即ちＡＴＰ産生系を持つ。ＡＴ
Ｐは化３の様なヌクレオチドである。[0019] All bacteria have an ATP production system, a process that extracts the chemical energy contained in organic compounds by decomposing them and captures them in the form of adenosine triphosphate (hereinafter abbreviated as "ATP"). have A.T.
P is a nucleotide as shown in chemical formula 3.
【００２０】[0020]
【化３】[C3]
【００２１】ＡＴＰは水に易溶で、非常に不安定な化合
物である。ＡＴＰの特異な反応として、発光酵素ルシフ
ェラーゼと発光基質ルシフェリンがＡＴＰと反応してＡ
ＴＰ１分子当り１フオトンの発光をする。即ち、ATP is easily soluble in water and is a very unstable compound. As a unique reaction of ATP, the luminescent enzyme luciferase and the luminescent substrate luciferin react with ATP to produce ATP.
One photon of light is emitted per TP molecule. That is,
【００
２２】ルシフェリンはルシフェラーゼ、Ｍｇ２＋の存在
下でＡＴＰを必要とするアデニル基転移を受けて活性化
され、ルシフェリルアデニル酸となる。00
22] Luciferin is activated in the presence of luciferase and Mg2+ by undergoing adenyl group transfer requiring ATP, and becomes luciferyl adenylate.
【００２３】[0023]
【００２４】これはルシフェラーゼの活性中心に固く結
合しており、酸素分子と反応して発光し、ＡＭＰとＣＯ
２を放出してオキシルシフェリンとなる。[0024] This is tightly bound to the active center of luciferase, reacts with oxygen molecules, emits light, and generates AMP and CO.
2 to become oxyluciferin.
【００２５】
この反応を生物発光という。生物発光の一般的な特徴は
、熱の発生を全く伴わない冷光である点にある。発光極
大波長は発光細菌で４９０ｎｍ、ウミホタルで４６０ｎ
ｍ、ホタルで５４４〜５８２ｎｍである。[0025] This reaction is called bioluminescence. The general characteristic of bioluminescence is that it is a cold light without any heat generation. The maximum wavelength of light emission is 490nm for luminescent bacteria and 460n for sea fireflies.
m, 544 to 582 nm for fireflies.
【００２６】この反応を利用して、即ち発光光子の量を
測定することによってＡＴＰ濃度を測定し、ＡＴＰ濃度
と細胞数との相関関係より相対的な生細胞数を求めるこ
とが出来る。Utilizing this reaction, that is, by measuring the amount of emitted photons, the ATP concentration can be measured, and the relative number of living cells can be determined from the correlation between the ATP concentration and the number of cells.
【００２７】この反応を利用して、例えば水中の生菌数
を測定する場合被試験水の採取から測定、データ処理ま
で自動化しない限り上述の細菌発光反応を効果的に利用
したとは云えない。そこで、本発明者等は鋭意検討した
結果下記に詳述する方法を策定した。When using this reaction to measure, for example, the number of viable bacteria in water, it cannot be said that the above-mentioned bacterial luminescence reaction has been effectively utilized unless the process from collection of test water to measurement and data processing is automated. As a result of intensive study, the inventors of the present invention have formulated the method described in detail below.
【００２８】即ち、本発明によって、■ 被試験水及
び第１試薬を分注すること、■ 第２試薬を分注する
こと、■ 第３試薬を分注すること、■ ＡＴＰと
第３試薬との反応による発光量を測定すること、■
使用した反応管及びパイプラインの洗浄をすること、■
使用した反応管の水ブランクテストをすることから
主として構成されるＡＴＰと第３試薬との反応によって
発生する発光量を測定してＡＴＰ濃度を測定し、ＡＴＰ
濃度と細胞数との相関関係により被試験水中の生菌数を
測定する方法が提供される。That is, according to the present invention, (1) dispensing the water to be tested and the first reagent, (2) dispensing the second reagent, (2) dispensing the third reagent, and (2) dispensing ATP and the third reagent. Measuring the amount of light emitted by the reaction,■
Cleaning used reaction tubes and pipelines, ■
The ATP concentration was measured by performing a water blank test on the reaction tube used, and measuring the amount of luminescence generated by the reaction between ATP, which mainly consists of ATP, and the third reagent.
A method is provided for measuring the number of viable bacteria in test water based on the correlation between concentration and cell number.
【００２９】本発明の基本的な方法は上述した通りであ
るが、本発明の最も重要な特徴はこの方法を自動的且つ
連続的に行う点である。そのために本発明は被試験水を
収容する装置から被試験水を自動的且つ連続的に移送し
反応管に分注し測定部に搬送することを特徴とする。Although the basic method of the present invention has been described above, the most important feature of the present invention is that this method is performed automatically and continuously. To this end, the present invention is characterized in that the water to be tested is automatically and continuously transferred from a device containing the water to be tested, dispensed into reaction tubes, and transported to the measuring section.
【００３０】即ち、本発明によって、（１） 被試験
水移送部、（２） 反応管搬送部、（３） 測光部
および（４） オペレーションコンソール部から主と
して構成されている水中の生菌数の自動・連続測定装置
にして；（１）−１；前記被試験水移送部が、
■ 被試験水を収容する装置、
■ 被試験水を一時的に貯溜する一時貯溜槽および■
該被試験水を収容する装置と該一時貯溜槽の間を被
試験水を自動・連続的に還流させる装置から構成されて
おり、
（１）−２；前記反応管搬送部が、
■ サンプル供給系、
■ 第１試薬供給系および
■ 第２試薬供給系から構成されていて、該サンプル
供給系がパイプラインを備えていて、該パイプラインが
、前記被試験水を一時的に貯溜する一時貯溜槽に導入さ
れていて被試験水を吸引、分注及び吐出する手段を備え
ており且つ搬送部から測光部へ自動・連続的に搬送され
る反応管と接続していて更に該パイプラインを洗浄する
様に該パイプラインに洗浄水を供給する手段を備えてい
ることを各々特徴とし、
（１）−３；前記測光部が、
■ 第３試薬供給系および
■ 水中の生菌の保有するＡＴＰが第３試薬と反応し
て発生する光子の量を測定する測光系を備えていること
を特徴とする水中の生菌数を自動・連続的に測定する装
置が提供される。That is, according to the present invention, it is possible to determine the number of viable bacteria in water, which is mainly composed of (1) a test water transfer section, (2) a reaction tube transfer section, (3) a photometry section, and (4) an operation console section. In an automatic/continuous measuring device; (1)-1; the test water transfer section includes: (1) a device for storing the test water; (2) a temporary storage tank for temporarily storing the test water; and (2)
It consists of a device that automatically and continuously circulates the test water between a device that accommodates the test water and the temporary storage tank, (1)-2; system, consisting of a first reagent supply system and a second reagent supply system, the sample supply system is equipped with a pipeline, and the pipeline is a temporary storage for temporarily storing the water to be tested. It is introduced into the tank and is equipped with means for suctioning, dispensing, and discharging the water to be tested, and is connected to the reaction tube that is automatically and continuously transported from the transport section to the photometry section, and is also capable of cleaning the pipeline. (1)-3; The photometric section is characterized by: (1) a third reagent supply system; and (2) ATP possessed by viable bacteria in the water. Provided is an apparatus for automatically and continuously measuring the number of viable bacteria in water, which is equipped with a photometric system that measures the amount of photons generated by the reaction with a third reagent.
【００３１】第１図は本発明の被試験水移送部及び反応
管搬送部の一部を示す概念図である。第１図を参考にし
て装置の動作原理を解説する。FIG. 1 is a conceptual diagram showing part of the test water transfer section and reaction tube transfer section of the present invention. The operating principle of the device will be explained with reference to Figure 1.
【００３２】被試験水は被試験水を収容する装置１から
被試験水を一時的に貯溜する一時貯溜槽２ヘ移送され且
つ一時貯溜槽２から被試験水を収容する装置１へ還流さ
せられる。The water to be tested is transferred from the device 1 for storing the water to be tested to the temporary storage tank 2 for temporarily storing the water to be tested, and from the temporary storage tank 2 to the device 1 for storing the water to be tested. .
【００３３】被試験水を収容する装置１から被試験水を
一時貯溜槽２へ移送するには被試験水を収容する装置１
と一次貯溜槽２の間を繊維質が付着しない様に比較的太
いパイプ３で往路、帰路と還流する様に連結させ、介在
させたポンプ４の作動によって実施される。被試験水を
被試験水を収容する装置１と一次貯溜槽２の間を還流さ
せる替わりに被試験水を収容する装置１から一次貯溜槽
２へ移送された被試験水を溢流させてもよい。その場合
、ポンプ移送でもあるいは被試験水を収容する装置１と
一次貯溜槽２の間を傾斜パイプで連結して落差移送して
もよい。To transfer the test water from the test water storage device 1 to the temporary storage tank 2, the test water storage device 1 is used to transfer the test water to the temporary storage tank 2.
A relatively thick pipe 3 is connected between the primary storage tank 2 and the primary storage tank 2 so that the outgoing and return flow can be carried out to prevent fibers from adhering, and an interposed pump 4 is operated. Instead of circulating the test water between the device 1 that stores the test water and the primary storage tank 2, it is also possible to overflow the test water transferred from the device 1 that stores the test water to the primary storage tank 2. good. In that case, the water may be transferred by a pump or by connecting the apparatus 1 containing the water to be tested and the primary storage tank 2 with an inclined pipe to transfer the water by head.
【００３４】一次貯溜槽２にはパイプ５が導入されてい
て、そのパイプにはバルブ６および７およびシリンジ８
が設置されている。一方、図１に図示されていないが搬
送部でサンプル供給系、第１試薬供給系及び第２試薬供
給系へ搬送される反応管９がバルブ６を介してパイプラ
イン５に接続されている。更に、バルブ７と接続しポン
プ１０を介して無菌水槽１１が設置されている。A pipe 5 is introduced into the primary storage tank 2, and valves 6 and 7 and a syringe 8 are connected to the pipe.
is installed. On the other hand, although not shown in FIG. 1, a reaction tube 9 that is transported to a sample supply system, a first reagent supply system, and a second reagent supply system in a transport section is connected to the pipeline 5 via a valve 6. Furthermore, a sterile water tank 11 is connected to the valve 7 via a pump 10.
【００３５】一次貯溜槽２に一次的に貯溜された被試験
水はシリンジ８によってバルブ６とバルブ７の間まで吸
引した後バルブ６を搬送部のサンプル供給系にセットさ
れた反応管９の方向へ変え所定量分シリンジ８を作動さ
せて反応管９へ分注される。The test water temporarily stored in the primary storage tank 2 is sucked into the space between the valves 6 and 7 by the syringe 8, and then the valve 6 is moved in the direction of the reaction tube 9 set in the sample supply system of the transport section. The predetermined amount is dispensed into the reaction tube 9 by operating the syringe 8.
【００３６】被試験水が分注された反応管３は搬送部の
第１試薬供給系へ搬送セットされ、そこでサンプル同様
試薬シリンジと試薬バルブの動作により正確に所定量が
分注される。The reaction tube 3 into which the water to be tested has been dispensed is conveyed and set to the first reagent supply system of the conveyance section, where a predetermined amount is accurately dispensed by the operation of the reagent syringe and reagent valve, similar to the sample.
【００３７】被試験水及び第１試薬が分注された反応管
３は搬送系の第２試薬供給部へ搬送セットされ、そこで
第１試薬と同様にして第２試薬が分注される。The reaction tube 3 into which the test water and the first reagent have been dispensed is transported and set to the second reagent supply section of the transport system, where the second reagent is dispensed in the same manner as the first reagent.
【００３８】被試験水、第１試薬及び第２試薬が分注さ
れた反応管３は測光系の第３試薬供給系へ搬送セットさ
れ、そこで第２試薬と同様に第３試薬が分注される。The reaction tube 3 into which the test water, the first reagent, and the second reagent have been dispensed is transported and set to the third reagent supply system of the photometric system, where the third reagent is dispensed in the same manner as the second reagent. Ru.
【００３９】被試験水、第１試薬、第２試薬及び第３試
薬が分注された反応管は測光系へ搬送され水中の生菌の
保有するＡＴＰと第３試薬の反応によって発生する光子
の量が測定される。The reaction tube into which the test water, the first reagent, the second reagent, and the third reagent have been dispensed is transported to a photometric system, where photons generated by the reaction between the ATP possessed by living bacteria in the water and the third reagent are measured. quantity is measured.
【００４０】本発明の別の態様として被試験水移送部、
反応管搬送部および測光部をパイプラインで接続され且
つ該パイプラインがパルス・モータ制御サンプリング・
ポンプ、サンプリング・バルブ、サンプリング・ピペッ
ト、切換バルブ、パルス・モータ制御試薬ポンプ、バル
ブカット方式試薬バルブ、試薬ボトル、試薬ピペット、
洗浄バルブ、無菌水ポンプ等を備えている装置のサンプ
リング動作及び試薬の吸引・吐出洗浄の動作原理をステ
ップ別に解説する。
Ａ．サンプリング動作[0040] Another aspect of the present invention includes a water transfer section to be tested,
The reaction tube conveyance section and the photometry section are connected by a pipeline, and the pipeline is connected to a pulse motor control sampling section.
Pumps, sampling valves, sampling pipettes, switching valves, pulse motor-controlled reagent pumps, valve-cut reagent valves, reagent bottles, reagent pipettes,
We will explain step-by-step the operating principles of sampling operations and reagent suction/discharge cleaning of equipment equipped with cleaning valves, sterile water pumps, etc. A. Sampling operation
【００４１】一時貯溜槽に一時的に貯溜された被試験水
はパルス・モータ制御により正確に必要量が吸引され、
バルブ・カット方式のサンプリング・バルブによって分
注時の液切れをよくして正確に秤量される。[0041] The test water temporarily stored in the temporary storage tank is sucked in exactly the required amount by pulse motor control.
The valve-cut sampling valve allows for easy liquid drainage during dispensing and accurate weighing.
【００４２】ステップ１；被試験水吸引被試験水は一時
貯溜槽からサンプリング・ピペットにより吸引される。Step 1: Aspirating the water to be tested The water to be tested is aspirated from the temporary reservoir using a sampling pipette.
【００４３】ステップ２；被試験水セットサンプリング
・ピペットにより吸引された被試験水はサンプリング・
ポンプによりバルブカットに最適な位置にセットされる
。Step 2; Test water set The test water aspirated by the sampling pipette is set as the sampling pipette.
The pump will set it in the optimal position for valve cutting.
【００４４】ステップ３；分注吐出準備サンプリング・
バルブが切換わりサンプリング・ポンプのバックラッシ
ュをとり分注吐出の準備がされる。Step 3; Dispensing and dispensing preparation sampling
The valve switches to remove the backlash of the sampling pump and prepare for dispensing and dispensing.
【００４５】ステップ４；分注
さらにサンプリング・バルブが切り換わりサンプリング
・ポンプにより測定に必要な量が分注される。Step 4: Dispensing Further, the sampling valve is switched and the amount necessary for measurement is dispensed by the sampling pump.
【００４６】ステップ５；連続測定の場合の被試験水吸
引準備
サンプリング・バルブが切換わり連続して被試験水が吸
引出来るように、サンプリング・ポンプのバックラッシ
ユをとり被試験水吸引の準備をする。Step 5: Preparation for suction of test water in case of continuous measurement: Prepare for suction of test water by removing the backlash of the sampling pump so that the sampling valve can be switched and test water can be sucked continuously. .
【００４７】ステップ６；連続測定の場合の被試験水吸
引
サンプリング・バルブを元の位置にもどし、測定に必要
な量の被試験水をサンプリングポンプにより吸引する。
また、分注された被試験水は反応管に移送される。Step 6: In the case of continuous measurement, the test water suction sampling valve is returned to its original position, and the amount of test water required for measurement is sucked in by the sampling pump. Further, the dispensed test water is transferred to a reaction tube.
【００４８】ステップ７；被試験水洗浄サンプリング・
バルブ、サンプリング・ポンプは元の位置にもどり、サ
ンプリング・パイプは無菌水により洗浄される。また、
サンプリング・ポンプは元の位置にもどつた後バックラ
ッシュをとって次の被試験水の吸引の準備をする。Step 7; Test water cleaning sampling.
The valve and sampling pump are returned to their original positions and the sampling pipe is flushed with sterile water. Also,
After the sampling pump returns to its original position, it takes off the backlash and prepares to suck the next sample of water to be tested.
【００４９】Ｂ．試薬の吸引・吐出・洗浄の動作試薬ラ
インは無菌水、空気にて洗浄した後、残留無菌水を追い
出し乾燥させる。試薬はパルスモータ制御の試薬ポンプ
及びバルブ・カット方式の試薬バルブにより高精度に分
注することが出来る。B. Reagent suction, discharge, and cleaning operations After cleaning the reagent line with sterile water and air, remove residual sterile water and dry it. Reagents can be dispensed with high precision using a pulse motor-controlled reagent pump and a valve-cut type reagent valve.
【００５０】ステップ１；試薬の吸引
試薬は、試薬ボトルから試薬ピペット及び試薬ポンプに
より適正量が吸引される。Step 1: Aspiration of reagent An appropriate amount of reagent is aspirated from the reagent bottle using a reagent pipette and a reagent pump.
【００５１】ステップ２；試薬のセット試薬ピペットに
より吸引された試薬は、試薬ポンプによりバルブカット
に最適な位置へ移送される。Step 2: Setting the reagent The reagent aspirated by the reagent pipette is transferred to the optimal position for valve cutting by the reagent pump.
【００５２】スチップ３；試薬の送液準備試薬吸引ライ
ン、洗浄バルブを各々切り換え試薬ポンプのバックラッ
シュをとり、試薬を反応管へ移送する準備をする。同時
に洗浄用チューブに無菌水を洗浄ポンプによりセットす
る。Step 3: Preparation for transferring reagents. Switch the reagent suction line and cleaning valve to remove backlash from the reagent pump, and prepare to transfer the reagent to the reaction tube. At the same time, set sterile water in the cleaning tube using the cleaning pump.
【００５３】ステップ４；試薬の送液
試薬バルブを切り換え、試薬ポンプにより試薬を反応管
まで移送する。この時送液ライン洗浄用チューブに洗浄
ポンプにより無菌水をセットする。Step 4: Switch the reagent valve to transfer the reagent to the reaction tube using the reagent pump. At this time, sterile water is set in the liquid supply line cleaning tube using a cleaning pump.
【００５４】ステップ５；試薬ラインの洗浄及び試薬の
吸引準備
試薬ライン洗浄用バルブを切り換え、試薬ポンプを経由
して試薬の吸引ラインに無菌水を流す。ここでステップ
１にもどり繰り返し動作する。Step 5: Cleaning of reagent line and preparation for suction of reagent Switch the reagent line cleaning valve and flow sterile water into the reagent suction line via the reagent pump. At this point, the process returns to step 1 and repeats the operation.
【００５５】以上で本発明の全体の構成及び主要部の動
作原理を解説したが、以下に主要部の特徴を解説する。The overall structure of the present invention and the principle of operation of the main parts have been explained above, and the features of the main parts will be explained below.
【００５６】■ 第１試薬及び第２試薬の保存及び調
整第１試薬及び第２試薬は電子冷却により４℃に冷却保
存され、測定動作中には２０℃の恒温状態下で使用され
る。■ Storage and adjustment of the first and second reagents The first and second reagents are stored at 4° C. by electronic cooling, and are used at a constant temperature of 20° C. during measurement operations.
【００５７】■ 第３試薬（ルシフェリン、ルシフェ
ラーゼ、Ｍｇ２＋）の保存及び調整
第３試薬は予め電子冷却又は冷凍庫にて保存されており
測定動作が開始されると冷凍庫が開放され第３試薬が収
容されている容器が自動的に取り出され、完全に解凍す
るまで２０乃至３０℃の恒温下に放置される。解凍され
た第３試薬は室温で使用され、それ以外は４℃で保存さ
れる。■ Storage and adjustment of the third reagent (luciferin, luciferase, Mg2+) The third reagent is stored in electronic cooling or a freezer in advance, and when the measurement operation is started, the freezer is opened and the third reagent is stored. The container is automatically taken out and left at a constant temperature of 20 to 30° C. until completely thawed. The thawed third reagent is used at room temperature, and the rest is stored at 4°C.
【００５８】■ 無菌水の保存
パイプラインを洗浄するための無菌水は純水に紫外線照
射等により無菌状態にされる。[0058] Storing Sterile Water Sterile water for cleaning pipelines is rendered sterile by irradiating pure water with ultraviolet rays or the like.
【００５９】■ 測 定
適当なインターバルで、第１試薬、無菌水、第２試薬及
び第３試薬を反応管へ分注してブランクテストを行って
得たデータを入力処理する。一方、被試験水、第１試薬
、第２試薬無菌水及び第３試薬が分注された反応管は測
定し得たデータを入力する。水ブランクテス及び被試験
水テストは各々２回行う。① Measurement At appropriate intervals, the first reagent, sterile water, second reagent, and third reagent are dispensed into reaction tubes and a blank test is performed, and the data obtained is input and processed. On the other hand, the reaction tube into which the test water, the first reagent, the second reagent sterile water, and the third reagent are dispensed receives measured data. The water blank test and the tested water test are each performed twice.
【００６０】主要測定仕様は、計測法−微弱発光計測、
測定時間−３０秒、測定モード−積分である。The main measurement specifications are: measurement method - weak luminescence measurement;
Measurement time - 30 seconds, measurement mode - integration.
【００６１】■ 制 御
本発明は各種試薬の保存、取出し及び調整→被試験水の
サンプリング→反応管の搬送→試薬の分注→測定→全パ
イプラインの洗浄→データ処理を自動・連続制御する。
特に、被試験水を収容している各種装置、例えば製紙工
場の抄紙白水槽から白水を自動・連続的に反応管へサン
プリングすることによって全自動・連続測定が可能にな
った。■ Control The present invention automatically and continuously controls storage, removal, and adjustment of various reagents → sampling of test water → transportation of reaction tubes → dispensing of reagents → measurement → cleaning of all pipelines → data processing. . In particular, fully automatic and continuous measurements have become possible by automatically and continuously sampling white water from various devices containing test water, such as white water tanks in paper mills, into reaction tubes.
【００６２】以下、実施例を掲げ本発明を具体的に説明
する。[0062] The present invention will be specifically explained below with reference to Examples.
【００６３】[0063]
【実施例】試 薬
下記の試薬を使用した。
イ． 第１試薬；ルーマックインターナショナル社製
“生物学的緩衝剤”ＨＥＰＥＳ▲Ｒ▼”ロ． 第２試
薬；ルーマックインターナショナル社製、エトキシル化
アルキルフェノール系非イオン性界面活性剤。
ハ． 第３試薬；ターナー社製、“ルシフェリン、ル
シフェラーゼ及びＭｇ２＋”から成るＡＴＰ抽出液。[Example] Reagents The following reagents were used. stomach. First reagent: "Biological buffer" HEPES▲R▼, manufactured by Lumack International Co., Ltd. B. Second reagent: Ethoxylated alkylphenol nonionic surfactant, manufactured by Lumac International Co., Ltd. C. Third reagent; Manufactured by Turner, ATP extract consisting of "luciferin, luciferase and Mg2+".
【００６４】ＡＴＰ発光測定装置
高感度光電子増倍管を使用した微弱発光計測装置を使用
した。装置はオプチカルユニット、デジタルフォトメー
タ、インテグレータータイマー、デジタルプリンター等
を備えている。ATP luminescence measurement device A weak luminescence measurement device using a highly sensitive photomultiplier tube was used. The device is equipped with an optical unit, digital photometer, integrator timer, digital printer, etc.
【００６５】被試験水移送部及び反応管搬送部某製紙会
社の上質紙抄造工程中の白水、即ち被試験水を収容して
いる装置と被試験水を一時的に貯溜する容量５ｌの一時
貯溜槽を直径２５ｍｍのテフロン製パイプで連結して、
１０ｌ／分の流速で被試験水を循環させた。Test water transfer section and reaction tube transfer section A device containing white water, ie, test water, during the high-quality paper manufacturing process of a certain paper company, and a temporary storage with a capacity of 5 liters for temporarily storing the test water. Connect the tanks with a Teflon pipe with a diameter of 25 mm,
The water to be tested was circulated at a flow rate of 10 l/min.
【００６６】直径１３ｍｍ、長さ４０ｍｍの反応管４０
０本をスネークチェーン方式のホルダーにセットし反応
管搬送部から測光部へ連続自動的に移送されるように設
計した。一方、一次貯溜槽に導入されたパイプラインに
被試験水を吸引するシリンジ及び分注、吐出するバルブ
を設け搬送されてくる反応管に連続・自動的に分注され
るように設計した。[0066] Reaction tube 40 with a diameter of 13 mm and a length of 40 mm.
The tube was set in a snake chain type holder and designed to be continuously and automatically transferred from the reaction tube transport section to the photometry section. On the other hand, a syringe for aspirating the test water and valves for dispensing and discharging the test water were installed in the pipeline introduced into the primary storage tank, and the test water was designed to be continuously and automatically dispensed into the reaction tube being transported.
【００６７】更に、第１試薬、及び第２試薬を各々収容
するボトルを設置し、このボトルに各試薬を吸引するシ
リンジ及び反応管に分注、吐出するバルブを設けたパイ
プラインを導入した。Furthermore, bottles containing the first reagent and the second reagent were installed, and a pipeline provided with a syringe for sucking each reagent and a valve for dispensing and discharging the reagent into the reaction tube was introduced into the bottle.
【００６８】第３試薬はルシフェリン、ルシフェラーゼ
及びＭｇ２＋を第１試薬に溶解した後１日の使用量であ
る１．６ｍｌずつボトルに区分けし、装置にビルドイン
されている冷凍庫にて冷凍保存し、使用１時間前に冷凍
庫より自動的に取り出し室温下で解凍した後、自動的に
吸引して反応管に分注、吐出した。After dissolving luciferin, luciferase, and Mg2+ in the first reagent, the third reagent is divided into bottles each containing 1.6 ml, which is the daily usage amount, and stored frozen in the built-in freezer of the device before use. The solution was automatically taken out from the freezer one hour before, thawed at room temperature, and then automatically suctioned, dispensed, and discharged into reaction tubes.
【００６９】無菌水は装置の系外のタンク内に収容した
純水に紫外線を照射して無菌水とし、タンク内にはパイ
プラインを導入し、吸引、分注手段を設けて試薬及び被
試験水が経由した全パイプラインを洗浄出来る設計とし
た。Sterile water is made sterile water by irradiating ultraviolet rays to pure water stored in a tank outside the system of the device.A pipeline is introduced into the tank, and suction and dispensing means are installed to collect reagents and test objects. The design is such that all pipelines that water passes through can be cleaned.
【００７０】測 定
測定動作を開始する約１時間前に冷凍庫を開け第３試薬
が収容されているボトルを自動的に取り出し２０℃の恒
温雰囲気で６０分間放置して解凍しボトルを開栓して第
３試薬の送液動作に備えた。Measurement: Approximately 1 hour before starting the measurement operation, open the freezer, automatically take out the bottle containing the third reagent, leave it in a constant temperature atmosphere at 20°C for 60 minutes to thaw, and open the bottle. and prepared for the third reagent feeding operation.
【００７１】一方、測定動作を開始すると先ず無菌水で
サンプルラインを洗浄した後反応管搬送部より反応管を
搬送して第１試薬、無菌水、第２試薬及び第３試薬を各
々１００μｌずつ順次３０秒のインターバルで分注し水
ブランク測定を行い得たデータを入力処理した。On the other hand, when the measurement operation is started, first, the sample line is washed with sterile water, and then the reaction tube is transported from the reaction tube transport section, and 100 μl each of the first reagent, sterile water, second reagent, and third reagent are sequentially delivered. The data obtained by dispensing at 30 second intervals and performing water blank measurements was input and processed.
【００７２】次いで、３０秒後搬送されてくる反応管に
第１試薬、被試験水、第２試薬及び第３試薬を各々１０
０μｌずつ順次３６秒のインターバルで分注した後ＡＴ
Ｐ発光量を測定し得たデータを入力処理し、自動解析し
た結果、下記の結果を得た。測定後無菌水でパイプライ
ンを洗浄した後同じ動作を各々２回繰返して１サイクル
を終了した。Next, the first reagent, the water to be tested, the second reagent, and the third reagent were added at 10 liters each into the reaction tube that was transferred after 30 seconds.
After dispensing 0 μl at intervals of 36 seconds, AT
As a result of input processing and automatic analysis of the data obtained by measuring the amount of P emission, the following results were obtained. After the measurement, the pipeline was washed with sterile water, and the same operation was repeated twice to complete one cycle.
【００７３】本装置において得られた結果及び同時に採
取した被試験水の生菌数を混和培養法により測定した結
果を表１に示す。[0073] Table 1 shows the results obtained with this apparatus and the results of measuring the number of viable bacteria in the test water sampled at the same time using the mixed culture method.
【００７４】[0074]
【表１】[Table 1]
【００７５】この結果より、本発明の装置によって、従
来より行なわれている混和培養法において得られた結果
と殆んど差の無い結果を得る事ができる事が確認された
。[0075] From these results, it was confirmed that the apparatus of the present invention can obtain results that are almost the same as those obtained with the conventional mixed culture method.
【００７６】[0076]
【発明の効果】イ． 本発明の装置によって、水中の
生菌数が自動・連続的に測定される。
ロ． 本発明の装置によって、従来から行なわれてい
る混和培養法において得られる結果と殆んど差の無い結
果を得る事ができる。[Effect of the invention] a. With the device of the present invention, the number of viable bacteria in water is automatically and continuously measured. B. With the apparatus of the present invention, it is possible to obtain results that are almost the same as those obtained with conventional mixed culture methods.
【図１】本発明の装置の一部の概念図である。FIG. 1 is a conceptual diagram of a part of the apparatus of the present invention.
１：被試験水収容装置 ２：一時貯溜槽 ３，５；パイプ ４，１０；ポンプ ６，７：バルブ ８：シリンジ ９：反応管 １１：無菌水槽 1: Test water storage device 2: Temporary storage tank 3,5; pipe 4,10; pump 6,7: Valve 8: Syringe 9: Reaction tube 11: Sterile water tank
Claims (1)
搬送部、（３）測光部及び（４）オペレーションコンソ
ール部から主として構成されている水中の生菌数の自動
・連続測定装置にして； （１）−１；前記被試験水移送部が、 ■ 被試験水を収容する装置、 ■ 被試験水を一時的に貯溜する一時貯溜槽及び■
被試験水を、被試験水を収容する装置と一時貯溜槽の
間を自動・連続的に還流させる装置から構成されており
、 （１）−２；前記反応管搬送部が、 ■ サンプル供給系、 ■ 第１試薬供給系および ■ 第２試薬供給系から構成されていて、該サンプル
供給系がパイプラインを備えていて、該パイプラインが
前記被試験水を一時的に貯溜する一時貯溜槽に導入され
ていて被試験水を吸引、分注及び吐出する手段を備えて
おり、且つ搬送部から測光部へ自動・連続的に搬送され
る反応管と接続していて更に該パイプラインを洗浄する
様に該パイプラインに洗浄水を供給する手段を備えてい
ることを各々特徴とし、 （１）−３；前記測光部が、 ■ 第３試薬供給系および ■ 水中の生菌の保有するＡＴＰが第３試薬と反応し
て発生する光子の量を測定する測光系を備えていること
を特徴とする水中の生薗数を自動・連続的に測定する装
置。Claim 1: Automatic and continuous measurement of the number of viable bacteria in water, which mainly consists of (1) test water transfer section, (2) reaction tube transfer section, (3) photometry section, and (4) operation console section. As an apparatus: (1)-1; The test water transfer section comprises: (1) a device for storing the test water; (2) a temporary storage tank for temporarily storing the test water; and (2)
It consists of a device that automatically and continuously circulates the test water between a device that accommodates the test water and a temporary storage tank, (1)-2; , consisting of a first reagent supply system and a second reagent supply system, the sample supply system is equipped with a pipeline, and the pipeline is connected to a temporary storage tank for temporarily storing the water to be tested. It is equipped with means for suctioning, dispensing, and discharging the water to be tested, and is connected to the reaction tube that is automatically and continuously transported from the transport section to the photometry section, and further cleans the pipeline. (1)-3; The photometric section is characterized by: (1) a third reagent supply system and (2) a means for supplying washing water to the pipeline; 1. A device for automatically and continuously measuring the number of green beans in water, characterized by being equipped with a photometric system that measures the amount of photons generated by reaction with a third reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15081191A JPH04287681A (en) | 1991-03-15 | 1991-03-15 | Automatic and continuous measuring device of number of viable microorganism in water |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15081191A JPH04287681A (en) | 1991-03-15 | 1991-03-15 | Automatic and continuous measuring device of number of viable microorganism in water |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04287681A true JPH04287681A (en) | 1992-10-13 |
Family
ID=15504939
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP15081191A Pending JPH04287681A (en) | 1991-03-15 | 1991-03-15 | Automatic and continuous measuring device of number of viable microorganism in water |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04287681A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008178420A (en) * | 2004-12-28 | 2008-08-07 | Univ Nagoya | Culture apparatus |
| JP2009232744A (en) * | 2008-03-27 | 2009-10-15 | Hitachi Plant Technologies Ltd | System for measuring microorganisms |
-
1991
- 1991-03-15 JP JP15081191A patent/JPH04287681A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008178420A (en) * | 2004-12-28 | 2008-08-07 | Univ Nagoya | Culture apparatus |
| JP2009232744A (en) * | 2008-03-27 | 2009-10-15 | Hitachi Plant Technologies Ltd | System for measuring microorganisms |
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