JP3327768B2 - A rapid method for the determination of phosphorus-accumulating microorganisms - Google Patents
A rapid method for the determination of phosphorus-accumulating microorganismsInfo
- Publication number
- JP3327768B2 JP3327768B2 JP06844596A JP6844596A JP3327768B2 JP 3327768 B2 JP3327768 B2 JP 3327768B2 JP 06844596 A JP06844596 A JP 06844596A JP 6844596 A JP6844596 A JP 6844596A JP 3327768 B2 JP3327768 B2 JP 3327768B2
- Authority
- JP
- Japan
- Prior art keywords
- phosphorus
- particles
- accumulating
- stained
- dna
- 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.)
- Expired - Fee Related
Links
- 244000005700 microbiome Species 0.000 title claims description 59
- 238000000034 method Methods 0.000 title claims description 40
- 239000002245 particle Substances 0.000 claims description 53
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 25
- 229910052698 phosphorus Inorganic materials 0.000 claims description 25
- 239000011574 phosphorus Substances 0.000 claims description 25
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 claims description 17
- 239000007850 fluorescent dye Substances 0.000 claims description 14
- 238000011002 quantification Methods 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 10
- 229920000388 Polyphosphate Polymers 0.000 claims description 9
- 239000001205 polyphosphate Substances 0.000 claims description 9
- 235000011176 polyphosphates Nutrition 0.000 claims description 9
- 238000010186 staining Methods 0.000 claims description 7
- 238000000684 flow cytometry Methods 0.000 claims description 5
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 claims description 4
- 229960001570 ademetionine Drugs 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 claims 6
- 108091092742 A-DNA Proteins 0.000 claims 1
- 238000003556 assay Methods 0.000 claims 1
- 239000012085 test solution Substances 0.000 claims 1
- 229920000137 polyphosphoric acid Polymers 0.000 description 9
- 238000005259 measurement Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 4
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000002073 fluorescence micrograph Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000000007 visual effect Effects 0.000 description 4
- 239000000833 heterodimer Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000010802 sludge Substances 0.000 description 3
- QTANTQQOYSUMLC-UHFFFAOYSA-O Ethidium cation Chemical compound C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 QTANTQQOYSUMLC-UHFFFAOYSA-O 0.000 description 2
- 241001509405 Gordonia amarae Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 241000589774 Pseudomonas sp. Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 2
- 239000010865 sewage Substances 0.000 description 2
- VXAADOHKCZYPNU-ANNFTPPJSA-N (2S)-2-amino-4-[[(2S,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl-methylsulfonio]butanoate Chemical group O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1.O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 VXAADOHKCZYPNU-ANNFTPPJSA-N 0.000 description 1
- KLLLJCACIRKBDT-UHFFFAOYSA-N 2-phenyl-1H-indole Chemical compound N1C2=CC=CC=C2C=C1C1=CC=CC=C1 KLLLJCACIRKBDT-UHFFFAOYSA-N 0.000 description 1
- 241000588624 Acinetobacter calcoaceticus Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、下水などの廃水処
理におけ生物脱リン処理方法に応用されるリン蓄積微生
物を定量する方法に関する。[0001] The present invention relates to a method for quantifying a phosphorus-accumulating microorganism applied to a biological phosphorus removal treatment method in treating wastewater such as sewage.
【０００２】[0002]
【従来の技術】廃水処理における生物脱リン処理方法
は、活性汚泥の嫌気的および好気的条件下でリンの放
出、吸収現象を利用して、下水中のリン分を生物学的に
除去する方法であり、処理施設の構造が簡単で薬剤添加
が不要などの利点から広く利用されているリン除去方法
である。ところで、この生物脱リン処理方法の運転を管
理、制御するには、脱リンのメカニズムに関与している
リン蓄積微生物が機能する環境を効果的に制御すること
が必要であり、それには、先ず、リン蓄積微生物の個体
数を正確に測定することが重要となっている。2. Description of the Related Art A biological dephosphorization method in wastewater treatment uses a phosphorus release and absorption phenomenon under anaerobic and aerobic conditions of activated sludge to biologically remove phosphorus components in sewage. This is a phosphorus removal method that is widely used because it has a simple structure of a treatment facility and does not require addition of a chemical. By the way, in order to manage and control the operation of this biological dephosphorization treatment method, it is necessary to effectively control the environment in which the phosphorus-accumulating microorganisms involved in the dephosphorization mechanism function. It is important to accurately measure the population of phosphorus-accumulating microorganisms.
【０００３】ところで、このようなリン蓄積微生物の個
体数を定量する方法として、確率論手法を応用したＭＰ
Ｎ法（最確数法）が最も正確な方法として用いられてい
る。しかしながら、このＭＰＮ法は、煩雑な手順を必要
とする長期間にわたる生物試験法であるため、リン蓄積
微生物の現存量を定量するのに１週間から１か月の試験
期間が必要であった。従って、この方法は生物脱リン処
理方法の管理制御に使用することができないという不都
合があった。[0003] As a method of quantifying the number of such phosphorus-accumulating microorganisms, an MP using a stochastic technique has been proposed.
The N method (most probable number method) is used as the most accurate method. However, since the MPN method is a long-term biological test method requiring a complicated procedure, a test period of one week to one month was required to determine the existing amount of the phosphorus accumulating microorganism. Therefore, this method has a disadvantage that it cannot be used for management and control of the biological dephosphorization treatment method.
【０００４】[0004]
【発明が解決しようとする課題】本発明は、上記の問題
点を解決するためになされたものであり、生物脱リン処
理システムの活性汚泥などに存在するリン蓄積微生物の
個体数の定量法として、また個体数のほかリン蓄積微生
物のリン含有量の定量法として、システムの運転をリア
ルタイムに管理、制御するための指標を迅速に精度よく
定量する改良された方法を提供する。SUMMARY OF THE INVENTION The present invention has been made in order to solve the above-mentioned problems, and has been developed as a method for quantifying the number of phosphorus-accumulating microorganisms present in activated sludge of a biological dephosphorization treatment system. In addition, the present invention provides an improved method for quickly and accurately quantifying an index for managing and controlling the operation of a system in real time as a method for quantifying the phosphorus content of a phosphorus-accumulating microorganism in addition to the number of individuals.
【０００５】[0005]
【課題を解決するための手段】上記の問題は、次の
（１）の手段により解決することができる。 （１）被測定液中のリン蓄積微生物を含む粒子を、後記
ＤＡＰＩ蛍光染料と同じＤＮＡのＡ−Ｔ領域に優先的で
あるＤＮＡ蛍光染料で染色した後、ＤＡＰＩ蛍光染料で
染色するステップと、染色して得られたＤＮＡ染色粒子
とＤＡＰＩにより染色されたポリリン酸染色粒子とを蛍
光波長の差によって識別するステップと、ＤＮＡ染色粒
子であってかつポリリン酸染色粒子でもある粒子をカウ
ントするステップを含むことを特徴とするリン蓄積微生
物の迅速定量法。The above problem can be solved by the following means (1). (1) Particles containing phosphorus-accumulating microorganisms in the liquid to be measured are described below.
Preferentially to the AT region of the same DNA as the DAPI fluorescent dye
Staining with a certain DNA fluorescent dye and then staining with a DAPI fluorescent dye, identifying a DNA stained particle obtained by staining and a polyphosphate stained particle stained with DAPI by a difference in fluorescence wavelength, A method for rapidly quantifying a phosphorus-accumulating microorganism, comprising a step of counting particles that are both stained particles and polyphosphate stained particles.
【０００６】なお、前記の本発明のリン蓄積微生物の迅
速定量法は、次の（２）、（３）、および（４）記載の
方法によって好ましく具体化することができる。 （２）ＤＡＰＩ蛍光染料がＳアデノシルメチオニン（S-
adenosylmethionine）と併用される前記（１）に記載の
リン蓄積微生物の迅速定量法。[0006] The method for rapid quantification of phosphorus-accumulating microorganisms of the present invention can be preferably embodied by the following methods (2), (3) and (4). (2) DAPI fluorescent dye is S-adenosylmethionine (S-
adenosylmethionine) as described in (1) above.
【０００７】（３）蛍光顕微鏡下において、ＤＮＡ染色
粒子を赤色光に蛍光発光させるとともに、ポリリン酸染
色粒子を緑色光に蛍光発光させて各々を識別する前記
（１）または（２）に記載のリン蓄積微生物の迅速定量
法。(3) The method according to the above (1) or (2), wherein the DNA stained particles are caused to emit red light under a fluorescence microscope and the polyphosphate stained particles are caused to emit green light under a fluorescence microscope to identify each of them. A rapid quantification method for phosphorus accumulating microorganisms.
【０００８】（４）フローサイトメトリーの観測視野に
染色された粒子を含有する被測定液を通過させ、ＤＮＡ
染色粒子を赤色光に蛍光発光させるとともに、ポリリン
酸染色粒子を緑色光に蛍光発光させて各々を識別する前
記（１）または（２）に記載のリン蓄積微生物の迅速定
量法。(4) The solution containing the stained particles is passed through the observation field of view of flow cytometry, and the DNA
The rapid quantification method of the phosphorus-accumulating microorganism according to the above (1) or (2), wherein the stained particles are caused to emit fluorescent light with red light, and the polyphosphoric acid stained particles are caused to emit fluorescent light with green light to identify each of them.
【０００９】[0009]
【００１０】さらに、前記（３）、（４）の方法では、
リン蓄積微生物の個体数のほか、リン蓄積微生物のリン
含有量を定量する方法として、次の（５）のように具体
化することができる。（５） 前記（３）、（４）に記載のリン蓄積微生物の迅
速定量法において、染色処理されたリン蓄積微生物粒子
の面積と蛍光強度を測定してその粒子毎にリン含有量を
算出するステップを含むリン蓄積微生物の迅速定量法。Further, in the above methods (3) and (4 ) ,
As a method for quantifying the phosphorus content of the phosphorus-accumulating microorganism in addition to the number of phosphorus-accumulating microorganisms, the following (5) can be embodied. (5) In the rapid quantification method of the phosphorus accumulating microorganism according to the above (3) or (4 ), the area and the fluorescence intensity of the stained phosphorus accumulating microorganism particles are measured, and the phosphorus content is calculated for each particle. Method for rapid quantification of phosphorus-accumulating microorganisms including steps.
【００１１】[0011]
【発明の実施の形態】本発明は、リン蓄積微生物であ
る、アシネトバクター カルコアセチカス（Acinetobac
ter calcoaceticus ）、ノカルディア アマラエ (Noca
rdia amarae)、またはシュードモナス エスピー（Pseu
domonas sp.)などが体内にポリリン酸を蓄積するという
性質があることと、一方、遺伝子（ＤＮＡ）と結合して
蛍光を発する蛍光染料である４’，６−ジアミディノ−
２−フェニリンドール（4',6-diamidino-2-phenylindol
e 、ＤＡＰＩと略称する）をポリリン酸と結合させると
長波長側にシフトした蛍光を発するようになる現象に着
目して、完成したものである。BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a phosphorus-accumulating microorganism, Acinetobacter calcoaceticus.
ter calcoaceticus), Nocardia amarae (Noca
rdia amarae) or Pseudomonas sp. (Pseu
domonas sp.) has the property of accumulating polyphosphate in the body, while 4 ', 6-diamidino-, a fluorescent dye that emits fluorescence when bound to a gene (DNA).
2-phenylindole (4 ', 6-diamidino-2-phenylindol
e, abbreviated as DAPI), which is completed by focusing on the phenomenon that, when polyphosphoric acid is combined with polyphosphoric acid, fluorescence shifted to a longer wavelength side is emitted.
【００１２】以下に、請求項１の発明に係る定量方法の
フローの実施形態例を、次の（１）被測定液の前処理、
（２）ＥＡＨ添加、（３）ＤＡＰＩ、ＳＡＭの添加およ
び（４）蛍光発光測定の順に説明する。 （１）被測定液の前処理。 リン蓄積微生物を含む活性汚泥を適当量サンプリング
し、ホルマリンを終濃度２％（または、エタノールを終
濃度５０％）になるように添加し、固定する。次いで、
ホルマリンまたはエタノールが残っているとバックグラ
ウンド蛍光が高くなるのを避けるため、ＰＢＳ（０．１
５Ｍ ＮａＣｌを含むリン酸緩衝液）または生理食塩水
で洗浄して被測定液の前処理を行う。An embodiment of the flow of the quantification method according to the first aspect of the present invention will be described below with reference to the following (1) pretreatment of the liquid to be measured.
(2) Addition of EAH, (3) Addition of DAPI and SAM, and (4) Measurement of fluorescence emission will be described in this order. (1) Pretreatment of the liquid to be measured. An activated sludge containing a phosphorus accumulating microorganism is sampled in an appropriate amount, and formalin is added to a final concentration of 2% (or ethanol to a final concentration of 50%) and fixed. Then
To prevent background fluorescence from increasing when formalin or ethanol remains, PBS (0.1
The sample is washed with 5M NaCl-containing phosphate buffer) or physiological saline to perform pretreatment of the liquid to be measured.
【００１３】（２）ＥＡＨの添加。 次いで、この適当量の前処理液にＤＮＡ蛍光染料であり
ＤＡＰＩと同じＤＮＡのＡ−Ｔ領域に優先的であるエチ
ジウム アクリジン ヘテロダイマー (Ethidium Acrid
ine Heterodimer 、ＥＡＨと略称する）の濃度１０μｇ
／ｍリットルの水溶液を終濃度５μｇ／ｍリットルにな
るように添加し、３７℃、３０分遮光保持して、前記処
理液中のリン蓄積微生物を含む全ての微生物について、
そのＤＮＡを前記ＥＡＨにて染色する。なお、ＤＮＡ染
料には、ＥＡＨの他に、プロピジウム アイオダイド
（Propidium Iodide）、エチジウム ブロマイド (Ethi
diumBromide) なども使用可能である。(2) Addition of EAH. Then, an appropriate amount of the pretreatment liquid was added to a DNA fluorescent dye, Ethidium Acridin heterodimer (Ethidium Acridin heterodimer) which is preferential to the AT region of the same DNA as DAPI.
ine Heterodimer, abbreviated as EAH)
/ Ml of an aqueous solution to a final concentration of 5 µg / ml, and kept at 37 ° C for 30 minutes under light shielding, for all microorganisms including phosphorus-accumulating microorganisms in the treatment solution.
The DNA is stained with the EAH. In addition, in addition to EAH, propidium iodide (Propidium Iodide), ethidium bromide (Ethi
diumBromide) can also be used.
【００１４】（３）ＤＡＰＩ、ＳＡＭの添加。 さらに、前記（２）の処理を行った被測定液にＤＡＰＩ
水溶液（濃度１０μｇ／ｍリットル）を終濃度１μｇ／
ｍリットルとなるように添加し、３７℃、２０分遮光保
持して、前記前処理液中のリン蓄積微生物が蓄積してい
るポリリン酸を前記ＤＡＰＩにて染色する。この場合、
Ｓアデノシルメチオニン（S-adenosylmethionine ＳＡ
Ｍと略称する）をＤＡＰＩに対して５〜２０％加えて併
用すると、後記の蛍光発光測定において発光強度が増強
される利点がある。(3) Addition of DAPI and SAM. Further, DAPI is added to the liquid to be measured after the treatment of (2).
The aqueous solution (concentration: 10 μg / ml) was converted to a final concentration of 1 μg / ml.
The solution is added so that the volume of the solution becomes 0.1 ml, and the light is maintained at 37 ° C. for 20 minutes. in this case,
S-adenosylmethionine SA
M) (abbreviated as M) is added to DAPI in an amount of 5 to 20%, which is advantageous in that the emission intensity is enhanced in the fluorescence emission measurement described later.
【００１５】（４）蛍光発光測定。 このようにして得られる被測定液中には、全ての微生物
のそのＤＮＡが染色されたところのＤＮＡ染色粒子とリ
ン蓄積微生物が蓄積しているポリリン酸が染色されたと
ころのポリリン酸染色粒子とが併存していることにな
る。そして、ＤＮＡ染色粒子は波長６００〜６４０ｎｍ
の赤色蛍光を発光するのに対して、ポリリン酸染色粒子
は波長５１０〜５４０ｎｍの緑色蛍光を発光する特性が
あるので、照射された測定光により両者を蛍光色によっ
て識別したうえ、前記の赤色蛍光を発すると同時に緑色
蛍光を発光する粒子をカウントする。この場合、リン蓄
積微生物が蓄積しているポリリン酸のように、重合度が
５以上のときは緑色蛍光が特に顕著となる。このように
カウントされた粒子は、ＤＮＡを有するとともにポリリ
ン酸を含有するところから、リン蓄積微生物であると認
識することができ、被測定液中のリン蓄積微生物の個体
数を知ることができるのである。(4) Fluorescence measurement. In the liquid to be measured thus obtained, DNA stained particles where the DNA of all the microorganisms were stained and polyphosphate stained particles where the polyphosphate accumulated by the phosphorus accumulating microorganism were stained were included. Will coexist. The DNA stained particles have a wavelength of 600 to 640 nm.
Since the phosphoric acid stained particles have a characteristic of emitting green fluorescence having a wavelength of 510 to 540 nm while emitting red fluorescent light, the two particles are distinguished from each other by a fluorescent color based on the irradiated measurement light, and the above-described red fluorescent light is emitted. And the particles emitting green fluorescence at the same time are counted. In this case, when the degree of polymerization is 5 or more, such as polyphosphoric acid accumulated by a phosphorus accumulating microorganism, green fluorescence is particularly remarkable. Since the particles counted in this way have DNA and contain polyphosphate, they can be recognized as phosphorus-accumulating microorganisms, and the number of phosphorus-accumulating microorganisms in the liquid to be measured can be known. is there.
【００１６】蛍光顕微鏡を用いた前記蛍光発光測定の１
実施形態について、図１により説明する。図１（１ａ）
（１ｂ）（１ｃ）は、いづれも蛍光顕微鏡で観測される
視野を模式的に示したものである。視野（１ａ）は、蛍
光染色を行わなかった場合を仮定して例示したもので、
観察される全ての粒子を輪郭線で表現してある。これら
粒子には、リン蓄積微生物を含む全ての微生物以外に、
被測定液中の有機質、無機質の微粒子が含まれるのであ
る。視野（１ｂ）は、赤色蛍光を発光するＤＮＡ染色粒
子の像、すなわち微生物の像が観測される視野を示し、
視野（１ｃ）は、緑色蛍光を発光するポリリン酸を含む
粒子の像が観測される視野を示している。In the fluorescence emission measurement 1 using a fluorescence microscope,
An embodiment will be described with reference to FIG. Fig. 1 (1a)
(1b) and (1c) schematically show the fields of view observed with a fluorescence microscope. The field of view (1a) is an example assuming that fluorescent staining was not performed.
All observed particles are represented by contour lines. These particles include, in addition to all microorganisms, including phosphorus-accumulating microorganisms,
The liquid to be measured contains organic and inorganic fine particles. The field of view (1b) indicates the field of view of the image of the DNA stained particles emitting red fluorescence, that is, the field of view of the image of the microorganism,
The field of view (1c) indicates the field of view in which an image of particles containing polyphosphoric acid that emits green fluorescence is observed.
【００１７】ここで、リン蓄積微生物は、ＤＮＡを有す
るが故に視野（１ｂ）において観察されるとともに、ポ
リリン酸を含むために視野（１ｃ）においても観察され
ることから、視野（１ｃ）において、粒子ｃ１はリン蓄
積微生物である、粒子ｃ２は微生物であるが緑色蛍光が
弱いのでリン蓄積微生物ではない、粒子ｃ３は赤色蛍光
が見られないので無生物粒子である、というように認識
できる。そこで粒子ｃ１をカウントすれば、リン蓄積微
生物の個体数を定量することができるのである。そして
このような、蛍光顕微鏡を用いた蛍光発光測定で得られ
る赤色蛍光像と緑色蛍光像とを比較する画像処理は、コ
ンピュータを用いて電子的に行うことができるから、全
微生物個体数、リン蓄積微生物個体数等を自動的に正確
かつ極めて迅速にカウントすることが可能となるのであ
る。Here, the phosphorus-accumulating microorganism is observed in the visual field (1b) because it has DNA, and is also observed in the visual field (1c) because it contains polyphosphoric acid. It can be recognized that the particle c1 is a phosphorus accumulating microorganism, the particle c2 is a microorganism, but is not a phosphorus accumulating microorganism because green fluorescence is weak, and the particle c3 is an inanimate particle because no red fluorescence is observed. Therefore, if the number of particles c1 is counted, the number of phosphorus-accumulating microorganisms can be quantified. Such image processing for comparing a red fluorescence image and a green fluorescence image obtained by fluorescence emission measurement using a fluorescence microscope can be performed electronically using a computer. The number of accumulated microorganisms can be automatically and accurately and extremely quickly counted.
【００１８】また、フローサイトメトリーを用いた前記
蛍光発光測定の第２の実施形態について、図２により説
明する。図２（２ａ）（２ｂ）（２ｃ）は、いづれも被
測定粒子を個別にフローサイトメトリーの観測視野を通
過させたときに観測される視野を模式的に示したもので
ある。視野（２ａ）は、蛍光染色を行わなかった場合を
仮定して例示したもので、前記の図１の視野（１ａ）の
相当するものである。視野（２ｂ）は、赤色蛍光を発光
する微生物の像が観測される視野を示し、視野（２ｃ）
は、緑色蛍光を発光するポリリン酸を含む粒子の像が観
測される視野を示している。A second embodiment of the fluorescence emission measurement using flow cytometry will be described with reference to FIG. FIGS. 2 (2a), (2b), and (2c) schematically show the field of view observed when the particles to be measured individually pass through the observation field of flow cytometry. The field of view (2a) is exemplified assuming that no fluorescent staining is performed, and corresponds to the field of view (1a) in FIG. 1 described above. The field of view (2b) indicates the field of view in which the image of the microorganism that emits red fluorescence is observed, and the field of view (2c)
Indicates a field of view in which an image of particles containing polyphosphoric acid that emits green fluorescence is observed.
【００１９】ここで、リン蓄積微生物は、ＤＮＡを有し
ているので視野（２ｂ）において観察されるとともに、
同時にポリリン酸を含むために視野（２ｃ）においても
観察されることから、前記蛍光顕微鏡の場合と同じく、
視野（２ｃ）における粒子ｃ１をリン蓄積微生物として
カウントして、リン蓄積微生物の個体数を定量すること
ができるのである。この場合においても、蛍光顕微鏡を
用いた場合と同じく、赤色蛍光像と緑色蛍光像との比較
画像処理を電子的に行うことができるから、全微生物個
体数、リン蓄積微生物個体数等を自動的に、正確にかつ
迅速にカウントすることできるのである。Here, since the phosphorus-accumulating microorganism has DNA, it is observed in the visual field (2b).
At the same time, it is also observed in the visual field (2c) because it contains polyphosphoric acid.
By counting the particles c1 in the field of view (2c) as phosphorus-accumulating microorganisms, the number of phosphorus-accumulating microorganisms can be quantified. In this case, as in the case of using a fluorescence microscope, the comparison image processing of the red fluorescence image and the green fluorescence image can be performed electronically. In addition, it is possible to count accurately and quickly.
【００２０】[0020]
【００２１】[0021]
【００２２】[0022]
【００２３】[0023]
【００２４】[0024]
【００２５】[0025]
【００２６】[0026]
【００２７】以上、リン蓄積微生物粒子数をカウントす
る事例に基づいて説明したが、第４の実施形態につい
て、図４に基づいて説明する。上記の顕微鏡の撮影画面
の画像処理またはフローサイトメトリーによれば、リン
蓄積微生物の粒子数のみならず、それぞれの粒子の大き
さ、例えば投影面積とＤＡＰＩ蛍光染料に由来するポリ
リン酸の蛍光強度の強弱を測定することができる。そし
て、その結果、濃度既知の標準粒子と比較する手法によ
って、リン蓄積微生物の粒子個々の大きさとリンの濃度
を知ることができるから、リン蓄積微生物の粒子毎にリ
ン含有量を算出することが可能となる。The above description has been made based on the case where the number of phosphorus-accumulating microorganism particles is counted. The fourth embodiment will be described with reference to FIG. According to the image processing or flow cytometry of the photographing screen of the microscope, not only the number of particles of the phosphorus-accumulating microorganism, but also the size of each particle, for example, the projected area and the fluorescence intensity of polyphosphoric acid derived from the DAPI fluorescent dye. Strength can be measured. Then, as a result, the size of each of the particles of the phosphorus-accumulating microorganism and the concentration of phosphorus can be known by a method of comparing with the standard particles of which the concentration is known, so that the phosphorus content can be calculated for each particle of the phosphorus-accumulating microorganism. It becomes possible.
【００２８】図４において、左側の像を蛍光顕微鏡で撮
影した個体の観察画像とした場合、この画像をデジタル
ＣＣＤカメラの画面に転写して、コンピュータで画像解
析を行う。例えば、右側の拡大像のように、電子的枡目
（画素またはピクセル）で再構成されたデジタル画像の
枡目の数をカウントすることにより面積を測定すること
ができ、また、それぞれの枡目の明るさ（グレイ値）
を、例えば、明るさを数値化した階層レベルａ、ｂ、
ｃ、〜ｎに当てはめて累計することにより個体個々の平
均蛍光強度を知ることができる。そして、リンの濃度既
知の標準粒子について同様に行い基準値を設定しておけ
ば、リン含有量の絶対値を求めることができるのであ
る。In FIG. 4, when the image on the left is an observation image of an individual photographed with a fluorescence microscope, this image is transferred to the screen of a digital CCD camera and image analysis is performed by a computer. For example, as shown in the enlarged image on the right side, the area can be measured by counting the number of meshes of a digital image reconstructed by electronic meshes (pixels or pixels). Brightness (gray value)
Are, for example, hierarchical levels a, b,
The average fluorescence intensity of each individual can be obtained by applying the values to c and to n and accumulating them. Then, if the reference value is set in the same manner for the standard particles having a known phosphorus concentration, the absolute value of the phosphorus content can be obtained.
【００２９】上記のように本発明の実施形態のいくつか
を説明したが、生物脱リン処理においては、様々な種類
のリン蓄積微生物の存在が報告されており、それぞれの
種によりリンの蓄積量が異なっていることも指摘されて
いる。そこで、前記したように、本発明の定量方法によ
り、リン蓄積微生物の全個体数を測定することにより従
来よりも効率的に生物脱リン処理を制御可能となるが、
前記第４の実施形態の方法によればリン蓄積微生物のリ
ン含有量の相違をも把握できるので、より正確で精密な
制御が可能となるのである。Although some of the embodiments of the present invention have been described above, in the biological dephosphorization treatment, the existence of various types of phosphorus-accumulating microorganisms has been reported, and the amount of accumulated phosphorus by each species has been reported. Are different. Therefore, as described above, the quantification method of the present invention makes it possible to control the biological dephosphorization treatment more efficiently than before by measuring the total number of phosphorus-accumulating microorganisms.
According to the method of the fourth embodiment, the difference in the phosphorus content of the phosphorus-accumulating microorganism can be grasped, so that more accurate and precise control is possible.
【００３０】[0030]
【発明の効果】本発明のリン蓄積微生物の迅速定量法
は、以上に説明したように構成されているので、リン蓄
積微生物の個体数を迅速に精度よく定量することができ
るとともに、同定量操作の自動化も可能となった。さら
に、リン蓄積微生物のリン含有量を知ることも可能にで
き、生物脱リン処理システムの運転管理指標としてより
有効に応用できるようになるという優れた効果がある。
よって本発明は従来の問題点を解消したリン蓄積微生物
の定量法として、その工業的価値は極めて大なるものが
ある。The rapid quantification method for phosphorus-accumulating microorganisms of the present invention is constituted as described above, so that the number of phosphorus-accumulating microorganisms can be quantified quickly and accurately and the amount of identification can be controlled. Can also be automated. Furthermore, it is also possible to know the phosphorus content of the phosphorus-accumulating microorganism, and there is an excellent effect that it can be more effectively applied as an operation management index of the biological phosphorus removal treatment system.
Therefore, the present invention has an extremely large industrial value as a method for quantifying a phosphorus-accumulating microorganism which has solved the conventional problems.
【図１】本発明の１実施形態の説明図。FIG. 1 is an explanatory diagram of one embodiment of the present invention.
【図２】第２の実施形態の説明図。FIG. 2 is an explanatory diagram of a second embodiment.
【図３】第３の実施形態の説明図。FIG. 3 is an explanatory diagram of a third embodiment.
【図４】第４の実施形態の説明図。FIG. 4 is an explanatory diagram of a fourth embodiment.
フロントページの続き (56)参考文献 Ａｐｐｌｉｅｄ ａｎｄ Ｅｎｖｉｒ ｏｎｍｅｎｔａｌ Ｍｉｃｒｏｂｉｏｌ ｏｇｙ，1997，Ｖｏｌ．60，Ｎｏ．３, ｐ．792−800 Ｂｉｏｃｈｉｍｉｃａ ｅｔ Ｂｉｏ ｐｈｙｓｉｃａ Ａｃｔａ，1982，Ｖｏ ｌ．721，ｐ．394−398 Ａｐｐｌｉｅｄ ａｎｄ Ｅｎｖｉｒ ｏｎｍｅｎｔａｌ Ｍｉｃｒｏｂｉｏｌ ｏｇｙ，1991，Ｖｏｌ．57，Ｎｏ．12, ｐ．3585−3592 食品工業，1995，Ｖｏｌ．38，Ｎｏ. 24，ｐ．25−31 Ｃａｎ．Ｊ．Ｍｉｃｒｏｂｉｏｌ．, 1980，Ｖｏｌ．26，ｐ．912−920 (58)調査した分野(Int.Cl.7，ＤＢ名) C12Q 1/68 Continuation of the front page (56) References Applied and Environmental Microbiology, 1997, Vol. 60, no. 3, p. 792-800 Biochimica et Biophysica Acta, 1982, Vol. 721, p. 394-398 Applied and Environmental Microbiology, 1991, Vol. 57, No. 12, p. 3585-3592 Food Industry, 1995, Vol. 38, No. 24, p. 25-31 Can. J. Microbiol. , 1980, Vol. 26, p. 912-920 (58) Field surveyed (Int. Cl. 7 , DB name) C12Q 1/68
Claims (5)
する粒子を、後記ＤＡＰＩ蛍光染料と同じＤＮＡのＡ−
Ｔ領域に優先的であるＤＮＡ蛍光染料で染色処理した
後、ＤＡＰＩ蛍光染料で染色処理するステップと、染色
して得られたＤＮＡ染色粒子とＤＡＰＩにより染色され
たポリリン酸染色粒子とを蛍光波長の差によって識別す
るステップと、ＤＮＡ染色粒子であって、かつポリリン
酸染色粒子でもあるリン蓄積微生物粒子をカウントする
ステップを含むことを特徴とするリン蓄積微生物の迅速
定量法。1. A method according to claim 1, wherein particles containing phosphorus-accumulating microorganism particles in the liquid to be measured are mixed with A-DNA of the same DNA as a DAPI fluorescent dye.
After staining with a DNA fluorescent dye which is preferential to the T region , staining with a DAPI fluorescent dye, and staining the DNA stained particles obtained by staining with the polyphosphate stained particles stained with DAPI at a fluorescence wavelength A method for rapid quantification of phosphorus-accumulating microorganisms, comprising a step of distinguishing by difference and a step of counting phosphorus-accumulating microorganism particles that are DNA stained particles and are also polyphosphate stained particles.
ニン（S-adenosylmethionine）と併用される請求項１に
記載のリン蓄積微生物の迅速定量法。2. The method according to claim 1, wherein the DAPI fluorescent dye is used in combination with S-adenosylmethionine.
を赤色光に蛍光発光させるとともに、ＤＡＰＩ染色粒子
を緑色光に蛍光発光させて各々を識別する請求項１また
は２に記載のリン蓄積微生物の迅速定量法。3. The phosphorous-accumulating microorganism according to claim 1 or 2, wherein the DNA-stained particles are caused to emit fluorescent light with red light and the DAPI-stained particles are caused to emit fluorescent light with green light under a fluorescence microscope to identify each of them. Assay method.
された粒子を含有する被測定液を通過させ、ＤＮＡ染色
粒子を赤色光に蛍光発光させるとともに、ＤＡＰＩ染色
粒子を緑色光に蛍光発光させて各々を識別する請求項１
または２に記載のリン蓄積微生物の迅速定量法。4. A test solution containing the stained particles is passed through the observation field of view of flow cytometry to cause the DNA stained particles to emit red light and the DAPI stained particles to emit green light. Claim 1 for identifying
Or the rapid quantification method of a phosphorus-accumulating microorganism according to 2.
ン蓄積微生物の迅速定量法において、染色処理されたリ
ン蓄積微生物粒子の面積と蛍光強度を測定してその粒子
毎にリン含有量を算出するステップを含むリン蓄積微生
物の迅速定量法。5. The method for rapidly quantifying a phosphorus-accumulating microorganism according to claim 3 or 4, wherein the area and the fluorescence intensity of the stained phosphor-accumulating microorganism particles are measured, and the phosphorus content of each particle is measured. A rapid quantification method for phosphorus-accumulating microorganisms, comprising a calculating step.
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Non-Patent Citations (5)
| Title |
|---|
| Ａｐｐｌｉｅｄ ａｎｄ Ｅｎｖｉｒｏｎｍｅｎｔａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ，１９９１，Ｖｏｌ．５７，Ｎｏ．１２，ｐ．３５８５−３５９２ |
| Ａｐｐｌｉｅｄ ａｎｄ Ｅｎｖｉｒｏｎｍｅｎｔａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ，１９９７，Ｖｏｌ．６０，Ｎｏ．３，ｐ．７９２−８００ |
| Ｂｉｏｃｈｉｍｉｃａ ｅｔ Ｂｉｏｐｈｙｓｉｃａ Ａｃｔａ，１９８２，Ｖｏｌ．７２１，ｐ．３９４−３９８ |
| Ｃａｎ．Ｊ．Ｍｉｃｒｏｂｉｏｌ．，１９８０，Ｖｏｌ．２６，ｐ．９１２−９２０ |
| 食品工業，１９９５，Ｖｏｌ．３８，Ｎｏ．２４，ｐ．２５−３１ |
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