JP4500206B2 - Biofilm evaluation method - Google Patents
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- JP4500206B2 JP4500206B2 JP2005136341A JP2005136341A JP4500206B2 JP 4500206 B2 JP4500206 B2 JP 4500206B2 JP 2005136341 A JP2005136341 A JP 2005136341A JP 2005136341 A JP2005136341 A JP 2005136341A JP 4500206 B2 JP4500206 B2 JP 4500206B2
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- 238000011156 evaluation Methods 0.000 title claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000007850 fluorescent dye Substances 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 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 claims description 3
- 229960005542 ethidium bromide Drugs 0.000 claims description 3
- 239000006285 cell suspension Substances 0.000 claims description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 13
- 229910001220 stainless steel Inorganic materials 0.000 description 10
- 239000010935 stainless steel Substances 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 7
- 230000032770 biofilm formation Effects 0.000 description 6
- 229910052697 platinum Inorganic materials 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 239000000725 suspension Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000003746 surface roughness Effects 0.000 description 2
- 239000010409 thin film Substances 0.000 description 2
- 241001673062 Achromobacter xylosoxidans Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- HJUFTIJOISQSKQ-UHFFFAOYSA-N fenoxycarb Chemical compound C1=CC(OCCNC(=O)OCC)=CC=C1OC1=CC=CC=C1 HJUFTIJOISQSKQ-UHFFFAOYSA-N 0.000 description 1
- 239000010408 film Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000008155 medical solution Substances 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 229910001120 nichrome Inorganic materials 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Description
この発明は、バイオフィルム評価方法に関するものである。 The present invention relates to a biofilm evaluation method.
例えば浴室の洗い場に設けた排水口や、台所のシンクに設けた排水口等にはヌメリが発生する。 For example, slime is generated at a drain outlet provided in a bathroom washroom, a drain outlet provided in a kitchen sink, or the like.
このヌメリの薄膜は、微生物が排出するスライムで囲まれた微生物の集合体であるバイオフィルムであることが分かっているが、このバイオフィルムの定量検査は未だなされていない。 This slime thin film is known to be a biofilm that is a collection of microorganisms surrounded by slime that is discharged by microorganisms, but the biofilm has not yet been quantitatively tested.
この発明は、上述の事柄に留意してなされたもので、その目的は、台所や洗面台などの一般的な生活環境におけるバイオフィルム形成菌を定量可能なバイオフィルム評価方法を提供することである。 The present invention has been made in consideration of the above-described matters, and an object thereof is to provide a biofilm evaluation method capable of quantifying biofilm-forming bacteria in a general living environment such as a kitchen or a washstand. .
上記目的を達成するために、この発明のバイオフィルム評価方法は、滅菌シャーレに入れたバイオフィルム担体に菌懸濁液を加え、前記バイオフィルム担体を別の滅菌シャーレに移し替え、蛍光染色剤を前記バイオフィルム担体の表面を覆うように加え、続いて、前記バイオフィルム担体の洗浄操作を行った後、共焦点レーザー走査蛍光顕微鏡システムを用いて前記バイオフィルム担体表面のバイオフィルム形成量を測定してヌメリの発生を評価するにあたり、
前記蛍光染色剤が臭化エチジウム水溶液である一方、
前記洗浄操作として、前記別の滅菌シャーレに前記バイオフィルム担体が浸るように蒸留水を加えて振盪し、着色した水を除去する操作を複数回繰り返す工程が施されることを特徴としている（請求項１）。
In order to achieve the above object, the biofilm evaluation method of the present invention adds a bacterial suspension to a biofilm carrier placed in a sterile petri dish, transfers the biofilm carrier to another sterile petri dish, and adds a fluorescent stain. in addition to cover the surface of the biofilm carrier, followed by the after washing operation of a biofilm carrier, biofilm formation amount of the biofilm support surface was measured using a confocal laser scanning fluorescence microscope system In evaluating the occurrence of slime ,
While the fluorescent stain is an aqueous ethidium bromide solution,
The washing operation is characterized in that a step of adding distilled water and shaking so that the biofilm carrier is immersed in the other sterile petri dish and removing the colored water is repeated a plurality of times (claims) Item 1).
ここでバイオフィルムとは、微生物が排出するスライムで囲まれた微生物の集合体で、ヌメリの薄膜である。 Here, a biofilm is an aggregate of microorganisms surrounded by slime discharged from microorganisms, and is a thin film of slime.
この発明では、滅菌シャーレに入れたバイオフィルム担体に菌懸濁液を加え、前記バイオフィルム担体を別の滅菌シャーレに移し替え、蛍光染色剤を前記バイオフィルム担体の表面を覆うように加え、続いて、前記バイオフィルム担体の洗浄操作を行った後、共焦点レーザー走査蛍光顕微鏡システムを用いるので、前記バイオフィルム担体表面のバイオフィルム形成量を測定してヌメリの発生を評価することができる。 In this invention, the cell suspension was added to biofilm carrier into a sterile Petri dish, transferred the biofilm carrier to another sterile petri dish, adding a fluorescent dye so as to cover the surface of the biofilm support, Subsequently, since the confocal laser scanning fluorescence microscope system is used after the cleaning operation of the biofilm carrier, the amount of biofilm formation on the surface of the biofilm carrier can be measured to evaluate the occurrence of slime.
以下、この発明の実施例について説明する。なお、それによってこの発明は限定されるものではない。 Examples of the present invention will be described below. The present invention is not limited thereby.
（１）使用菌株
バイオフィルム測定方法を行うにあたり、使用菌株として、台所や洗面台などの一般的な生活環境におけるバイオフィルム形成菌であるAlcaligenes xylosoxydans IFO 15126を供試菌として用いた。
(1) Strain used In carrying out the biofilm measurement method, Alcaligenes xylosoxydans IFO 15126, which is a biofilm-forming bacterium in a general living environment such as a kitchen or a wash basin, was used as a test bacterium.
（２）培養および菌懸濁液の調製
供試菌を、Nutrient Broth（Beef extract 0.3％、Peptone 0.5 ％、pH 6.6-7.0）５ｍｌに一白金耳（いちはっきんじ）植菌し、３７℃、１８時間静置培養を行った。この培養液を、２０倍希釈したNutrient Broth-Sabouraud液体培地（１：１）混合培地で適宜希釈して、使用するまで氷冷しておいた。
なお、一白金耳（いちはっきんじ）とは、菌液または菌叢（コロニー）の「１白金耳量」を１単位分（一回分）ということを意味しており、「１白金耳量」とは、「白金耳（はっきんじ）」（植菌のための器具）の先端部の金属製の小さな輪（直径４ミリ程度）に菌液の液膜を張った状態、または菌叢の塊を付着させた状態で、新しい培地に１回移植した量のことを指す。ちなみに、従来、前記先端部の金属に白金線を使用していたので、白金耳という名称がついているが、最近ではニクロム線で代用することも多い。前記一白金耳（いちはっきんじ）は、ＪＩＳ（Ｚ２９１１、Ｌ１９０２）に掲載されているほか、臨床検査関係のマニュアルにも記載されている。
(2) Cultivation and preparation of the bacterial suspension The test bacteria were inoculated into 5 ml of Nutrient Broth (Beef extract 0.3%, Peptone 0.5%, pH 6.6-7.0) at 37 ° C. Then, static culture was performed for 18 hours. This culture solution was appropriately diluted with a 20-fold diluted Nutrient Broth-Sabouraud liquid medium (1: 1) mixed medium and kept on ice until used.
One platinum ear means “one platinum ear amount” of the bacterial solution or bacterial flora (colony) as one unit (one time), and “one platinum ear amount”. Is a state in which a liquid film of bacterial solution is stretched on a small metal ring (about 4 mm in diameter) at the tip of "platinum ear" (instrument for inoculation), or the bacterial flora This refers to the amount transplanted once into a new medium with the lump of lump attached. Incidentally, since a platinum wire is conventionally used for the metal at the tip, it is named platinum ear, but recently, a nichrome wire is often used instead. In addition to being published in JIS (Z2911, L1902), the one platinum ear is also described in clinical laboratory related manuals.
（３）使用薬剤
また、バイオフィルムの形成予防薬剤として、「お酢」を使用し、薬剤をカートリッジのままガラスシャーレに入れ、カートリッジがすべて浸るように３００ｍｌの蒸留水を加え、２あるいは１２時間、静置した後、水をよくかき混ぜてサンプリングし、薬剤溶液とした。
(3) Drugs used “Vinegar” is used as a biofilm formation-preventing drug, and the drug is placed in a glass petri dish with the cartridge as it is, and 300 ml of distilled water is added so that the entire cartridge is immersed for 2 or 12 hours. After standing, water was mixed well and sampled to obtain a drug solution.
（４）バイオフィルム担体
バイオフィルム担体は、表面粗さの異なる４種類のステンレス鋼製円盤（直径２８ｍｍ、質量２０ｇ、1,▽▽▽ ;2,▽▽ ;3,▽ ;4,〜）とした。
(4) Biofilm carrier The biofilm carrier consists of four types of stainless steel disks (diameter 28 mm, mass 20 g, 1, ▽▽▽; 2, ▽▽; 3, ▽; 4, ~) with different surface roughness. did.
（５）バイオフィルム形成試験
乾熱滅菌したステンレス鋼製円盤をクリーンベンチ内で滅菌シャーレ（直径９０ｍｍ、深さ２０ｍｍ）に入れ、あらかじめ調製しておいた菌懸濁液（１０6 cells ／シャーレ）を加えた。
なお、前記薬剤溶液を加える場合は、２０倍希釈となるようにシャーレに加えた。そのシャーレを２８℃、７日間培養した。
(5) Biofilm formation test A stainless steel disc sterilized by dry heat was placed in a sterile petri dish (diameter 90 mm, depth 20 mm) in a clean bench, and a bacterial suspension (10 6 cells / petri dish) prepared in advance. Was added.
In addition, when adding the said chemical | medical solution, it added to the petri dish so that it might become 20 times dilution. The petri dish was cultured at 28 ° C. for 7 days.
（６）バイオフィルム形成の観察
ステンレス鋼製円盤を、エタノール消毒したピンセットで取り出し、別の滅菌シャーレに移した。蛍光染色剤（臭化エチジウム水溶液、終濃度１００μg ／ｍｌ）をステンレス鋼製円盤の表面を覆うように加えて３分間静置した。滅菌シャーレにステンレス鋼製円盤が十分に浸るように蒸留水を加え、約５秒間穏やかに振盪し、着色した水を除去した。再び蒸留水を加え、同様にしてステンレス鋼製円盤の洗浄操作を５回繰り返した。ステンレス鋼製円盤の表面に触れないように注意して余分な水分を除去した後、下記の共焦点レーザー走査蛍光顕微鏡システムでステンレス鋼製円盤表面の写真を撮影し、バイオフィルムの形成を観察した。また、同共焦点レーザー走査蛍光顕微鏡システムのアプリケーションにより輝度計算を行った。コントロールとして、乾熱滅菌したステンレス鋼製円盤に同様の染色処理を施したものを観察に供した。
(6) Observation of biofilm formation The stainless steel disk was taken out with tweezers sterilized with ethanol and transferred to another petri dish. A fluorescent staining agent (ethidium bromide aqueous solution, final concentration 100 μg / ml) was added so as to cover the surface of the stainless steel disk, and allowed to stand for 3 minutes. Distilled water was added so that the stainless steel disk was sufficiently immersed in the sterilized petri dish, and gently shaken for about 5 seconds to remove the colored water. Distilled water was added again, and the washing operation of the stainless steel disk was repeated 5 times in the same manner. After removing excess water, taking care not to touch the surface of the stainless steel disc, the following confocal laser scanning fluorescence microscope system was used to take a picture of the surface of the stainless steel disc and observe the formation of the biofilm. . In addition, the brightness was calculated by the application of the confocal laser scanning fluorescence microscope system. As a control, a stainless steel disk that had been sterilized by dry heat was subjected to the same dyeing treatment for observation.
なお、共焦点レーザー走査蛍光顕微鏡システム（BioRad、MRC-1024）は、顕微鏡としてECLIPSE E800(Nikon) 、励起光源として水銀ランプ（１００Ｗ）を用い、励起フィルター、ダイクロイックミラーおよび吸収フィルターの組み合わせとして、Ex510-560 、DM575 、BA590 を使用した。 The confocal laser scanning fluorescence microscope system (BioRad, MRC-1024) uses an ECLIPSE E800 (Nikon) as a microscope and a mercury lamp (100 W) as an excitation light source. -560, DM575, BA590 were used.
測定結果は以下のようになった。 The measurement results were as follows.
輝度測定結果（表１）より、ステンレス鋼製円盤の表面粗さは輝度に影響を及ぼし、また、表面が粗すぎる場合（4,〜）輝度は低くなることから、ある程度の粗さ（2,▽▽ ;3,▽）があった方が高い輝度、すなわち、バイオフィルム形成量が得られることが分かった。さらに、本方法でバイオフィルムの形成予防効果も確認することができた。 From the luminance measurement results (Table 1), the surface roughness of the stainless steel disk affects the luminance, and if the surface is too rough (4, ~), the luminance will be low, so some roughness (2, It was found that the brightness with ▽▽; 3, ▽), that is, the amount of biofilm formation can be obtained. Furthermore, the biofilm formation preventive effect could be confirmed by this method.
Claims (1)
前記蛍光染色剤が臭化エチジウム水溶液である一方、
前記洗浄操作として、前記別の滅菌シャーレに前記バイオフィルム担体が浸るように蒸留水を加えて振盪し、着色した水を除去する操作を複数回繰り返す工程が施されることを特徴とするバイオフィルム評価方法。 The cell suspension was added to biofilm carrier into a sterile Petri dish, transferred the biofilm carrier to another sterile petri dish, adding a fluorescent dye so as to cover the surface of the biofilm carrier, followed by the Bio After performing the washing operation of the film carrier, in order to evaluate the occurrence of slime by measuring the amount of biofilm formed on the surface of the biofilm carrier using a confocal laser scanning fluorescence microscope system ,
While the fluorescent stain is an aqueous ethidium bromide solution,
The biofilm is characterized in that, as the washing operation, a step of repeating a plurality of operations of adding distilled water and shaking to remove the colored water so that the biofilm carrier is immersed in the another sterile petri dish is performed a plurality of times. Evaluation methods.
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| JP5174408B2 (en) * | 2007-09-25 | 2013-04-03 | 花王株式会社 | Numeli inhibitor composition |
| KR100884935B1 (en) | 2007-10-29 | 2009-02-23 | 재단법인서울대학교산학협력재단 | Method of integratedly analyzing a microorganism and a biofilm chip used for the same |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10201496A (en) * | 1997-01-21 | 1998-08-04 | Canon Inc | Counting of environmental microorganism |
| JP2001208690A (en) * | 2000-01-27 | 2001-08-03 | Matsushita Electric Ind Co Ltd | Method for quantifying slime |
| JP2001333796A (en) * | 2000-05-29 | 2001-12-04 | Chisso Corp | Method of wipe examination for microorganism |
| JP2003534235A (en) * | 1999-05-12 | 2003-11-18 | ザ ゼネラル ホスピタル コーポレーション | Making biofilms transparent |
| WO2003095995A1 (en) * | 2002-05-14 | 2003-11-20 | Amersham Biosciences Uk Limited | Method for assessing biofilms |
| JP2005065624A (en) * | 2003-08-26 | 2005-03-17 | Nitto Denko Corp | Method and kit for measuring microbial amount |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10201496A (en) * | 1997-01-21 | 1998-08-04 | Canon Inc | Counting of environmental microorganism |
| JP2003534235A (en) * | 1999-05-12 | 2003-11-18 | ザ ゼネラル ホスピタル コーポレーション | Making biofilms transparent |
| JP2001208690A (en) * | 2000-01-27 | 2001-08-03 | Matsushita Electric Ind Co Ltd | Method for quantifying slime |
| JP2001333796A (en) * | 2000-05-29 | 2001-12-04 | Chisso Corp | Method of wipe examination for microorganism |
| WO2003095995A1 (en) * | 2002-05-14 | 2003-11-20 | Amersham Biosciences Uk Limited | Method for assessing biofilms |
| JP2005065624A (en) * | 2003-08-26 | 2005-03-17 | Nitto Denko Corp | Method and kit for measuring microbial amount |
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