JP4331582B2 - Method for detecting E. coli and / or coliforms - Google Patents
Method for detecting E. coli and / or coliforms Download PDFInfo
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- JP4331582B2 JP4331582B2 JP2003418901A JP2003418901A JP4331582B2 JP 4331582 B2 JP4331582 B2 JP 4331582B2 JP 2003418901 A JP2003418901 A JP 2003418901A JP 2003418901 A JP2003418901 A JP 2003418901A JP 4331582 B2 JP4331582 B2 JP 4331582B2
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- 241000588724 Escherichia coli Species 0.000 title claims description 36
- 238000000034 method Methods 0.000 title description 8
- 150000003839 salts Chemical class 0.000 claims description 32
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 10
- 238000001514 detection method Methods 0.000 claims description 9
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 8
- YUDPTGPSBJVHCN-DZQJYWQESA-N 4-methylumbelliferyl beta-D-galactoside Chemical compound C1=CC=2C(C)=CC(=O)OC=2C=C1O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O YUDPTGPSBJVHCN-DZQJYWQESA-N 0.000 claims description 6
- DHJFFLKPAYHPHU-BYNIDDHOSA-N 5-bromo-4-chloro-3-indolyl beta-D-glucuronide Chemical compound O1[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 DHJFFLKPAYHPHU-BYNIDDHOSA-N 0.000 claims description 6
- 241000283690 Bos taurus Species 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 6
- 210000000941 bile Anatomy 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 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 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 229940054269 sodium pyruvate Drugs 0.000 claims description 5
- YUDPTGPSBJVHCN-CHUNWDLHSA-N 4-methylumbelliferyl alpha-D-galactoside Chemical compound C1=CC=2C(C)=CC(=O)OC=2C=C1O[C@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O YUDPTGPSBJVHCN-CHUNWDLHSA-N 0.000 claims description 4
- 235000010333 potassium nitrate Nutrition 0.000 claims description 4
- 239000004323 potassium nitrate Substances 0.000 claims description 4
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 claims 1
- 239000002609 medium Substances 0.000 description 29
- 239000000758 substrate Substances 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 238000004040 coloring Methods 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 6
- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 description 4
- QSUILVWOWLUOEU-GOVZDWNOSA-N 4-nitrophenyl beta-D-glucuronide Chemical compound O1[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1OC1=CC=C([N+]([O-])=O)C=C1 QSUILVWOWLUOEU-GOVZDWNOSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000006173 Good's buffer Substances 0.000 description 3
- 102000005936 beta-Galactosidase Human genes 0.000 description 3
- 108010005774 beta-Galactosidase Proteins 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000000306 component Substances 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 235000020681 well water Nutrition 0.000 description 3
- 239000002349 well water Substances 0.000 description 3
- GAZVSOQUFLIBJQ-BYNIDDHOSA-N (2s,3s,4s,5r,6s)-6-[(5-bromo-6-chloro-1h-indol-3-yl)oxy]-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound O1[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1OC1=CNC2=CC(Cl)=C(Br)C=C12 GAZVSOQUFLIBJQ-BYNIDDHOSA-N 0.000 description 2
- QZTKDVCDBIDYMD-UHFFFAOYSA-N 2,2'-[(2-amino-2-oxoethyl)imino]diacetic acid Chemical compound NC(=O)CN(CC(O)=O)CC(O)=O QZTKDVCDBIDYMD-UHFFFAOYSA-N 0.000 description 2
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 2
- XCBLFURAFHFFJF-UHFFFAOYSA-N 3-[bis(2-hydroxyethyl)azaniumyl]-2-hydroxypropane-1-sulfonate Chemical compound OCCN(CCO)CC(O)CS(O)(=O)=O XCBLFURAFHFFJF-UHFFFAOYSA-N 0.000 description 2
- JXCKZXHCJOVIAV-UHFFFAOYSA-N 6-[(5-bromo-4-chloro-1h-indol-3-yl)oxy]-3,4,5-trihydroxyoxane-2-carboxylic acid;cyclohexanamine Chemical compound [NH3+]C1CCCCC1.O1C(C([O-])=O)C(O)C(O)C(O)C1OC1=CNC2=CC=C(Br)C(Cl)=C12 JXCKZXHCJOVIAV-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- DBXNUXBLKRLWFA-UHFFFAOYSA-N N-(2-acetamido)-2-aminoethanesulfonic acid Chemical compound NC(=O)CNCCS(O)(=O)=O DBXNUXBLKRLWFA-UHFFFAOYSA-N 0.000 description 2
- JOCBASBOOFNAJA-UHFFFAOYSA-N N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid Chemical compound OCC(CO)(CO)NCCS(O)(=O)=O JOCBASBOOFNAJA-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- KUYNOZVWCFXSNE-BYNIDDHOSA-N inodxyl glucuronide Chemical compound O1[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1OC1=CNC2=CC=CC=C12 KUYNOZVWCFXSNE-BYNIDDHOSA-N 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 229910052749 magnesium Inorganic materials 0.000 description 2
- 239000012533 medium component Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- AJTVSSFTXWNIRG-UHFFFAOYSA-N 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid Chemical compound OCC[NH+](CCO)CCS([O-])(=O)=O AJTVSSFTXWNIRG-UHFFFAOYSA-N 0.000 description 1
- 239000007991 ACES buffer Substances 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 241000588923 Citrobacter Species 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241001646719 Escherichia coli O157:H7 Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 241000588653 Neisseria Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 102100040653 Tryptophan 2,3-dioxygenase Human genes 0.000 description 1
- 101710136122 Tryptophan 2,3-dioxygenase Proteins 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000000721 bacterilogical effect Effects 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- PAFZNILMFXTMIY-UHFFFAOYSA-N cyclohexylamine Chemical class NC1CCCCC1 PAFZNILMFXTMIY-UHFFFAOYSA-N 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
本発明は水等の検体中に大腸菌（Ｅ．ｃｏｌｉ）及び／又は大腸菌群が存在するか否かを簡便、迅速かつ正確に判定する方法に関する。 The present invention relates to a method for simply, rapidly and accurately determining whether E. coli and / or coliforms are present in a sample such as water.
水、飲食品等の細菌汚染を未然に防止するために、これらの検体中のＥ．ｃｏｌｉ及び大腸菌群の検出は必須である。大腸菌群は、グラム陰性の無芽胞桿菌で、乳糖を分解して酸とガスを産生する好気性又は通性嫌気性菌の一群の細菌であり、Escherichia、Enterobacter、Citrobacter、Klebsiellaなどが含まれる。このうち、細菌学の分類により大腸菌と称されるEscherichia coli(E.coli）は、大腸菌群の中でも病原性が強い。従って、水、飲食品等の検体中のＥ．ｃｏｌｉを検出することは特に重要である。 In order to prevent bacterial contamination of water, food and drink, etc., E. Detection of E. coli and coliforms is essential. The Escherichia coli group is a group of aerobic or facultative anaerobic bacteria that decomposes lactose to produce acid and gas, and is a gram-negative gonococcus, and includes Escherichia, Enterobacter, Citrobacter, Klebsiella, and the like. Among these, Escherichia coli (E. coli), which is called E. coli by bacteriological classification, is highly pathogenic among the coliforms. Therefore, E. coli in samples such as water and foods and drinks. It is particularly important to detect E. coli.
Ｅ．ｃｏｌｉの迅速検出法としては、着色性基質である５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−グルクロニドを添加した培地を用いる方法（特許文献１）が知られている。しかしこの培地を用いる方法では、Ｅ．ｃｏｌｉ以外のShigellaやSalmonellaなども陽性になってしまうという問題があった。 E. As a rapid detection method for E. coli, a method using a medium supplemented with 5-bromo-4-chloro-3-indolyl-β-D-glucuronide which is a coloring substrate is known (Patent Document 1). However, in the method using this medium, E. There was a problem that Shigella and Salmonella other than E.coli were also positive.
また、ＭＭＯ−ＭＵＧ培地やピルビン酸添加ＸＧａｌ−ＭＵＧ培地は、生成した蛍光物質に紫外線を照射して励起された蛍光を目視で判定する必要があり、簡便性の面で充分満足できるものではなかった。さらに、ピルビン酸添加ＸＧａｌ−ＭＵＧ培地は、２価イオン、特にカルシウムやマグネシウムを多く含む井戸水などを検体とした場合には白濁して、Ｅ．ｃｏｌｉの有無の判定が困難であった。また、水中のＥ．ｃｏｌｉを同定するための改良された方法（特許文献２）はβ−Ｄ−ガラクトシダ−ゼ基質とトリプトファナ−ゼ基質が含まれている培地を用いる方法で、培養後Ｅ．ｃｏｌｉを確認するためにはインド−ル試薬（コバック試薬）を添加する必要があり煩雑であった。
従って、本発明の目的は簡便な操作で、迅速かつ正確にＥ．ｃｏｌｉ及び大腸菌群を検出する方法を提供することにある。 Therefore, the object of the present invention is to quickly and accurately perform the E. coli operation with a simple operation. It is to provide a method for detecting E. coli and coliforms.
本発明者らは上記実状に鑑み、簡便性、迅速性を求めて培地成分、着色性基質及び蛍光性基質を種々検討した結果、特定の培地成分、特定の着色性基質、特定の蛍光性基質及び特定のpHのグッドの緩衝剤（Good's buffer)を組み合せて用いれば、水を検体に用いた場合でも濁りを生じず、短時間の簡便な操作で高感度にＥ．ｃｏｌｉ及び大腸菌群を検出できることを見出し、本発明を完成するに至った。 In light of the above circumstances, the present inventors have conducted various studies on medium components, coloring substrates and fluorescent substrates for simplicity and rapidity. As a result, specific medium components, specific coloring substrates, and specific fluorescent substrates are obtained. In addition, when a good pH's buffer (Good's buffer) is used in combination, even when water is used as a sample, turbidity does not occur, and E.C. The inventors have found that E. coli and coliforms can be detected, and have completed the present invention.
すなわち、本発明は、（１）ペプトン０．１〜２．０ｗ／ｖ％、（２）塩化ナトリウム０．１〜２．０ｗ／ｖ％、（３）pHが６．５〜７．５のグッドの緩衝剤０．０１〜１mol／Ｌ、（４）ピルビン酸ナトリウム０．０５〜５．０ｗ／ｖ％、（５）硝酸カリウム０．０５〜５．０ｗ／ｖ％、（６）５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−グルクロニド（Ｘ−Ｇｌｕｃ）もしくはその塩、５−ブロモ−６−クロロ−３−インドリル−β−Ｄ−グルクロニドもしくはその塩、５−ブロモ−３−インドリル−β−Ｄ−グルクロニドもしくはその塩、６−クロロ−３−インドリル−β−Ｄ−グルクロニドもしくはその塩、３−インドリル−β−Ｄ−グルクロニドもしくはその塩、４−ニトロフェニル−β−Ｄ−グルクロニドもしくはその塩又はｐ−ニトロフェニル−β−Ｄ−グルクロニドもしくはその塩、（７）４−メチル−ウンベリフェリル−β−Ｄ−ガラクトピラノシドもしくはその塩又は４−メチル−ウンベリフェリル−α−Ｄ−ガラクトピラノシドもしくはその塩、並びに（８）ドデシル硫酸ナトリウム０．００５〜１．０ｗ／ｖ％及び／又は牛胆汁末０．５〜２．０ｗ／ｖ％を含有する培地に被検体を加えて培養し、当該培地の着色の有無を検出することを特徴とするＥ．ｃｏｌｉの検出法を提供するものである。
また、前記培地に被検体を加えて培養し、当該培地の着色を検出し、さらに紫外線照射により蛍光の有無を検出することを特徴とするＥ．ｃｏｌｉ及び大腸菌群の検出法を提供するものである。
さらには、前記培地を含有するＥ．ｃｏｌｉ及び／又は大腸菌群検出用培地を提供するものである。
That is, the present invention is (1) peptone 0.1-2.0 w / v%, (2) sodium chloride 0.1-2.0 w / v%, (3) pH is 6.5-7.5. Good's buffer 0.01-1 mol / L, (4) sodium pyruvate 0.05-5.0 w / v%, (5) potassium nitrate 0.05-5.0 w / v%, (6) 5-bromo -4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) or a salt thereof, 5-bromo-6-chloro-3-indolyl-β-D-glucuronide or a salt thereof, 5-bromo-3- Indolyl-β-D-glucuronide or a salt thereof, 6-chloro-3-indolyl-β-D-glucuronide or a salt thereof, 3-indolyl-β-D-glucuronide or a salt thereof, 4-nitrophenyl-β-D- Glucuronide or its salt or -Nitrophenyl-β-D-glucuronide or a salt thereof, (7) 4-methyl-umbelliferyl-β-D-galactopyranoside or a salt thereof or 4-methyl-umbelliferyl-α-D-galactopyra Cultivated by adding a test substance to a medium containing noside or a salt thereof and (8) sodium dodecyl sulfate 0.005-1.0 w / v% and / or bovine bile powder 0.5-2.0 w / v% And the presence or absence of coloring of the medium is detected. E. coli detection method is provided.
In addition, E., wherein the subject is added to the medium and cultured, the color of the medium is detected, and the presence or absence of fluorescence is detected by ultraviolet irradiation. E. coli and coliform group detection methods are provided.
Furthermore, an E. coli containing the medium is used. E. coli and / or coliform group detection medium is provided.
本発明の培地を用いれば、肉眼観察により簡便かつ迅速にＥ．ｃｏｌｉを正確に検出でき、同時に大腸菌群も検出できる。本発明の培地は、カルシウムやマグネシウムを多く含む井戸水等の水を被検体とした場合でも白濁が生じず、正確な検出が可能である。 By using the culture medium of the present invention, E. coli can be easily and quickly observed with the naked eye. E. coli can be detected accurately, and at the same time, coliforms can be detected. The medium of the present invention does not cause white turbidity even when water such as well water containing a large amount of calcium or magnesium is used as a test sample, and can be accurately detected.
本発明の培地には、前記（１）〜（８）の成分が含まれる。このうち、（１）ペプトンは、栄養成分であり、培地中に０．１〜２．０ｗ／ｖ％、特に０．１〜１．０ｗ／ｖ％含有するのがより好ましい。ここで、培地中の濃度は、培養時の濃度である。（２）塩化ナトリウムは、培地中に０．１〜２．０ｗ／ｖ％、特に０．１〜１．０ｗ／ｖ％が好ましい。 The medium of the present invention contains the components (1) to (8). Among these, (1) Peptone is a nutrient component, and it is more preferable to contain 0.1 to 2.0 w / v%, particularly 0.1 to 1.0 w / v% in the medium. Here, the density | concentration in a culture medium is a density | concentration at the time of culture | cultivation. (2) Sodium chloride is preferably 0.1 to 2.0 w / v%, particularly 0.1 to 1.0 w / v% in the medium.
（３）pHが６．５〜７．５のグッドの緩衝剤としては、ピペラジン−１，４−ビス（２−エタンスルホン酸）（PIPES）、２−〔４−（２−ヒドロキシエチル）−１−ピペラジニル〕エタンスルホン酸（HEPES）、Ｎ−トリス（ヒドロキシメチル）メチル−２−アミノエタンスルホン酸（TES）、３−モルホリノプロパンスルホン酸（MOPS）、Ｎ−（２−アセトアミド）−２−アミノエタンスルホン酸（ACES）、Ｎ−（２−アセトアミド）イミノジ酢酸（ADA）、Ｎ，Ｎ−ビス（２−ヒドロキシエチル）−２−アミノエタンスルホン酸（BES）、ビス（２−ヒドロキシエチル）イミノトリス（ヒドロキシメチル）メタン（Bis-Tris）、３−〔Ｎ，Ｎ−ビス（２−ヒドロキシエチル）アミノ〕−２−ヒドロキシプロパンスルホン酸（DIPSO）、２−ヒドロキシ−３−モルホリノプロパンスルホン酸（MOPSO）、２−ヒドロキシ−Ｎ−トリス（ヒドロキシメチル）メチル−３−アミノプロパンスルホン酸及びこれらの塩等が挙げられる。これらの緩衝剤は、培地中に、それぞれ０．０１〜１mol／Ｌ、好ましくは０．０２〜１mol／Ｌ含有する。本発明の培地が、井戸水等を被検体とした場合でも白濁が生じないのは、これらの緩衝剤がカルシウムイオンやマグネシウムイオンとの間で水不溶性物質を生成しないためと考えられる。 (3) As a good buffer having a pH of 6.5 to 7.5, piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), 2- [4- (2-hydroxyethyl)- 1-piperazinyl] ethanesulfonic acid (HEPES), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), 3-morpholinopropanesulfonic acid (MOPS), N- (2-acetamido) -2- Aminoethanesulfonic acid (ACES), N- (2-acetamido) iminodiacetic acid (ADA), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), bis (2-hydroxyethyl) Iminotris (hydroxymethyl) methane (Bis-Tris), 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 2-hydroxy-3- Le morpholino propane sulfonic acid (MOPSO), 2-hydroxy -N- tris (hydroxymethyl) methyl-3-amino-propanesulfonic acid and salts thereof. These buffers are contained in the medium in an amount of 0.01 to 1 mol / L, preferably 0.02 to 1 mol / L, respectively. The reason why the white turbidity does not occur in the culture medium of the present invention even when using well water or the like as a test sample is that these buffer agents do not generate water-insoluble substances with calcium ions or magnesium ions.
（４）ピルビン酸ナトリウムは、培地中に０．０５〜５．０ｗ／ｖ％、より好ましくは０．０５〜２ｗ／ｖ％含有する。（５）硫酸カリウムは、培地中に０．０５〜５．０ｗ／ｖ％、より好ましくは０．０５〜２ｗ／ｖ％含有する。 (4) Sodium pyruvate is contained in the medium in an amount of 0.05 to 5.0 w / v%, more preferably 0.05 to 2 w / v%. (5) Potassium sulfate is contained in the medium in an amount of 0.05 to 5.0 w / v%, more preferably 0.05 to 2 w / v%.
（６）５−ブロモ−４−クロロ−３−インドリル−β−Ｄ−グルクロニド（Ｘ−Ｇｌｕｃ）又はその塩、５−ブロモ−６−クロロ−３−インドリル−β−Ｄ−グルクロニド又はその塩、５−ブロモ−３−インドリル−β−Ｄ−グルクロニド又はその塩、６−クロロ−３−インドリル−β−Ｄ−グルクロニド又はその塩、３−インドリル−β−Ｄ−グルクロニド又はその塩、４−ニトロフェニル−β−Ｄ−グルクロニド又はその塩、ｐ−ニトロフェニル−β−Ｄ−グルクロニド又はその塩は、着色性基質であり、Ｅ．ｃｏｌｉが産生する酵素、グルクロニダーゼにより分解し、色素を生成する。これらの塩としては、シクロヘキシルアンモニウム塩が好ましい。これらの着色性基質の培地中の含有量は、０．００１〜０．１ｗ／ｖ％が好ましい。 (6) 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) or a salt thereof, 5-bromo-6-chloro-3-indolyl-β-D-glucuronide or a salt thereof, 5-bromo-3-indolyl-β-D-glucuronide or a salt thereof, 6-chloro-3-indolyl-β-D-glucuronide or a salt thereof, 3-indolyl-β-D-glucuronide or a salt thereof, 4-nitro Phenyl-β-D-glucuronide or a salt thereof, p-nitrophenyl-β-D-glucuronide or a salt thereof is a coloring substrate; It is decomposed by glucuronidase, an enzyme produced by E. coli, to produce a pigment. As these salts, cyclohexylammonium salts are preferred. The content of these coloring substrates in the medium is preferably 0.001 to 0.1 w / v%.
（７）４−メチル−ウンベリフェリル−β−Ｄ−ガラクトピラノシド（４ＭＵＧＡＬ）又はその塩、４−メチル−ウンベリフェリル−α−Ｄ−ガラクトピラノシド又はその塩は、蛍光性基質であり、大腸菌群が産生する酵素、β−Ｄ−ガラクトシダーゼにより分解し、蛍光色素を生成する。これらの蛍光性基質の培地中の含有量は０．００１〜０．１ｗ／ｖ％が好ましい。 (7) 4-methyl-umbelliferyl-β-D-galactopyranoside (4MUGAL) or a salt thereof, 4-methyl-umbelliferyl-α-D-galactopyranoside or a salt thereof is a fluorescent substrate And is degraded by β-D-galactosidase, an enzyme produced by coliforms, to produce a fluorescent dye. The content of these fluorescent substrates in the medium is preferably 0.001 to 0.1 w / v%.
（８）ドデシル硫酸ナトリウム及び／又は牛胆汁末は、界面活性剤であり、グラム陽性菌の発育を抑制する作用を有する。ドデシル硫酸ナトリウムは培地中に０．００５〜１．０ｗ／ｖ％、特に０．００５〜０．５ｗ／ｖ％含有するのが好ましい。また牛胆汁末の場合は、０．５〜２．０ｗ／ｖ％、特に１．０〜２．０ｗ／ｖ％含有するのが好ましい。 (8) Sodium dodecyl sulfate and / or bovine bile powder is a surfactant and has an action of suppressing the growth of Gram-positive bacteria. Sodium dodecyl sulfate is preferably contained in the medium in an amount of 0.005 to 1.0 w / v%, particularly 0.005 to 0.5 w / v%. In the case of bovine bile powder, it is preferable to contain 0.5 to 2.0 w / v%, particularly 1.0 to 2.0 w / v%.
さらに、本発明の培地には、酵母エキス、アミノ酸類、グリセロール（０．５〜１．５ｗ／ｖ％）等が含まれていてもよい。また、イソプロピル−β−Ｄ−チオガラクトピラノシドを０．００１〜０．１ｗ／ｖ％含有するのが、大腸菌群が産生する酵素β−ガラクトシダーゼの産生を促進し、酵素反応をより正確にする点から好ましい。 Furthermore, the culture medium of the present invention may contain yeast extract, amino acids, glycerol (0.5 to 1.5 w / v%) and the like. In addition, containing isopropyl-β-D-thiogalactopyranoside in an amount of 0.001 to 0.1 w / v% promotes the production of the enzyme β-galactosidase produced by Escherichia coli and makes the enzyme reaction more accurate. This is preferable.
本発明の培地は、使用時のpHが６．５〜７．５、特に６．８〜７．３とするのが好ましい。 The culture medium of the present invention preferably has a pH of 6.5 to 7.5, particularly 6.8 to 7.3.
本発明の培地は、前記成分を混合して液体培地として用いることもできるが、これに寒天０．１〜１．５ｗ／ｖ％配合して寒天培地として用いることもできる。 The medium of the present invention can be used as a liquid medium by mixing the above components, but it can also be used as an agar medium by blending 0.1 to 1.5 w / v% of agar.
本発明の培地を用いて、被検体中のＥ．ｃｏｌｉの存在を検出するには、当該培地に被検体を加えて３０〜４４．５℃で数時間〜十数時間培養した後、当該培地の着色の有無を肉眼観察すればよい。 Using the culture medium of the present invention, E. In order to detect the presence of E. coli, a test sample is added to the medium, and cultured at 30 to 44.5 ° C. for several hours to tens of hours, and then the presence or absence of coloring of the medium may be visually observed.
さらに、Ｅ．ｃｏｌｉを確認するために紫外線を照射し、蛍光を確認できれば大腸菌群であることを認め、さらにＥ．ｃｏｌｉが存在する確立を高めることができる。 In addition, E.I. In order to confirm E. coli, ultraviolet rays were irradiated. The probability that E. coli exists can be increased.
ここで被検体としては、水、飲食品等が挙げられるが、本発明は水を被検体とする場合に特に有用である。被検体を予めトリプトソーヤブイヨン等の培地で培養した培養液として用いることもできる。 Examples of the subject include water, food and drink, and the like, but the present invention is particularly useful when water is the subject. It can also be used as a culture solution in which the subject is cultured in advance in a medium such as tryptosome broth.
以下、実施例を挙げてさらに詳細に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, although an example is given and explained in detail, the present invention is not limited to these.
実施例１
表２〜６記載の微生物６３株をトリプトソーヤブイヨン又はブドウ等ペプトン水で３５℃、２４時間培養した。この培養液１０μＬを下記表１の培地１０mLに接種後、３５℃、２４時間培養し、培地の着色の有無並びに紫外線照射による蛍光の有無を肉眼観察した。その結果を表２〜６に示す。
Example 1
63 strains of microorganisms listed in Tables 2 to 6 were cultured in peptone water such as tryptosome broth or grape at 35 ° C. for 24 hours. 10 μL of this culture solution was inoculated into 10 mL of the medium shown in Table 1 below, followed by culturing at 35 ° C. for 24 hours. The results are shown in Tables 2-6.
表２〜６から明らかなように、Ｅ．ｃｏｌｉ（E.coli O157:H7を除く）は１００％着色及びＥ．ｃｏｌｉを含む大腸菌群は１００％蛍光が観察された。他の菌株では全く着色が見られず、本発明方法が正確であることが確認された。 As is clear from Tables 2-6, E.I. E. coli (except E. coli O157: H7) is 100% colored and E. coli. 100% fluorescence was observed in the coliform group containing E. coli. Other strains did not show any coloration, confirming that the method of the present invention was accurate.
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