In situ hybridization of microcolonies using catalyzed reporter deposition with tetramethylbenzidine: a method for detecting low numbers of bacterial cells in drinking water
In this study, catalyzed reporter deposition in situ hybridization (CARD--ISH) with tetramethylbenzidine (TMB) was used for rapid detection of the food pathogens Salmonella typhimurium and Escherichia coli. The bacteria in a sample were concentrated by membrane filtration. The filter membranes with...
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| Published in | European food research & technology Vol. 227; no. 4; pp. 995 - 999 |
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| Main Authors | , , , , , , , |
| Format | Journal Article |
| Language | English German |
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Berlin/Heidelberg
Berlin/Heidelberg : Springer-Verlag
01.08.2008
Springer-Verlag Springer Springer Nature B.V |
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| Abstract | In this study, catalyzed reporter deposition in situ hybridization (CARD--ISH) with tetramethylbenzidine (TMB) was used for rapid detection of the food pathogens Salmonella typhimurium and Escherichia coli. The bacteria in a sample were concentrated by membrane filtration. The filter membranes with the cells thus removed were incubated on nutrient agar for 4-5 h to allow the formation of microcolonies. Instead of fluorescent tyramide, 3,3',5,5'-tetramethylbenzidine (TMB), which yields a blue precipitate, was used for signal amplification after in situ hybridization. Microcolonies amplified with TMB produced blue signals, which were sufficiently intense to allow visual evaluation either using a stereomicroscope, or even with the naked eye. Therefore even low cell numbers of hygienically critical bacteria can be detected on the filter membrane without a protracted examination. This enables the detection of low cell numbers (<10 cfu) in a sample of 100 ml tap water within 9-10 h. |
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| AbstractList | In this study, catalyzed reporter deposition in situ hybridization (CARD--ISH) with tetramethylbenzidine (TMB) was used for rapid detection of the food pathogens Salmonella typhimurium and Escherichia coli. The bacteria in a sample were concentrated by membrane filtration. The filter membranes with the cells thus removed were incubated on nutrient agar for 4-5 h to allow the formation of microcolonies. Instead of fluorescent tyramide, 3,3',5,5'-tetramethylbenzidine (TMB), which yields a blue precipitate, was used for signal amplification after in situ hybridization. Microcolonies amplified with TMB produced blue signals, which were sufficiently intense to allow visual evaluation either using a stereomicroscope, or even with the naked eye. Therefore even low cell numbers of hygienically critical bacteria can be detected on the filter membrane without a protracted examination. This enables the detection of low cell numbers (<10 cfu) in a sample of 100 ml tap water within 9-10 h. [PUBLICATION ABSTRACT] In this study, catalyzed reporter deposition in situ hybridization (CARD--ISH) with tetramethylbenzidine (TMB) was used for rapid detection of the food pathogens Salmonella typhimurium and Escherichia coli. The bacteria in a sample were concentrated by membrane filtration. The filter membranes with the cells thus removed were incubated on nutrient agar for 4-5 h to allow the formation of microcolonies. Instead of fluorescent tyramide, 3,3',5,5'-tetramethylbenzidine (TMB), which yields a blue precipitate, was used for signal amplification after in situ hybridization. Microcolonies amplified with TMB produced blue signals, which were sufficiently intense to allow visual evaluation either using a stereomicroscope, or even with the naked eye. Therefore even low cell numbers of hygienically critical bacteria can be detected on the filter membrane without a protracted examination. This enables the detection of low cell numbers (<10 cfu) in a sample of 100 ml tap water within 9-10 h. In this study, catalyzed reporter deposition in situ hybridization (CARD—ISH) with tetramethylbenzidine (TMB) was used for rapid detection of the food pathogens Salmonella typhimurium and Escherichia coli . The bacteria in a sample were concentrated by membrane filtration. The filter membranes with the cells thus removed were incubated on nutrient agar for 4–5 h to allow the formation of microcolonies. Instead of fluorescent tyramide, 3,3′,5,5′-tetramethylbenzidine (TMB), which yields a blue precipitate, was used for signal amplification after in situ hybridization. Microcolonies amplified with TMB produced blue signals, which were sufficiently intense to allow visual evaluation either using a stereomicroscope, or even with the naked eye. Therefore even low cell numbers of hygienically critical bacteria can be detected on the filter membrane without a protracted examination. This enables the detection of low cell numbers (<10 cfu) in a sample of 100 ml tap water within 9–10 h. In this study, catalyzed reporter deposition in situ hybridization (CARD-ISH) with tetramethylbenzidine (TMB) was used for rapid detection of the food pathogens Salmonella typhimurium and Escherichia coli. The bacteria in a sample were concentrated by membrane filtration. The filter membranes with the cells thus removed were incubated on nutrient agar for 4-5h to allow the formation of microcolonies. Instead of fluorescent tyramide, 3,3',5,5'-tetramethylbenzidine (TMB), which yields a blue precipitate, was used for signal amplification after in situ hybridization. Microcolonies amplified with TMB produced blue signals, which were sufficiently intense to allow visual evaluation either using a stereomicroscope, or even with the naked eye. Therefore even low cell numbers of hygienically critical bacteria can be detected on the filter membrane without a protracted examination. This enables the detection of low cell numbers (<10cfu) in a sample of 100ml tap water within 9-10h. |
| Author | Boschke, Elke Ludwig, Frank Röske, Isolde Symank, Kristin Eschenhagen, Ursula Eschenhagen, Martin Kiessling, Angelika Bley, Thomas |
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| Cites_doi | 10.1128/AEM.64.8.3079-3083.1998 10.1093/nar/gkg016 10.1128/AEM.68.6.3094-3101.2002 10.1128/jb.172.2.762-770.1990 10.4315/0362-028X-66.10.1935 10.1016/S0076-6879(05)97021-3 10.1006/fmic.1999.0317 10.1177/002215549904700302 10.4315/0362-028X-61.11.1425 10.1128/AEM.70.9.5426-5433.2004 10.1128/JCM.38.2.814-817.2000 10.1128/MMBR.59.1.143-169.1995 10.1128/IAI.64.9.3811-3817.1996 10.1128/AEM.68.2.661-667.2002 10.1111/j.1365-2672.1993.tb02767.x 10.1128/AEM.44.4.809-813.1982 |
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| Keywords | FISH Tetramethylbenzidine Catalyzed reporter deposition Rapid detection Microcolonies Drinking water Analysis method In situ Control method Detection |
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| References_xml | – volume: 64 start-page: 3079 year: 1998 ident: 811_CR6 publication-title: Appl Environ Microbiol doi: 10.1128/AEM.64.8.3079-3083.1998 contributor: fullname: KF Eckner – volume: 8 start-page: 368 year: 2002 ident: 811_CR9 publication-title: BIOspektrum contributor: fullname: W Köster – volume: 31 start-page: 514 year: 2003 ident: 811_CR11 publication-title: Nucleic Acids Res doi: 10.1093/nar/gkg016 contributor: fullname: A Loy – volume: 68 start-page: 3094 year: 2002 ident: 811_CR14 publication-title: Appl Environ Microbiol doi: 10.1128/AEM.68.6.3094-3101.2002 contributor: fullname: A Pernthaler – volume: 172 start-page: 762 year: 1990 ident: 811_CR2 publication-title: J Bacteriol doi: 10.1128/jb.172.2.762-770.1990 contributor: fullname: RI Amann – volume: 66 start-page: 1935 year: 2003 ident: 811_CR4 publication-title: J Food Prot doi: 10.4315/0362-028X-66.10.1935 contributor: fullname: PW Benoit – volume: 7 start-page: 80 year: 1993 ident: 811_CR7 publication-title: Food Sci Technol Today contributor: fullname: KA Grant – ident: 811_CR13 doi: 10.1016/S0076-6879(05)97021-3 – volume: 17 start-page: 305 year: 2000 ident: 811_CR18 publication-title: Food Microbiol doi: 10.1006/fmic.1999.0317 contributor: fullname: ML Tortorello – volume: 47 start-page: 281 year: 1999 ident: 811_CR17 publication-title: J Histochem Cytochem doi: 10.1177/002215549904700302 contributor: fullname: EJ Speel – volume: 61 start-page: 1425 year: 1998 ident: 811_CR19 publication-title: J Food Prot doi: 10.4315/0362-028X-61.11.1425 contributor: fullname: ML Tortorello – volume: 70 start-page: 5426 year: 2004 ident: 811_CR12 publication-title: Appl Environ Microbiol doi: 10.1128/AEM.70.9.5426-5433.2004 contributor: fullname: A Pernthaler – volume: 38 start-page: 814 year: 2000 ident: 811_CR8 publication-title: J Clin Microbiol doi: 10.1128/JCM.38.2.814-817.2000 contributor: fullname: GJ Jansen – volume: 59 start-page: 143 year: 1995 ident: 811_CR3 publication-title: Microbiol Rev doi: 10.1128/MMBR.59.1.143-169.1995 contributor: fullname: RI Amann – volume: 64 start-page: 3811 year: 1996 ident: 811_CR10 publication-title: Infect Immun doi: 10.1128/IAI.64.9.3811-3817.1996 contributor: fullname: TR Licht – ident: 811_CR1 – volume: 68 start-page: 661 year: 2002 ident: 811_CR15 publication-title: Appl Environ Microbiol doi: 10.1128/AEM.68.2.661-667.2002 contributor: fullname: A Pernthaler – volume: 75 start-page: 199 year: 1993 ident: 811_CR5 publication-title: J Appl Bacteriol doi: 10.1111/j.1365-2672.1993.tb02767.x contributor: fullname: CW Blackburn – volume: 10 start-page: 809 year: 1982 ident: 811_CR16 publication-title: Appl Environ Microbiol doi: 10.1128/AEM.44.4.809-813.1982 contributor: fullname: GL Pettipher |
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| Title | In situ hybridization of microcolonies using catalyzed reporter deposition with tetramethylbenzidine: a method for detecting low numbers of bacterial cells in drinking water |
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