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In situ hybridization of microcolonies using catalyzed reporter deposition with tetramethylbenzidine: a method for detecting low numbers of bacterial cells in drinking water
Authors:Ursula Eschenhagen  Martin Eschenhagen  Frank Ludwig  Angelika Kiessling  Kristin Symank  Elke Boschke  Isolde Röske  Thomas Bley
Affiliation:1. Institute of Food Technology and Bioprocess Engineering, Technische Universit?t Dresden, Bergstrasse 120, 01069, Dresden, Germany
2. Institute of Microbiology, Technische Universit?t Dresden, Zellescher Weg 20 b, 01217, Dresden, Germany
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.
Keywords:Rapid detection  FISH  Catalyzed reporter deposition  Tetramethylbenzidine  Microcolonies
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