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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Authors: | Ursula Eschenhagen Martin Eschenhagen Frank Ludwig Angelika Kiessling Kristin Symank Elke Boschke Isolde Röske Thomas Bley |
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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
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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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Keywords: | Rapid detection FISH Catalyzed reporter deposition Tetramethylbenzidine Microcolonies |
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