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 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.