In situ hybridization of microcolonies using catalyzed reporter deposition with tetramethylbenzidine: a method for detecting low numbers of bacterial cells in drinking water

• Published in: European food research & technology Vol. 227; no. 4; pp. 995 - 999
• Main Authors: Eschenhagen, Ursula, Eschenhagen, Martin, Ludwig, Frank, Kiessling, Angelika, Symank, Kristin, Boschke, Elke, Röske, Isolde, Bley, Thomas
• Format: Journal Article
• Language: English; German
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.08.2008; Springer-Verlag; Springer; Springer Nature B.V
• Subjects: Agriculture; Analytical Chemistry; Bacteria; Biological and medical sciences; Biotechnology; Chemistry; Chemistry and Materials Science; Drinking water; E coli; Escherichia coli; Food; Food industries; Food Science; Forestry; Fundamental and applied biological sciences. Psychology; General aspects; Hybridization; Membrane filtration; Membranes; Methods; Methods of analysis, processing and quality control, regulation, standards; Microorganisms; Original Paper; Process controls; Salmonella; Salmonella typhimurium; Studies; FISH; Tetramethylbenzidine; Catalyzed reporter deposition; Rapid detection; Microcolonies; Drinking water; Analysis method; In situ; Control method; Detection
• ISSN: 1438-2377; 1438-2385
• DOI: 10.1007/s00217-007-0811-9

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.