Rapid and Sensitive Quantification of Vibrio cholerae and Vibrio mimicus Cells in Water Samples by Use of Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Solid-Phase Cytometry

A new protocol for rapid, specific, and sensitive cell-based quantification ofVibrio cholerae /Vibrio mimicus in water samples was developed. The protocol is based on catalyzed reporter deposition fluorescencein situ hybridization (CARD-FISH) in combination with solid-phase cytometry. For pure cultures, we were able to quantify down to 6V. cholerae cells on one membrane with a relative precision of 39% and down to 12 cells with a relative precision of 17% after hybridization with the horseradish peroxidase (HRP)-labeled probe Vchomim1276 (specific forV. cholerae andV. mimicus ) and signal amplification. The corresponding position of the probe on the 16S rRNA is highly accessible even when labeled with HRP. For the first time, we were also able to successfully quantifyV. cholerae /V. mimicus via solid-phase cytometry in extremely turbid environmental water samples collected in Austria. Cell numbers ranged from 4.5 × 101 cells ml−1 in the large saline lake Neusiedler See to 5.6 × 104 cells ml−1 in an extremely turbid shallow soda lake situated nearby. We therefore suggest CARD-FISH in combination with solid-phase cytometry as a powerful tool to quantifyV. cholerae /V. mimicus in ecological studies as well as for risk assessment and monitoring programs.