Experimental improvements in combining CARD-FISH and flow cytometry for bacterial cell quantification

Area of Science:

• Aquatic microbiology
• Molecular biology
• Cytometry

Background:

• Flow cytometry and Fluorescence In Situ Hybridization (FISH) are key techniques for bacterial identification and quantification.
• Combining these methods offers rapid, multi-parametric, and phylogenetically specific analysis.
• This approach is particularly promising for aquatic microbiology research.

Purpose of the Study:

• To optimize a combined flow cytometry and CARD-FISH protocol for analyzing marine bacteria.
• To improve cell detachment and detection efficiency in natural samples.
• To assess the applicability of the protocol for quantifying specific phylogenetic groups.

Main Methods:

• In-solution hybridization protocol tested on E. coli pure culture and marine bacteria.
• Established CARD-FISH protocol adapted for filter-based analysis in a closed system.
• Optimized cell detachment procedure for re-suspending hybridized cells from membrane filters.
• Flow cytometric enumeration of hybridized marine bacteria.

Main Results:

• High efficiency hybridization signal observed for E. coli pure culture.
• Lower, but detectable, hybridization signal for marine bacteria in initial tests.
• Successful re-suspension of hybridized cells from filters.
• Cytometric enumeration achieved 85.7%±18.1% of total events for hybridized marine bacteria.
• High-quality cytograms indicating protocol effectiveness.

Conclusions:

• The optimized CARD-FISH protocol coupled with flow cytometry enhances bacterial cell detection and quantification in aquatic environments.
• The procedure demonstrates applicability for enumerating specific phylogenetic groups within natural microbial communities.
• This integrated approach represents a powerful tool for advancing aquatic microbiology.