In situ detection of bacteria in calcified biofilms using FISH and CARD-FISH

Modified protocols of fluorescence in situ hybridization (FISH) and catalyze reporter deposition fluorescence in situ hybridization (CARD-FISH) were developed in order to detect bacteria in situ in calcified stromatolite biofilms. Smooth, well-preserved thin sections of calcified biofilms (approximately 5 microm thin, vertical sectioning of approximately 1 cm deep) were obtained by cryo-sectioning using the adhesive tape-stabilization technique. A modified hybridization buffer was applied during hybridization to prevent calcite dissolution as well as false binding of oligonucleotide probes to the charged mineral surfaces. Particularly, bright and specific CARD-FISH signals allowed the detection of bacteria in intensively calcified biofilms even at low magnification, which is suitable for investigating millimeter- to centimeter-scale vertical distribution patterns of bacteria.