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 (~ 5 µm thin, vertical sectioning of ~ 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.