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.
Geoscience Centre, University of Göttingen, Goldschmidtstrasse 3, D-37077 Göttingen, Germany. fshirai@gwdg.de
Fumito
F
Shiraishi
Barbara
B
Zippel
Thomas R
TR
Neu
Gernot
G
Arp
D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification; D001431:Bacteriological Techniques; D018441:Biofilms; D005618:Fresh Water / Q000382:microbiology; D019015:Geologic Sediments / Q000382:microbiology*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics
D015345:Oligonucleotide Probes