An improved protocol for quantification of freshwater Actinobacteria by fluorescence in situ hybridization.
We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml(-1)) followed by achromopeptidase (60 U ml(-1)) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton.
Sekar
Raju
R
Max Planck Institute for Marine Microbiology, Celciusstrasse 1, D-28359 Bremen, Germany.
Pernthaler
Annelie
A
Pernthaler
Jakob
J
Warnecke
Falk
F
Posch
Thomas
T
Amann
Rudolf
R
D039903:Actinobacteria / Q000235:genetics* / Q000302:isolation & purification*; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification; D002463:Cell Membrane Permeability; D015169:Colony Count, Microbial / Q000379:methods; D005618:Fresh Water / Q000382:microbiology*; D006094:Gram-Positive Bacteria / Q000235:genetics / Q000302:isolation & purification; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009113:Muramidase; D010933:Plankton / Q000302:isolation & purification; D012697:Serine Endopeptidases
D009113:Muramidase; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase