Efficiency of fluorescence in situ hybridization for bacterial cell identification in temporary river sediments with contrasting water content.
We studied the efficiency of two hybridization techniques for the analysis of benthic bacterial community composition under varying sediment water content. Microcosms were set up with sediments from four European temporary rivers. Wet sediments were dried, and dry sediments were artificially rewetted. The percentage of bacterial cells detected by fluorescence in situ hybridization with fluorescently monolabeled probes (FISH) significantly increased from dry to wet sediments, showing a positive correlation with the community activity measured via incorporation of (3)H leucine. FISH and signal amplification by catalyzed reporter deposition (CARD-FISH) could significantly better detect cells with low activity in dried sediments. Through the application of an optimized cell permeabilization protocol, the percentage of hybridized cells by CARD-FISH showed comparable values in dry and wet conditions. This approach was unrelated to (3)H leucine incorporation rates. Moreover, the optimized protocol allowed a significantly better visualization of Gram-positive Actinobacteria in the studied samples. CARD-FISH is, therefore, proposed as an effective technique to compare bacterial communities residing in sediments with contrasting water content, irrespective of differences in the activity state of target cells. Considering the increasing frequencies of flood and drought cycles in European temporary rivers, our approach may help to better understand the dynamics of microbial communities in such systems.
Fazi
Stefano
S
Water Research Institute (IRSA-CNR), Roma, Italy. fazi@irsa.cnr.it
Amalfitano
Stefano
S
Pizzetti
Ilaria
I
Pernthaler
Jakob
J
D039903:Actinobacteria / Q000235:genetics / Q000302:isolation & purification*; D001419:Bacteria / Q000166:cytology / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D015373:Bacterial Typing Techniques; D004777:Environment; D019015:Geologic Sediments / Q000737:chemistry / Q000382:microbiology*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D045483:Rivers / Q000737:chemistry