Recovery and quantification of bacterial cells associated with streambed sediments.
Efficient detachment and purification of bacterial cells associated with streambed sediments are required in order to quantify cell abundance and to assess community composition through the application of epifluorescence microscopy techniques. We applied chemical (i.e., sodium pyrophosphate and polysorbate) and physical treatments (i.e., shaking and sonication), followed by Nycodenz density gradient centrifugation to efficiently recover benthic bacteria. This procedure resulted in a highly purified cell suspension allowing for a precise cell quantification through the application of fluorescent dyes. About 93% of total cells were recovered from the original sediment, with higher recovery from the finer grain-size class (90%) in comparison to the coarse fraction (69%). The potential damaging effects of the applied procedures on cell integrity were assessed on planktonic bacteria in a pre-filtered water control. As a consequence of the high purity of the extracted bacteria, flow cytometry was successfully applied as counting method for sediment cell suspension. However, a significant decrease of protein synthesis in purified samples was measured by estimating the (3)H-Leucine incorporation rates, rising uncertainties on the possibility to apply potential metabolic assays after Nycodenz purification.
Amalfitano
Stefano
S
Water Research Institute (IRSA-CNR), via Reno 1, 00198 Roma, Italy. amalfitano@irsa.cnr.it
Fazi
Stefano
S
D001419:Bacteria / Q000737:chemistry / Q000254:growth & development* / Q000302:isolation & purification*; D001431:Bacteriological Techniques; D002499:Centrifugation, Density Gradient / Q000379:methods*; D011756:Diphosphates / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D005618:Fresh Water / Q000382:microbiology*; D019015:Geologic Sediments / Q000382:microbiology*; D008856:Microscopy, Fluorescence / Q000379:methods; D011136:Polysorbates / Q000737:chemistry; D013010:Sonication
D011756:Diphosphates; D011136:Polysorbates; C003319:sodium pyrophosphate