Enumerating bacterial cells on bioadhesive coated slides.

Quantifying bacterial abundance and biomass is fundamental to many microbiological studies. Directly counting via epifluorescence microscopy has become the method of choice, especially for environmental samples, and conventional techniques require filtration of cells onto black polycarbonate membrane filters. We investigated the utility of instead capturing stained bacterial suspensions on bioadhesive slides, performing tests using pure cultures of bacteria, mixtures of cultured bacteria, and environmental samples from five habitat types. When compared to the standard filtration and flow cytometric approaches, bioadhesive slides were found to be an accurate and precise platform for rapid enumeration of bacteria. Total bacterial counts made using the three methods were positively correlated for acridine orange and Live/Dead® (L/D) staining (0.81≤r≤0.95, all p≤0.002). All platforms had similar precision, though counts obtained using bioadhesive slides were significantly higher than those made with polycarbonate filters and flow cytometry. The specific bioadhesive slides we used resulted in substantial cell mortality for certain pure cultures and river water samples, limiting their use for L/D determination. Cell enumeration using bioadhesive slides is particularly effective because it is highly precise at a wide range of cell concentrations, allows observation of cells that are not readily discernible on filters, reduces the number of steps and processing materials associated with sample analysis, and increases throughput.

Effects of age and composition of field-produced biofilms on oyster larval setting.

Lack of success in restoring the native Eastern oyster, Crassostrea virginica, to Chesapeake Bay has been linked to the low occurrence of oyster larval setting in tributaries to the Bay. Among the many potential factors that could affect efforts to produce oysters through aquaculture or supplementation of shell beds is substratum condition. The present study examined larval setting on field-produced biofilms from Little Wicomico River (Virginia, USA) to assess whether bacterial community structure (examined by terminal restriction fragment length polymorphism, T-RFLP) or other characteristics of contemporary biofilms in this tributary (biofilm age and mass, algal abundance, and percentage organic matter) inhibited setting of larval oysters. The structure of the natural and heterogenous bacterial community in the biofilms and the success of oyster set were correlated, suggesting that specific microbial species may play a role in oyster setting. Larval set increased with biofilm age and mass, suggesting that established field-produced biofilms have no inhibitory effect. In contrast, the percentage of organic matter was negatively correlated with oyster set, whereas chlorophyll a concentration had no observed effect. The study expands prior knowledge by providing a more realistic timeframe for biofilm development (weeks as opposed to days), recounting effects of biofilms that are more representative of the natural dynamic and disturbance processes that would be expected to occur on submerged structures, and by incorporating seasonal and spatial variation. An important negative effect observed during the study period was heavy predation by Stylochus ellipticus on newly set oysters. Overall, the results of this study, which is the first assessment of the effects of biofilms produced naturally within a Chesapeake Bay tributary, suggest that the absence of large numbers of oysters in Little Wicomico River is not related to microbes or other specific characteristics of biofilms that develop on suitable setting substrata, but rather to heavy predation of newly set larvae.