Assessment of Factors Influencing Direct Enumeration of Viruses within Estuarine Sediments

Abstract

Accurate enumeration of viruses within environmental samples is critical for investigations of the ecological role of viruses and viral infection within microbial communities. This report evaluates differences in viral and bacterial direct counts between estuarine sediment samples which were either immediately processed onboard ship or frozen at ؊20°C and later processed. Viral and bacterial abundances were recorded at three stations spanning the length of the Chesapeake Bay in April and June 2003 within three sediment fractions: pore water (PW), whole sediment (WS), and sediment after pore water removal (AP). No significant difference in viral abundance was apparent between extracts from fresh or frozen sediments. In contrast, bacterial abundance was significantly lower in the samples subjected to freezing. Both bacterial and viral abundance showed significant differences between sediment fractions (PW, WS, or AP) regardless of the fresh or frozen status. Although pore water viral abundance has been used in the past as a measurement of viral abundance in sediments, this fraction accounted for only ca. 5% of the total sediment viral abundance across all samples. The effect of refrigerated storage of sediment viral extracts was also examined and showed that, within the first 2 h, viral abundance decreased ca. 30% in formalin-fixed extracts and 66% in unfixed extracts. Finally, the reliability of direct viral enumeration via epifluorescence microscopy was tested by using DNase treatment of WS extractions. These tests indicated that a large fraction (>86%) of the small SYBR gold fluorescing particles are likely viruses.

In situ structuring of virioplankton through ­bacterial exoenzymatic activity: interaction with phytoplankton

“…Drops of 20 to 35% in viral abundance have been reported after a few hours in the presence of fixatives (glutaraldehyde or formaldehyde) and storage at 4°C with pelagic samples (Wen et al 2004), as well as with viruses from estuarine sediments (Helton et al 2006). However, in the present study, all samples were treated in the same way, allowing valuable cross-comparisons.…”

Viral infections stimulate the metabolism and shape prokaryotic assemblages in submarine mud volcanoes

“…This was because it is widely recognised that the use of formaldehyde or glutaraldehyde for sample storage can result in a rapid loss of viruses in sediment samples (Helton et al, 2006;Dell'Anno et al, 2009).…”

Counting Viruses and Bacteria in Photosynthetic Microbial Mats

“…Solutions used for extraction were made with Milli-Q water (18.2 M⍀) and added only after fixation of the sample, therefore avoiding osmotic shock. To promote the release of particle-associated viruses (and bacteria) in the microbial mat samples, chemical treatment was first tested with tetrasodium pyrophosphate (TSPP) (14) and EDTA (18,21) in combination with water bath sonication (14). TSPP is commonly used to extract viruses and bacteria from sediment particles (14).…”

“…In microbial mats, EPS bind microorganisms, viruses, and particles together in a complex matrix (17), making it more challenging to extract viruses and bacteria than from bulk sediments. To allow detailed studies of viruses in microbial mats, modifications to protocols currently used for quantitative assessment of benthic viruses are necessary (14,18,19). Here, we report an improved assay allowing efficient extraction and enumeration by epifluorescence microscopy (EFM) or flow cytometry (FCM) of viruses from photosynthetic microbial mats, as well as intertidal sediments.…”