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 degrees 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.

FIG. 1.

Regression analysis of virus (slope = 0.6, r² = 0.2, P = 0.02) (A) and bacterium (slope = 0.3, r² = 0.6, P < 0.0001) (B) abundances comparing fresh to frozen storage conditions. The CI values are based on original abundances and are shown at a 99% CI.

FIG. 2.

Mean viral (A) and bacterial (B) abundances according to sediment fraction and storage condition. Bars: □, freshly extracted samples; ▪, samples frozen prior to extraction.

FIG. 3.

Effect of multiple extractions on viral recovery from Chesapeake Bay sediment samples. The percentages of viruses recovered are based on summed viral abundances from all four extractions. Extraction results from stations 908 (□), 804 (░⃞), and 724 (▪) are shown.

FIG. 4.

DNase test on WS samples: untreated viral extracts (□), DNase-treated viral extracts (▧), and heated and DNase-treated viral extracts (▪).

FIG. 5.

Effects of refrigerated storage and formalin fixation loss of viral particles from sediment extracts for the first 10 h (A) and from 0 to 408 h (B). Symbols: •, fixed viral extracts; ○, unfixed viral extract samples.