Imaging and quantifying virus fluorescence signals on aquatic aggregates: a new method and its implication for aquatic microbial ecology

Abstract

The development of accurate methods to detect and enumerate viruses is an important issue in aquatic microbial ecology. In particular, viruses attached to floating aggregates are a largely ignored field both in marine and inland water ecology. Data on the total abundance and the colonization of aggregates by viruses are rare, mainly due to methodological difficulties. In the present study, we used confocal laser scanning microscopy (CLSM) to resolve fluorescence signals of single viruses and bacterial cells in a complex three-dimensional matrix of riverine aggregates. CLSM in combination with different fluorochromes is a very promising approach for obtaining information both on the aggregate architecture and on the spatial distribution of viruses attached to fully hydrated aggregates. Aggregates from the Danube River harbored up to 5.39 x 10(9) viruses cm(-3). We discuss the problems associated with different methods such as sonication or directly counting viruses on aggregates, both combined with epifluorescence microscopy and CLSM, to quantify viruses on suspended particles.

Fig. 1

CLSM maximum intensity projection showing particle-derived viruses and bacteria on a filter after sonication. White arrows point to viruses, and yellow ones to bacteria. Calibration bar: 5 μm.

Fig. 2

CLSM image series of a riverine aggregate in the axial direction. Dual channel presentation of specific glycoconjugates, bacteria and viruses. Arrow points to viruses. Color allocation: green, nucleic acid; red, glycoconjugates; yellow, bacteria and viruses in or in contact with lectin-specific EPS. Calibration bar: 30 μm.

Fig. 3

3D volume reconstructions from a riverine aggregate. (a) and (b) represent different views of the aggregate. Arrows point to viruses. Color allocation: green, nucleic acid; red, glycoconjugates; yellow, bacteria and viruses in or in contact with lectin-specific EPS. Calibration grid: 5 μm.

Fig. 4

(a) Single scan of a cryo-section showing the distribution of viruses, bacteria and glycoconjugates inside an aggregate. (b) View of one channel to present bacteria and viruses. White arrows point to viruses. Color allocation: green, nucleic acid; red, glycoconjugates; yellow, bacteria and viruses in or in contact with lectin-specific EPS. Calibration bar: 10 μm.

Fig. 5

3D volume reconstruction from a riverine aggregate. For deconvolution, the method of classic maximum likelihood estimation was applied. The program huygens 3.0.0, SVI (Scientific Volume Imaging B.V., the Netherlands) was used. Arrows point to viruses. Color allocation: green, nucleic acid; red, glycoconjugates; yellow, bacteria and viruses in or in contact with lectin-specific EPS. Calibration grid: 5 μm.