Evaluation of confocal laser scanning microscopy for enumeration of virus-like particles in aquatic systems
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Abundances of virus-like particles (VLPs, mostly bacteriophages) are high in aquatic environments; therefore, techniques for precise enumeration are essential in ecological monitoring. VLPs were determined after staining with SYBR Gold by conventional epifluorescence microscopy and compared to enumerations performed by confocal laser scanning microscopy (CLSM). In order to assess the potential of CLSM for viral direct counts (VDCs), we processed samples from different freshwater and marine systems. Optical sectioning by CLSM and production of an overlay picture of multiple scans enables the often uneven whole investigated filter area to be brought to the plane of focus. This allows for subsequent image analysis of digitally created high-quality images. Another advantage using the CLSM was that the short spot excitation of the stain via laser beam minimized fading of the stain. The VDC results show that there is no significant difference between the two methods. Regarding the known difficulties of viral abundance estimates on particulate material, CLSM was further applied to enumerate VLPs on a small set of marine transparent exopolymeric particles sampled from the Atlantic Ocean. Our data suggest that CLSM is a useful tool to count viruses in water samples as well as attached to certain types of aquatic aggregates.
KeywordsViruses Confocal laser scanning microscopy Epifluorescence microscopy SYBR Gold Microaggregates
We are thankful to R. Agis, G. Winkler, and M.G. Weinbauer for providing surface samples from Egypt, Kenya, and The Netherlands and C.R. Fisher, M. Bright, and A. Nussbaumer for the deep-sea samples from the Pacific Ocean. Special thanks are due to M.G. Weinbauer for helpful discussions and comments on the manuscript. This work was supported by a grant from the Austrian Science Foundation FWF to P.P. (FWF P 17798-B03).
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