Thus, Nuclepore membrane pore sizes were analyzed using OSI-027 in vitro scanning electron micrographs as described in the methods section. Pore sizes were consistent in membranes pre- BTSA1 order and post-filtration. However, the pore sizes for Nuclepore 30 membranes were not uniform and ranged from 20 to 50 nm in size with the majority of pores being < 40 nm (78%)(Figure 2B); the Nuclepore 15 membranes were

also not uniform and ranged from 10 to 30 nm in size with the majority of pores being < 20 nm (69%) (Figure 2C). Figure 2 Pore size distribution of untreated Nuclepore™ filters determined by SEM analysis. (A) SEM image of Nuclepore™ 30 membrane. Scale bar is 200 nm. (B) Pore size range check details of Nuclepore 30 membrane. (C) Pore size range of Nuclepore 15 membrane. Conclusions Modifications of existing protocols allow the reliable use of Anodisc 13 membranes for enumeration of VLP using epifluorescence microscopy. In parallel studies, we found that Nuclepore filters (polycarbonate, 0.03 & 0.015 μm pore sizes) consistently

yielded lower observable VLP. These low counts may be attributed to non-uniform pore sizes that were evident by scanning electron microscopy of these filters (Figure 2). However, more rigorous parallel comparisons of the Nuclepore and Anodisc membranes are necessary to determine this conclusively. Differences in VLP abundance estimates between Anodisc 13 and 25 membranes were evident with

environmental samples if a post-rinse step was not included in sample processing. While rinsing of membranes gave the most consistent results across the two Anodisc membranes, it may result in loss of enumeration of VLP depending upon the environment from which the sample was derived. Given the heterogeneity of natural virus populations, individual aminophylline investigators will need to consider the issue of applying a post-rinse on a case-by-case basis. Methods Sample collection and preparation Viral lysate was made using cyanophage S-PWM1, which infects Synechococcus sp. WH7803 (aka DC2) [21]. The lysate was filtered through a 0.2-μm Durapore™ filter and stored at 4°C – this filtered material served as the lysate standard. Open ocean water samples were collected from the Sargasso Sea (May 28, 2005; 36.343° N, 51.315° W) and coastal water samples were collected off the coast of Georgia, USA (Nov 18, 2007; 31.372° N, 80.561° W). Multiple seawater aliquots (2 mL) were uniformly distributed, fixed in 0.5% glutaraldehyde and frozen at -80°C at the start of this study to ensure reproducibility. Enumeration of viruses using 25 mm Anodisc membranes The protocol using 25 mm Anodisc membranes follows that published by Ortmann and Suttle (2009), with minor modifications. Briefly, filtration was performed on a Hoefer® filtration manifold (Hoefer, Holliston, MA) without chimney weights. After the backing (0.