The filter was back-stained by placement sample side up onto 100 μL of SYBR Gold stain (25 × concentration, Invitrogen, Carlsbad, CA) and incubated for 15 min followed by application of a vacuum to remove the stain. Samples were also prepared with a post-stain rinse of 850 μL of 0.02 μm filtered media or seawater. For direct comparison to the Anodisc 13 membranes, parallel samples GF120918 were also pre-stained

in a microcentrifuge tube prior to filtration. Filtration time using the above protocol was < 5 min per mL of sample. Determination of filterable area for Anodisc membranes The filterable area of the Anodisc membranes was determined by passage of a cell culture of the naturally pigmented bacterium Synechococcus sp. WH7803 through them. Digital images were analyzed with Adobe® Photoshop® CS4 (Adobe Systems Incorporated, San Jose, CA) to calculate the area containing pigmented cells. The data reported is a range of the averages obtained from triplicate filters. Enumeration of viruses using Nuclepore membranes As pre-stained black Nuclepore membranes with pore sizes of 15 and 30 nm are not commercially available, membranes were stained using 0.2% Irgalan Black (Acid black

107, Organic Dyestuffs Corporation, East Providence, RI) dissolved in 2% acetic acid as previously described [8], with the exceptions that staining time was reduced from 3 hours find more to 15 minutes and SC79 in vivo filters were Fossariinae used immediately. Polyester drain discs (Whatman), which are designed to improve flow rate and provide a flat surface to eliminate rupturing were used as backing filters. Filters were placed in 25 mm Swinnex filter holders for filtration and processed using the same reagents and solutions described for the Anodisc membranes. The filtration time required for the Nuclepore 15 and 30 membranes using the above protocol was < 60 min and < 10 min per mL, respectively. SEM imaging of Nuclepore membranes To assess whether the filtration protocol could be damaging or altering membrane pore size, scanning electron micrographs

of the Nuclepore membranes were taken before and after filtrating media (0.02 μM filtered AN) or seawater (0.02 μM filtered Sargasso Sea water) using a LEO 1525 field emission scanning electron microscope (Carl Zeiss Inc., Thornwood, NY, USA). Avoiding lateral stress, the membranes were cut, mounted on a stub and viewed. No coating was applied so as to not obscure the pores. At least 3 regions of each filter were viewed and at least 50 pores measured from each filter. Filtration did not appear to damage the filters or change pore size. Initial attempts at preparing the filters for SEM did suggest that lateral stress (excessive stretching or twisting) of the membranes could drastically increase pore size (data not shown).