The funnel was necessary as the inlet portion could only hold ~150 μL of liquid and
the surface tension caused by the Luer-lock was too great to permit an even passage of liquid under vacuum. Briefly, the Luer-lock was cut off of the Swinnex fitting inlet to maximize the opening. Next, the tip of the 15 mL tube was removed and the end of the tube subsequently finely sanded so that when inserted into the inlet and assembled with the outlet it would not come in contact with the filter membrane. The two pieces were bonded using a cyanoacrylate-type glue and allowed to cure for 24 hours. For filtration, the inlet/funnel was screwed onto the outlet portion of the Swinnex, which was see more connected to a vacuum source. This filtration apparatus is inexpensive (< $20 USD) and in combination with a manifold, allows for high throughput filtration. Figure 1 Custom-built 13 mm filter
funnel. Funnel was assembled from a Swinnex® inlet bonded to the conical end of a 15 ml polypropylene tube. Enumeration of VLP using 13 mm Anodisc membranes Our protocol for preparing virus slides using 13 mm Anodisc membranes is based on that of Ortmann and Suttle (2009), with check details modifications of the staining procedure. JAK inhibitor back-staining is the standard protocol for Anodisc 25 membranes and involves placing the membrane sample side up onto a drop Adenosine of stain, incubating, then removing excess stain by either wicking  or applying vacuum . However, back-staining is technically challenging due to the small size and absence of a support ring on the 13 mm membranes. Thus, samples were pre-stained prior to filtration. The detailed protocol is as follows: i) A virus sample was brought up to a final volume of 900 μL using 0.02-μm filtered diluent (AN media or seawater). ii) 100 μL of SYBR Gold (25 ×, 0.02 μm filtered) was added to the sample and then incubated for 15 min in the dark. iii) A backing filter (0.2 μm, polyethersulfone, Pall Corporation, Port Washington, NY)
was placed onto the screen of the Swinnex outlet and overlaid with sterile MilliQ water (~2 mL). Vacuum pressure (5 in Hg) was applied to pull the water through and stopped immediately so not to dry out the filter. iv) The backing filter was overlaid with MilliQ water (~2 mL) again and a 13 mm Anodisc placed on top of the water. v) The vacuum was then applied to pull the water through and sandwich the filters together. vi) With the vacuum still on, the modified Swinnex inlet (containing a gasket) was carefully screwed on and tightened with sufficient torque; excessive torque would crack the membrane and insufficient torque caused particles to be preferentially filtered towards the periphery of the membrane. vii) The sample was added to the center of the funnel.