Now that I've gotten a few replies, and a working solution - so I thought I should post a followup. One suggestion was to use SYBR Green instead of YOYO-1. Although there is some literature using SYBR Green as a DNA stain for enumeration of viruses, it is unclear (at least to me) what advantage SYBR would offer over YOYO-1 in terms of non-specific binding to the beads. We have now actually tried counting cowpox particles on the Calibur, and you can do this so long as the virus is *alone* - when mixed with Pseudomonas for test sprays we could not discriminate by size or YOYO-1 fluorescence between cowpox and bacteria (which was the point of the spray - to see if we could). Smaller viruses would probably not have this problem, but in biological samples I would still expect to see problems with specificity. The microspheres still add specificity so we can be sure we are looking at cowpox and not something else. Doug Redelman pointed out that dry milk could give some rather odd results due to the impurity of the milk that might give us even more fits than the BSA. Jan Keij came up with what appears to be the game-winner, however - suggesting that we switch the order of staining and do the YOYO-1 first followed by adding the beads. We've actually tried this, incubating the virus with YOYO-1 for one hour at 37 degrees Celsius followed by a shorter incubation with the beads (20 minutes at room temp) followed by two washes and then fixing with formadelhyde ( a safety requirement - even for cowpox ). In practice, this worked very well giving us nice data that shows a linear relationship between fluorescence and viral pfu concentration - allowing us to detect down to 100 pfu per milliliter (and hopefully lower, once we tweak the system some). We do still see staining of the beads with out virus present, but we can discriminate now between the background staining of the beads and staining when virus is present. I believe that this technique works better than our original because previously we would incubate with the beads, wash twice, and then add YOYO-1. We are probably losing beads during each wash - and that resulted in our odd staining patterns. By switching the order and eliminating those washes between the YOYO-1 and the beads, we've eliminated the source of the variablity - and therefore the staining we get now looks so much better. Many thanks to all who replied - and MOST ESPECIALLY to Jan Keij!! -Doug Reed Douglas S. Reed, Ph.D. Microbiologist Department of Aerobiology and Product Evaluation Division of Toxinology and Aerobiology U.S. Army Medical Research Institute of Infectious Disease 1425 Porter St. Ft. Detrick Frederick, MD 21702-5011 301-619-6728 301-619-2541 fax Doug.Reed@det.amedd.army.mil
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