A simple methodological approach for counting and identifying culturable viruses adsorbed to cellulose nitrate membrane filters

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they cov ered the entire surface of each filter and penetrated through the pores. Wh en such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method a nd a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruse s in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step whic h is necessary when the commonly used methods are employed. The pore size o f the membrane filter, the sample contents, and the sample volume were opti mized for tap mater, seawater, and a 0.25 M glycine buffer solution. The nu mbers of viruses recovered under the optimized conditions were more than 50 % greater than the numbers counted by the standard plaque assay. When cefta zidime was added to the assay medium in addition to the antibiotics which a re typically used, the method could be used to study natural samples with l ow and intermediate levels of microbial pollution without decontamination o f the samples. This methodological approach also allowed plaque hybridizati on either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.