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Applied and Environmental Microbiology, January 2000, p. 194-198, Vol. 66, No. 1
Microbiology Section, State General
Laboratory, Ministry of Health,1 and
Molecular Virology Department, The Cyprus Institute of
Neurology and Genetics,3 Nicosia, Cyprus, and
Department de Microbiologia, Universitat de Barcelona,
Barcelona, Spain2
Received 30 July 1999/Accepted 7 October 1999
We identified conditions under which Buffalo green monkey cells
grew on the surfaces of cellulose nitrate membrane filters in such a
way that they covered the entire surface of each filter and penetrated
through the pores. When such conditions were used, poliovirus that had
previously been adsorbed on the membranes infected the cells and
replicated. A plaque assay method and a quantal method (most probable
number of cytopathic units) were used to detect and count the viruses
adsorbed on the membrane filters. Polioviruses in aqueous suspensions
were then concentrated by adsorption to cellulose membrane filters and
were subsequently counted without elution, a step which is necessary
when the commonly used methods are employed. The pore size of the
membrane filter, the sample contents, and the sample volume were
optimized for tap water, seawater, and a 0.25 M glycine buffer
solution. The numbers of viruses recovered under the optimized
conditions were more than 50% greater than the numbers counted by the
standard plaque assay. When ceftazidime was added to the assay medium
in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels
of microbial pollution without decontamination of the samples. This
methodological approach also allowed plaque hybridization either
directly on cellulose nitrate membranes or on Hybond N+ membranes
after the preparations were transferred.
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Simple Methodological Approach for Counting and Identifying
Culturable Viruses Adsorbed to Cellulose Nitrate Membrane
Filters
*
Corresponding author. Mailing address: Department de
Microbiologia, Universitat de Barcelona, Avinguda Diagonal, 645, 08028 Barcelona, Spain. Phone: 34 93 402 14 87. Fax: 34 93 411 05 92. E-mail:
joan{at}porthos.bio.ub.es.
Applied and Environmental Microbiology, January 2000, p. 194-198, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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