1.1

Free viruses (vlps) inhabit the aquatic environment in concentrations of many millions per millilitre of water. Virus population densities of>10⁸ vlps/ml^-1 have been recorded from sea water, and indications are that similar numbers exist in many freshwater habitats. All living species can be infected by viruses, and all viruses are obligate parasites that require a host for their replication. The infected host cell is destroyed in the process. The size and composition, therefore, of communities of free viruses is of concem to suppliers, consumers and users of freshwater.

1.2

The aims of the present study are (a) to develop a low-cost, easy to use fluorescence; microscopy technique that can be used in laboratories to enumerate free viruses in fresh water, and (b) to use transmission electron microscopy (TEM) to evaluate this technique and to categorise free viruses sampled seasonally from a range of freshwater habitats. The current report covers methodology developed.

1.3

The nucleic acid specific, cyanine-based fluorescent stain YO-PRO-1 was selected from 7 stains chosen for their fluorescent brilliance and/or specificity. YO-PRO-1 enabled precise (low variance), reproducible vlp counts to be made from photographic transparencies of randomly selected areas of the sample, photographed by fluorescence microscopy at 500x magnification.

1.4

Fluorescence vlp counts were compared with TEM counts, made from test waters with virus densities ranging from 10⁶ -10⁸ vlps/ml^-1. TEM enabled accurate counts to be made of virus numbers, and individual viruses to be categorised by their morphological characteristics. However, TEM vlp counts were always found to be lower by 13-27% than counts by fluorescence microscopy.

1.5

Experimentation demonstrated that drying of TEM sample preparations concentrated viruses at the peripheral, uncountable areas of the specimen grid, and that this effect, rather than falsely elevated fluorescent counts, was the likely cause of the counting discrepency.

1.6

A fluorescence technique using YO-PRO-1 and a TEM technique using uranyl acetate negative stain (with adjustment for the avoidance of drying effects) have been standardised for the enumeration of viruses and will be used in the virus survey in the final part of the project. Staining protocols are given together with experimental details, representative micrographs, and a tabulated summary of the test water virus communities analysed.