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Applied and Environmental Microbiology, July 2004, p. 3862-3867, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.3862-3867.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Accurate Estimation of Viral Abundance by Epifluorescence Microscopy

Kevin Wen,1 Alice C. Ortmann,2 and Curtis A. Suttle1,2,3*

Departments of Microbiology and Immunology,1 Earth and Ocean Sciences,2 Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z43

Received 23 December 2003/ Accepted 19 March 2004

Virus enumeration by epifluorescence microscopy (EFM) is routinely done on preserved, refrigerated samples. Concerns about obtaining accurate and reproducible estimates led us to examine procedures for counting viruses by EFM. Our results indicate that aldehyde fixation results in rapid decreases in viral abundance. By 1 h postfixation, the abundance dropped by 16.4% ± 5.2% (n = 6), and by 4 h, the abundance was 20 to 35% lower. The average loss rates for glutaraldehyde- and formaldehyde-fixed samples over the first 2 h were 0.12 and 0.13 h–1, respectively. By 16 days, viral abundance had decreased by 72% (standard deviation, 6%; n = 6). Aldehyde fixation of samples followed by storage at 4°C, for even a few hours, resulted in large underestimates of viral abundance. The viral loss rates were not constant, and in glutaraldehyde- and formaldehyde-fixed samples they decreased from 0.13 and 0.17 h–1 during the first hour to 0.01 h–1 between 24 and 48 h. Although decay rates changed over time, the abundance was predicted by using separate models to describe decay over the first 8 h and decay beyond 8 h. Accurate estimates of abundance were easily made with unfixed samples stained with Yo-Pro-1, SYBR Green I, or SYBR Gold, and slides could be stored at –20°C for at least 2 weeks or, for Yo-Pro-1, at least 1 year. If essential, samples can be fixed and flash frozen in liquid nitrogen upon collection and stored at –86°C. Determinations performed with fixed samples result in large underestimates of abundance unless slides are made immediately or samples are flash frozen. If protocols outlined in this paper are followed, EFM yields accurate estimates of viral abundance.

* Corresponding author. Mailing address: Department of Earth and Ocean Science, Room 1321 Bioscience Building, 6270 University Blvd., Vancouver, BC V6T 1Z4, Canada. Phone: (604) 822-8610. Fax: (604) 822-6091. E-mail: csuttle{at}eos.ubc.ca.

Applied and Environmental Microbiology, July 2004, p. 3862-3867, Vol. 70, No. 7
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.7.3862-3867.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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