A comparison of methods for counting viruses in aquatic systems

Abstract

In this study, we compared different methods-including transmission electron microscopy-and various nucleic acid labeling methods in which we used the fluorochromes 4',6'-diamidino-2-phenylindole (DAPI), 4-[3-methyl-2,3-dihydro-(benzo-1, 3-oxazole)-2-methylmethyledene]-1-(3'-trimethyl ammoniumpropyl)-quinilinium diioide (YOPRO-1), and SYBR Green I, which can be detected by epifluorescence microscopy (EM), for counting viruses in samples obtained from freshwater ecosystems whose trophic status varied and from a culture of T7 phages. From a quantitative and qualitative viewpoint, our results showed that the greatest efficiency for all ecosystems was obtained when we used the EM counting protocol in which YOPRO-1 was the label, as this fluorochrome exhibited strong and very stable fluorescence. A modification of the original protocol in which YOPRO-1 was used is recommended, because this modification makes the protocol faster and allows it to be used for routine analysis of fixed samples. Because SYBR Green I fades very quickly, the use of this fluorochrome is not recommended for systems in which the viral content is very high (>10(8) particles/ml), such as treated domestic sewage effluents. Experiments in which we used DNase and RNase revealed that the number of viruses determined by EM was slightly overestimated (by approximately 15%) because of interference caused by the presence of free nucleic acids.

FIG. 1

Viral concentrations in the different environments tested, as determined by TEM and by EM after staining with various fluorochromes. ND, not determined. The table shows the results obtained by a one-way analysis of variance in which Fisher's least-significant-difference test was used. An asterisk indicates significance at a level of P < 0.05. NS, not significant. ME(DAPI), EM-DAPI protocol; ME(O-YOPRO), original EM-YOPRO protocol; ME(M-YOPRO), modified EM-YOPRO protocol; ME(SYBR I), EM-SYBR Green I protocol; TEM, TEM protocol; O-YOPRO, original YOPRO; M-YOPRO, modified YOPRO; SYBR I, SYBR Green I.

FIG. 2

Comparison of fading times for the various fluorochromes used with EM. The numbers in parentheses are the coefficients of variation for the fading times obtained with six samples that originated from the different bodies of water sampled. The table shows the results obtained by a one-way analysis of variance in which Fisher's least-significant-difference test was used. An asterisk indicates significance at a level of P < 0.05. NS, not significant. O-YOPRO, original YOPRO; M-YOPRO, modified YOPRO; SYBR I, SYBR Green I.

FIG. 3

EM-SYBR Green I protocol: relationship between the mean viral concentrations and the corresponding coefficients of variation (CV) for the six environments tested.