AME 43:223-231 (2006)  -  doi:10.3354/ame043223

Comparison of SYBR Green I and SYBR Gold stains for enumerating bacteria and viruses by epifluorescence microscopy

Akira Shibata1,4,*, Yoichi Goto1, Hiroaki Saito2, Tomohiko Kikuchi3, Tatuki Toda1, Satoru Taguchi1

1Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan
2Tohoku National Fisheries Research Institute, Shinhama-cho 3-27-5, Shiogama 985-0001, Japan
3Faculty of Education and Human Sciences, Yokohama National University, 792 Tokiwadai, Hodogaya, Yokohama 240-8501, Japan
4Present address: Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

ABSTRACT: SYBR Gold staining is used for enumerating bacteria and viruses in aquatic samples. However, its suitability for epifluorescence microscopy has not been sufficiently investigated. Thus we compared bacterial and viral counts using SYBR Gold and SYBR Green I stains. Variables for both bacterial and viral counts included season and ocean depths of sample collection and the period of sustained excitation under epifluorescence microscopy. We also examined the storage period and procedures for preservation of samples with formaldehyde for bacterial counts. Natural seawater samples were used for all experiments. Ratios of counts obtained with SYBR Gold to those with SYBR Green I staining were 0.99 ± 0.09 (mean ± SD, n = 58) for bacteria and 1.0 ± 0.1 (n = 38) for viruses, which indicated no significant differences between stains. In samples fixed with 0.74% formaldehyde that were stored at 4°C, bacterial counts obtained with SYBR Gold staining decreased over time in parallel with those obtained with SYBR Green I staining. However, counts from fixed samples with both SYBR stains did not decrease significantly after 30 d when glass slides were prepared immediately and stored at –20°C, or when samples were flash-frozen in liquid nitrogen and stored at –80°C. Under sustained excitation, counts of bacteria and viruses stained with SYBR Gold decreased less than with SYBR Green I, suggesting greater persistence of the fluorescence signal with SYBR Gold. These results indicate the suitability of SYBR Gold staining for use in the determination of bacterial and viral abundance in natural seawater.

KEY WORDS: SYBR Green I · SYBR Gold · Bacteria · Virus · Enumeration · Epifluorescence microscopy

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