TABLE 1.

Treatments to which virus samples were subjected in order to optimize enumeration of viruses in solution by flow cytometrya

TreatmentLevels
Fixative glutaraldehyde (%)0.1, 0.5, 1, or 2
Fixative formaldehyde (%)0.4, 0.8, 2, or 4
Storage temp (°C)4, −20, −80, or −196 (N2)
Storage time at 4, −20, and −80°C1 h or 1 mo
Storage time at 4°C1 h, 2 wk, or 1 or 6 mo
Dye SYBR Green I (10−5)0, 0.5, 1, 5, 10, 15, or 25 (of the commercial stock)
Dye SYBR Gold (10−5)5, 10, or 25 (of the commercial stock)
Incubation temp20, 40, 60, 80, or 90°C
Incubation time (min)1, 4, 7, 10, 13, 16, or 19
Dilution solutionTE, Tris, PBS, dH2O, or seawater
pH of TE buffer7, 7.4, 7.8, 8, 8.4, or 8.85
Dilution with seawater (%)0, 1, 2, 5, 10, 20, or 50
Addition of detergentsTriton X-100, Tween 80, NP-40, and SDS at a 0.1% (vol/vol) final concn
Addition of citrate (concn [mM])0, 1, 2, 5, 10, 20, or 50
  • a Viruses used included four marine phytoplankton viruses (CeV, MpV, PoV, and PpV), one freshwater phytoplankton virus (PBCV), one virus infecting a cyanobacterium (S-PMS2), four characterized bacteriophages (Lambda, T2, T4, and T7), three uncharacterized marine bacteriophages (T-φHS1C A, φ16, and T-φD1B), and two natural marine virus communities. dH2O, distilled water.