Estimation of viral-induced phytoplankton mortality using the modified dilution method
To create the grazer-free diluent whole seawater is gravity filtered through acid-washed 0.2–0.45 µm filters (e.g., PALL Acropak™ Supor® membrane capsules) into a clean carboy.
After gravity filtration half of the grazer-free diluent is passed through a tangential flow filtration system with kilodalton (kDa) pore size to create the virus-free diluent.
The grazer-free and virus-free diluents are added to 10-L polycarbonate bottles in the correct proportions to create the parallel t0 dilution series, e.g., 20%, 40%, 70%, and 100% whole water.
The mesoplankton-free whole water is then gently added by siphoning.
From each of these t0 bottles, triplicate 1-L polycarbonate bottles were rinsed twice and then gently filled by siphoning (to minimize physical damage to the grazers, viruses, and phytoplankton populations), ensuring that bottles are filled completely to avoid trapping air bubbles inside upon closure.
After filling, triplicate experimental bottles are placed randomly into experimental conditions.
For determination of phytoplankton composition and abundance and virus abundance, triplicate subsamples (5 mL) are taken from the 10 L t0 dilution bottles and the 0.2–0.45-µm and kDa diluents.
Initial (t0) and final (t24) phytoplankton composition and abundance estimates are typically determined by analysis of samples using flow cytometry
Apparent phytoplankton growth rates (µ, d–1) are calculated from each experimental bottle as the changes in abundance during the incubation using the equation:µ = ln(Pt/Po)/t.
The actual dilution rate is calculated by dividing the t0 phytoplankton abundance in each bottle by the averaged abundance of the replicate t0 100% counts (3–6 bottles).
