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 ) .
