subsample bottles were rinsed three times with 0.2 pm filtered sea water to remove any 
residues. 
2.3 Subsample Analysis 
Subsamples were analyzed in parallel using the analytic methods described in Table 1; 
detailed methodology for each analytic method is provided in Appendix B. The indicative 
methods fall into three major groups: those that detect chlorophyll fluorescence activity (CFA), 
those that rely on FDA as a viability probe, and those that measure ATP. CFA devices (Walz 
WATER-PAM, bbe lOcells, TD Ballast-Check2, and Hach BW680) measure baseline fluorescence 
under dark adaptation (F 0 ), and maximal fluorescence (F m ) under saturating light to estimate 
total active chlorophyll fluorescence (F v ) in the subsample (F v =F m -F 0 ) (Wright et al., 2015). The 
Hach BW680 and the bbe lOcells determine relative 'active' chlorophyll biomass estimates 
based on the F v value, whereas the TD BallastCheck-2™ estimates organism concentration using 
corrected fluorescence measurements (see Appendix B for details). The F v measurements from 
the CFA devices can be converted into a cell number using an instrument-specific calibration 
value. In contrast, FDA methods rely on the conversion of FDA to fluorescein by viable cells, 
which makes them appear green when excited by blue light. Thus, FDA is not a 'traditional' 
stain, in the sense that it does not 'bind' or 'bond' with internal cellular compounds (as would a 
nuclear 'stain' like SYBR Green). In the case of the Satake Pulse Counter, the amount of 
fluorescence within each cell is used to estimate cell size, whereas the Moss Landing Marine 
Labs (MLML) bulk FDA method relies on measuring the absolute fluorescein production rate, as 
measured extra-cellularly after it diffuses out of the cell.