Dataset: protist cell counts
Deployment: 64PE325

Excerpt from Morgan-Smith et al. (2013):

Samples were collected using a rosette sampler fitted with 25 L Niskin bottles. Samples were immediately fixed with 0.2 micron-filtered methanol-stabilized formaldehyde (final concentration ~2%) and kept overnight at room temperature to allow outgassing and prevent bubble formation on filters. Subsamples of 100-250 ml were then filtered under gentle vacuum (-200mbar) onto Millipore 0.2 micron white polycarbonate filters for DAPI-FITC. A pore size of 0.2 micron is preferred for enumeration of total eukaryotes because some smaller eukaryotes are lost through the pores of 0.8 micron filters, leading to underestimation of eukaryote abundance (Morgan-Smith et al., 2011). Filters were washed with phosphate buffered saline (PBS) and ultra-pure water, then immediately stored at -80 C.

DAPI-FITC double staining was used to enumerate total eukaryotes. Pie-shaped slices of approximately 1/16-1/8 of filter were cut and 3-4 of these slices were placed atop a 25 mm diameter, 0.2 micron pore size polycarbonate filter on a glass frit and covered with a 10-ml filter tower, which was then flooded with 1ml FITC staining solution (2.5 ml 0.5 M sodium carbonate buffer, pH 9; 11 ml 0.01 M potassium phosphate buffer, pH 7.2; 11 ml 0.85 % sodium chloride; 10 mg FITC), and incubated for 10 min in the dark. Then vacuum was applied (-200 mbar) and filters were washed twice with 10 ml ice-cold carbonate buffer (0.5M, pH9). Filter sections were mounted on slides using Vectashield mounting medium with DAPI and stored at -20 C until analysis. Samples were counted on Olympus BX51 and BX50 epifluorescence micro scopes.