Glutaraldehyde-fixed Heterotrophic Bacterial Abundance and Biomass (Process Cruise)

Methods

Microscopy based Heterotrophic bacterial abundance

Preserved samples (3 ml SW + 25% glutaraldehyde, 1% final) were frozen in liquid N2 and stored at -80°C until analysis (<3 months). Aliquots from defrosted samples were filtered on polycarbonate 0.22 µm pore size 25-mm diameter filters with 0.45 µm backing filters, dried at r.t., stained using VECTASHIELD with DAPI (4', 6-diamidino-2-phenylindole), and imaged at 100X magnification using a Nikon C1 upright microscope. Slides were imaged at 1000X magnification on a Nikon TE2000-U inverted epifluorescence microscope for P0704. Stained slides were stored at -20°C until microscopy (<1 week for original processing, up to 1 year for additional processing as needed). Images were analyzed using Nikon Advanced Research 3.2 software. Either 20 image fields or at least 200 cells per filter, whichever was smaller, was processed to measure cell abundance, line length, and width using the signal thresholding. Bacterial counts were manually corrected by removing auto fluorescent cells identified in the TRICT channel. The line length cutoff 0.20-2.0μm was used to exclude non-bacterial cells. The average cell number per field was converted to abundance based on volumes filtered and the surface area of the filter and field of view.

Microscopy based Heterotrophic bacterial biomass

Preserved samples (3 ml SW + 25% glutaraldehyde, 1% final) were frozen in liquid N2 and stored at -80°C until analysis (<3 months). Aliquots from defrosted samples were filtered on polycarbonate 0.22 µm pore size 25-mm diameter filters with 0.45 µm backing filters, dried at r.t., stained using VECTASHIELD with DAPI (4', 6-diamidino-2-phenylindole), and imaged at 100X magnification using a Nikon C1 upright microscope. Slides were imaged at 1000X magnification on a Nikon TE2000-U inverted epifluorescence microscope for P0704. Stained slides were stored at -20°C until microscopy (<1 week for original processing, up to 1 year for additional processing as needed). Images were analyzed using Nikon Advanced Research 3.2 software. Either 20 image fields or at least 200 cells per filter, whichever was smaller, was processed to measure cell abundance, line length, and width using the signal thresholding. The line length cutoff 0.20-2.0μm was used to exclude non-bacterial cells. Biovolumes were calculated from the line length and width based on the equation V=(π/4)×W^2×(L-W/3) (Bratbak, 1985). The cell-specific carbon was calculated as described by Simon and Azam (1989) using the equation Cell specific carbon = 0.86×88.6×V^0.59. To calculate biomass, cell specific carbon was multiplied by bacterial cell abundance.

FCM based Heterotrophic bacterial abundance and biomass

Preserved samples (3 ml SW + 25% glutaraldehyde, 1% final) were frozen in liquid N2, stored at -80°C, and sent to the University of Hawaii’s School of Ocean and Earth Science and Technology (SOEST) Flow Cytometry Facility for analysis. Prior to analysis, samples were thawed and stained with Hoescht 33342 in the dark at r.t. for 1 h (Monger & Landry, 1993) and analyzed with a Beckman-Coulter EPICS Altra flow cytometer with dual lasers (tuned to 488 nm and the UV range) to estimate abundances of heterotrophic bacteria (HBac), Prochlorococcus (Pro), Synechococcus (Syn), pico-eukaryotes (PEuk). All samples are spiked with fluorescent beads to normalize fluorescence and scattering properties. Raw data (listmode files) are processed using the software FlowJo (Treestar Inc., www.flowjo.com) with correction factors of 0.95 for preservative, 0.10 run volume and 0.82 coincidence. Abundance estimates were converted to carbon biomass equivalents using constant factors of 11, 32, and 100 fg C cell-1 for HBac, Pro, and Syn, respectively. PEuks abundance estimates were not converted to carbon biomass equivalents.