Dataset: ETNP OMZ 2023 Total Prokaryoplankton Cell Count Metadata R/V Atlantis AT50-08 in the Eastern Tropical Pacific Minimum Oxygen Zone from February to March 2023 (MicroPro project)

Methods + Sampling:

Shipboard Protocol:

Whole water samples of either 13mL or 45mL were collected in 15mL or 50mL Falcon tubes, respectively, on deck at the rosette. Samples were fixed by adding borate-buffered formaldehyde (2% final concen.) and stored at 4 °C.

 

Laboratory Protocol:

Storage: Samples were stored at 4°C before processing.

 

Sample Processing: Black-stained (Millipore Sigma) polycarbonate membrane filters (0.2 µm pore size, 25 mm diameter) were washed with dd-H2­O then briefly dipped into very dilute Triton X-100 detergent for better cell dispersion (range between 0­.001-0.01%). Washed membranes were placed on top of moistened glass fiber backing filters (Whatman GF/C) on the filtration base and funnel was secured. 14-16mL of sample was filtered, vacuum was applied until approx. 2 mL of the sample remained in funnel. Samples were then stained with DAPI (1µg/ml final concen.) and incubated for 5 min. The remaining 2mL of stained sample was passed through the filter and rinsed with ~5mL distilled water. Filters were dried for >15 minutes at room temperature. Filters were mounted on slides with 4:1 Citifluor:Vectashield. Before enumeration, slides were stored at -20 °C. Separate reagent blanks were prepared during each filtration session.

 

Slide Enumeration: At least 10 grids and >300 cells were enumerated per filter to minimize analytical error (Kirchman et al., 1982). Individual fields/grids were randomly selected along north-south and east-west transects before viewing to minimize operator bias. Manual counting and statistical calculations were facilitated by our NewCount macro within Excel which, once initialized, transforms a computer keyboard into blind data entry portal that tallies a single count from any keystroke (except two control keys), recognizes end of a single field count and tabulates cell concentrations, S.D., S.E., C.O.V. and 95% confidence limits once designated number of fields is counted (developed by G. Taylor and D. Zheng; available upon request). Cells/L were calculated by using the following formula, where dilution factor compensates for the volume of formaldehyde fixative, and multiplication factor is the constant required to convert average grid/field count to the total count for the entire filter area exposed to sample: (((adjusted mean cells per grid)/(dilution factor*mL filtered))*multiplication factor)*1000 = cells/L. Coefficient of variation was calculated using the following formula: (standard error(cells/L)*100)/average(cells/L)=CoV. Total prokaryotic counts were corrected for storage decay between sample collection and filter preparation via the following exponential decay curve: Ncorr=Nobs/e-0.000605(ts-tc), where Ncor is the corrected cell abundance at the time of sample collection, Nobs is the observed cell abundance after sample preparation, ts is date of sample preparation, and tc is date of sample collection. Raw cell count data available from N. Butkevich or G. Taylor by request. The correction factor is based on empirical observations of stored samples from the CARIACO time series. Details are available within Scranton and Taylor (2019) BCO-DMO repository metadata.