Dataset: flow cytometry
Deployment: FK141215

This data set is associated with PI Douglas Bartlett (NSF OCE-1536776) and Schmidt Ocean Institute R/V Falkor cruise FK141215. The cruise occurred December 15-21, 2014 in the Challenger Deep within the territorial waters of the Federated States of Micronesia. During this cruise the Leggo lander was deployed multiple times and drops 1 and 3 recovered seawater samples that were analyzed. Additional details can be found at: https://schmidtocean.org/cruise/expanding-mariana-trench-perspectives/ and https://scripps.ucsd.edu/labs/dbartlett/contact/challenger-deep-cruise-2014/

Leggo Lander Drop 1:
Time (in Guam) deployed/recovered: December 16, 9:00/19:26.
Position at deployment: 11° 21.9836 N 142° 25.9533 E, middle section of the Challenger Deep.
Greatest depth of dive: approximately ~10,900 m.
In situ temperature on seafloor: 2.6°C.
Notes: This drop recovered seawater samples from about a meter off the seafloor. This included a 3 L Niskin bottle of seawater and ~ 150 mls of seawater collected in a pressure-retaining seawater sampler. The PRS sampler held more than 81% of the in situ pressure. 

Flow Cytometry:
Direct counts (flow cytometry) on the microbes obtained in the Leggo drop 1 Niskin bottle and the Leg drop 1 pressure-retaining sampler. Samples were fixed with ~1% PFA and frozen. Later samples were removed from the -80 freezer and thawed in the dark.

The Attune was started and a Performance Test was run with the "Performance Tracking Beads" to check that all lasers and filters were working, and that voltages were correct. Cleaning and decontamination was also done using the Attune Wash solution and MilliQ water. Random samples selected from Logan's samples were run using Instrument Settings in the Attune software. This allows for real-time adjustments of voltages and thresholds in the different channels to get the best resolution for that day’s run as well as allows for quantification of instrument noise for a given day.

Once samples were thawed, 300 uL of each sample was loaded into a 96-well U-bottom plate. 3 uL of Invitrogen Sybr-green stain (diluted to 100x in MilliQ water) was then added to each well. The plate then incubated in the dark for 30 minutes before being run. The Sybr-green stain stains any DNA within a cell which then fluoresces when passing the BL1 channel laser, allowing for counts of cells.

Once instrument settings were determined for the day, the plate was loaded into the NxT autosampler and the run was started. Samples were run at "Standard" sensitivity at 100 uL/min for a total volume of 250 uL. Counts were delayed for 15 seconds to avoid any noise or dilution that can occur when sample starts being sipped. Once entire plate was run, I used the Attune software to correct for noise and gate various populations within the samples.

Note: Leggo1 is the first drop of the Leggo Lander and the values reflect the flow cytometric cell counts from its 2 liter Niskin bottle. Leggo1_PRS_day_2 is the flow cytometric cell count obtained from the pressure retaining sampler used on the first Leggo drop, following incubation in ice water for about 36 hours after recovery.

Colony Identification:
Data from the identification of bacteria cultured from the Leggo drop 1 and 3 Niskin bottles are available as a supplemental file (.txt). These identifications were performed using standard methods associated with PCR amplification of the 16S rRNA gene followed by dideoxy sequencing at Retrogen Inc.