http://lod.bco-dmo.org/id/dataset/964619
eng; USA
utf8
dataset
Highest level of data collection, from a common set of sensors or instrumentation, usually within the same research project
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
2025-06-11
ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
ISO 19115-2:2009(E)
Microbial fluid cell count data collected on Falkor (too), cruise Fkt231202 with ROV fluid sampler in the Central Eastern Pacific offshore Costa Rica in Dec 2023
2025-06-12
publication
2025-06-12
revision
Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)
2025-07-23
publication
https://doi.org/10.26008/1912/bco-dmo.964619.1
Julie Huber
Woods Hole Oceanographic Institution
principalInvestigator
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
publisher
Cite this dataset as: Huber, J. (2025) Microbial fluid cell count data collected on Falkor (too), cruise Fkt231202 with ROV fluid sampler in the Central Eastern Pacific offshore Costa Rica in Dec 2023. Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2025-06-12 [if applicable, indicate subset used]. doi:10.26008/1912/bco-dmo.964619.1 [access date]
Microbial Fluid Cell Counts Dataset Description: Methods and Sampling: <p>Instrument used to collect the sampler was a SUPR (Suspended Underwater Particulate Rosette) sampler modified for ROV SuBastian.&nbsp;Low temperatre venting fluids as well as background seawater were collected from the seafloor using the ROV-mounted SUPR water sampler with integrated temperature probe. Fluids were pumped at a rate of 1 L/min into 2 L bottles after flushing 5X the volume of the bottle. Once shipboard, fluids were preserved with 18.75 mL of sample fluid and 1.25 mL of 32% paraformaldehyde and stored at 4 deg C. In the lab at WHOI, samples were stained, visualized, and enumerated as described below.</p>
<p><strong>SYBR Gold Staining Protocol of bacteria and virus particles&nbsp;</strong></p>
<p>*Adapted from Thurber et al. 2009 Nature Protocols</p>
<ol>
<li>Vortex samples prior to fixation to ensure samples are evenly mixed and cells are not clumping.<br />
&nbsp;</li>
<li>Prepare a sterile, 1x PBS solution (pH7.4) with nuclease-free H20.<br />
&nbsp;</li>
<li><strong>Fixation:</strong> Fix samples with Paraformaldehyde (PFA) to make a 2% PFA final solution.<br />
&nbsp;</li>
<li>Vortex sample well and let incubate for 1hr.&nbsp; Keep samples in dark. During fixation incubation time – vortex samples briefly throughout 4x-6x<br />
&nbsp;</li>
<li>Store samples at 4°C in the dark until SYBR staining and microscopy.<br />
&nbsp;</li>
<li><strong>SYBR Gold Staining:</strong> Into an eppie or 15mL conical, aliquot part of the diluted 2% PFA fixed sample and resuspend into a final volume of <em>at least</em> 1mL nuclease-free PBS. (i.e. 100µL fixed sample into 900µL nuclease-free, PBS or scale as needed). Vortex sample well.
<ul>
<li>A final volume of <em>at least</em> 1mL is needed to ensure even spreading of sample onto filter.</li>
<li>Amount of sample to aliquot into a final volume will vary between samples depending on cell/viral density of environmental sample.&nbsp; May need to try various volumes of sample to find the perfect dilution amount.<br />
&nbsp;</li>
</ul>
</li>
<li>Add 1µl of 1000x SYBR Gold to each 1mL of sample.&nbsp; Gently vortex.<br />
&nbsp;</li>
<li>Incubate samples in dark for 30mins. During SYBR incubation – vortex samples briefly throughout incubation to prevent clumping (2x-3x).<br />
&nbsp;</li>
<li>While samples are incubating in SYBR gold, wash Millipore 12-sample filtration system with MilliQ ddH<sub>2</sub>0.
<ul>
<li>Clean well to prevent crossover of bacterial contamination from previous users.<br />
&nbsp;</li>
</ul>
</li>
<li><strong>Filtration:</strong> Filter sample onto 0.2µm filters (for bacterial counts) or 0.02µm Anodiscs (for viral and bacterial counts).<br />
&nbsp;</li>
<li>Set up 0.2µm or 0.02µm filters with glass support pads beneath each filtration frit – 12 frits max. Lock filters and support pads into filtration system.<br />
&nbsp;</li>
<li>Turn on vacuum and wash filter with 0.3 mL Nuclease-free ddH<sub>2</sub>0 to ensure filtration is working properly.
<ul>
<li>Glass support pads are reusable.&nbsp; Dry and reuse until re-use is not possible.<br />
&nbsp;</li>
</ul>
</li>
<li>Label which sample is placed on which frit.<br />
&nbsp;</li>
<li>Filter SYBR gold-stained sample onto anodisc filters, filtering ~1-2 mL at a time.<br />
&nbsp;</li>
<li>If not using all frits, make sure unused frits are closed with stopper.&nbsp; Once sample is filtered through, close frit with stopper until all samples have been passed through the anodisc filter discs.<br />
&nbsp;</li>
<li>Once sample is fully filtered through, wash each filter 3x with PBS to ensure removal of background SYBR stain.<br />
&nbsp;</li>
<li>Once all samples are filtered, remove filters carefully (do not let them flip over). Transfer to a kimwipe to let anodiscs airdry in the dark for ~15-30mins.<br />
&nbsp;</li>
<li><strong>Slide Preparation:</strong> While drying, prep large glass slides and label accordingly.<br />
&nbsp;</li>
<li>Once filters are dry, place 10µL of pure Vectashield onto the middle of the slide.<br />
&nbsp;</li>
<li>Place anodic filter (Sample side-up) on top of the Vectashield.<br />
&nbsp;</li>
<li>Place a single drop of vectashield OR 1:1 TE:Vectashield OR immersion oil on top of anodisc filter.&nbsp; &nbsp;(<em>I prefer vetcashield</em>).<br />
&nbsp;</li>
<li>Place circular glass coverslip onto anodisc filter.&nbsp; Pressing down gently to remove bubbles.<br />
&nbsp;</li>
<li>After 5 mins. Slides are ready for imaging.
<ul>
<li>Or slides can be sealed with clear nail polish.&nbsp; Dry for 15-20 mins.<br />
&nbsp;</li>
</ul>
</li>
<li>Once Dry, image slides same day with the 100x oil objective (488 FITC channel) or store in dark at -20°C until imaging.</li>
</ol>
Funding provided by NSF Office of International Science and Engineering (NSF OISE) Award Number: OISE-2114593 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=2114593
completed
Julie Huber
Woods Hole Oceanographic Institution
(508) 289-2556
Woods Hole Oceanographic Institution Marine Chemistry & Geochemistry, Watson Building, MS51
Woods Hole
MA
02543
USA
jhuber@whoi.edu
pointOfContact
asNeeded
Dataset Version: 1
Unknown
Cruise_Number
Sample_Number
Sample_Type
ROV_Storage
Sample_code
Description
Dive
Dive_Site
Time_UTC
Date
LAT
LON
Depth
Cells_per_mL
standard_deviation
ISO_DateTime_UTC
microscopy
SUPR for ROV SuBastian
Vortex
theme
None, User defined
Cruise identfier
sample identification
sample type
instrument
sample description
dive_id
site
time of day
date
latitude
longitude
depth
abundance
standard deviation
ISO_DateTime_UTC
featureType
BCO-DMO Standard Parameters
Microscope - Optical
ROV SuBastian
Shaker
instrument
BCO-DMO Standard Instruments
FKt231202
service
Deployment Activity
Central Eastern Pacific Ocean, offshore Costa Rica
place
Locations
otherRestrictions
otherRestrictions
Access Constraints: none. Use Constraints: Please follow guidelines at: http://www.bco-dmo.org/terms-use Distribution liability: Under no circumstances shall BCO-DMO be liable for any direct, incidental, special, consequential, indirect, or punitive damages that result from the use of, or the inability to use, the materials in this data submission. If you are dissatisfied with any materials in this data submission your sole and exclusive remedy is to discontinue use.
Crustal Ocean Biosphere Research Accelerator
https://osprey.bco-dmo.org/program/856233
Crustal Ocean Biosphere Research Accelerator
NSF Abstract:
The deep seafloor covers two-thirds of Earth’s surface area, but there is limited understanding of the deep-ocean ecosystems and resources and the ability of these ecosystems to withstand human impacts. Human uses such as deep-sea mining and carbon sequestration are poised to fundamentally alter physical, chemical, and biological conditions of the seafloor and surrounding environments. These activities have the potential to rival negative effects from bottom fishing and other human impacts to the deep sea, yet the science to inform and evaluate the impacts of these new industries is lacking. The Crustal Ocean Biosphere Research Accelerator (COBRA) project connects diverse stakeholders and experts – interdisciplinary academic and government scientists, private institutions, policy makers, industry experts and other stakeholders – through virtual meetings to coordinate efforts. The goal is to generate new knowledge and inform decision-making relating to emergent industrial uses of the deep ocean and decrease the likelihood of serious harm to the environment while maintaining the broad benefits that society currently enjoys.
The COBRA network of networks has nine key partners that bring access to international science and crustal ocean exploration assets (Ocean Exploration Trust, Schmidt Ocean Institute, Ocean Networks Canada, Cluster Ocean Floor at MARUM, and C-DeepSea), to experts that provide science-based recommendations to policy makers (Deep Ocean Stewardship Initiative working groups, including the Challenger 150 program), to governmental groups responsible for assessing crustal ocean resources (USGS Global Marine Minerals Group), and to experts in team science (CREDITS program). COBRA unites these partners in a common mission to accelerate research on the structure, function, resilience, and ecosystem services of the crustal ocean biosphere to inform decision making. COBRA will help to close knowledge gaps by facilitating dedicated and coordinated expedition and observatory efforts combined with emergent characterization approaches. In parallel, COBRA will train at least 50 globally distributed early-career researchers in ocean exploration, science, and policy through innovative virtual expedition leadership training and support two dozen international research exchanges that promote team science collaboration, diversity, equity, and inclusivity. COBRA will also establish a web-based search portal that points to all data types deposited in appropriate internationally accessible data repositories to promote data discovery and accelerate knowledge transfer and collaboration.
The Accelerating Research through International Network-to-Network Collaborations (AccelNet) program is designed to accelerate the process of scientific discovery and prepare the next generation of U.S. researchers for multiteam international collaborations. The AccelNet program supports strategic linkages among U.S. research networks and complementary networks abroad that will leverage research and educational resources to tackle grand scientific challenges that require significant coordinated international efforts.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
Description:
The mission of the Crustal Ocean Biosphere Research Accelerator (COBRA) is to accelerate research on the structure, function, resilience, and ecosystem services of the crustal ocean biosphere to inform decision making. The goal is to generate new knowledge and inform decision-making relating to emergent industrial uses of the deep ocean, such as deep-sea mining and subseafloor carbon sequestration, and decrease the likelihood of serious harm to the environment while maintaining the broad benefits that society currently enjoys. COBRA will help to close knowledge gaps by facilitating dedicated and coordinated expedition and observatory efforts combined with emergent characterization approaches. In parallel, COBRA will train at least 50 globally distributed early-career researchers in ocean exploration, science, and policy through innovative virtual expedition leadership training and support two dozen international research exchanges that promote team science collaboration, diversity, equity, and inclusivity. COBRA will also establish a web-based search portal that points to all data types deposited in appropriate internationally accessible data repositories to promote data discovery and accelerate knowledge transfer and collaboration.
Affiliated Programs:
C-DEBI, IODP, OOI, DOSI, Schmidt Ocean Institute, Ocean Exploration Trust, Ocean Networks Trust
COBRA
largerWorkCitation
program
Octopus Odyssey
https://schmidtocean.org/cruise/octopus-odyssey/
Octopus Odyssey
<p><strong>Brief Overview:</strong><br />
The first Octopus Odyssey expedition took place from June 2 to June 21, 2023 on R/V Falkor (too). The second expedition, Octopus Odyssey (too) took place from December 2 to December 15, 2023. Both expeditions explored The Dorado Outcrop, one of Costa Rica's "Off-Axis seamounts on the complex Cocos Plate. These two cruises featured early career training activities and international capacity-sharing elements that were integrated into the NSF-funded COBRA program. In addition to the NSF award, this project was also supported by Schmidt Ocean Institute, Blue Nature Alliance, and Bigelow Laboratory for Ocean Sciences.</p>
<p>More information is available from Schmidt Ocean Institute at:<br />
<a href="https://schmidtocean.org/cruise/octopus-odyssey/" target="_blank">https://schmidtocean.org/cruise/octopus-odyssey/</a><br />
and<br />
<a href="https://schmidtocean.org/cruise/octopus-odyssey-too/" target="_blank">https://schmidtocean.org/cruise/octopus-odyssey-too/</a></p>
<p><strong>Detailed Description:</strong><br />
Seamount ecosystems support highly diverse animal communities on the seafloor and the surrounding ocean, yet the diversity, connectivity and ecosystem services of these environments is poorly understood. The Pacific Ocean margin of Costa Rica contains a range of seamount habitats, from the rough terrain of the southwestern margin to the sparser terrain of the northwest margin. While the southwestern terrain has previously been surveyed (including by R/V Falkor in 2019) and some seamount areas are already protected, far less is known about the ecosystems of the northwestern terrain. In 2013/2014 unique animal behaviors and hydrothermal venting were discovered using ROV Jason and HOV Alvin on a small feature in the northwestern terrain. Namely, extensive aggregations of octopus were observed at a place called the Dorado Outcrop, located in areas of diffuse venting of slightly warmed hydrothermal fluids. At the time of discovery, it was unclear if these aggregations could be considered nurseries, since no viable eggs were observed with brooding mothers.</p>
<p>Two expeditions of the RV Falkor (too) were planned for 2023 to return to this region to ask new questions about the connection of life, rocks, and fluids around these seafloor features. The team wanted to answer questions, such as:</p>
<ul>
<li>Are there viable octopus nurseries hosted on seamounts offshore Costa Rica?</li>
<li>If yes, are the octopus nurseries active at a different time of year?</li>
<li>Do octopus brooding in hydrothermal springs have different microbiomes as compared to other octopus, and are those microbiomes connected to the microbes in the hydrothermal springs or surrounding rocks?</li>
<li>Are the hydrothermal spring fluids unique, representing different trends in fluid-rock-life reactions, or do they represent a single altered fluid?</li>
<li>Are there seasonal trends in biodiversity on the seafloor or in the water above?</li>
</ul>
<p>In June of 2023, an international team traveled to this region aboard R/V Falkor (too) for the Octopus Odyssey Leg 1 expedition Fkt230602 with a major goal to determine if the eggs at the nursery were viable, as past expeditions to the outcrop had never seen evidence of developing embryos. From 2-21 June 2023, we conducted 14 dives with ROV SuBastian to explore six seafloor features (only one of which had ever been explored before), augmented by 13 full-water-column CTD Niskin Rosette casts and six multibeam surveys. We had roughly 229 hours of ROV operations in the water (172 hours on the seafloor + 57 hours of ascent/descent), resulting in 208 hours of video. The longest ROV dive was approximately 35 hours and the deepest depth of ROV exploration was 3178 m. We had 285 sampling events during the ROV dives: 150 primary biological specimens (plus associates), 66 sediment push cores, 28 ROV Niskin samples of bottom water, 13 squeezer fluid samples, 30 rock samples. This also included deployments of 22 different experiments planned for recovery in December 2023, and recovery of 2 experiments from the Dorado Outcrop deployed in 2014. We also conducted 31 video transects. Operations went very smoothly, although some transit between sites had to be diverted due to long line fishing in the area, and one medical evacuation required transit to port before returning to site. On the first ROV dive at the nursery in June, we witnessed baby octopus hatching, confirming our primary hypothesis that there are viable octopus nurseries in this region. We also found the fifth known octopus nursery in the world on a different seafloor feature 30 nautical miles away. Exploration of the six seafloor features on the expedition revealed an incredibly rich biodiversity and biogeography of life on ancient volcanoes offshore Costa Rica. We also documented additional evidence of the hydrogeology of the region – how water moves in, out, and through oceanic crust. This data can inform why volcanoes and earthquakes in Costa Rica vary as different types of seamounts and oceanic crust subducts beneath overriding plates.</p>
<p>In December 2023, the Octopus Odyssey (too) Leg 2 team returned to this region on RV Falkor (too) on expedition Fkt231202 to ask new questions about biodiversity in the region and to recover experiments to track the hydrogeology of the area. From 1-15 December 2023, Octopus Odyssey (too) conducted twelve full-ocean depth ROV dives with ROV SuBastian, augmented by five full-ocean depth CTD Niskin Rosette casts, and multibeam operations resulting in 7416 km2 of coverage in Costa Rican waters. We had roughly 104 hours of ROV operations (55 hours on the seafloor + 49 hours of ascent/descent). This has resulted in approximately 141 hours of video. The longest ROV dive was a little over 16 hours and the deepest depth of ROV exploration was 3179 mbsl. We had 241 sampling events with the ROV in the water: 93 primary biological specimens, 14 sediment push cores, 21 ROV Niskin samples, 20 rock samples, and 51 fluid samples collected with a third-party SUPR sampler. On the ship, we collected an additional 66 secondary associate biological samples from primary specimens, bringing the total number of samples to 307 (this does not include subsamples). We also conducted 23 video transects. For the most part, our operations went according to schedule. No ROV operations were ended early due to operational issues, although one dive was aborted on launch due to a ground fault in a third-party instrument; this was quickly resolved and the dive restarted. One dive ended early due to a fishing long-line drifting towards the vessel; we recovered early then re-dove on the site after the long-line passed by. Communications with fishing boats and the fisheries ministry, enabled by the Berth-of-Opportunity Observer from Instituto Costarricense de Pesca y Acuacultura (INCOPESCA), helped prevent further issues in the area. The biggest finding of the return expedition was confirmation that the octopus nurseries offshore Costa Rica support baby octopus throughout the year, not just in the summer rainy season. Scientists onboard witnessed spectacular scenes of the first moments of life, as baby octopus emerged from their eggs, including traveling with one hatchling for an epic journey over 150 m up into the water. Immature eggs were also observed to have tiny octopus embryos inside. Having two expeditions to the same region in one year was essential for confirming this finding. Moreover, the seamounts offshore Costa Rica support at least four new species of deep-sea octopuses, based on the collection of specimens from both Octopus Odyssey expeditions in June and December 2023. This is an unprecedented biodiversity of octopus in this small area especially at these depths.</p>
<p>Equally as important as achieving the scientific objectives was the objective to continue the theme of capacity sharing, early career development, and raising awareness of deep-sea heritage in Latin America. The international Octopus Odyssey and Octopus Odyssey (too) teams gathered to achieve collaborative co-production of knowledge and training with Costa Ricans, honoring the work in Costa Rica’s waters. Spanish-speaking scientists were given priority for dive lead watches to enable livestream narration in Spanish, and priority for leadership experience. Ship-to-shore engagements were also prioritized for Spanish-speaking audiences, particularly in Costa Rica. These efforts were intended to raise the profile of the deep-sea heritage in Costa Rica ahead of the 2024 UN Ocean Conference meeting taking place in Costa Rica in June 2024. Over 300 biological specimens collected on the two expeditions are archived at the Museum of Zoology at the University of Costa Rica, enabling current and future generations of students and researchers to develop expertise in regional deep-sea animals. It is likely that many of the specimens collected represent new species and new records of known species for the region. Rock and sediment samples collected on the expeditions are revolutionizing the understanding of the complex geological origins and processes occurring on this part of the seafloor. Surprisingly, initial analysis of microfossils in sediments reveals that seafloor sediments are millions-of-years old, indicating strong currents, dissolution and scouring. In addition, fossils of beaked whales were found on numerous outcrops. All microfossils and macrofossils are archived in the Paleontology collection at the Central American School of Geology at the University of Costa Rica for continued study, with additional mineralogical samples shared with the Global Marine Minerals Program at the U.S. Geological Society. Finally, bathymetric and subbottom profile mapping data conducted on the OctoOdyssey expeditions was used to define the diverse seafloor features in this region to then propose official names to GEBCO. This naming effort is being led by Costa Rican scientists in consultation with the Costa Rican Committee on Nomenclature; the new proposed names were unanimously approved by the committee and will now be included on Costa Rican maps.</p>
OctoOdyssey
largerWorkCitation
project
eng; USA
biota
oceans
Central Eastern Pacific Ocean, offshore Costa Rica
-87.279746
-86.943568
8.621694
9.120658
2023-12-04
2023-12-12
Central Eastern Pacific offshore Costa Rica
0
BCO-DMO catalogue of parameters from Microbial fluid cell count data collected on Falkor (too), cruise Fkt231202 with ROV fluid sampler in the Central Eastern Pacific offshore Costa Rica in Dec 2023
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
http://lod.bco-dmo.org/id/dataset-parameter/968867.rdf
Name: Cruise_Number
Units: unitless
Description: <p>Cruise number for sample collection</p>
http://lod.bco-dmo.org/id/dataset-parameter/968868.rdf
Name: Sample_Number
Units: unitless
Description: <p>Sample number for sample collection</p>
http://lod.bco-dmo.org/id/dataset-parameter/968869.rdf
Name: Sample_Type
Units: unitless
Description: <p>"F" stands for fluid samples</p>
http://lod.bco-dmo.org/id/dataset-parameter/968870.rdf
Name: ROV_Storage
Units: unitless
Description: <p>SUPR is the fluid sampler used to collect the samples on the ROV</p>
http://lod.bco-dmo.org/id/dataset-parameter/968871.rdf
Name: Sample_code
Units: unitless
Description: <p>Sample code for sample collection</p>
http://lod.bco-dmo.org/id/dataset-parameter/968872.rdf
Name: Description
Units: unitless
Description: <p>Description of sample collected, included bottle number and in some cases, temperature or marker</p>
http://lod.bco-dmo.org/id/dataset-parameter/968873.rdf
Name: Dive
Units: unitless
Description: <p>ROV dive number</p>
http://lod.bco-dmo.org/id/dataset-parameter/968874.rdf
Name: Dive_Site
Units: unitless
Description: <p>Name of dive site for sample collected</p>
http://lod.bco-dmo.org/id/dataset-parameter/968875.rdf
Name: Time_UTC
Units: unitless
Description: <p>Time utc for sample collection</p>
http://lod.bco-dmo.org/id/dataset-parameter/968876.rdf
Name: Date
Units: unitless
Description: <p>Date of for sample collection</p>
http://lod.bco-dmo.org/id/dataset-parameter/968877.rdf
Name: LAT
Units: decimal degrees
Description: <p>Latitude for sample collection</p>
http://lod.bco-dmo.org/id/dataset-parameter/968878.rdf
Name: LON
Units: decimal degrees
Description: <p>Longitude for sample collected</p>
http://lod.bco-dmo.org/id/dataset-parameter/968879.rdf
Name: Depth
Units: meters (m)
Description: <p>Depth of sample collection</p>
http://lod.bco-dmo.org/id/dataset-parameter/968880.rdf
Name: Cells_per_mL
Units: cells/mL
Description: <p>Concentration of microbial cells per mL of fluid collected</p>
http://lod.bco-dmo.org/id/dataset-parameter/968881.rdf
Name: standard_deviation
Units: unitless
Description: <p>± 2 standard deviation for sample enumeration</p>
http://lod.bco-dmo.org/id/dataset-parameter/968882.rdf
Name: ISO_DateTime_UTC
Units: unitless
Description: <p>Datetime for sample collection in ISO format</p>
GB/NERC/BODC > British Oceanographic Data Centre, Natural Environment Research Council, United Kingdom
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
2425
https://darchive.mblwhoilibrary.org/server/api/core/bitstreams/39a24608-48ea-41cc-b0fc-842911a089c5/content
download
https://doi.org/10.26008/1912/bco-dmo.964619.1
download
onLine
dataset
<p>Instrument used to collect the sampler was a SUPR (Suspended Underwater Particulate Rosette) sampler modified for ROV SuBastian.&nbsp;Low temperatre venting fluids as well as background seawater were collected from the seafloor using the ROV-mounted SUPR water sampler with integrated temperature probe. Fluids were pumped at a rate of 1 L/min into 2 L bottles after flushing 5X the volume of the bottle. Once shipboard, fluids were preserved with 18.75 mL of sample fluid and 1.25 mL of 32% paraformaldehyde and stored at 4 deg C. In the lab at WHOI, samples were stained, visualized, and enumerated as described below.</p>
<p><strong>SYBR Gold Staining Protocol of bacteria and virus particles&nbsp;</strong></p>
<p>*Adapted from Thurber et al. 2009 Nature Protocols</p>
<ol>
<li>Vortex samples prior to fixation to ensure samples are evenly mixed and cells are not clumping.<br />
&nbsp;</li>
<li>Prepare a sterile, 1x PBS solution (pH7.4) with nuclease-free H20.<br />
&nbsp;</li>
<li><strong>Fixation:</strong> Fix samples with Paraformaldehyde (PFA) to make a 2% PFA final solution.<br />
&nbsp;</li>
<li>Vortex sample well and let incubate for 1hr.&nbsp; Keep samples in dark. During fixation incubation time – vortex samples briefly throughout 4x-6x<br />
&nbsp;</li>
<li>Store samples at 4°C in the dark until SYBR staining and microscopy.<br />
&nbsp;</li>
<li><strong>SYBR Gold Staining:</strong> Into an eppie or 15mL conical, aliquot part of the diluted 2% PFA fixed sample and resuspend into a final volume of <em>at least</em> 1mL nuclease-free PBS. (i.e. 100µL fixed sample into 900µL nuclease-free, PBS or scale as needed). Vortex sample well.
<ul>
<li>A final volume of <em>at least</em> 1mL is needed to ensure even spreading of sample onto filter.</li>
<li>Amount of sample to aliquot into a final volume will vary between samples depending on cell/viral density of environmental sample.&nbsp; May need to try various volumes of sample to find the perfect dilution amount.<br />
&nbsp;</li>
</ul>
</li>
<li>Add 1µl of 1000x SYBR Gold to each 1mL of sample.&nbsp; Gently vortex.<br />
&nbsp;</li>
<li>Incubate samples in dark for 30mins. During SYBR incubation – vortex samples briefly throughout incubation to prevent clumping (2x-3x).<br />
&nbsp;</li>
<li>While samples are incubating in SYBR gold, wash Millipore 12-sample filtration system with MilliQ ddH<sub>2</sub>0.
<ul>
<li>Clean well to prevent crossover of bacterial contamination from previous users.<br />
&nbsp;</li>
</ul>
</li>
<li><strong>Filtration:</strong> Filter sample onto 0.2µm filters (for bacterial counts) or 0.02µm Anodiscs (for viral and bacterial counts).<br />
&nbsp;</li>
<li>Set up 0.2µm or 0.02µm filters with glass support pads beneath each filtration frit – 12 frits max. Lock filters and support pads into filtration system.<br />
&nbsp;</li>
<li>Turn on vacuum and wash filter with 0.3 mL Nuclease-free ddH<sub>2</sub>0 to ensure filtration is working properly.
<ul>
<li>Glass support pads are reusable.&nbsp; Dry and reuse until re-use is not possible.<br />
&nbsp;</li>
</ul>
</li>
<li>Label which sample is placed on which frit.<br />
&nbsp;</li>
<li>Filter SYBR gold-stained sample onto anodisc filters, filtering ~1-2 mL at a time.<br />
&nbsp;</li>
<li>If not using all frits, make sure unused frits are closed with stopper.&nbsp; Once sample is filtered through, close frit with stopper until all samples have been passed through the anodisc filter discs.<br />
&nbsp;</li>
<li>Once sample is fully filtered through, wash each filter 3x with PBS to ensure removal of background SYBR stain.<br />
&nbsp;</li>
<li>Once all samples are filtered, remove filters carefully (do not let them flip over). Transfer to a kimwipe to let anodiscs airdry in the dark for ~15-30mins.<br />
&nbsp;</li>
<li><strong>Slide Preparation:</strong> While drying, prep large glass slides and label accordingly.<br />
&nbsp;</li>
<li>Once filters are dry, place 10µL of pure Vectashield onto the middle of the slide.<br />
&nbsp;</li>
<li>Place anodic filter (Sample side-up) on top of the Vectashield.<br />
&nbsp;</li>
<li>Place a single drop of vectashield OR 1:1 TE:Vectashield OR immersion oil on top of anodisc filter.&nbsp; &nbsp;(<em>I prefer vetcashield</em>).<br />
&nbsp;</li>
<li>Place circular glass coverslip onto anodisc filter.&nbsp; Pressing down gently to remove bubbles.<br />
&nbsp;</li>
<li>After 5 mins. Slides are ready for imaging.
<ul>
<li>Or slides can be sealed with clear nail polish.&nbsp; Dry for 15-20 mins.<br />
&nbsp;</li>
</ul>
</li>
<li>Once Dry, image slides same day with the 100x oil objective (488 FITC channel) or store in dark at -20°C until imaging.</li>
</ol>
Specified by the Principal Investigator(s)
- Imported "FKt231202_Huber_CellCounts_ForBCODMO.xlsx" into BCO-DMO system
- Removed extra zeros that imported with date
- Created new parameter with date and time in ISO format
- Exported file as "964619_v1_microbial_fluid_cell_counts.csv"
Specified by BCO-DMO Data Managers
asNeeded
7.x-1.1
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
microscopy
microscopy
PI Supplied Instrument Name: microscopy PI Supplied Instrument Description:Store samples at 4°C in the dark until SYBR staining and microscopy. Instrument Name: Microscope - Optical Instrument Short Name: Instrument Description: Instruments that generate enlarged images of samples using the phenomena of reflection and absorption of visible light. Includes conventional and inverted instruments. Also called a "light microscope". Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB05/
SUPR for ROV SuBastian
SUPR for ROV SuBastian
PI Supplied Instrument Name: SUPR for ROV SuBastian PI Supplied Instrument Description:Instrument used to collect the sampler was a SUPR (Suspended Underwater Particulate Rosette) sampler modified for ROV SuBastian. Instrument Name: ROV SuBastian Instrument Short Name: Instrument Description: ROV SuBastian is operated from the research vessel Falkor and the R/V Falkor(too). The ROV is outfitted with a suite of sensors and scientific equipment to support scientific data and sample collection, as well as interactive research, experimentation, and technology development. More information available at https://schmidtocean.org/technology/robotic-platforms/4500-m-remotely-operated-vehicle-rov/
Vortex
Vortex
PI Supplied Instrument Name: Vortex PI Supplied Instrument Description:Vortex samples prior to fixation to ensure samples are evenly mixed and cells are not clumping. Instrument Name: Shaker Instrument Short Name: Instrument Description: A Shaker is a piece of lab equipment used to mix, blend, or to agitate substances in tube(s) or flask(s) by shaking them, which is mainly used in the fields of chemistry and biology. A shaker contains an oscillating board which is used to place the flasks, beakers, test tubes, etc.
Cruise: FKt231202
FKt231202
R/V Falkor (too)
Community Standard Description
International Council for the Exploration of the Sea
R/V Falkor (too)
vessel
FKt231202
Beth N. Orcutt
Bigelow Laboratory for Ocean Sciences
R/V Falkor (too)
Community Standard Description
International Council for the Exploration of the Sea
R/V Falkor (too)
vessel