http://lod.bco-dmo.org/id/dataset/897371
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
2023-06-09
ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
ISO 19115-2:2009(E)
Optical density and cell counts from flow cytometry of Ruegeria pomeroyi laboratory cultures
2023-06-09
publication
2023-06-09
revision
Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)
2023-06-12
publication
https://doi.org/10.26008/1912/bco-dmo.897371.1
Julia Diaz
University of California-San Diego Scripps
principalInvestigator
Solange Duhamel
University of Arizona
principalInvestigator
Jamee Adams
University of California-San Diego Scripps
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: Diaz, J., Duhamel, S., Adams, J. (2023) Optical density and cell counts from flow cytometry of Ruegeria pomeroyi laboratory cultures. Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2023-06-09 [if applicable, indicate subset used]. doi:10.26008/1912/bco-dmo.897371.1 [access date]
Methods and Sampling: <p>Experimental Procedures</p>
<p>Culture conditions and growth tracking</p>
<p>R. pomeroyi&nbsp;DSS-3 was cultured in media modified from the recipe of Rivers et al.&nbsp;(2016). Briefly, media (100 mL) were prepared using 0.2 μm-filtered natural seawater collected from the Scripps Institution of Oceanography pier that was autoclaved (121°C, 20 min) in 125 mL acid-washed glass Erlenmeyer flasks. Sterile-filtered (0.2 μm) glucose and nutrient stocks, including P sources, were aseptically added to the sterile seawater base in a laminar flow hood. Phosphate-replete media (+Pi) contained 18 μM P. P depleted media (-P) were prepared by adding phosphate to a final concentration of 1.8 μM P. ATP (Millipore Sigma), AMP (Fisher Scientific), 3polyP (Millipore Sigma), or 45polyP (Millipore Sigma) were added to -P media at a final concentration of 18 μM P. All media were inoculated with 50 μL of&nbsp;R. pomeroyi&nbsp;grown to stationary phase in +Pi media, in order to limit the carryover of P. Cultures were grown in a Thermo shaker/incubator at 30˚C with shaking at 150rpm for 10 days. Samples for optical density (600 nm) and flow cytometry were taken daily. Flow cytometry samples were prepared by sampling 2ml of cultures into cryovials, preserved with a final concentration of 0.5% glutaraldehyde at 4˚C for 10 minutes, and frozen at -80°C until analysis. Growth rates were calculated over the interval of log-linear growth in +Pi cultures. Growth rates in -P cultures were calculated over the same time period as +Pi cultures. All growth experiments were performed in triplicate.</p>
<p>DOP hydrolysis and&nbsp;APA competition plates (see related "Ruegeria pomeroyi DOP hydrolysis rates" dataset&nbsp;https://www.bco-dmo.org/dataset/897359)</p>
<p>Flow Cytometry</p>
<p>For cell counts, culture samples were preserved in filtered (0.22 μm) glutaraldehyde (0.5% final concentration), left to fix at 4˚C for 10 minutes, and frozen at -80°C until analysis. Preserved samples were thawed and counted on a Guava EasyCyte HT flow cytometer (Millipore), and instrument calibration was performed using instrument-specific beads (Luminex). Prior to running on the flow cytometer, samples were prepared in clear, round-bottom 96 well plates (Fisher Scientific) and diluted with filtered (0.22 μm) seawater either 100X (T0, T1) or 1000X (T2 - T8). Triplicate blanks prepared with filtered (0.22 μm) seawater and glutaraldehyde (0.5% final concentration) were run with samples, and the average blank cell count was subtracted from all samples. Blanks and diluted samples were stained with diluted SYBR Green nucleic acid gel stain (diluted in deionized water to 100X; Fisher Scientific) and left in the dark for 30 minutes. After staining, bacterial cell concentrations were analyzed at a low flow rate (0.24 μL s-1) for 3 minutes, and cells were counted based on diagnostic forward scatter versus green fluorescence signals.&nbsp;<br />
<br />
Taxonomic Identifiers (Species, LSID):<br />
Ruegeria pomeroyi, urn:lsid:marinespecies.org:taxname:567965<br />
<br />
Time ranges: Experiments were performed 3/9/21 - 3/17/21. Fixed flow cytometry samples were run from 3/18/21 - 3/23/21</p>
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-1736967 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=1736967
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-1737083 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=1737083
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-2001212 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=2001212
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-1948042 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=1948042
completed
Julia Diaz
University of California-San Diego Scripps
j2diaz@ucsd.edu
pointOfContact
Solange Duhamel
University of Arizona
520-621-6047
1007 E Lowell Street Life Sciences South 315
Tucson
AZ
85721
USA
duhamel@arizona.edu
pointOfContact
Jamee Adams
University of California-San Diego Scripps
jcadams@ucsd.edu
pointOfContact
asNeeded
Dataset Version: 1
Unknown
Media_Type
Growth_Day
Optical_Density
FCM
Guava EasyCyte flow cytometer (Luminex)
theme
None, User defined
treatment
time_elapsed
optical_density
cell_concentration
featureType
BCO-DMO Standard Parameters
Flow Cytometer
instrument
BCO-DMO Standard Instruments
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.
Collaborative Research: Assessing the role of compound-specific phosphorus hydrolase transformations in the marine phosphorus cycle
https://osprey.bco-dmo.org/project/747715
Collaborative Research: Assessing the role of compound-specific phosphorus hydrolase transformations in the marine phosphorus cycle
<p><em>NSF Award Abstract:</em><br />
Phosphorus (P) is an essential building block for life. Because P is in short supply over vast areas of the ocean, P availability may control biological productivity, such as photosynthesis and carbon fixation, which has implications for uptake of the greenhouse gas carbon dioxide and thus climate regulation. Marine microorganisms must satisfy their nutritional requirement for P by obtaining it from seawater, where P is present in a variety of chemical forms, from simple phosphate ions (Pi) to complex dissolved organic phosphorus (DOP) molecules. The concentration of DOP vastly exceeds Pi over most ocean areas, therefore DOP is a critically important source of P for marine microbial nutrition and productivity. However, much remains unknown about the contribution of specific DOP compounds to the P nutrition, productivity, and structure of marine microbial communities. In this project, the investigators will conduct field experiments in the Atlantic Ocean and perform a series of controlled laboratory studies with pure enzymes and microbial cultures to determine how and to what extent different DOP compounds are degraded to Pi in the marine environment. Furthermore, the contribution of these compound-specific DOP molecules to microbial P nutrition, carbon fixation, and community structure will be determined, thus advancing the current state of knowledge regarding the factors that control the activity and distribution of microbial species in the ocean, and the ocean?s role in the climate system. This project will support two female junior investigators, a postdoctoral researcher, and graduate and undergraduate students. The undergraduate students will be recruited from the Marine Sciences program at Savannah State University, an Historically Black Colleges and Universities. In addition, results will be incorporated into new hands-on K-12 educational tools to teach students about microbial P biogeochemistry, including a digital game and formal lesson plans with hands-on demos. These tools will be validated with K-12 educators and will be widely accessible to the public through various well-known online platforms. These activities will thus reach a broad audience including a significant fraction of underrepresented groups.</p>
<p>P is a vital nutrient for life. Marine microorganisms utilize P-hydrolases, such as alkaline phosphatase (AP), to release and acquire phosphate (Pi) from a wide diversity of dissolved organic P (DOP) compounds, including P-esters (P-O-C bonds), phosphonates (P-C), and polyphosphates (P-O-P). Compound-specific DOP transformations have the potential to exert critical and wide-ranging impacts on marine microbial ecology (e.g. variable DOP bioavailability among species), biogeochemistry (e.g. P geologic sequestration via formation of calcium Pi), and global climate (e.g. aerobic production of the greenhouse gas methane by dephosphorylation of methylphosphonate). However, the mechanisms and comparative magnitude of specific DOP transformations, in addition to their relative contributions to microbial community-level P demand, productivity, and structure, are not completely understood. This study will fill these knowledge gaps by tracking the fate of specific DOP pools in the marine environment. Specifically, this project will test four hypotheses in the laboratory using recombinant enzymes and axenic cultures representative of marine eukaryotic and prokaryotic plankton from high and low nutrient environments, and in the field using observational and experimental approaches along natural Pi gradients in the Atlantic Ocean. In particular, the investigators will reveal potential differences in the hydrolysis and utilization of specific DOP compounds at the community- (bulk enzymatic assays), taxon- (cell sorting of radiolabeled cells in natural samples), species- (axenic cultures) and molecular-levels (pure enzyme kinetic studies and cell-associated proteomes and exoproteomes). Results from our proposed work will provide a robust understanding of the enzymatic basis involved in the transformation of specific forms of DOP and create new knowledge on the relative contribution of these specific P sources to Pi production, marine microbial nutrition, community structure, primary productivity, and thus global carbon cycling and climate. In particular, our refined measurements of the concentration of bioavailable DOP and our unique estimates of DOP remineralization fluxes will provide critical new information to improve models of marine primary production and P cycling.</p>
P-hydrolase
largerWorkCitation
project
eng; USA
oceans
2021-03-09
2021-03-23
0
BCO-DMO catalogue of parameters from Optical density and cell counts from flow cytometry of Ruegeria pomeroyi laboratory cultures
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/897391.rdf
Name: Media_Type
Units: unitless
Description: <p>Media type used in culture</p>
http://lod.bco-dmo.org/id/dataset-parameter/897392.rdf
Name: Growth_Day
Units: unitless
Description: <p>Growth day (numeric, 0..n).</p>
http://lod.bco-dmo.org/id/dataset-parameter/897393.rdf
Name: Optical_Density
Units: optical_density
Description: <p>Optical Density (OD)</p>
http://lod.bco-dmo.org/id/dataset-parameter/897394.rdf
Name: FCM
Units: cells per milliliter (cells/ml)
Description: <p>Flow Cytometry cell concentration</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
4629
https://darchive.mblwhoilibrary.org/server/api/core/bitstreams/33a4b496-cd73-4122-84d7-048cc98106b4/content
download
https://doi.org/10.26008/1912/bco-dmo.897371.1
download
onLine
dataset
<p>Experimental Procedures</p>
<p>Culture conditions and growth tracking</p>
<p>R. pomeroyi&nbsp;DSS-3 was cultured in media modified from the recipe of Rivers et al.&nbsp;(2016). Briefly, media (100 mL) were prepared using 0.2 μm-filtered natural seawater collected from the Scripps Institution of Oceanography pier that was autoclaved (121°C, 20 min) in 125 mL acid-washed glass Erlenmeyer flasks. Sterile-filtered (0.2 μm) glucose and nutrient stocks, including P sources, were aseptically added to the sterile seawater base in a laminar flow hood. Phosphate-replete media (+Pi) contained 18 μM P. P depleted media (-P) were prepared by adding phosphate to a final concentration of 1.8 μM P. ATP (Millipore Sigma), AMP (Fisher Scientific), 3polyP (Millipore Sigma), or 45polyP (Millipore Sigma) were added to -P media at a final concentration of 18 μM P. All media were inoculated with 50 μL of&nbsp;R. pomeroyi&nbsp;grown to stationary phase in +Pi media, in order to limit the carryover of P. Cultures were grown in a Thermo shaker/incubator at 30˚C with shaking at 150rpm for 10 days. Samples for optical density (600 nm) and flow cytometry were taken daily. Flow cytometry samples were prepared by sampling 2ml of cultures into cryovials, preserved with a final concentration of 0.5% glutaraldehyde at 4˚C for 10 minutes, and frozen at -80°C until analysis. Growth rates were calculated over the interval of log-linear growth in +Pi cultures. Growth rates in -P cultures were calculated over the same time period as +Pi cultures. All growth experiments were performed in triplicate.</p>
<p>DOP hydrolysis and&nbsp;APA competition plates (see related "Ruegeria pomeroyi DOP hydrolysis rates" dataset&nbsp;https://www.bco-dmo.org/dataset/897359)</p>
<p>Flow Cytometry</p>
<p>For cell counts, culture samples were preserved in filtered (0.22 μm) glutaraldehyde (0.5% final concentration), left to fix at 4˚C for 10 minutes, and frozen at -80°C until analysis. Preserved samples were thawed and counted on a Guava EasyCyte HT flow cytometer (Millipore), and instrument calibration was performed using instrument-specific beads (Luminex). Prior to running on the flow cytometer, samples were prepared in clear, round-bottom 96 well plates (Fisher Scientific) and diluted with filtered (0.22 μm) seawater either 100X (T0, T1) or 1000X (T2 - T8). Triplicate blanks prepared with filtered (0.22 μm) seawater and glutaraldehyde (0.5% final concentration) were run with samples, and the average blank cell count was subtracted from all samples. Blanks and diluted samples were stained with diluted SYBR Green nucleic acid gel stain (diluted in deionized water to 100X; Fisher Scientific) and left in the dark for 30 minutes. After staining, bacterial cell concentrations were analyzed at a low flow rate (0.24 μL s-1) for 3 minutes, and cells were counted based on diagnostic forward scatter versus green fluorescence signals.&nbsp;<br />
<br />
Taxonomic Identifiers (Species, LSID):<br />
Ruegeria pomeroyi, urn:lsid:marinespecies.org:taxname:567965<br />
<br />
Time ranges: Experiments were performed 3/9/21 - 3/17/21. Fixed flow cytometry samples were run from 3/18/21 - 3/23/21</p>
Specified by the Principal Investigator(s)
<p>BCO-DMO Data Processing Notes:<br />
* Excel file "R. pom OD &amp; FCM_BCO-DMO.xlsx" loaded into the bco-dmo data system.&nbsp;<br />
* Column names modified to match BCO-DMO naming conventions to support broad interoperability.&nbsp;</p>
Specified by the Principal Investigator(s)
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
Guava EasyCyte flow cytometer (Luminex)
Guava EasyCyte flow cytometer (Luminex)
PI Supplied Instrument Name: Guava EasyCyte flow cytometer (Luminex) Instrument Name: Flow Cytometer Instrument Short Name:Flow Cytometer Instrument Description: Flow cytometers (FC or FCM) are automated instruments that quantitate properties of single cells, one cell at a time. They can measure cell size, cell granularity, the amounts of cell components such as total DNA, newly synthesized DNA, gene expression as the amount messenger RNA for a particular gene, amounts of specific surface receptors, amounts of intracellular proteins, or transient signalling events in living cells.
(from: http://www.bio.umass.edu/micro/immunology/facs542/facswhat.htm) Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB37/