http://lod.bco-dmo.org/id/dataset/925050
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
2024-04-16
ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
ISO 19115-2:2009(E)
Microbial cellular abundance growth response through epifluorescent microscopy from the Neuse River Estuary, North Carolina USA from 2021-2022
2024-07-25
publication
2024-07-25
revision
Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)
2024-07-26
publication
https://doi.org/10.26008/1912/bco-dmo.925050.1
Scott M. Gifford
University of North Carolina at Chapel Hill
principalInvestigator
Melanie R. Cohn
University of North Carolina at Chapel Hill
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: Cohn, M. R., Parker, S., Gifford, S. M. (2024) Microbial cellular abundance growth response through epifluorescent microscopy from the Neuse River Estuary, North Carolina USA from 2021-2022. Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 1) Version Date 2024-07-25 [if applicable, indicate subset used]. doi:10.26008/1912/bco-dmo.925050.1 [access date]
Methods and Sampling: <p><strong>Sample Collection.&nbsp;</strong>Sample water was collected from the microcosms every couple of days during the experiment incubation for enumeration of the microbial community via epiflorescent microscopy. The water was sampled from the microcosms using a 25-mL serological pipette (VWR) with the tip snapped off to widen the opening. The serological pipette was rinsed three times with Milli-Q water before drawing sample water from the center of the microcosm near the mixer.&nbsp;&nbsp;A new serological pipette was used for each treatment. To a 50-mL falcon tube, 25 mL of sample was collected.&nbsp;</p>
<p><strong>Staining Procedure.&nbsp;</strong>For Ecocosm I (Fall 2021) 10 uL of 100x SYBR Green I was added to duplicate eppendorf tubes with 1 mL of sample water for a final stain concentration of 10x and vortexed gently for 5 seconds. The sample was incubated in the dark at room temperature for 45 minutes. For Ecocosm II, DAPI stain was used to allow for cell-sizing analysis in addition to enumeration; 50 uL of 50x DAPI solution was added to 1 mL of sample as above and incubated in the dark for 10 minutes prior to slide preparation.</p>
<p><strong>Slide Preparation.</strong>&nbsp;Samples were filtered down onto black, 25-mm filters (GTBP, Millipore Sigma, 0.22 um) with a backing filter (PVDF, Millipore Sigma, 25-mm, 0.45 um) on a 25-mm multifilter manifold (Millipore). Each filter well was filled with c.a. 5 mL of Milli-Q water then 1 mL of stained sample was added to a filtering well and filtered down under gentle (5-10 in Hg) vacuum pressure. The GTBP filters were mounted to slides with microscopy oil and a cover slip.</p>
<p><strong>Imaging. </strong>The slides were imaged on a Nikon Ti2 inverted microscope using the NIS Elements software. SYBR-stained slides were imaged at 60x magnification at 470 nm and 620 nm excitation to capture autofluorescence. DAPI-stained slides were similarly counted at 60x under 395 nm and 620 nm excitation. Manual counts calculated from 10 fields of view are reported in this dataset. Further details on imaging techniques, analysis, and automated image analysis are found in Parker (2022) in the publications section.&nbsp;</p>
<p><strong>List of equipment used</strong></p>
<p>25-mL serological pipette for sampling (VWR, sterile)</p>
<p>50-mL Falcon tube (Corning)</p>
<p>1.7-mL eppendorf tubes (Eppendorf)</p>
<p>SYBR Green I in DMSO stain, 10,000x (Invitrogen): diluted to 100x (flow cytometry) with Milli-Q water, 0.2-um filtered, and frozen in 0.2 mL aliquots to prevent freeze-thaw cycles.</p>
<p>DAPI, 1 mL (1 mg/mL) 4',5-diamidina-2-phenylindole (Fisher Scientific)</p>
<p>25-mm PVDF filter (Millipore Sigma)</p>
<p>25-mm, 0.22 um GTBP filter (Millipore Sigma)</p>
<p>Filter manifold (Millipore Sigma 1225: 12, 25-mm wells)</p>
<p>1mm glass microscope slide (Vista-Vision)</p>
<p>Glass cover slips&nbsp; No. 1.5 (Slip-Rite)</p>
<p>Microscopy immersion oil&nbsp;</p>
<p>Nikon Ti2 inverted microscope with excitation filters for: DAPI (395/25 nm), SYBR (470/40 nm), and autofloresence (620/60 nm)</p>
<p>Nikon Elements Acquisition Software</p>
<p>&nbsp;</p>
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-1850692 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=1850692
completed
Scott M. Gifford
University of North Carolina at Chapel Hill
614-406-0117
4202G Murray Hall 123 South Rd
Chapel Hill
NC
27599
USA
sgifford@email.unc.edu
pointOfContact
Melanie R. Cohn
University of North Carolina at Chapel Hill
440-840-7829
3105K Murray Hall, CB#3300
Chapel Hill
NC
27559
USA
mcohn@email.unc.edu
pointOfContact
asNeeded
Dataset Version: 1
Unknown
Experiment_Name
lat
lon
Date
Incubation_Day
Treatment
Tank_ID
Method
Stain
Excitation
Magnification
Size_Fraction
cells_per_L_avg
cells_per_L_sd
ratio_620_tot
ratio_err
Nikon Ti2 inverted microscope
theme
None, User defined
experiment id
latitude
longitude
start_date
incubation time or duration
treatment
replicate
No BCO-DMO term
wavelength
cell_concentration
standard deviation
ratio
featureType
BCO-DMO Standard Parameters
Inverted Microscope
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.
Bacteria as Biosensors of Carbon and Energy Flow in Marine Ecosystems: Quantitative Links Between Substrates, Transcripts, and Metabolism
https://osprey.bco-dmo.org/project/753813
Bacteria as Biosensors of Carbon and Energy Flow in Marine Ecosystems: Quantitative Links Between Substrates, Transcripts, and Metabolism
<p><em>NSF Award Abstract:</em><br />
The formation and flux of organic material is the foundation of ocean ecosystems, which in turn, substantially influences the global carbon cycle. As such, a fundamental goal in the ocean sciences is increasing our ability to identify marine organic matter's sources, transformations, and sinks, as well as how these components may change due to anthropogenic activities. Understanding these components is especially important in estuarine and coastal systems given these ecosystems are critical zones of organic carbon transformations. However, the dissolved organic carbon (DOC) pool in these systems consists of numerous different compounds from a multitude of sources that can turn over at vastly different rates (minutes to millennia). This makes it difficult to identify which DOC compounds support microbial growth, limiting the incorporation of microbial metabolism into predictive ecosystem models. Novel approaches are therefore needed to identify the DOC substrates driving microbial metabolism in ocean ecosystems. This project is premised on the idea that the bacterial cellular system is the ultimate chemical sensor of the organic environment and that the information recorded in the cell's active gene pool (transcripts) can be leveraged to make insights into DOC composition when the relationships between organic substrate availability, gene activity, and metabolism are known. This project identifies substrate-transcript relationships for a model marine bacterium, as well as the growth and metabolic outcomes of substrate availability. These insights are used to identify the biologically active DOC substrates in coastal environments when the model organism is added directly to coastal samples, and to interpret both historical and current environmental RNA and DNA data sets. This work provides novel insights into the substrates driving the ocean's carbon cycle and how marine bacterial cellular systems are regulated. Bioassays are developed that can be applied in many different aquatic environment settings. The project trains graduate and undergraduate students directly involved in the research and minority undergraduates will be recruited to use research modules for hands-on study of cell cultivation, bioinformatics, and microbial metabolism. High school students will be engaged through a module developed for an aquatic microbiology field trip and subsequent sample and data analysis.</p>
<p>Bacterial processing of dissolved organic carbon (DOC) mediates the flux of gigatons of carbon in the ocean, yet a significant hurdle to incorporating bacterial metabolism into ocean models is the inability to quantify the DOC substrates supporting bacterial metabolism and their transformation. Metatranscriptomics (sequencing of community mRNAs) has the potential to be a sensitive method for surveying bacterioplankton responses to the DOC pool and making insights into its composition but is currently limited by insufficient knowledge as to how transcriptional patterns relate to substrate availability. This project will identify carbon substrates supporting microbial metabolism and their transformation in estuarine-coastal ecosystems by elucidating the relationships between transcript abundances and carbon substrate availability. It aims to bridge the gap between model organism and environmental -omic studies by creating quantitative inventories of transcripts in response to defined substrates, and then using these calibrated transcriptional signals to interpret environmental DOC bioassays and metatranscriptomes. The first component of the project will establish genome-wide transcript-substrate relationships in a model marine bacterium in response to individual, environmentally-relevant carbon substrates. The second component will determine the extent to which transcription and metabolism are altered when the bacterium is exposed to complex mixtures of defined and undefined substrates, revealing the potential for transcription to identify individual substrates within a complex DOC pool and how metabolic processing may shape the DOC pools labile and refractory components. Finally, these calibrated transcriptional responses will be used to identify the DOC substrates driving bacterial metabolism in an estuarine-coastal system via DOC drawdown bioassays in which the model organism is added to natural seawater samples, as well as community wide bacterioplankton responses to the extant DOC pool via metatranscriptomics.</p>
Bacterial DOC Sensor
largerWorkCitation
project
eng; USA
biota
inlandWaters
-76.365069
-76.365069
34.925672
34.925672
2021-09-02
2022-05-23
Estuaries and Coastal Ecosystems of North Carolina
0
BCO-DMO catalogue of parameters from Microbial cellular abundance growth response through epifluorescent microscopy from the Neuse River Estuary, North Carolina USA from 2021-2022
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/925196.rdf
Name: Experiment_Name
Units: unitless
Description: <p>Name of microcosm incubation experiment. Eco1 was initiated September 2, 2021 and Eco2 was initiated April 4, 2022</p>
http://lod.bco-dmo.org/id/dataset-parameter/925197.rdf
Name: lat
Units: decimal degrees
Description: <p>Sampling location latitude, south is negative</p>
http://lod.bco-dmo.org/id/dataset-parameter/925198.rdf
Name: lon
Units: decimal degrees
Description: <p>Sampling location longitude, west is negative</p>
http://lod.bco-dmo.org/id/dataset-parameter/925199.rdf
Name: Date
Units: unitless
Description: <p>Respiration assay start date</p>
http://lod.bco-dmo.org/id/dataset-parameter/925200.rdf
Name: Incubation_Day
Units: days
Description: <p>Days elapsed since microcosm incubation initiation</p>
http://lod.bco-dmo.org/id/dataset-parameter/925201.rdf
Name: Treatment
Units: unitless
Description: <p>Light treatment applied to incubation: 12 h PAR/dark (L), 12 h UV-B/dark (V), dark (D), or in situ at time of collection (in situ)</p>
http://lod.bco-dmo.org/id/dataset-parameter/925202.rdf
Name: Tank_ID
Units: unitless
Description: <p>Identifier for microcosm replicate (two tanks per light treatment)</p>
http://lod.bco-dmo.org/id/dataset-parameter/925203.rdf
Name: Method
Units: unitless
Description: <p>Method of acquisition</p>
http://lod.bco-dmo.org/id/dataset-parameter/925204.rdf
Name: Stain
Units: unitless
Description: <p>Type of fluorecent dye used to stain cells. The flag -8888 represents no stain applied.</p>
http://lod.bco-dmo.org/id/dataset-parameter/925205.rdf
Name: Excitation
Units: nanometers (nm)
Description: <p>The excitation wavelength/ filter cassette used on a Nikon Ti2 microscope for cell enumeration</p>
http://lod.bco-dmo.org/id/dataset-parameter/925206.rdf
Name: Magnification
Units: unitless
Description: <p>The magnification of the objective lense used for cell enumeration</p>
http://lod.bco-dmo.org/id/dataset-parameter/925207.rdf
Name: Size_Fraction
Units: micrometers (um)
Description: <p>The filter fraction used for slide preperation.</p>
http://lod.bco-dmo.org/id/dataset-parameter/925208.rdf
Name: cells_per_L_avg
Units: cells L-1
Description: <p>The average cellular abundance from 10 fields of view. The flag -7777 denotes below detection and the flag -9999 denotes a missing value.</p>
http://lod.bco-dmo.org/id/dataset-parameter/925209.rdf
Name: cells_per_L_sd
Units: cells L-1
Description: <p>The standard deviation of cellular abundance from 10 fields of view. The flag -7777 denotes below detection and the flag -9999 denotes a missing value.</p>
http://lod.bco-dmo.org/id/dataset-parameter/925210.rdf
Name: ratio_620_tot
Units: unitless
Description: <p>The ratio of cells that autofluoreced under 620 nm excitation over the total number of SYBR- or DAPI-stained cells enumerated. The flag -9999 denotes a missing value.</p>
http://lod.bco-dmo.org/id/dataset-parameter/925211.rdf
Name: ratio_err
Units: unitless
Description: <p>The error propagation of the 620:tot ratio (ratio_620_tot). The flag -9999 denotes a missing value.</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
24518
https://darchive.mblwhoilibrary.org/server/api/core/bitstreams/2508aa6d-3220-40cb-8282-c8880e336c6c/content
download
https://doi.org/10.26008/1912/bco-dmo.925050.1
download
onLine
dataset
<p><strong>Sample Collection.&nbsp;</strong>Sample water was collected from the microcosms every couple of days during the experiment incubation for enumeration of the microbial community via epiflorescent microscopy. The water was sampled from the microcosms using a 25-mL serological pipette (VWR) with the tip snapped off to widen the opening. The serological pipette was rinsed three times with Milli-Q water before drawing sample water from the center of the microcosm near the mixer.&nbsp;&nbsp;A new serological pipette was used for each treatment. To a 50-mL falcon tube, 25 mL of sample was collected.&nbsp;</p>
<p><strong>Staining Procedure.&nbsp;</strong>For Ecocosm I (Fall 2021) 10 uL of 100x SYBR Green I was added to duplicate eppendorf tubes with 1 mL of sample water for a final stain concentration of 10x and vortexed gently for 5 seconds. The sample was incubated in the dark at room temperature for 45 minutes. For Ecocosm II, DAPI stain was used to allow for cell-sizing analysis in addition to enumeration; 50 uL of 50x DAPI solution was added to 1 mL of sample as above and incubated in the dark for 10 minutes prior to slide preparation.</p>
<p><strong>Slide Preparation.</strong>&nbsp;Samples were filtered down onto black, 25-mm filters (GTBP, Millipore Sigma, 0.22 um) with a backing filter (PVDF, Millipore Sigma, 25-mm, 0.45 um) on a 25-mm multifilter manifold (Millipore). Each filter well was filled with c.a. 5 mL of Milli-Q water then 1 mL of stained sample was added to a filtering well and filtered down under gentle (5-10 in Hg) vacuum pressure. The GTBP filters were mounted to slides with microscopy oil and a cover slip.</p>
<p><strong>Imaging. </strong>The slides were imaged on a Nikon Ti2 inverted microscope using the NIS Elements software. SYBR-stained slides were imaged at 60x magnification at 470 nm and 620 nm excitation to capture autofluorescence. DAPI-stained slides were similarly counted at 60x under 395 nm and 620 nm excitation. Manual counts calculated from 10 fields of view are reported in this dataset. Further details on imaging techniques, analysis, and automated image analysis are found in Parker (2022) in the publications section.&nbsp;</p>
<p><strong>List of equipment used</strong></p>
<p>25-mL serological pipette for sampling (VWR, sterile)</p>
<p>50-mL Falcon tube (Corning)</p>
<p>1.7-mL eppendorf tubes (Eppendorf)</p>
<p>SYBR Green I in DMSO stain, 10,000x (Invitrogen): diluted to 100x (flow cytometry) with Milli-Q water, 0.2-um filtered, and frozen in 0.2 mL aliquots to prevent freeze-thaw cycles.</p>
<p>DAPI, 1 mL (1 mg/mL) 4',5-diamidina-2-phenylindole (Fisher Scientific)</p>
<p>25-mm PVDF filter (Millipore Sigma)</p>
<p>25-mm, 0.22 um GTBP filter (Millipore Sigma)</p>
<p>Filter manifold (Millipore Sigma 1225: 12, 25-mm wells)</p>
<p>1mm glass microscope slide (Vista-Vision)</p>
<p>Glass cover slips&nbsp; No. 1.5 (Slip-Rite)</p>
<p>Microscopy immersion oil&nbsp;</p>
<p>Nikon Ti2 inverted microscope with excitation filters for: DAPI (395/25 nm), SYBR (470/40 nm), and autofloresence (620/60 nm)</p>
<p>Nikon Elements Acquisition Software</p>
<p>&nbsp;</p>
Specified by the Principal Investigator(s)
<p>The number of cells counted across 10 fields of view at 60x magnification for each sample was averaged, multiplied by the dilution factor, and corrected for the percent of filter area counted. The results in this datafile include the 60x SYBR, DAPI, and autofluorescent manual microscopy counts reported in cells/L with and standard deviation for each microcosm. Additionally, the ratio of stained to autofluorecent cells is reported.&nbsp;</p>
<p>For Ecocosm I, microscopy counts were not taken on experiment days 3, 4, and 14: some associated data sets under this project were sampled on these days. In order to provide cell counts on these additional sampling days, the abundances were interpolated for the missing days using a model 1 linear regression of the surrounding timepoints to calculate the "missing" days. These dates are denoted by "micoscopy_interpolated" under the "Method" parameter.</p>
Specified by the Principal Investigator(s)
For the submitted file Results_BacAbundance_microscopy2.csv, the file was modified using the BCO-DMO laminar tool described below.
Converted the location information from the submission tool to decimal degrees to use with laminar to add latitude and longitude columns to the dataset.
Latitude: 34°55'32.42" N -> 34.925672
Longitude: 76°21'54.25" W -> -76.365069
Kept 6 decimal points precision from conversation with submitter.
With laminar pipeline:
Loaded the most recent dataset, which was attached to an email on 4/29/2024, named Results_Microbial_Abundance_microscopy_v2.csv.
Skipped loading row 2 which is a line with the following note
Note: -7777=below detection, -8888=NA, -9999=missing
Renamed headers to match BCO-DMO naming convention. Replaced periods and colons with underscores. Removed units from Excitation header because it will be noted in the Osprey parameters section. Removde µm from Size.Fraction because it will be noted in the Osprey parameters section. Replaced / with _per_. For BCO-DMO naming scheme, parameter names must start with a letter and not a number, so renamed the column name 620:tot to ratio_620_tot.
In the columns cells_per_L_avg and cells_per_L_sd, replaced the values (9,999) with -9999, replaced the values (7,777) with -7777 and replaced the values (7,777,000) with -7777 to match the flag format noted in the excel dataset submitted.
Removed commas from the numbers in columns cells_per_L_avg and cells_per_L_sd in order to convert the column into a number format.
Added latitude (lat) and longitude (lon) columns with static values from converted lat and lon submitted location information using 6 digit precision.
Converted the date format from %Y%m%d into an ISO date format of %Y-%m-%d.
Reordered the columns so that the metadata lat, lon, and Date columns are at the front of the table, right after the column Experiment_Name.
Saved the updated dataset to a file named 925050_v1_microbial_cellular_abundance.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
Nikon Ti2 inverted microscope
Nikon Ti2 inverted microscope
PI Supplied Instrument Name: Nikon Ti2 inverted microscope PI Supplied Instrument Description:Nikon Ti2 inverted microscope with excitation filters for: DAPI (395/25 nm), SYBR (470/40 nm), and autofloresence (620/60 nm).
Instrument Name: Inverted Microscope Instrument Short Name: Instrument Description: An inverted microscope is a microscope with its light source and condenser on the top, above the stage pointing down, while the objectives and turret are below the stage pointing up. It was invented in 1850 by J. Lawrence Smith, a faculty member of Tulane University (then named the Medical College of Louisiana).
Inverted microscopes are useful for observing living cells or organisms at the bottom of a large container (e.g. a tissue culture flask) under more natural conditions than on a glass slide, as is the case with a conventional microscope. Inverted microscopes are also used in micromanipulation applications where space above the specimen is required for manipulator mechanisms and the microtools they hold, and in metallurgical applications where polished samples can be placed on top of the stage and viewed from underneath using reflecting objectives.
The stage on an inverted microscope is usually fixed, and focus is adjusted by moving the objective lens along a vertical axis to bring it closer to or further from the specimen. The focus mechanism typically has a dual concentric knob for coarse and fine adjustment. Depending on the size of the microscope, four to six objective lenses of different magnifications may be fitted to a rotating turret known as a nosepiece. These microscopes may also be fitted with accessories for fitting still and video cameras, fluorescence illumination, confocal scanning and many other applications. Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB05/