Dataset: Microbial Cellular Abundance Epifluorescent Microscopy

Name: Microbial cellular abundance growth response through epifluorescent microscopy from the Neuse River Estuary, North Carolina USA from 2021-2022
Published: 2024-07-25
Keywords: Cellular Abundance, microscopy, bacteria, phytoplankton, estaury, biota, inlandWaters

Cite This Dataset

DOI:10.26008/1912/bco-dmo.925050.1

Spatial Extent: N:34.925672 E:-76.365069 S:34.925672 W:-76.365069

West Bay of the Neuse River Estuary in North Carolina, USA. Surface water.

Temporal Extent: 2021-09-02 - 2022-05-23

Project:

Bacteria as Biosensors of Carbon and Energy Flow in Marine Ecosystems: Quantitative Links Between Substrates, Transcripts, and Metabolism (Bacterial DOC Sensor)

Principal Investigator:

Scott M. Gifford (University of North Carolina at Chapel Hill, UNC-Chapel Hill)

Scientist:

Melanie R. Cohn (University of North Carolina at Chapel Hill, UNC-Chapel Hill)

Student:

Sierra Parker (University of North Carolina at Chapel Hill, UNC-Chapel Hill)

BCO-DMO Data Manager:

Lynne M. Merchant (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Version:

Version Date:

2024-07-25

Restricted:

Validated:

Yes

Current State:

Final no updates expected

Microbial cellular abundance growth response through epifluorescent microscopy from the Neuse River Estuary, North Carolina USA from 2021-2022

Abstract:

Microbial cellular abundance was enumerated for two microcosm incubation experiments to track the growth response of the microbial community. This dataset targets bacterial and phytoplankton abundance through epiflorescent microscopy. Sample water originated from the West Bay of the Neuse River Estuary, North Carolina USA in 2021 and 2022. The microcosms were 60-L, conducted in biological duplicates under three light treatment incubations: 12 h light-dark cycle of photosynthetically active radiation (PAR), 12 h light-dark cycle of UV-B radiation, or darkness. Unfiltered sample water was stained with SYBR Green I (Fall 2021 experiment) or DAPI (Spring 2022 experiment) for enumeration at 60x for bacterial and cyanobacterial abundance. Autofluorescence from excitation at 620 nm and 60x was used to enumerate small phytoplankton and cyanobacteria. This dataset highlights patterns in microbial growth across treatments over the course of the incubation and is used to generate cell-specific, normalized rates for associated datasets in the project.

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Archival Copy

Version Date	Archive	Persistent Identifier (PID)	Date PID Issued
2024-07-25	Marine Biological Laboratory/Woods Hole Oceanographic Institution Library (MBLWHOI DLA)	10.26008/1912/bco-dmo.925050.1	2024-07-26

Methods & Sampling

Sample Collection. 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. A new serological pipette was used for each treatment. To a 50-mL falcon tube, 25 mL of sample was collected.

Staining Procedure. 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.

Slide Preparation. 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.

Imaging. 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.

List of equipment used

25-mL serological pipette for sampling (VWR, sterile)

50-mL Falcon tube (Corning)

1.7-mL eppendorf tubes (Eppendorf)

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.

DAPI, 1 mL (1 mg/mL) 4',5-diamidina-2-phenylindole (Fisher Scientific)

25-mm PVDF filter (Millipore Sigma)

25-mm, 0.22 um GTBP filter (Millipore Sigma)

Filter manifold (Millipore Sigma 1225: 12, 25-mm wells)

1mm glass microscope slide (Vista-Vision)

Glass cover slips No. 1.5 (Slip-Rite)

Microscopy immersion oil

Nikon Ti2 inverted microscope with excitation filters for: DAPI (395/25 nm), SYBR (470/40 nm), and autofloresence (620/60 nm)

Nikon Elements Acquisition Software

Data Processing Description

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.

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.

BCO-DMO Processing

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.

Data Files

File
925050_v1_microbial_cellular_abundance.csv (Comma Separated Values (.csv), 23.94 KB) MD5:965d9548ef8c0ab8b28934ce8923921f Primary data file for dataset ID 925050, version 1

More Information about this dataset

Funding Sources

Funding Source	Award Number
NSF Division of Ocean Sciences	OCE-1850692

Deployments

None available yet

Instruments

Inverted Microscope

Supplied Name: Nikon Ti2 inverted microscope

Supplied Description:

Nikon Ti2 inverted microscope with excitation filters for: DAPI (395/25 nm), SYBR (470/40 nm), and autofloresence (620/60 nm).

Instrument Type

Generic Name: Inverted Microscope

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB05/

Generic 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.

Parameters

Date and time formats are described in python strftime() syntax.

Supplied Name	Supplied description	Supplied Units	Standard Name
Experiment_Name	Name of microcosm incubation experiment. Eco1 was initiated September 2, 2021 and Eco2 was initiated April 4, 2022	unitless	exp_id
lat	Sampling location latitude, south is negative	decimal degrees	lat
lon	Sampling location longitude, west is negative	decimal degrees	lon
Date	Respiration assay start date Format: %Y-%m-%d	unitless	start_date
Incubation_Day	Days elapsed since microcosm incubation initiation	days	time_inc
Treatment	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)	unitless	treatment
Tank_ID	Identifier for microcosm replicate (two tanks per light treatment)	unitless	replicate
Method	Method of acquisition	unitless	no_bcodmo_term
Stain	Type of fluorecent dye used to stain cells. The flag -8888 represents no stain applied.	unitless	no_bcodmo_term
Excitation	The excitation wavelength/ filter cassette used on a Nikon Ti2 microscope for cell enumeration	nanometers (nm)	wavelength
Magnification	The magnification of the objective lense used for cell enumeration	unitless	no_bcodmo_term
Size_Fraction	The filter fraction used for slide preperation.	micrometers (um)	no_bcodmo_term
cells_per_L_avg	The average cellular abundance from 10 fields of view. The flag -7777 denotes below detection and the flag -9999 denotes a missing value.	cells L-1	cell_concentration
cells_per_L_sd	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.	cells L-1	std_dev
ratio_620_tot	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.	unitless	ratio
ratio_err	The error propagation of the 620:tot ratio (ratio_620_tot). The flag -9999 denotes a missing value.	unitless	no_bcodmo_term