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

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