Abstract:

A mesocosm experiment was carried out in and October of 2015 (Mesocosm#2) using coastal water collected near Galveston Bay, Texas. Four mesocosm treatments were prepared in triplicate for this study: (1) Control (seawater only); (2) WAF (oil + seawater); (3) Chemically-enhanced WAF (CE-WAF; oil + Corexit + seawater); (4) Diluted CE-WAF (1:10 dilution). Initial mesocosm volumes were 79L. Prior to experiment initiation, phytoplankton were collected from coastal Galveston, TX waters and added to each tank to stimulate interactions between phytoplankton, oil, and Corexit. Starting at time-point zero and every 12 hours thereafter, water samples were collected from each mesocosm in clean, opaque, Nalgene bottles. For cell and aggregate counts, 10mL of water was collected from each mesocosm and fixed with 2% formalin. Fixed samples were stained with DAPI (45uM final concentration), filtered onto 25mm, 0.2um black polycarbonate filters, and counted directly via fluorescent microscopy. For beta-galactosidase measurements, parallel samples of 10um-filtered and unfiltered water were amended with 4-methylumbelliferyl beta-D-galactopyranoside. Florescence was quantified using a Tecan Spark 10M multimode microplate reader.

Suggested Citation:

Shawn Doyle, Emily Whitaker, Jason Sylvan. 2017. Coastal water with coastal microbial concentrate (COAST): Microbial cell and micro-aggregate counts with parallel beta-galactosidase activity measurements. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7R78C8T

Publications:

Doyle, S. M., Whitaker, E. A., De Pascuale, V., Wade, T. L., Knap, A. H., Santschi, P. H., … Sylvan, J. B. (2018). Rapid Formation of Microbe-Oil Aggregates and Changes in Community Composition in Coastal Surface Water Following Exposure to Oil and the Dispersant Corexit. Frontiers in Microbiology, 9. doi:10.3389/fmicb.2018.00689

Purpose:

Quantify the number of microbial cells and micro-aggregates within the mesocosms and also measure rates of the exoenzyme, beta-galactosidase.

Data Parameters and Units:

Data is in .csv table format. Cell and aggregate counts are in cells/mL and aggregates/mL, respectively. Fraction indicates whether the sample was filtered (10um pore-size) or unfiltered (whole) prior to measurement. Cell counts were only performed with unfiltered (whole) samples. Beta-galactosidase measurements are in nM/day. Blank cells indicate that data is not available; blank cells are not zero.

Methods:

Direct cell counts were performed with an epifluorescence microscope (Zeiss Axio Imager.M2) after staining the fixed samples with DAPI (45uM final concentration) for 5 minutes in the dark and filtering them onto 25mm, 0.2um black polycarbonate filters. Each filter was mounted on a glass microscope slide with coverslip using two drops of antifade. The filter-sterilized anti-fade solution consisted of 90 mM p-phenylenediamine and 45% glycerol dissolved in phosphate buffered saline. A 100um×100um ocular counting grid was used at 1000× magnification for cell counts. Due to their much larger size, aggregates were instead counted for each field of view at 400× magnification. Beta-galactosidase measurements were performed on whole water and <10 um size fractions by collecting 10 ml sample and adding 4-methylumbelliferyl beta-D-galactopyranoside (Sigma-Aldrich). Immediately after substrate addition and at two subsequent time points, typically an hour and two hours, 1 ml sample was removed and frozen for later analysis. Upon thawing, fluorescence was quantified using a Tecan Spark 10M multimode microplate reader with excitation and emission wavelengths set at 380/20nm and 454/20nm, respectively. Amount of substrate cleaved was calculated by fitting raw fluorescence to a standard curve of 4-methylumbellierone (Sigma-Aldrich) included in every plate read. The estimated oil equivalents (EOE) were determined using Macondo surrogate oil as the calibration standard for the fluorescence analyses (Horiba Scientific Aqualog Fluorometer). The EOE mean concentration of the three mesocosms for the control, WAF, DCEWAF and CEWAF at the start of the experiments were 0 mg/L, 0.26 mg/L, 2.74 mg/L and 41.5 mg/L, respectively.

Instruments:

Microscope: Zeiss Axio Imager.M2 Plate Reader: Tecan Spark 10M multimode microplate reader

Error Analysis:

The estimated oil equivalents (EOE) were determined using Macondo surrogate oil as the calibration standard for the fluorescence analyses (Horiba Scientific Aqualog Fluorometer). The EOE mean concentration of the three mesocosms for the control, WAF, Diluted CE-WAF and CE-WAF at the start of the experiments were 0 mg/L, 0.26 mg/L, 2.74 mg/L and 41.5 mg/L, respectively.

Individual Files: