Abstract:

Transparent exopolymer particles (TEP) and Coomassie stainable particles (CSP) represent distinct groups of microbial exopolymer particles (EPS) that play a role in e.g. oil dispersion and MOS formation. We used the well-established spectrophotometrical methods as well as a recently published method that allows direct observations of TEP and CSP in whole water samples using a portable flow-through microscope (FlowCAM). Water samples were taken at different depths at three sites (Taylor Energy, OC26, and GC767) onboard R/V Point Sur (PS17-05).

Suggested Citation:

Kai Ziervogel. 2017. Testing a new method for the quantification of exopolymeric substances in the waters (TEP and CSP). Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N72V2D62

Purpose:

The purpose of this study was to test the newly developed FlowCAM method for TEP and CSP analysis. If applicable, the method would provides an onboard tool to analyze concentration and particle properties of EPS (size, volume etc.) shortly after sampling.

Data Parameters and Units:

Worksheet* (number following the bottle # indicates analytical replicates); Worksheet** ("_filt" means water was filtered through 0.2um syringe filter prior to CBB staining); CBB refers to the stain Coomassie brilliant blue, AB refers to the stain Alcian blue Water depth (m) Count (total number of particle); Particles / ml (particles X per millileters); Summary Stats (summary of the statistics of the particle property detmerined by VisualSpreadsheet software); Mean (average number of particles); Min (minimum number of particles); Max (maximum number of particles); StdDev (standard deviation); % CV (% coefficient of variation); Area (ABD) (number of pixels in the threshold binary grayscale image converted to a measure of area by use of the calibration factor, i.e. the conversion factor from pixels to microns -- units are microns per pixel; a fixed instrument factor); Area (Filled) (area represented by the particle edge and all the pixels inside the edge); Aspect Ratio (the ration of the length of the axis of the Legendre ellipse of inertia of the particle); Average Blue (average pixel value of the blue color plane); Average Green (average pixel value of the green color plane); Average Red (average pixel value of the red color plane); Capture X (the leftmost x coordinate in pixels of the particle in the original image); Capture Y (the top y coordinate in pixels of the particle in the original image); Circle Fit (deviation of the particle edge fom a best-fit circle, normalized to the rang [0,1] where a perfect fit has a value of 1); Circularity (a shape parameter computed from the perimeter and the filled area; a circel has a value of 1.0); Circularity (Hu) (an alternative measure of circularity that often provides a better indication of a circular shape of a particle); Compactness (a shape parameter derived from the perimeter of the filled area); Convex Perimeter (perimeter of the filled area); Convexity (a shape parameter that is computed as the ratio of filled area and convex perimeter); D[3 2](ABD) (these number -- the volume moment mean of the particle -- are the mean diameter computed by weighing the the area based diameter -- ABD -- of each particle by the ABD volume of the particle); D[3 2](ESD) (these number -- the volume moment mean of the particle -- are the mean diameter computed by weighing the the equivalent sphereical diamter -- ESD -- of each particle by the ESD volume of the particle); D[4 3](ABD) (these number -- the volume moment mean of the particle -- are the mean diameter computed by weighing the the area based diameter -- ABD -- of each particle by the ABD volume of the particle); D[4 3](ESD) (these number -- the volume moment mean of the particle -- are the mean diameter computed by weighing the the equivalent sphereical diamter -- ESD -- of each particle by the ESD volume of the particle); Diameter (ABD) (area based diameter; the diamter based on a circle with an area that is equal to the ABD area); Diameter (ESD) (equivalent sphereical diamter; the mean value of 36 ferret measurements, i.e. perpendicular distance between parallel tangents touching opposite sides of the particle); Diameter (FD) (Feret diameter); Edge Gradient (Average intensity of the pixels making up the outside boarder of a particle); Elapsed Time (time that the image was captured); Elongation (the inverse of geodesic aspect ration); Fiber Curl (a shape parameter computed from geodesic length and length, also known as curl index; (geodesic length/length)-1); Fiber Straightness (a shape parameter computed from geodesic length and length; length/geodesic length); Geodesic Aspect Ratio (the ratio of geodesic thickness and to geodesic length; elongation is the inverse of this ratio); Geodesic Length (value obtained by modeling the particle as a rectangle and computing length by solving the equations: area=geodesic length*geodesic thickness, perimeter= 2*(geodesic length*geodesic thickness)); Geodesic Thickness (value obtained by modeling the particle as a rectangle and computing thickness by solving the equations: area=geodesic length*geodesic thickness, perimeter= 2*(geodesic length*geodesic thickness); Image Height (image height in pixels of the particle in the saved collage image); Image Width (image width in pixels of the particle in the saved collage image); Intensity (average grayscale value of the pixels making up a particle); Length (maximum value of 36 feret measurements); Particles Per Chain (the number of particles that were grouped into one particle based on the nearest neighboor distance); Perimeter (length of the particle edge not including the lengths of edges of holes in the paricle); Ratio Blue/Green (average blue/average green); Ratio Red/Blue (average red/average blue); Ratio Red/Green (average red/average green); Roughness (a measure of the uneveness or irregularity of the particle's surface -- the ratio of perimeter to convex perimeter); Sigma Intensity (standard deviation of greyscale values); Sum Intensity (sum of greyscale pixel values); Symmetry (a measure of the symetrie of the particle about its center); Transparency (1-(ABD diameter - ESD diamter); Volume (ABD) (sphere volume calculated from ABD diameter); Volume (ESD) (sphere volume calculated from ESD diameter); Width (the minimum value of 36 feret measurements); TEP (raw data from colorimetric analysis of TEP - transparent exopolymer particles); CSP (raw data from colorimetric analysis of CSP - Coomassie stainable particles); Date (sampling date MM/DD/YYYY); # (number of particles); Sample (Site) (ID for the sampling site where the water was taken); Sample (niskin bottle) (number of niskin bottle that was subsampled); Sample (depth m) (water depth in meters where the niskin bottle was triggered); mL Filtered (volume of the water that was filtered in milliliters); L Filtered (volume of the water that was filtered in liters); Abs@787 (absorbance reading at 787 nanometers); Abs-Blk (blank corrected absorbance reading at 787 nanometers); Avg Abs-Blk (the average blank corrected absorbance reading at 787 nanometers); Stdev Abs-Blk (the standard deviation of the blank corrected absorbance reading at 787 nanometers); CV (coefficient of variation = stdev Abs-Blk/Avg Abs-Blk); µg Gxan eq/L (Gum Xanthan equivalents in micrograms/liter); Avg µg Gxan eq/L (average Gum Xanthan equivalents in micrograms/liter); Stdev µg Gxan eq/L (standard deviation Gum Xanthan equivalents in micrograms/liter); µg BSA eq/L (bovine serum albumin equivalents in micrograms/liter); Avg µg BSA eq/L (average bovine serum albumin equivalents in micrograms/liter); Stdev µg BSA eq/L (standard deviation bovine serum albumin equivalents in micrograms/liter); Sample Sites: Taylor Energy: 28.9350, -88.9720 OC26: 28.7020, -88.3620 GC767: 27.2130, -91.0030

Methods:

TEP and CSP were analyzed using the colorimetric method with Gum Xanthan and BSA as standards, respectively. Water samples were filtered onto 0.4um polycarbonate filter and stained with either Alcian Blue (AB for TEP) or Coomassie brilliant blue (CBB for CSP). The absorbance of the stained and extracted TEP and CSP at 787 nm and 615 nm, respectively, were analyzed on spectrophotometer in the Passow lab at UCSB. FlowCAM analysis of TEP and CSP in whole water samples were conducting following the method as described in Cisternas-Novoa et al. (2015), Mar Chem 175, 56-71 pp. AB and CBB stain were directly added to the whole water sample and transferred into a flow-through microscope (FlowCAM) equipped with a camera system. We used a 4x objective and a 300umx3000um flow cell; the camera takes pictures of all particles in the field of view (particle diameter 10-10000 um, distance to nearest neighbor: 1 um). The auto image rate was set to 20 frames/sec; the flow rate was 1.7 mL/min, and the sample volume was 4 mL (run time per sample: 2:35 min). At this setting the efficiency of the system to capture all particles was 41%. All of the pictures were checked manually and duplicates as well as unstained particles were deleted; results from image analysis (particle counts and properties) with the software VisualSpreadsheet were downloaded into excel for further analysis.

Instruments:

VS1 FlowCam spectrometer

Individual Files: