Determination of extracellular polymeric substances (EPS) using a modified phenol-sulfuric acid (PSA) assay for sugars

Using a DI-rinsed graduated cylinder, sub-sample 300–500 ml of the sample of interest (reduce the volume if a high biomass is known or suspected, as the filter will clog). Record the volume filtered. (Marine examples: for seawater, > 600 ml is typical; for melted bottom sea ice, 25 ml may be adequate if a visible algal band was present.) DI = 0.2-µm-filtered deionized water.

Filter volume onto PC membrane filter using a backing disk and DI-water-rinsed filtration tower/base (and filtration flask if dEPS is being collected) using gentle vacuum pressure (<15 inches Hg on the gauge). Record the volume filtered. PC = polycarbonate; dEPS = dissolved EPS.

Using forceps fold the filter in half twice (contents inwards) and place the folded filter in a labeled plastic vial or tube and freeze until processed in the laboratory.

If sampling dEPS, remove enough filtrate from the filter flask (1–5 ml for organic-rich samples and 125 ml for typical seawater samples) to an appropriate clean container (e.g., sterile 125-mL glass bottle or 50-cc disposable plastic tube) and label/freeze until processed in the laboratory.

Prepare a glucose standard curve using DI water. (We have compared artificial seawater solutions with DI water and found little difference.).

Transfer frozen filters into glass tubes to thaw at room temperature. Add 250 µL of DI water to the tube and mix well by shaking manually.

Add 250 µL of 5% phenol to the tube and vortex, then add 1,250 µL of the sulfuric acid (5 x 250 µL increments using the repeater and combitips). Add acid increments cautiously but in quick succession in a fume hood. (This reaction is highly exothermic, gets very hot, and can become frothy or foam over if the sample salt concentration is high, as in winter sea-ice brines; in that case, a temporary loose parafilm cover over the tubes is advised.)

Let glass tube sit to cool, then cap with parafilm and mix by vortexing at low speed (cautiously).

Pipet 200 µL reaction mixture from the tube into a 96-well microplate (acid is corrosive to the metal pistons of classic pipetmen; use barrier tips for this step.)

Incubate the tubes or microplates at 30°C for 30 minutes before measuring absorption at 490 nm.

Read using Spectramax M2 Spectrophotometer/Plate Reader

Obtain the linear function describing the relationship between absorption (OD490) and glucose equivalent (in µg) of the standard curve. Use linear regression, resulting in a standard curve function of y = mx + b. Account for the reaction volume to convert µg glucose/mL to µg glucose. OD = optical density.

Using the function established above, convert from OD490 (y) to glucose equivalents (x) as follows: x = (y – b) / m.

If measuring pEPS (x), account for the volume of sample filtered (Vf). The formula therefore becomes: x = (y – b) / (m * Vf).

For pEPS analyses, give some thought to carbohydrates contributed by cells captured on the filter.By estimating cell volume and C content and accounting for cell numbers in our samples, we have found the intracellular C contribution of EPS to be negligible.

Measure 250 µL of thawed sample filtrate directly, following the pEPS protocol above but without the addition of DI water.If no EPS is detected, then the sample can be concentrated for a second analysis, if desired; see Ewert et al. (2013) in protocol description.