Extraction and Analysis of Extracellular Polymeric Substances: Comparison of Methods and Extracellular Polymeric Substance Levels in Salmonella pullorum SA 1685

Abstract

Abstract

Extracellular polymeric substances (EPS) production and composition for Salmonella pullorum SA 1685 exposed to artificial groundwater (AGW) has been examined utilizing three EPS extraction methods: lyophilization, ethanol, and sonication. Experiments were carried out to evaluate the robustness of three EPS extraction methods and the sensitivity of each to subtle changes in solution ionic strength (IS) and duration of exposure. EPS extraction and analysis was conducted via sugar and protein analyses using the phenol sulfuric acid and Lowry methods, respectively, after 0-, 6-, 12-, 18-, and 24-h incubation times in AGW with 10^−2.5, 10⁻², and 10^−1.5 M IS. Lyophilization and ethanol methods resulted in a greater amount of EPS extracted than the sonication method (mass of EPS/cell), yet these methods fluctuated to a greater extent in the total amount—or level—of EPS extracted under the various test conditions. Systematic comparisons and extensive statistical analyses were conducted between the various experimental conditions. To our knowledge, this is the first study systematically comparing EPS extraction techniques utilizing Salmonella. As we investigated the relative EPS content in Salmonella SA1685 under conditions simulating groundwater, our results provide insight into the suitability of each method for detection of environmentally induced changes in bacteria suspended in simulated or real subsurface aquatic systems.