Anthrone reagent

The anthrone reaction was used to quantify the amount of polysaccharide in a tested solution [33 ]. Anthrone Reagent (Fisher Scientific, USA) was added (0.2 grams) to 100 ml concentrated sulfuric acid. Known concentrations of glucose (0, 25, 50, 100, and 200 mg/L) in water was used as a standard. The culture was grown to OD600nm = 0.15 and diluted 1:1 with THB; grew for 4 hr. Cells were harvested (750ul), washed twice with 1ml of PBS and resuspended in 150 μl of PBS. Cell concentrations were measured at OD 600 nm. Fifty microliter of the solution (test sample, standard, or PBS for a blank) was transferred into centrifuge tubes. Five hundred microliters of Anthrone Reagent was added into each tube and mixed well. Tubes were boiled at 100°C water bath for 10 min and were cooled for ~10 minutes at room temperature. After that, 100 microliters was used to measure OD 630 nm. The absorbance was normalized with the corresponding culture density. By comparing the ODs of an unknown to those of a standard, the polysaccharide concentration was determined. The results were analyzed by Student T test for statistical significance.

Planktonic bacteria were pelleted via centrifugation, and the culture supernatant was lyophilized in a lyophilizer (ScanVac freeze dryer). The bacterial pellet, lyophilized supernatant, and pellicle biofilm were resuspended in 300 μL of an acetic-nitric reagent and incubated for 30 min at boiling temperatures. The pellets were then washed twice with sterile water, followed by adding 67% sulfuric acid with intermittent mixings and incubated at RT for 1 h. The samples were placed on an ice bath, and 1 mL of cold anthrone reagent (Fisher Scientific) was added and mixed gently. The tubes were incubated in a boiling water bath for 15 min, after which they were placed on ice. The absorbance at 620 nm was recorded with Multiskan GO.

Planktonic bacteria were pelleted via centrifugation, and the culture supernatant was lyophilized in a lyophilizer (ScanVac freeze dryer). The bacterial pellet, lyophilized supernatant, and pellicle biofilm were resuspended in 300 μL of an acetic-nitric reagent and incubated for 30 min at boiling temperatures. The pellets were then washed twice with sterile water, followed by adding 67% sulfuric acid with intermittent mixings and incubated at RT for 1 h. The samples were placed on an ice bath, and 1 mL of cold anthrone reagent (Fisher Scientific) was added and mixed gently. The tubes were incubated in a boiling water bath for 15 min, after which they were placed on ice. The absorbance at 620 nm was recorded with Multiskan GO.