Quantification of Bacterial Alginate Using the Uronic Acid Carbazole Assay

Take a culture of the bacterium Pseudomonas aeruginosa in broth.

Plate the culture onto isolation agar and incubate to promote bacterial growth and the production of alginate, an exopolysaccharide composed of uronic acids.

Add isotonic saline to the bacterial lawn and scrape to collect the bacterial cells along with alginate.

Transfer the suspension into a tube, vortex to disperse the contents, and place it on ice to prevent enzymatic degradation.

Measure the optical density, or OD, to determine bacterial density.

Add the bacterial suspension to the acid-borate solution and mix to hydrolyze alginate into uronic acid monomers.

Add carbazole, a chromogenic reagent, and incubate. Under acidic conditions, the reagent reacts with uronic acids to form colored complexes.

Measure the absorbance to quantify the uronic acid levels, which reflect alginate concentration.

Using a standard curve, determine alginate concentration, then divide it by the measured bacterial cell density to calculate alginate production per unit of biomass.

To begin the uronic acid carbazole assay, identify a single colony from a pure culture of the desired strain to be tested and pick the colony using a sterile toothpick.

Place the toothpick in a culture test tube containing five milliliters of PIB. Grow in a shaker incubator at 37 degrees Celsius for 24 hours. Following incubation, add an aliquot of the cultured PIB broth onto a pre-warmed PIA plate.

Using a sterile cell spreader, spread the broth over the plate and grow at 37 degrees Celsius for 24 hours. Using a pipette controller and a sterile 50 milliliter pipette, add 0.85% sodium chloride to the grownup lawn and collect the sample by scraping the plate using a cell spreader. Aspirate the sample using a fresh 50 milliliter pipette and transfer into a 50 milliliter collection tube.

Vortex the sample on high to mix and place the samples on ice. To measure the OD 600 of the samples, first blank the spectrophotometer using one milliliter of 0.85% sodium chloride. Then add one milliliter of the sample to a new disposable cuvette and read the OD. Repeat this step twice to obtain a triplicate of reads for each sample.

Now add three milliliters of the sulfuric acid borate solution into culture tubes and let sit on ice. Add 350 microliters of the collected sample slowly to the acid mixture in the test tubes. After briefly vortexing on low, add 100 microliters of 0.1% carbazole and ethanol solution to the acid sample mixture.

Cap the tube and vortex on the medium setting for five seconds. Then place in a dry bath at 55 degrees Celsius for 30 minutes. After incubation, vortex the tubes briefly on high and allow to cool for five minutes.

Use the tube with 0.85% sodium chloride as a blank to the spectrophotometer. Then add one milliliter of the mixture to a clean cuvette and read the OD of the samples at 530 nanometers on a spectrophotometer. Prepare a standard curve by measuring the OD 530 of serial dilutions of known concentrations of d-mannuronic acid.

Repeat twice and extract a linear equation from these readings. Calculate the concentration of the alginate in each sample using the standard curve and divide the alginate concentration from linear equation by OD 600 to obtain the total amount of alginate per OD 600.