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.

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