Standardized chemical synthesis of Pseudomonas aeruginosa pyocyanin

Preparation of the toxin pyocyanin from the bacterium Pseudomonas aeruginosa is an exacting procedure. Pyocyanin is expensive to commercially purchase. The sellers do not give out the extraction procedure. Classically, pyocyanin preparation involves complicated multi-step P. aeruginosa culturing and solvent transfer extractions. The chemical synthesis first used (1979) has not been adequately described. We devised an easily reproducible protocol which consistently decreases the time taken for synthesis, extraction and purification of pyocyanin, and increases the pure pyocyanin proportion produced. Our procedure: • Involves more purification steps (chloroform/methanol/acidification/alkalinization). • Starts with a different pH (7.4 instead of 7), and lesser concentration of phenazine methosulfate; and retrenches a rotary evaporation step. • Removes 2 lyophilization steps, and entails different solvent proportions for thin layer chromatography. As we have extracted pyocyanin both from P. aeruginosa cultures, and via chemical synthesis; we know the procedural and product-quality differences. We endorse the relative ease, safety, and convenience of using the chemical synthesis described here. Crucially, our “naturally endotoxin-free” pyocyanin can be extracted easily without using infectious bacteria.