Dandekar & Chandler:Pyocyanin Extraction

Pyocyanin Extraction

Pyocyanin [1] is a secondary metabolite secreted by the Gram negative bacterium Pseudomonas aeruginosa[2]. This protocol was established to quantify the pyocyanin produced by a P. aeruginosa culture in broth media.

Principle

Pyocyanin is an extracellular redox-active phenazine that contains a distinctive blue-green pigment. This compound can be isolated from cellular contaminants by isolating it in an aqueous suspension formed by the addition of chloroform. Because of the unique pigmentation this compound can then quantified with a spectrophotometer.

Materials Needed

• Spectrophotometer capable of reading 520 nm
• Vortex
• 10mL test tube
• 1.5ml eppendorf tubes
• Sterile Cuvets
• Nanopure H20
• 0.2m HCl
• Chloroform

Protocol

This is the protocol for a P. aeruginosa culture grown in LB + 50mM MOPS

1. Transfer 5ml of of desired culture In a 10ml glass test tube.
2. Add 3ml of chloroform using a glass pipet.
3. Vortex vigorously for 15 seconds.
4. Let the sample settle for 5-10 minutes to allow for the aqueous phase to separate out. After the phases have separated out, a top pink layer should be visible. This is the aqueous layer that will be pipetted into a cuvette and read.
5. Carefully transfer aqueous layer into cuvette without sucking up any of the other layers.
6. Bring cuvette samples, including a cuvette filled with (water?) to use as a blank, to the spectrophotometer and take measurements at 520nm
7. To get ng/ul from this 520nm reading multiply OD reading by 17.3.