Dandekar & Chandler:Pyocyanin Extraction

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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 grown in LB + 50mM MOPS.

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
• Sterile Cuvets
• 5ml glass pipet
• Nanopure H20
• 0.2m HCl
• Chloroform

Protocol

1. Extract the culture by adding Chloroform to a total of 50% total volume. (1ml chloroform for 2ml culture)
2. Vortex vigorously for 30 seconds.
3. Using a glass Pasteur pipette, transfer the organic layer (the bottom portion, it should be blueish if the culture is making pyocyanin) to an eppendorf tube or glass tube.
4. Spin for 5 minutes at full speed, or let the sample settle for 10 minutes to allow for the aqueous phase and cellular debris to separate out.
5. Carefully transfer .7 - 1ml of the chloroform layer into a fresh tube being careful not to transfer any of the aqueous layers.
6. Add 20% of the total volume .01N HCl to the chloroform fraction and vortex for for 30 seconds. The blue chloroform fraction should become clear and the small aqueous fraction from the added acid to turn pink.
7. Remove the aqueous fraction and measure the absorbance at 520 nm.

• Multiply OD ₅₂₀ by 17.072 to get μg/mL (Essar et al 1990)
• If the amount of this is too small, dilute it to 1:10 into water and have larger to supply to measure in a cuvette.