Dandekar & Chandler:Pyocyanin Extraction

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(Protocol)
(Protocol)
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===Protocol===
===Protocol===
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# Transfer 5ml of of desired culture In a 10ml glass test tube.
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# Extract the culture by adding Chloroform to a total of 50% total volume. (1ml chloroform for 2ml culture)
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# Add 3ml of chloroform using a glass pipet.
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# Vortex vigorously for 30 seconds.
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# Vortex vigorously for 15 seconds.
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# 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.
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# 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.   
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# 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.  
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# Carefully transfer aqueous layer into cuvette without sucking up any of the other layers.  
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# Carefully transfer .7 - 1ml of the chloroform layer into a fresh tube being careful not to transfer any of the aqueous layers.
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# Bring cuvette samples, including a cuvette filled with (water?) to use as a blank, to the spectrophotometer and take measurements at 520nm
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# 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.   
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# Remove the aqueous fraction and measure the absorbance at 520 nm.  
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 +
 
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* Multiply OD <sub>520</sub> by 17.072 to get μg/mL (Essar et al 1990)
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* 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.

Revision as of 14:53, 18 October 2013

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Contents

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.
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