The selected Pseudomonas isolates were grown first in the nutrient broth at 37C for 48 hours for pigment production. Pigment rich broth culture was then centrifuged at 10000 rpm for 15 min and the supernatant was collected. It was then filtered through 0.45 μm pore sized membrane filter and used as crude extract. Extraction of pigment from crude extract was done using chloroform and HCl as Ingledew and Campbell, 1969 with some modification. Chloroform was added in the culture broth at the ratio of (1:1) and after vortexing a blue solvent layer was produced. The blue layer was collected, then 0.1N HCl solution (20% of the blue layer’s volume), was added and vortexed, which produced an acidified pink upper layer. The pink layer was then neutralized with Tris-Base and the neutralized layer was again treated with chloroform. The whole procedure was repeated several times to make it pure . After final neutralization with tris-base it was acidified with .05N HCl and the pink layer is neutralized with .1 N NaOH
Hello chandana, if your question is just to obtain crytalline form of pyocyanin, then first check the pH of the extract before crystallization. Whatever it is adjust it to 7.5 with 0.1 N NaOH and allow to stand for 1-3 h. For details see the attached.
If you are interested in the entire protocol of pyocyanin extraction, purification and quantification, then I will reproduce, verbatim, what Dr. Hazel Salistre of Pasteur Institut, France recommended for a similar question 5 years ago.
"This is the protocol we follow for pyocyanin extraction from P. aeruginosa strains and even though I am using simply LB to grow cultures, I get sufficient pyocyanin for comparison between my wild-type and mutant strains.
-Overnight cultures are standardized to OD600 1.0 before inoculating in 25 mL of LB with 1:100 dilution
-Cultures are grown at 37C, 200rpm (for whatever amount of time you want, my colleague collects supernatants after 8 hrs and 24 hrs, I do only after 18 hrs) in 250 mL flasks
-Supernatants are collected after centrifugation at 10000 rpm for 10 min. and filter-sterilized
-4.5 mL of chloroform is added to 7.5 mL of supernatant (25 mL of initial culture is enough for triplicates of supernatant for each strain) and vortexed 10 x 2 seconds (chloroform sinks to the bottom of the tube, make sure the supernatant mixes with chl. when you vortex, you will see that color of chloroform is becoming green-blue)
-Samples are centrifuged for 10 min. at 10000 rpm.
-3 mL of the resulting blue layer at the bottom (chloroform + pyocyanin) is transferred to a new tube.
-1.5 mL of 0.2 M HCl is added to each tube and vortexed for 10 x 2 seconds. At this step, you will see the blue color will turn into pink (top layer).
-Samples are centrifuged for 2 min. at 10000 rpm and 1 mL from the pink layer is transferred to cuvettes. Spectrophotometric measurements are done at 520 nm.
-Pyocyanin concentration (uL/mL) is calculated by multiplying the value you get at 520 nm with 17.072, then multiplying it again by 1.5 (because only 3 mL from initial 4.5 mL of chloroform is used).
Note: All centrifugation steps take place at 4C. 0.2 M HCl is used as a blank in the spectrophotometer".
I personally have found this protocol particularly useful and have stuck to it over the years with commendable results.
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