I have no direct experience with exctraction, but pyocyanin production should be visible on Muller-Hinton agar plate in case the production is strong enough. If you do not see any pigmentation on MH agar, it means the strain definitely does not produce visible quantity. For detecting low concentrations that are not visible also see http://www.ncbi.nlm.nih.gov/pubmed/8912228

Also see http://idosi.org/ijpms/1(1)11/1.pdf

It is possible that your PAO1 strain has lost the ability to produce, because of repeated passaging on artificial media. Fresh clinical isolates may be better for you purposes, you can test several ones and use the strongest producer. From my experience, some are really keen pigment makers :-)

I believe that Pseudomonas Minimal Medium will upregulate the expression of the pyocyanin biosynthetic cluster.

Per Liter

Distilled water

985 ml

Glycerol

15 ml

L-Glutamine

5 g

K2HPO4

1.5 g

MgSO4

0.2 g

thank you all for your answers :-)

I missed "extraction" in the initial question. I have extracted beautiful greens and blues from all flavors of Pseudomonads (including P aeruginosa) in Defined Medium ph7 and PMM with 3x 1:1 ethyl acetate:culture extractions followed by Rotavapor drying (or SpeedVacs). You may notice changes in the color as it becomes more concentrated or if resuspended in DMSO.

It depends on growth condition. PAO1 grows well in LB media but it only produces pyocyanin when grown in flat-bottom container in a shaker incubator. You won't see pyocyanin when you grow it in a tube with tapered-bottom. You can try it. Good luck.

Try using cetrimide agar for initial subculturing, it promotes the production of pyocyanin. Once you observe the characteristic colour, you can transfer the culture onto liquid media like NB or preferably to a specific media In a flat bottom vessel under shaking conditions. I hope it works for you,  worked for me. Good luck 

Regards

The extent of pyocyanin produced depends on the strain of PAO1. The PAO1 used for genome sequencing does produce pyocyanin. A couple of important considerations:

- Make sure that you use a stationary-phase culture. Production is under QS control and does not turn on until stationary.

- Aeration is a critical factor. Ensure flask:media volume ratio. I typically use 1/5 or 1/10. Works great. Shake at 250 rpm or above. It produces the pigment in tubes too, just not as much.

Regular LB broth works fine. Don't bother with other media.

Also, if it doesn't look green, don't even bother with the assay. You won't see anything. Try using PA14 as a positive control. It produces a TON of pyocyanin. Good luck!

Yes P. aeruginosa produces pyocyanin pigment. I suggest you should try Tryptone water and Kings medium B for 48 hours at 30- 35 C (shaker incubator). You can see the greenish pigment after 48 hr of incubation. One more thing you can do, just strike the bacteria on Kings medium B agar plate and observe under UV light you can see the green colour flourishing under UV light. 

Otherwise check your purity of the culture.

Good luck!

As far as I know, PAO1 strain will produce more pyocyanin if you deplete iron in your medium. You can use an iron chelator with rich medium or use a minimal medium as proposed before.

Good luck with that!

Mahamudul,

I believe I first found the recipe for PMM from: Handbook of Media for Environmental Microbiology By Ronald M. Atlas, but I no longer have access to that book with the new job. I recently found the recipe on: http://wiki.bugwood.org/Minimal_medium_%28Pseudomonas%29  and the person who added the recipe is Robert L. Wick at UMass (https://stockbridge.cns.umass.edu/robert-wick). Perhaps you can contact him directly. 

Carter

is it necessary that all P.aeruginosa produce green colour in MH plate ? if not so what are the factors inhibit the pigment production or any other reasons