Hi,

No, pyocyanin is not a protein. It is a heterocyclic compound which easily diffuses through membranes. Here is some info: http://en.wikipedia.org/wiki/Pyocyanine

Pyocyanin is secreted from the bacteria into the medium. When it reaches high concentrations the medium turns dark green/blue. To measure pyocyanin levels in the culture sup:

1. spin down bacteria

2. add chloroform to the bacterial medium and shake it intensively (pyocyanin accumulates in the chloroform phase)

3. take the chloroform fraction into a new tube and add 0.2 M HCl and mix it heavily. Pyocyanin will be extracted into the HCl-phase.

4. Measure absorption at 520 nm.

Pyocyanin is not a protein. It is a small pigment (about 210 Da) from the phenazine family of compounds. Its concentration can be determined spectrophotometrically following published procedures (see Essar DW, Eberly L, Hadero A, Crawford IP: Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications.

J Bacteriol 1990, 172(2):884-900.)

Hi

pyocynin is a pigment... there are 4 different pigment present in the pesudomonas aruginosa...

some of the pseudomonas give dark green pigmentation dur to the more concentration of pigmentation of pyocynin and FLUROCYNIN also.. as you need to seperate the pyocynin, when you measure the pyocynin with photometric method at 520 mention by our friend RADA... some of the traces of flurocynin is also quantify..

so you need to do more experiment is that put the flourancent light on the bacteria with the 340 nm bulb light.

measure the concentration with drescent scale, this will help you to find the actual concentration of pyocynin and flurocynin, this will help you to avoide with a false positive result.

Only p.aerngenosa produce pigement dark . Ammount pigemment not determine only during remain bacteria in media in large time .only differential between aeruginosa and fluorescens it is litmus milk . First peptonization and second alkaline

hi

here is a paper related to your problem http://www.researchgate.net/publication/16399941_Antibiotic_action_of_pyocyanin

Article Antibiotic action of pyocyanin

I have to formulate a new media form that i have to narrow down my work by identifying the particular isolate for pyocyanin production ,which produces more pyocyanin .in what way i can categories

Hello,

Pyocyanine is not a protein. But the major compoud responsible for fluorescence is pyoverdine, a iron scavanging siderophore.

The problem is not only the concentration of green dye. But concentration of bacteria and number, which has a role in the intensity of the dye. Through an dilutions. As for how to extract the dye so I do not know

Http://www.ncbi.nlm.nih.gov/pubmed/20265520

If anyone ever observed robust pyocyanin production sensitive to light??

or some specific sugars

Light, agitation and carbon source all these matter

Iam sorry iam do not know

I dont think that the pigment is light sensitive..when we incubate in a incubator, there is not much light and the bacteria still produces pigment ..carbon source is very important, even agitation enhances its production..not sure about light

Carbon souces and physiological condition of media affects pigment production. Agitation enhance bacterial growth. If u want to isolate pigment produced by P. aeruginosa follow protocol to detect catecholates and hydroxamates siderophres. It might be helpful for you.

I have observed that pyocyanin production is favoured in dark

in my case i observed cultures turning green in 1-2minutes when exposed to light.. the mid flask shows the difference..

as well the cultures were always green if incubated in diffused light, but no such observation in dark..

i workd with the quorum sensing mutants of Pseudomonas, such phenotypes were negative in mutants

Only Maltose also showed such high induction when added in late log phase..

but yet the cultures need light

We know that the production of pigment pyocyanine from pseudomonas. Dye concentration increased, up the length of stay in the incubator. At a temperature of 42. Temperature is not 37. Any concentration of the dye and the increase depends on the ideal temperature for this bacteria. Perfect for the growth of bacteria is at a temperature of 42. And not 37

Yes the pyocyanine production is light dependent.. Most of the stains will produce pigment when it is exposed to light

Dark green color is produced due to the increase over the number of bacterial cells. Must rely on the survival time in the incubator. The green light as possible there is another kind of bacteria or bacterial isolate to make sure a way of planning. Purification. Isolation or stay in the refrigerator for several days. Then the dye concentration increased.

Dye produce depend on temperture and incubation

Yes dye production depend on temp...

But at 42 i get red pigment, degraded form of pyocyanin

How to characterize the pigment production?

solvent extraction, Lambda max & Rf could help you in characterizing and confirm pyocyanin production

Thankx...which protocol should i follow..?

Cox 1986 and Sanchez et al., 2002

this will do

Essar, D. W., L. Eberly, et al. (1990). "Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications." J Bacteriol 172(2): 884-900