Isolation and Identification of Pseudomonas
In this study, 35 clinically isolated
Pseudomonas sp. strains will be cultured for primary isolation into Eosin
Methylene Blue Agar (EMB). The Cetrimide Agar will be used as a selective and
differential media for Non-lactose fermenting colonies of P.aeruginosa
and will be incubated overnight at 37°C.
The colonies from Cetrimide Agar will be
inoculated into Pseudomonas Broth (PB) and incubated for 24hrs at 37°C and observe for the change of color.
Extraction of pigment
From the culture supernatants, Pyocyanin will
be extracted, measured according to its absorbance on an acidic solution at
520nm and centrifuged at 4000 rpm for 10 minutes. The aqueous phase will be
removed after extracting culture supernatants with chloroform (1:2) in a new
test tube, then 1 mL of 0.2N HCl will be re-extracted at the bottom layer until
change in color is noticed. Consequently, using spectrophotometer at 520nm, the
absorbance of the pigment solution will be measured.
Purification of Pigment
The total amount of extracted Pyocyanin will
be added with 0.4M Borate NaOH buffer (pH: 10) until blue color is observed.
Then it will be extracted repeatedly in chloroform for 2 times until a clear
blue solution of Pyocyanin is achieved. On a petri dish, the clear blue
solution will be transferred and left overnight to evaporate chloroform.
Finally, using sterile distilled water Pyocyanin powders will be dissolved.
Preparation of Chemicals
Accurate concentration of 0.5% of NAOCl will
be diluted and tested against Pyocyanin using Sodium thiosulfate. 1.13% for neutralization of 0.5% NAOCl and
3.86% for the neutralization of concentraton greater than 1.0% NaOCl whill be
Exposure to Test Chemical
Aliquotes (0.2mL) of pyocyanin pigment will be
added to 9.8 mL of 0.5% NaOCl. To ensure immediate exposure of pyocyanin to the
chemical, bottles will be mixed using a spinmix. After 120 seconds aliquots
will be removed and immediately transferred to 9mL Na2S2O3 for neutralization
of of NaOCl. The aliquots from neutralized solution will be transferred to agar
plates and spread by the use of sterile glass spreads.