**2.2. Cultivation of P. aeruginosa MTCC 2453**

A freeze dried culture of *P. aeruginosa* strains MTCC2453,741,1934,1942 was obtained from the Microbial type culture collection center, Chandigarh, India and was grown in a medium containing 7g yeast extract, l0g peptone, 20g of KNO3, 6.4g of KH2PO4, 3.6g of Na2HPO4 (anhydrous), 2.5g of NaCl, 5µg/ml of CuSO4 per liter. The initial pH was adjusted to 6.5 with NaOH. The strains were maintained on nutrient broth with 50% glycerol concentration and stored at -70oC for further study.

Inoculums' of *P. aeruginosa* strains was prepared by inoculating a loopful of colonies in individual 100 ml conical flasks with the exact constituents of the above prescribed media and incubated at 370C, in stirring mode at 100/rev for 21 hours. These inoculums was used to seed the bulk 500ml x 4 sterile medium in 2000 liter conical flasks (separate conical flask used for all four strains) which was also kept at stirred mode (100/rev) for 21 hours at 370C [11].

## **2.3. Impact of copper sulphate and potassium nitrate on culture medium**

All P*. aeruginosa* strains MTCC2453, 741, 1934, and 1942 are inoculated separately in a sterile medium. Impact of copper sulphate and potassium nitrate in azurin synthesis were observed by adding different concentration of copper sulphate (1µg/ml- 5µg/ml) and potassium nitrate (5µg/l – 20µg/l) at different flasks for each concentration distinctly. The azurin protein optimization and quantification was studied in UV spectrometer (Perkin Elmer, Massachusetts, USA) at 595 nm by Bradford's method. The azurin synthesized from P. aeruginosa MTCC2453 is significantly higher than other strains [11, 12].
