were selected based on the highest activity and consistent production of
anti-bacterial metabolite for further studies. Each isolate was scaled up for extraction
of the anti-bacterial metabolite(s) with organic solvents. For scaling up, each
culture was inoculated into 25 ml culture broth at 35 psu (being the optimum salt
concentration for antibiotic production) and grown for 7 days in a shaker incubator
at 140 rpm and 28C 2C temperature. This media was scaled up to 500 ml using
the above 25 ml as an inoculum and incubated for 2 weeks. The culture broth was
then centrifuged at 13,000 rpm for 5 min at 4C. The culture supernatant was
decanted and collected. Sequential extraction was carried out with six different
organic solvents ranging from non-polar to polar solvents, (petroleum
ether > hexane > diethyl ether > chloroform > ethyl acetate > butanol) in a volume
ratio of 1:3 (culture supernatant: organic solvent). The organic layer was evaporated
to dryness using a rotary vacuum evaporator (Equitron Roteva) at 45C and the
resultant residue was then dissolved in 1 ml of the respective organic solvent and
then checked for its bioactivity against pathogens.