Fast screening of antimicrobial compounds producing bacteria by colony picking

Abstract

This study was conducted to identify effective method for screening of antimicrobial compounds producing bacteria. Colonies of 20 target marine bacteria species were picked from their pure agar cultures, placed on the lawn cultures of 04 kinds of test bacteria species (Bacillus cereus, Bacillus subtilis, Halomonas smyrnensis, Vibrio sp.) and incubated for 16 hours at 30 oC. Target bacteria (P. piscicida, P. rubra, P. peptidolytica, Ruegeria mobilis, and Streptomyces badius) which given clear zones were cultured in marine broth and supernatants and cell pellets were separated by centrifugation. Filtered supernatants of bacteria cultures and supernatants of cell extractions were mixed (v/v, 1:1) with organic solvents (ethyl acetate, butanol, chloroform). Each target bacteria was co-cultured with each test bacteria. Same procedure was followed and finally supernatants of cultures and organic solvent fractions of supernatants were obtained. Supernatant of each culture, three organic solvent fractions of each supernatant, supernatants of each cell extraction, and three organic solvent fractions of each supernatant of cell extractions, supernatant of co-cultures and ethyl acetate fraction of supernatant of co culture cell extractions were used to check the antibacterial activities by disk diffusion assay(CLSI, 2012). Only the ethyl acetate fraction of cell extraction supernatant of co cultured H. smyrnensis with P. piscicinds of test bacteria species (Bacillus cereus, Bacillus subtilis, Halomonas smyrnensis, Vibrio sp.) and incubated for 16 hours at 30 oC. Target bacteria (P. piscicida, P. rubra, P. peptidolytica, Ruegeria mobilis, and Streptomyces badius) which given clear zones were cultured in marine broth and supernatants and cell pellets were separated by centrifugation. Filtered supernatants of bacteria cultures and supernatants of cell extractions were mixed (v/v, 1:1) with organic solvents (ethyl acetate, butanol, chloroform). Each target bacteria was co-cultured with each test bacteria. Same procedure was followed and finally supernatants of cultures and organic solvent fractions of supernatants were obtained. Supernatant of each culture, three organic solvent fractions of each supernatant, supernatants of each cell extraction, and three organic solvent fractions of each supernatant of cell extractions, supernatant of co-cultures and ethyl acetate fraction of supernatant of co culture cell extractions were used to check the antibacterial activities by disk diffusion assay(CLSI, 2012). Only the ethyl acetate fraction of cell extraction supernatant of co cultured H. smyrnensis with P. piscici