ISOLATION AND CHARACTERIZATION OF BACTERIOPHAGE FROM WASTE WATER AGAINST E. COLI, A FOOD BORNE PATHOGENVIPIN SINGH, PRANAY JAIN AND SWATI DAHIYAAbstract Bacteriophages are the entities that require hostÂ’s metabolic processes to replicate itself. In the present study, phage has been isolated from stagnant water by overlay method against host bacteria. Titer of phages was calculated to be 107 PFU/mL using 10-fold dilutions. Stability was determined at different parameters like pH, temperature and ultra violet radiation. Morphology and identification of phage was done using Transmission Electron Microscopy (TEM) by staining with uranyl acetate. The phage capsid was about 78 nanometers (nm) in diameter with tail of length 527 nm as compared to wild type lambda phage whose head is about 65 nm. Isolated phage is classified into order Caudovirales since it possesses a long non contractile tail and icosahedral capsid head, thus is a member of the family Siphoviridae. Also the phage identified followed a lytic life cycle since plaque reaction activity was observed within 4-6 h against host bacteria i.e. E.coli by spot test. Molecular characterization of isolated bacteriophage (dsDNA >33.5Kbp) was carried out and compared with standard lambda DNA by subjecting to restriction enzyme digestion, using BamHI, EcoRI, and HindIII. The results of restriction digestion were compared in-silco and were found to be similar. Sodium dodecyl sulfate-polyacryamide gel electrophoresis (10% SDS-PAGE) profiles for protein structure analysis indicated ten protein bands of different molecular weight that were stained with Coomassie blue, followed by de-staining. It is therefore, proposed that the isolated phage be classified as lambda like virus. This phage could infect and kill several potentially harmful bacteria i.e. E.coli, and could be used as a control agent. We propose that isolated phage could be utilized as a bio-component in biosensor development against food borne pathogenic bacteria.
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