Title: A NOVEL METHOD FOR ISOLATION AND LONG TERM STORAGE OF LYTIC PHAGES OF VIBRIO VULNIFICUS APPLICANT: SREE BALAJI MEDICAL COLLEGE & HOSPITAL, BIHER- BHARATH UNIVERSITY ABSTRACT In the present invention, Vibrio vulnificus lytic phages were found to cause lysis of the bacterial cells of Vibrio and the phages can be used for phage therapy of Vibriosis. Therefore bacterial cells of V. vulnificus were grown in Luria Bertani (LB) medium/Nutrient broth medium for 6 h shaking (200 rpm) at 37°C, the cells from the bacterial cultures were centrifuged at 1000 rpm for 30 min at 4C, filtered through a 0.22 m membrane filter and the filtrate consisted only of a purified population of V. vulnificus lytic phages and they were stored for long-term in a mixture of chloroform (10%) and dimethyl sulfoxide (DMSO) (10%). The frozen V. vulnificus lytic phage stocks were taken from storage, used for the Vibrio lytic experiments and again stored at -40C for long term use. Double layer agar plates were prepared and the plaque formation, inhibition of the growth and lysis of the infected Vibrio cells were determined. For phage isolation, purification and stock preparation of single plaques for large-scale purification, serial-dilution was carried out from positive phage containing samples. The lytic effects and stability of the phages on long term storage, treatment with enzymes, organic solvents, detergents, pH temperature, were determined by employing agar bioassay method and one-step growth experiment. Our studies have shown that chloroform, acetone, ethyl alcohol, methyl alcohol, ribonuclease (RNase), trypsin, protease, Triton-X100 treatments did not affect the biocontrol lytic effects of the phages. In contrast, the phages were inactivated completely and hence they could not show biocontrol lytic effects at temperatures over 60°C, pH ( 12), lysozyme and sodium dodecyl sulfate (SDS) treatment. One-step growth experiments indicated that the latent period was 3 hrs with
