Department of Microbiology, Soochow University, Taipei 111, Taiwan, R.O.C.

   Vibrio vulnificus is a marine bacterium that causes septicemia with high mortality via wound infection or seafood ingestion. Subspecies typing of V. vulnificus can be performed by pulsed-field gel electrophoresis (PFGE).  However, this method is hampered by the degradation of chromosomal DNA in about 12% of strains.  This study presents a modified PFGE procedure.  The outer membrane of V. vulnificus cells was lysed utilizing a sucrose-EDTA method and components in the periplasmic space were removed prior to making plugs.  Cytoplasmic digestive enzymes were inactivated by an extended proteinase K reaction.  Experimental results indicated that five out of eight strains exhibiting smeared DNA in a previous study (Appl. Environ. Microbiol. 70: 5153-5158. 2004) achieved clear banding patterns.  This modified PFGE procedure can be applied to improve PFGE typing of untypeable strains of V. vulnificus.