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Usually the choice of antibiotics for treatment of secondary peritonitis is based on the results of cultures of abdominal fluid. Despite adequate antimicrobial therapy residual or repeat infection occurs in approximately 30% of the cases. Culturing of abdominal fluid and peritoneum samples was performed by routine laboratory techniques. Fluorescence in situ hybridization was applied on human peritoneum samples to evaluate its usefulness in identifying bacteria in secondary peritonitis. Antibiotic therapy should be based on the results of the cultures of peritoneum samples rather than on the cultures of abdominal fluid.