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OBJECTIVESTo identify the microflora in the gallbladder of patients undergoing laparoscopic cholecystectomy for gallstones, and the antibiotic susceptibility pattern of the isolates, as well as the usefulness of Gram staining of bile at the time of operation. METHODSBile samples were obtained from 112 patients undergoing elective laparoscopic cholecystectomy for gallstones and inoculated directly into aerobic and anaerobic blood culture bottles in the operating theatre. Samples were also collected in sterile universal containers for Gram staining of a centrifuged deposit. Isolates were identified and their in-vitro susceptibilities determined by Kirby Bauer technique. RESULTSOf 112 bile samples examined, 28 (25%) were culture positive, four of which contained more than one organism. The most common organisms isolated were Escherichia coli 9 (28.1%), Enterococcus faecalis 5 (15.6%) and Pseudomonas aeruginosa 3 (9.4%). In one sample we found Aeromonas hydrophilia and Enterobacter cloacae. No anaerobes were detected but Candida albicans was isolated in one case. In 19 bile samples (67.8%) organisms were identified on Gram stain. Positive bile cultures were found statistically significant (P < 0.05) in patients over the age of 50 (13/32), in patients who developed post-operative fever (6/12) and patients who developed leucocytosis (5/6). CONCLUSION Age over 50 years was the only significant pre-operative factor associated with positive bile cultures (P < 0.05). In view of the microflora of the gallbladder and the susceptibility pattern of our isolates we would suggest that antibiotic prophylaxis recommended for laparoscopic cholecystectomy for gallstones needs to be reviewed and the role of bacteribilia in the surgical management of cholelithiasis requires further study.