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By using optimum sampling, transport, and culture techniques in patients with gangrenous or perforated appendicitis, we recovered larger numbers of bacteria with a more diverse flora than has previously been reported. Thirty patients older than 12 years with histologically documented gangrenous or perforated appendicitis had peritoneal fluid, appendiceal tissue, and abscess contents (if present) cultured. Appendiceal tissue was obtained so as to exclude the lumen. A total, of 223 anaerobes and 82 aerobic or faculatative bacteria were recovered, an average of 10.2 different organisms per specimen. Twenty-one different genera and more than 40 species were encountered. Bacteroides fragilis group and Escherichia coli were isolated from almost all specimens. Within the B. fragilis group, eight species were represented. Other frequent isolates included Peptostreptococcus (80%), Pseudomonas (40% [P. aeruginosa, 23.3%, other Pseudomonas spp., 16.7%]), B. splanchnicus (40%), B. intermedius (36.7%), and Lactobacillus (36.7%). Interestingly a previously undescribed fastidious gram-negative anaerobic bacillus was isolated from nearly one half of all patients. This organism was found to have low DNA homology (by dot blot) with the known organisms most closely resembling it.