Several species of anaerobic bacteria display variable Gram stain reactions which often make identification difficult. A simple, rapid method utilizing a 3% solution of potassium hydroxide to distinguish between gram-positive and gram-negative bacterial was tested on 213 strains of anaerobic bacteria representing 19 genera. The Gram stain reaction and KOH test results were compared with the antibiotic disk susceptibilities (vancomycin and colistin) the preliminary grouping of anaerobic bacteria. All three procedures were in agreement for the majority of strains examined. Some strains of clostridia, eubacteria, and bifidobacteria stained gram negative or gram variable; the KOH and antibiotic disk susceptibility tests correctly classified these strains as gram-positive. The KOH test incorrectly grouped some strains of Bacteroides sp., Fusobacterium sp., Leptotrichia buccalis, and Veillonella parvula, but all Gram stain results for these strains were consistent for gram-negative bacteria. The KOH test is a useful supplement to the Gram stain and antibiotic disk susceptibility testing for the initial classification of anaerobic bacteria.

The penicillin-binding proteins (PBPs) of ATTC Type Strains of nine species of the Bacteroides fragilis group were visualized by gel electrophoresis and subsequent fluorography. Each species had a distinctive PBP pattern, although variation within species was seen. Generally, five PBPs could be visualized, ranging in molecular weight from approximately 40,000 to approximately 90,000. A laboratory-derived cefoxitin-resistant mutant of B. distasonis was compared with its wild type parent and cefoxitin-sensitive revertant. The fluorograph of the resistant mutant indicated a marked reduction of labelling to the PBP-1 complex as compared with the wild type and revertant. Cefoxitin-resistant clinical isolates of B. thetaiotaomicron and B. uniformis also showed changes to the PBP-1 complex, in comparison with sensitive strains.

Bacterial agglutination and polyacrylamide gel electrophoresis (PAGE) were methods evaluated for typing strains of Clostridium difficile. A panel of four antisera, obtained by immunizing rabbits with washed whole cells of different strains of C. difficile, produced distinctive patterns of agglutination. Ethylenediaminetetraacetate (EDTA) extracts subjected to PAGE also produced distinctive protein profiles. Excellent correlation between the two methods was observed when geographically distant isolates were typed without knowledge of their clinical origin. Both typing methods should receive further evaluation for their value as tools for epidemiological studies.