MBCs for Staphylococcus aureus as determined by macrodilution and microdilution techniques

Abstract

MBC testing of clindamycin, methicillin, cephalothin, gentamicin, and vancomycin with 67 clinical isolates of Staphylococcus aureus was examined by both standard macrodilution tubes and commercial microdilution trays. Standard macrodilution failed to give reproducible (99.9% killing) MBC results, even when a strictly defined protocol was followed. Continuous shaking during incubation resulted in regrowth of more colonies than did stationary incubation. Vortexing of incubated tubes before subculture resulted in regrowth of more colonies than did careful transfer of the contents to sterile tubes before vortexing and subculture. No significant difference in MBCs was demonstrated by the use of log-phase versus stationary-phase inocula. Use of the multiprong inoculator for subculture from commercial microdilution trays was unsatisfactory because, although antibiotics evaluated were inactivated by subculture to a pH 5.5 agar plate coated with a beta-lactamase solution, the volume of broth transferred by the prongs was small and inconsistent, ranging from 0 to 3 microliter. Subcultures of commercial microdilution panels with a 1-microliter loop, 10-microliter pipette, and 100-microliter pipette were also evaluated. Results of MBC testing were most reproducible when the entire 100-microliter volume was aspirated from commercial microdilution wells after stirring and the contents of each well were spread over a separate sheep blood agar plate.