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The objectives of this research were to (i) optimize the concentration of cycloheximide for use in WL media used in the wine industry and (ii) evaluate Dekkera/Brettanomyces differential medium (DBDM) as a means to detect Dekkera.Dekkera bruxellensis and other yeasts were transferred into WL broths containing 0, 10, 50 or 100 mg l−1 of cycloheximide. While several grew in 10 mg l−1, only Hanseniaspora uvarum, Pichia guillermondii, Schizosaccharomyces pombe and D. bruxellensis tolerated ≥50 mg l−1 of the antibiotic. On solidified WL media after 8-days incubation, colony sizes of two strains of D. bruxellensis (B1b and ATCC 52905) decreased with increased concentrations of cycloheximide, while others (F3 and P2) were unaffected. Although D. bruxellensis B1b did not grow well on another selective medium, DBDM, colony development was improved by the addition of sterilized red wine.Of the concentrations tested, 50 mg l−1 cycloheximide inhibited many grape/wine yeasts yet generally yielded countable colonies of Dekkera (1–2·5 mm diameter). Several strains of Dekkera did not grow well on DBDM, probably due to the lack of an unidentified nutrient(s).Better media formulations will improve the detection of Dekkera, thereby increasing microbiological control during winemaking.