Cycloheximide is a dangerous chemical that is commonly used in the brewing industry to suppress yeast growth in microbiological platings. When handled appropriately cycloheximide is an effective inhibitor of brewery yeast, however, its laboratory use poses personnel safety issues and hazardous waste disposal concerns. Actidione affects the central nervous system, is teratogenic, and can be fatal in certain doses via inhalation. Nystatin is a common antibiotic prescribed for human yeast infections, including topical use in creams. Since Nystatin is less soluble and less stable (heat and light) than cycloheximide, modifications to the Inhibitor Medium standard method will likely be required. The objective of this work was to determine the important factors for effective Nystatin use in LMDA and document the inhibition of a variety of brewery-sourced yeast strains. The stock solution matrix has a dramatic affect on yeast inhibition. In a water suspension, the inhibitor is largely insoluble and has an effective concentration of approximately 400 UNITS/ml of media. A matrix of 95% ethanol allows complete solubility of the hydrophobic (MW=926.1) antibiotic and reduces the effective concentration tenfold (i.e. 45 UNITS/ml of media). Since activity declines during refrigerated storage in both matrices, the stock solution should be kept frozen during storage (-20 C). Preparing and dispensing the stock solution in micro-centrifuge tubes greatly facilitates the subsequent thawing of stock solution aliquots. This technique is suitable for craft brewers and contract laboratories that have infrequent, and/or small, plating sessions. A wide variety of brewers' yeast are effectively suppressed by Nystatin. We've identified thirteen biochemically distinct yeast strains that were isolated from brewery sources, including 2 ale strains, 6 lager strains, and 5 species of wild type yeast. The suspension and ethanol matrix methods have been in use at a craft brewery and a contract lab, respectively, for the last 2 years. We have successfully prepared aerobic and anaerobic LMDA plates with Nystatin using membrane filtration for a variety of process samples (fermenter, centrifuged beer, packaged beer, yeast slurries, carbonated water) without loss of sensitivity for common beer spoilage bacteria.