AimsThe aim of this study was to optimize conditions for preparation and cryopreservation of mycoplasma reference materials suitable to evaluate alternative nucleic acid testing (NAT)-based assays and to compare their limits of detection (LODs) with those of conventional culture-based methods. Methods and ResultsAcholeplasma laidlawii, Mycoplasma gallisepticum and Mycoplasma arginini stocks with low ratios of genomic copies to colony forming units (12, 8 and 4, respectively) harvested in early stationary phases of growth were preserved with different cryoprotective agents (CPAs) under slow (1 degrees Cmin(-1)), moderate (8 degrees Cmin(-1)), fast (13 degrees Cmin(-1)) and snapshot' (60 degrees Cmin(-1)) cooling rates. Depending on mycoplasma species, increasing the cooling rate from slow to snapshot enhanced cell survival up to 5-fold. The addition of 10% (v/v) dimethyl sulfoxide (DMSO) and 15% (v/v) glycerol significantly improved cell survival of all tested strains. Cryoprotected stocks maintained high and stable titres for at least 1year during storage at -80 degrees C. Sonication of cell cultures prior to cryopreservation enhanced cell dispersion and reduced of GC/CFU ratios. ConclusionsIt is feasible to prepare stable reference stocks of cryopreserved mycoplasma cells suitable to reliably compare NAT- and culture-based mycoplasma testing methods. Significance and Impact of the StudyThis study describes experimental results demonstrating the preparation and storage of highly viable and dispersed mycoplasma reference stocks suitable for comparing alternative NAT-and conventional culture-based mycoplasma detection methods.