Efficient early detection of mycoplasma contamination remains a challenging problem during the development and manufacture of cell-derived biologics and other pharmaceutical products (Razin et al. 1998; Salman et al. 1998; Drexler and Uphoff 2002; Uphoff et al. 2002). The culture-based methods established several decades ago, and still employed to detect contamination with mycoplasma, are laborious, cumbersome and time-consuming (Eldering et al. 2004; Deutschmann et al. 2010; Lawrence et al. 2010). Consequently, there is a high demand for advanced alternative methods, which are able to rapidly detect low levels of mycoplasma contamination in samples collected during stages of manufacturing that require immediate decisions regarding further processing. Several innovative technologies described recently (McGarrity and Kotani 1986; Mattsson and Johansson 1993; Uphoff and Drexler 2004; Baczynska et al. 2005; Sung et al. 2006; Mariotti et al. 2008) have been developed into alternative mycoplasma testing methods that might replace culture-based methods. Currently, there are several commercially available mycoplasma detection kits that rely on enzyme immunoassay (Roche Diagnostics GmbH, Penzberg, Germany, bioluminescence-based technology (MycoAlert™, Lonza, Walkersville, MD, USA), PCR (PromoCell GmbH, Heidelberg, Germany; Venor™GeM and LookOut®, Sigma-Aldrich, St Louis, MO, USA; Takara/Westburg BV, Leusden, Netherlands; PromoKine GmbH, Heidelberg, Germany; MycoTOOL®, Lonza/Roche, Walkersville, MD, USA), qPCR (PromoCell GmbH, Heidelberg, Germany; LookOut®, Sigma; MycoSEQ®, Life Technologies, Carlsbad, CA, USA), transcription-mediated amplification (MilliProbe®, EMD Millipore Corp., Billerica, MS, USA) or fluorescent probe hybridization (MycoProbe®, R&D Systems, Inc., Minneapolis, MN, USA). Among those methodologies, nucleic acid technique (NAT)-based methods appear to be the most advanced and promising for rapid and sensitive detection of mycoplasmas. However, prior to incorporation into mycoplasma testing protocols and implementation, any useful alternative method must demonstrate a limit of detection (LOD) comparable with those of conventional culture-based methods. It is noteworthy that comparability studies face serious technical challenges when the alternative and conventional methods measure different biological features of mycoplasmas, resulting in dissimilar read-outs difficult to compare directly. Thus, our previous attempts to compare NAT-based methods, which detect the presence of mycoplasma-specific nucleic acids (either genomic DNA or cellular RNA) regardless of cell viability, and culture-based methods, which detect only viable cells, led us to understand that unbiased comparisons require special reference materials with a high percentage of viable cells and a low degree of aggregation (Volokhov et al. 2011). From this standpoint, the ratio between genomic copies (GC) and colony forming units (CFU) represents a valuable parameter to assess both the viability of bacterial cells and their level of aggregation in cultures (Dabrazhynetskaya et al. 2011). In practice, the GC/CFU ratio varies over a wide range, being affected by many factors, for example, innate features of specific mycoplasma strains, culture/incubation conditions, growth phase at which samples are collected, conditions of freezing/thawing and storage, etc. (Razin 1969; Addey et al. 1970; Raccach et al. 1975; Biddle et al. 2004; Cheng et al. 2007; Boonyayatra et al. 2010). For that reason, comparability studies can yield compromised results leading to inadvertent overestimation of the LOD of NAT-based methods when mycoplasma reference materials used in the studies were prepared inappropriately. To avoid this problem, the mycoplasma stocks used for comparability assays should have the lowest possible GC/CFU ratios, reflecting high cell viability and dispersion.