European Pharmacopoeia 2.6.7 Mycoplasmas requires validation of sensitivity and robustness with respect to sample matrix. In addition, the method shall show a performance equal or better to the classical cultivation procedure. As most test and cell culture labs are frighten to cultivate mycoplasma, such comparison is not easy to accomplish. Microsart® Validation Standards are not infectious and therefore safe in use. They are titrated to 10 CFU/ml, the sensitivity limit for NAT-based methods like PCR to replace the traditional culture method. The mycoplasma cells used for the manufacture of Microsart® Validation Standard are low passage reference strains cultivated in culture broth described in EP 2.6.7. The cultures are harvested in the early logarithmic phase of the growth to avoid an atypical high ratio of dead mycoplasma particles, titrated immediately in culture broth and plated on Hayflick and Frey medium for quantification based on colony forming units (CFU).