We will explain how to work with reference strains and their application in culture media controls, validations, and quality controls, in accordance with UNE–EN ISO/IEC 17025:2017, taking into account UNE–EN ISO 11133:2014. We will also cover the different types of culture media and their controls, the various types of strains and reference materials, their preservation and handling, related procedures, and the records required to demonstrate traceability of the processes performed.