An integrated workflow for Mycoplasma detection and removal from cell cultures and viral stocks

Contamination of cell cultures and viral stocks with Mycoplasma is one of the most frequent and underdiagnosed problems in laboratories working with these materials, severely affecting experimental reproducibility, data reliability, and biosafety. This article presents an integrated set of robust, sensitive, and broad applicable protocols for the detection, treatment, and mitigation of Mycoplasma contamination in cellular and viral systems. Basic Protocol 1 describes a sensitive qPCR-based method for Mycoplasma detection using DNA extracted from cell cultures and viral suspensions, including clear interpretation criteria based on amplification and melting curve analyses. Basic Protocol 2 outlines a sequential antibiotic treatment strategy for eliminating Mycoplasma from contaminated cell cultures, combining different antimicrobial classes and continuous monitoring of cell viability and morphology. Basic Protocol 3 presents a complementary approach to reduce Mycoplasma burden in viral stocks through serial dilution and controlled replication, aiming to preserve viral infectivity while minimizing bacterial contaminants. Together, these protocols provide a practical and reproducible workflow that enables rapid identification of contamination, implementation of effective corrective measures, and molecular validation of decontamination. The procedures are suitable for virology, cell biology, and experimental virus production laboratories, thereby improving experimental quality and data reliability. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sensitive detection of Mycoplasma contamination in cell cultures and viral stocks by qPCR Basic Protocol 2: Treatment of cell cultures for Mycoplasma decontamination Basic Protocol 3: Elimination of Mycoplasma contamination in viral stocks using serial dilution.