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Detection of Mycoplasma in cell cultures


Mycoplasma is a prokaryotic organism that is a frequent and occult contaminant of cell cultures. This organism can modify many aspects of cell physiology, rendering experiments that are conducted with contaminated cells worthless. Because of their small size, Mycoplasmas can pass through filters used to prevent bacterial and fungal contamination and potentially spread to all the cultures in a laboratory. It is essential that all new cell cultures entering a laboratory and all cell banks are tested for the presence of Mycoplasma. It is recommended that two techniques be used, selected from a PCR-based method, indirect staining and an agar and broth culture. This protocol describes these three tests for detecting Mycoplasma, which take from 1 d to 3–4 weeks, and such tests should be an obligatory component of quality control in every tissue culture laboratory.

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Figure 1
Figure 2: Typical gel photo from direct PCR.


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The protocols for agar and broth culture and PCR have been used routinely in the Division of Cell Biology and Imaging at the National Institute for Biological Standards and Control (part of the UK Health Protection Agency) for primary cell cultures and human stem cell lines (

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J.S. provided the PCR and L.Y. the agar culture protocol; both contributed to the drafts. G.S. provided the indirect staining protocol and additional references and introductory scientific text and reviewed draft documents. J.R.M. wrote the drafts and is the corresponding author.

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Correspondence to John R Masters.

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The authors declare no competing financial interests.

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Young, L., Sung, J., Stacey, G. et al. Detection of Mycoplasma in cell cultures. Nat Protoc 5, 929–934 (2010).

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