ASTM-E1533 Standard Practice for Indirect Detection of Mycoplasma in Cell Culture by 4`-6-Diamidino-2-2 Phenylindole (DAPI) Staining (Withdrawn 2014)

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Standard Practice for Indirect Detection of Mycoplasma in Cell Culture by 4`-6-Diamidino-2-2 Phenylindole (DAPI) Staining (Withdrawn 2014)


1.1 This practice covers procedures used for the detection of mycoplasma contamination by indirect DNA staining.

1.2 This practice does not cover direct methods for the detection of mycoplasma or other indirect methods such as enzymatical detection or DNA probes.

1.3 This practice does not cover methods for the identification of mycoplasma organisms.

1.4 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.

This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

Significance and Use

Mycoplasma contamination of cell cultures is a common problem that can affect the growth, metabolism, and function of cultured animal cells. The ability to detect mycoplasma in cell cultures provides an opportunity to ensure that cells are free of contamination, and to replace those that are not. For additional information, see Practices E 1531, E 1532, and E 1536. Strict adherence to established, well-tested procedures is necessary. This practice was developed by Task Group E48.01.02 to assist in developing and maintaining an established regimen for mycoplasma detection by indirect 4-6-Diamidino-2-Phenylindole (DAPI) fluorochrome staining.

This practice is intended for use in examining cultured animal cells for the presence of mycoplasma contamination.

This practice is not intended for use in the detection of mycoplasma contamination in serum, culture media, or systems other than cultures of animal cells.

All cell cultures to be examined for mycoplasma should undergo a minimum of two passages in antibiotic-free tissue culture medium before testing.


DAPI staining; indicator cells; mycoplasma; ICS Number Code 07.100.10 (Medical microbiology)

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Document Number


Revision Level

2000 R06 EDITION



Modification Type


Publication Date

Dec. 1, 2006

Document Type


Page Count

3 pages

Committee Number