ASTM E1533-93
(Practice)Standard Practice for Indirect Detection of Mycoplasma in Cell Culture by 4`-6-Diamidino-2-2 Phenylindole (DAPI) Staining
Standard Practice for Indirect Detection of Mycoplasma in Cell Culture by 4`-6-Diamidino-2-2 Phenylindole (DAPI) Staining
SCOPE
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.
1.5 This standard does not purport to address all of the safety problems, 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.
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Designation: E 1533 – 93
Standard Practice for
Indirect Detection of Mycoplasma in Cell Culture by 4*-6-
Diamidino-2-2 Phenylindole (DAPI) Staining
This standard is issued under the fixed designation E 1533; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope containing 16s and 23s ribosomal RNAs. They can be found as
contaminants in cell cultures.
1.1 This practice covers procedures used for the detection of
mycoplasma contamination by indirect DNA staining.
4. Significance and Use
1.2 This practice does not cover direct methods for the
4.1 Mycoplasma contamination of cell cultures is a common
detection of mycoplasma or other indirect methods such as
problem that can affect the growth, metabolism, and function
enzymatical detection or DNA probes.
of cultured animal cells. The ability to detect mycoplasma in
1.3 This practice does not cover methods for the identifica-
cell cultures provides an opportunity to ensure that cells are
tion of mycoplasma organisms.
free of contamination, and to replace those that are not. For
1.4 The values stated in SI units are to be regarded as the
additional information, see Practices E 1531, E 1532, and
standard. The values given in parentheses are for information
E 1536. Strict adherence to established, well-tested procedures
only.
is necessary. This practice was developed by Task Group
1.5 This standard does not purport to address all of the
E48.01.02 to assist in developing and maintaining an estab-
safety problems, if any, associated with its use. It is the
lished regimen for mycoplasma detection by indirect 48-6-
responsibility of the user of this standard to establish appro-
Diamidino-2-Phenylindole (DAPI) fluorochrome staining.
priate safety and health practices and determine the applica-
4.2 This practice is intended for use in examining cultured
bility of regulatory limitations prior to use.
animal cells for the presence of mycoplasma contamination.
2. Referenced Documents 4.3 This practice is not intended for use in the detection of
mycoplasma contamination in serum, culture media, or sys-
2.1 ASTM Standards:
tems other than cultures of animal cells.
E 1531 Practice for Direct Detection of Mycoplasma in Cell
4.4 All cell cultures to be examined for mycoplasma should
Culture by Broth Enrichment and Agar Growth
undergo a minimum of two passages in antibiotic-free tissue
E 1532 Practice for Indirect Detection of Mycoplasma in
culture medium before testing.
Cell Culture by DNA Binding with Bisbenzamide Fluo-
rochrome Stain
5. Quality Control
E 1536 Practice for Large Volume Testing of Serum for
2 5.1 Visually examine the DAPI stain concentrate routinely
Mycoplasma Contamination
for contamination. Fresh stock should be prepared periodically.
3. Terminology 5.2 Indicator cells:
5.2.1 Indicator cells support the growth of mycoplasma
3.1 Definitions:
species and provide positive and negative controls.
3.1.1 DAPI staining—staining of DNA in particular by
5.2.2 Use continuous cell lines such as the African green
using DAPI fluorochrome stain.
monkey kidney cell line, Vero, American Type Culture Collec-
3.1.2 direct detection of mycoplasma—detection of myco-
tion (ATCC CCL81) as indicator cells as described in this
plasma by cultivation in culture media.
practice; 3T6 mouse fibroblast (ATCC CCL 96) may also be
3.1.3 indirect detection of mycoplasma—detection of my-
used.
coplasma by DNA staining or any method other than cultiva-
5.2.3 Do not use transformed cells as indicators since they
tion.
produce large amounts of extra nuclear fluorescence.
3.1.4 mycoplasma—the smallest prokaryotes capable of
living freely, lacking a cell wall, having a circular double-
6. Procedure
stranded DNA relatively rich in adenine and thymine, and
6.1 Preparation of DAPI Stain Concentrate:
6.1.1 Add 1.0 mg DAPI stain to 100 mL sterilized distilled
This practice is under the jurisdiction of ASTM Committee E-48 on Biotech-
water and mix thoroughly at room temperature.
nology and is the direct responsibility of Subcommittee E48.02 on Characterization
6.1.2 The stain is heat and light sensitive. Prepare the
and Identification of Biological Systems.
Current edition approved March 15, 1993. Published May 1993.
concentrate in a bottle wrapped completely in aluminum foil,
Annual Book of ASTM Standards, Vol 11.05.
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
E 1533
and store at 2 to 8°C. 6.6.2 Label each slide to identify the specimen being tested.
6.2 Preparation of Indicator Cell Cultures: 6.7 Observation and Recording of Results:
6.2.1 Sterilize Leighton tubes containing a glass cover slip 6.7.1 Observe each specimen, including both the positive
of 32 3 4 mm. and negative controls, by fluorescence microscopy at 2503
6.2.2 By trypsinization,
...
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