ASTM E1531-00

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation:E1531–00
Standard Practice for
Detection of Mycoplasma Contamination of Cell Cultures by
Growth on Agarose Medium
This standard is issued under the fixed designation E 1531; 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 3.1.1 direct mycoplasma detection, n—demonstration of
characteristic colonial growth on axenic agar medium.
1.1 Thispracticecoverstheproceduresusedfordetectionof
3.1.2 indirect detection of mycoplasma, n— detection of
mycoplasma contamination by direct microbiological culture.
mycoplasma by DNA staining or any method other than
1.2 This practice does not cover indirect methods for
cultivation.
detection of mycoplasma such as DNA staining, biochemical
3.1.3 mycoplasma (Mollicute), n—smallest prokaryotes ca-
detection, or genetic probes.
pable of self replication.
1.3 This practice does not cover methods for identification
of mycoplasma organisms.
4. Significance and Use
1.4 This practice will not detect cultivar a strains (1) of
4.1 The demonstration of characteristic colonial growth on
Mycoplasma hyorhinis.
axenic solid medium is a sensitive and specific method to
1.5 This practice is not intended for use in detection of
detect mycoplasma infection of cell cultures and it is the
mycoplasma contamination in sera, culture media, vaccines, or
standard detection method (2).
other systems.
4.2 When mycoplasmas contaminate cell cultures they usu-
1.6 This standard does not purport to address all of the
allygrowtohightiter(10 colonyformingunits/mL)andwhen
safety concerns, if any, associated with its use. It is the
inoculatedontoagarmediumtheyproduceabundantandeasily
responsibility of the user of this standard to establish appro-
detectable growth (3).
priate safety and health practices and determine the applica-
4.3 M. hyorhinis cultivar a strains do not grow on conven-
bility of regulatory limitations prior to use.
tional mycoplasma media (1) but require an indicator cell
2. Referenced Documents culture system to detect their presence (see Practice E 1532).
Alternatively, a specialized axenic medium is suitable for
2.1 ASTM Standards:
direct isolation of cultivar a from infected cell cultures (4).
E 1532 Practice for Detection of Mycoplasma Contamina-
4.4 Immunofluorescent procedures are used to identify my-
tion of Cell Cultures by the Use of the Bisbenzamide DNA
coplasma isolates (5).
Binding Fluorochrome
E 1533 Practice for Indirect Detection of Mycoplasma in
5. DM-1 Solid Medium Preparation
Cell Culture by 48–6–Diamidino–2–2 Phenylindole
5.1 Dissolve CMRL-1066 powder (CMRL-1066 powder
(DAPI) Staining
Formula No. 78–5156EF , packaged for 10L), in 5000 mL of
E 1536 Practice for Detection of Mycoplasma Contamina-
distilled water. This is one-half the volume of water specified
tion of Bovine Serum by the Large Volume Method
on the package. Add 47.6 g HEPES , and 9.35 g NaCl.
3. Terminology 5.2 Adjust the pH to 7.3 and filter sterilize (450 nm). Store
this 2X CMRL in the refrigerator in 500 mL amounts.
3.1 Definitions:
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5.3 Dissolve 10.0 g of Myosate and 12 g of agarose in 400
mLof distilled water.Autoclave at 121°C for 15 minutes. Cool
the autoclaved solution to about 50°C, and combine with 500
This practice is under the jurisdiction of ASTM Committee E-48 on Biotech-
mL of 2X CMRL and 100 mL of sterile horse serum (both
nology and is the direct responsibility of Subcommittee E48.02 on Characterization
ingredients also warmed to 50°C).
and Identification of Biological Systems.
Current edition approved May 10, 2000. Published June 2000. Originally
published as E 1531 – 93. Last previous edition E 1531 – 93.
The boldface numbers in parenthesis refer to the list of references at the end of
this standard. Available from Life Technologies, Gaithersburg, MD.
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For referenced ASTM standards, visit the ASTM website, www.astm.org, or Available from Research Organics, Cleveland, OH.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Available from BBL Microbiology Systems, Cockeysville, MD.
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