ASTM E1536-00(2006)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: E1536 − 00(Reapproved 2006)
Standard Practice for
Detection of Mycoplasma Contamination of Bovine Serum
by Large Volume Method
This standard is issued under the fixed designation E1536; 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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope 4. Significance and Use
4.1 Mycoplasmas of bovine origin are prevalent contami-
1.1 Thispracticecoverstheproceduresusedfordetectionof
nants of cell cultures. Contamination can be detected by the
mycoplasma contamination in serum by direct microbiological
3,4
large volume method.
culture.
4.2 Heat inactivated serum need not be tested for mycoplas-
1.2 This practice does not cover procedures used for detec-
mas. Heating serum to 56°C for 30 min will kill mycoplasmas.
tion of mycoplasma in cell cultures.
4.3 Mycoplasmas may be present in any particular lot of
1.3 This practice does not cover indirect methods for
serum but may not be detected because of inadequate sample
detection of mycoplasma contamination.
size; thus, negative test results do not provide absolute assur-
1.4 This practice does not cover methods for identification
ance that the test serum is free of mycoplasmas.
of mycoplasma cultures.
1.5 This standard does not purport to address all of the 5. Liquid Medium Preparation
safety concerns, if any, associated with its use. It is the
5.1 Add 105-g mycoplasma broth base, 5-g glucose, 5-g
responsibility of the user of this standard to establish appro-
arginine, and 20 mL of a 0.5 % solution of phenol red to 4080
priate safety and health practices and determine the applica-
mL of distilled water. Mix to dissolve ingredients.
bility of regulatory limitations prior to use.
5.2 Dispense medium, in 400-mL amounts into 500-mL
screw-capped bottles.
2. Referenced Documents
2 5.3 Autoclave.
2.1 ASTM Standards:
5.4 Sterile refrigerated medium is stable for four months.
E1531 Practice for Detection of Mycoplasma Contamination
of Cell Cultures by Growth on Agarose Medium
6. Quality Control.
3. Terminology
6.1 Prior to testing large volumes of bovine serum, check
sterility and ability of liquid medium to support mycoplasma
3.1 Definitions:
growth.
3.1.1 direct mycoplasma detection, n—demonstration of
characteristic colonial growth on axenic agar medium.
6.2 Strains used to test for growth support: M. arginini,
G230, M. bovis, Donetta; A. laidlawii, PG8.
3.1.2 large volume testing, n—using a large volume inocu-
lum in an enrichment culture.
6.3 For quality control, a portion of the base liquid medium
is supplemented with 20 % of newborn calf serum. This batch
3.1.3 mycoplasma (Mollicute), n—smallest prokaryotes ca-
of serum must be extensively tested to ensure that it is free of
pable of self replication.
mycoplasma contamination and it should be in sufficient
quantity to last for an extended period of time. Challenge
mycoplasma strains for the quality control test should be
This practice is under the jurisdiction of ASTM Committee E55 on Manufac-
ture of Pharmaceutical Products
...