ASTM E1536-00
(Practice)Standard Practice for Detection of Mycoplasma Contamination of Bovine Serum by the Large Volume Method
Standard Practice for Detection of Mycoplasma Contamination of Bovine Serum by the Large Volume Method
SCOPE
1.1 This practice covers the procedures used for detection of mycoplasma contamination in serum by direct microbiological culture.
1.2 This practice does not cover procedures used for detection of mycoplasma in cell cultures.
1.3 This practice does not cover indirect methods for detection of mycoplasma contamination.
1.4 This practice does not cover methods for identification of mycoplasma cultures.
1.5 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.
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Designation:E 1536–00
Standard Practice for
Detection of Mycoplasma Contamination of Bovine Serum
by the Large Volume Method
This standard is issued under the fixed designation E 1536; 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 3.1.3 mycoplasma (Mollicute), n—smallest prokaryotes ca-
pable of self replication.
1.1 Thispracticecoverstheproceduresusedfordetectionof
mycoplasma contamination in serum by direct microbiological
4. Significance and Use
culture.
4.1 Mycoplasmas of bovine origin are prevalent contami-
1.2 This practice does not cover procedures used for detec-
nants of cell cultures. Contamination can be detected by the
tion of mycoplasma in cell cultures.
,
3 4
large volume method.
1.3 This practice does not cover indirect methods for
4.2 Heat inactivated serum need not be tested for mycoplas-
detection of mycoplasma contamination.
mas. Heating serum to 56°C for 30 minutes will kill myco-
1.4 This practice does not cover methods for identification
plasmas.
of mycoplasma cultures.
4.3 Mycoplasmas may be present in any particular lot of
1.5 This standard does not purport to address all of the
serum but may not be detected because of inadequate sample
safety concerns, if any, associated with its use. It is the
size; thus, negative test results do not provide absolute assur-
responsibility of the user of this standard to establish appro-
ance that the test serum is free of mycoplasmas.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
5. Liquid Medium Preparation
2. Referenced Documents 5.1 Add 105-g mycoplasma broth base, 5-g glucose, 5-g
2 arginine, and 20 mL of a 0.5 % solution of phenol red to 4080
2.1 ASTM Standards:
mL of distilled water. Mix to dissolve ingredients.
E 1531 Practice for the Detection of Mycoplasma Contami-
5.2 Dispense medium, in 400-mL amounts into 500-mL
nation of Cell Cultures by Growth on Agarose Medium
screw-capped bottles.
E 1532 Practice for the Detection of Mycoplasma Contami-
5.3 Autoclave.
nation of Cell Cultures by the Use of the Bisbenzamide
5.4 Sterile refrigerated medium is stable for four months.
DNA-Binding Fluorochrome
E 1533 Practice for Indirect Detection of Mycoplasma in
6. Quality Control
Cell Culture by 48-6-Diamidino-2-2 Phenylindole (DAPI)
6.1 Prior to testing large volumes of bovine serum, check
Staining
sterility and ability of liquid medium to support mycoplasma
growth.
3. Terminology
6.2 Strains used to test for growth support: M. arginini,
3.1 Definitions:
G230, M. bovis, Donetta; A. laidlawii, PG8.
3.1.1 direct mycoplasma detection, n—demonstration of
6.3 For quality control, a portion of the base liquid medium
characteristic colonial growth on axenic agar medium.
is supplemented with 20 % of newborn calf serum. This batch
3.1.2 large volume testing, n—using a large volume inocu-
of serum must be extensively tested to ensure that it is free of
lum in an enrichment culture.
mycoplasma contamination and it should be in sufficient
quantity to
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SCOPE
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