ASTM E1532-00
(Practice)Standard Practice for Detection of Mycoplasma Contamination of Cell Cultures by Use of the Bisbenzamide DNA-Binding Fluorochrome
Standard Practice for Detection of Mycoplasma Contamination of Cell Cultures by Use of the Bisbenzamide DNA-Binding Fluorochrome
SCOPE
1.1 This practice covers procedures used for the detection of mycoplasma contamination by DNA staining.
1.2 This practice does not cover cultivation methods for the detection of mycoplasma or other methods such as biochemical detection, hybridization methodology, histochemical stains, or immunochemical methods.
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.
General Information
Relations
Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation:E1532–00
Standard Practice for
Detection of Mycoplasma Contamination of Cell Cultures by
Use of the Bisbenzamide DNA-Binding Fluorochrome
This standard is issued under the fixed designation E 1532; 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 4. Significance and Use
1.1 This practice covers the use of cell cultures and DNA- 4.1 Mycoplasma hyorhinis, cultivar a strains (1) do not
binding flurorochrome techniques to detect mycoplasma con- grow on any of the standard media used for mycoplasma
tamination of cell cultures. cultivation. These strains, which are found as contaminants in
1.2 This practice does not cover axenic cultivation or cell cultures, are detected by indirect methods.
identification of mycoplasmas. 4.2 A specialized medium has been described but it is not
1.3 This standard does not purport to address all of the yet in wide use (2).
safety concerns, if any, associated with its use. It is the 4.3 This practice should be used in conjunction with Prac-
responsibility of the user of this standard to establish appro- tice E 1531.
priate safety and health practices and determine the applica- 4.4 All cell cultures to be examined for mycoplasma should
bility of regulatory limitations prior to use. undergo a minimum of two passages in antibiototic-free tissue
culture medium before testing.
2. Referenced Documents
5. Indicator Cell Cultures
2.1 ASTM Standards:
E 1531 Practice for the Detection of Mycoplasma Contami- 5.1 BHK-21, 3T6, and Vero are the most widely used
nation of Cell Cultures by Growth on Agarose Medium indicator cell cultures. BHK 21 are maintained as monolayer
E 1533 Practice for Indirect Detection of Mycoplasma in cultures, which are trypsinized to prepare, cell suspensions as
Cell Culture by 48-6-Diamindino-2-2 Phenylindole (DAPI) needed (4-6).
Staining 5.2 Fetal bovine is heat inactivated at 56°C for 30 minutes
E 1536 Practice for the Detection of Mycoplasma Contami- before it is used in cell culture medium.
nation of Bovine Serum by the Large Volume Method 5.3 Place previously sterilized 11 x 22-mm coverslips in a
10 x 35-mm plastic culture dishes.
3. Terminology
5.4 Add 3.8 mL of cell suspension to each dish. The
3.1 Definitions: suspension should be dilute enough so that the resulting
3.1.1 axenic cultivation, n—cultivation free from other
monolayer is subconfluent in 2 to 3 days. Growth medium is
living organisms. replaced and the coverslip cultures are ready for use.
3.1.2 direct mycoplasma detection, n—demonstration of
5.5 Inoculate 0.1 mL of sample into each culture dish.
characteristic colonial growth on axenic agar medium. 5.6 For positive control, inoculate two dishes with M.
3.1.3 mycoplasma (Mollicute), n—smallest prokaryotes ca-
hyorhinis, strain DBS 1050 (ATCC 29052).Additional control
pable of self replication. strains of M. orale or M. pirum are useful.
5.7 Two uninoculated dishes serve as negative controls.
6. Materials for Staining
1 6.1 Carnoy’s Fixative—Mix one part glacial acetic acid
This practice is under the jurisdiction of ASTM Committee E48 on Biotech-
with three-parts methanol. This fixative may be made in
nology and is the direct responsibility of Subcommittee E48.02 on Characterization
and Identification of Biological Systems.
advance and stored at room temperature.
Current edition approved May 10, 2000. Published June 2000. Originally
6.2 McIlvane’s Citrate-Phosphate Buffer, pH 5.5:
published as E 1532 – 93. Last previous edition E 1532 – 93.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on The boldface numbers in parentheses refer to the list of references at the end of
the ASTM website. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E1532–00
6.2.1 0.1 M Citric Acid Monohydrate (MW 210.14)—Add 7.6 Let stand for ten minutes, then aspirate.
21 g to 1000 mL of deionized water. 7.7 Wash with distilled water, then air dry.
6.2.2 0.2 M Dibasic Sodium Phosphate (MW 141.96)—Add 7.8 Mount preparations by placing each coverslip (cell side
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.