ASTM_E_1533_-_00.pdf

Designation:E 1533 00Standard Practice forIndirect Detection of Mycoplasma in Cell Culture by 4*-6-Diamidino-2-2 Phenylindole(DAPI)Staining1This standard is issued under the fixed designation E 1533;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last revision.A number in parentheses indicates the year of last reapproval.Asuperscript epsilon(e)indicates an editorial change since the last revision or reapproval.1.Scope1.1 This practice covers procedures used for the detection ofmycoplasma contamination by indirect DNA staining.1.2 This practice does not cover direct methods for thedetection of mycoplasma or other indirect methods such asenzymatical detection or DNA probes.1.3 This practice does not cover methods for the identifica-tion of mycoplasma organisms.1.4 The values stated in SI units are to be regarded as thestandard.The values given in parentheses are for informationonly.1.5 This standard does not purport to address all of thesafety problems,if any,associated with its use.It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2.Referenced Documents2.1 ASTM Standards:E 1531 Practice for Detection of Mycoplasma Contamina-tion of Cell Cultures by Growth on Agrose Medium2E 1532 Practice for Detection of Mycoplasma Contamina-tion of Cell Cultures by Use of the Bisbenzamide DNA-Binding Flurorochrome2E 1536 Practice for Detection of Mycoplasma Contamina-tion of Bovine Serum by the Large Volume Method23.Terminology3.1 Definitions:3.1.1 DAPI stainingstaining of DNA in particular byusing DAPI fluorochrome stain.3.1.2 direct detection of mycoplasmadetection of myco-plasma by cultivation in culture media.3.1.3 indirect detection of mycoplasmadetection of my-coplasma by DNA staining or any method other than cultiva-tion.3.1.4 mycoplasmathe smallest prokaryotes capable ofliving freely,lacking a cell wall,having a circular double-stranded DNA relatively rich in adenine and thymine,andcontaining 16s and 23s ribosomal RNAs.They can be found ascontaminants in cell cultures.4.Significance and Use4.1 Mycoplasma contamination of cell cultures is a commonproblem that can affect the growth,metabolism,and functionof cultured animal cells.The ability to detect mycoplasma incell cultures provides an opportunity to ensure that cells arefree of contamination,and to replace those that are not.Foradditional information,see Practices E 1531,E 1532,andE 1536.Strict adherence to established,well-tested proceduresis necessary.This practice was developed by Task GroupE48.01.02 to assist in developing and maintaining an estab-lished regimen for mycoplasma detection by indirect 48-6-Diamidino-2-Phenylindole(DAPI)fluorochrome staining.4.2 This practice is intended for use in examining culturedanimal cells for the presence of mycoplasma contamination.4.3 This practice is not intended for use in the detection ofmycoplasma contamination in serum,culture media,or sys-tems other than cultures of animal cells.4.4 All cell cultures to be examined for mycoplasma shouldundergo a minimum of two passages in antibiotic-free tissueculture medium before testing.5.Quality Control5.1 Visually examine the DAPI stain concentrate routinelyfor contamination.Fresh stock should be prepared periodically.5.2 Indicator cells:5.2.1 Indicator cells support the growth of mycoplasmaspecies and provide positive and negative controls.5.2.2 Use continuous cell lines such as the African greenmonkey kidney cell line,Vero,American Type Culture Collec-tion(ATCC CCL81)as indicator cells as described in thispractice;3T6 mouse fibroblast(ATCC CCL 96)may also beused.5.2.3 Do not use transformed cells as indicators since theyproduce large amounts of extra nuclear fluorescence.6.Procedure6.1 Preparation of DAPI Stain Concentrate:6.1.1 Add 1.0 mg DAPI stain to 100 mL sterilized distilledwater and mix thoroughly at room temperature.6.1.2 The stain is heat and light sensitive.Prepare theconcentrate in a bottle wrapped completely in aluminum foil,1This practice is under the jurisdiction of ASTM Committee E-48 on Biotech-nology and is the direct responsibility of Subcommittee E48.02 on Characterizationand Identification of Biological Systems.Current edition approved May 10,2000.Published July 2000.2Annual Book of ASTM Standards,Vol 11.05.1Copyright ASTM,100 Barr Harbor Drive,West Conshohocken,PA 19428-2959,United States.and store at 2 to 8C.6.2 Preparation of Indicator Cell Cultures:6.2.1 Sterilize Leighton tubes containing a glass cover slipof 32 3 4 mm.6.2.2 By trypsinization,prepare a cell culture containing 105cells/mL.6.2.3 Dispense 2 mL of recently(same day)prepared cellculture in each Leighton tube,taking care that the glass coverslip is submerged totally in the medium.Number the tubes.6.2.4 Inoculation:6.2.4.1 Inoculate 0.2 mL of previously frozen cell sample,thawed immediat