ASTM_E_1532_-_00.pdf

Designation:E 1532 00Standard Practice forDetection of Mycoplasma Contamination of Cell Cultures byUse of the Bisbenzamide DNA-Binding Fluorochrome1This standard is issued under the fixed designation E 1532;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 the use of cell cultures and DNA-binding flurorochrome techniques to detect mycoplasma con-tamination of cell cultures.1.2 This practice does not cover axenic cultivation oridentification of mycoplasmas.1.3 This standard does not purport to address all of thesafety concerns,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 the Detection of Mycoplasma Contami-nation of Cell Cultures by Growth on Agarose Medium2E 1533 Practice for Indirect Detection of Mycoplasma inCell Culture by 48-6-Diamindino-2-2 Phenylindole(DAPI)Staining2E 1536 Practice for the Detection of Mycoplasma Contami-nation of Bovine Serum by the Large Volume Method23.Terminology3.1 Definitions:3.1.1 axenic cultivation,ncultivation free from otherliving organisms.3.1.2 direct mycoplasma detection,ndemonstration ofcharacteristic colonial growth on axenic agar medium.3.1.3 mycoplasma(Mollicute),nsmallest prokaryotes ca-pable of self replication.4.Significance and Use4.1 Mycoplasma hyorhinis,cultivar a strains(1)3do notgrow on any of the standard media used for mycoplasmacultivation.These strains,which are found as contaminants incell cultures,are detected by indirect methods.4.2 A specialized medium has been described but it is notyet in wide use(2).4.3 This practice should be used in conjunction with Prac-tice E 1531.4.4 All cell cultures to be examined for mycoplasma shouldundergo a minimum of two passages in antibiototic-free tissueculture medium before testing.5.Indicator Cell Cultures5.1 BHK-21,3T6,and Vero are the most widely usedindicator cell cultures.BHK 21 are maintained as monolayercultures,which are trypsinized to prepare,cell suspensions asneeded(4-6).5.2 Fetal bovine is heat inactivated at 56C for 30 minutesbefore it is used in cell culture medium.5.3 Place previously sterilized 11 x 22-mm coverslips in a10 x 35-mm plastic culture dishes.5.4 Add 3.8 mL of cell suspension to each dish.Thesuspension should be dilute enough so that the resultingmonolayer is subconfluent in 2 to 3 days.Growth medium isreplaced and the coverslip cultures are ready for use.5.5 Inoculate 0.1 mL of sample into each culture dish.5.6 For positive control,inoculate two dishes with M.hyorhinis,strain DBS 1050(ATCC 29052).Additional controlstrains of M.orale or M.pirum are useful.5.7 Two uninoculated dishes serve as negative controls.6.Materials for Staining6.1 Carnoys FixativeMix one part glacial acetic acidwith three-parts methanol.This fixative may be made inadvance and stored at room temperature.6.2 McIlvanes Citrate-Phosphate Buffer,pH 5.5:6.2.1 0.1 M Citric Acid Monohydrate(MW 210.14)Add21 g to 1000 mL of deionized water.6.2.2 0.2 M Dibasic Sodium Phosphate(MW 141.96)Add34.08 g to 1200 mL of deionized water.6.2.3 For working solution,combine 572 mL of 0.2 Mdibasic sodium phosphate with 450 mL of 0.1 M citric acid.6.2.4 Store buffer at 2 to 8C.6.3 Bisbenzamide(H-Stain)Stock Solution:dissolve 5.0mg Hoechst 33258(Calbiochem)in 100.0 ml deionized water.Portion in 0.5 ml amounts and store at 20C.6.4 Bisbenzamide(H-Stain)Working SolutionAdd 0.1 mLof Bisbenzamide stock solution to 100 mL of McIlvanes1This 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 June 2000.Originallypublished as E 1532 93.Last previous edition E 1532 93.2Annual Book of ASTM Standards,Vol 11.05.3The boldface numbers in parentheses refer to the list of references at the end ofthis standard.1Copyright ASTM,100 Barr Harbor Drive,West Conshohocken,PA 19428-2959,United States.citrate-phosphate buffer.Protect from light and use directly.6.5 MountantTo 50 mL McIlvanes citrate-phosphatebuffer,add 50.0 mL glycerol.Mix and dispense in smalldropper bottles.Store at 2 to 8C.7.Staining Procedure7.1 Fix cultures by adding approximately 0.5 mL to 1 mL ofCarnoys fixative to the cell culture dishes containing medium.7.2 Let stand two minutes then aspirate.7.3 Reapply fixative,and let stand five minutes.7.4 Aspirate and air-dry coverslips.7.5 Apply sufficient volume of DNA-stain to immersecoverslip.7.6 Let stand for ten minutes,then aspirate.7.7 Wash with distilled water,then air dr