ASTM_E_1531_-_00.pdf

Designation:E 1531 00Standard Practice forDetection of Mycoplasma Contamination of Cell Cultures byGrowth on Agarose Medium1This standard is issued under the fixed designation E 1531;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 procedures used for detection ofmycoplasma contamination by direct microbiological culture.1.2 This practice does not cover indirect methods fordetection of mycoplasma such as DNA staining,biochemicaldetection,or genetic probes.1.3 This practice does not cover methods for identificationof mycoplasma organisms.1.4 This practice will not detect cultivar a strains(1)2ofMycoplasma hyorhinis.1.5 This practice is not intended for use in detection ofmycoplasma contamination in sera,culture media,vaccines,orother systems.1.6 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 1532 Practice for Detection of Mycoplasma Contamina-tion of Cell Cultures by the Use of the Bisbenzamide DNABinding Fluorochrome3E 1533 Practice for Indirect Detection of Mycoplasma inCell Culture by 486Diamidino22 Phenylindole(DAPI)Staining3E 1536 Practice for Detection of Mycoplasma Contamina-tion of Bovine Serum by the Large Volume Method33.Terminology3.1 Definitions:3.1.1 direct mycoplasma detection,ndemonstration ofcharacteristic colonial growth on axenic agar medium.3.1.2 indirect detection of mycoplasma,n detection ofmycoplasma by DNA staining or any method other thancultivation.3.1.3 mycoplasma(Mollicute),nsmallest prokaryotes ca-pable of self replication.4.Significance and Use4.1 The demonstration of characteristic colonial growth onaxenic solid medium is a sensitive and specific method todetect mycoplasma infection of cell cultures and it is thestandard detection method(2).4.2 When mycoplasmas contaminate cell cultures they usu-ally grow to high titer(108colony forming units/mL)and wheninoculated onto agar medium they produce abundant and easilydetectable growth(3).4.3 M.hyorhinis cultivar a strains do not grow on conven-tional mycoplasma media(1)but require an indicator cellculture system to detect their presence(see Practice E 1532).Alternatively,a specialized axenic medium is suitable fordirect isolation of cultivar a from infected cell cultures(4).4.4 Immunofluorescent procedures are used to identify my-coplasma isolates(5).5.DM-1 Solid Medium Preparation5.1 Dissolve CMRL-1066 powder(CMRL-1066 powderFormula No.785156EF4,packaged for 10L),in 5000 mL ofdistilled water.This is one-half the volume of water specifiedon the package.Add 47.6 g HEPES5,and 9.35 g NaCl.5.2 Adjust the pH to 7.3 and filter sterilize(450 nm).Storethis 2X CMRL in the refrigerator in 500 mL amounts.5.3 Dissolve 10.0 g of Myosate6and 12 g of agarose7in 400mL of distilled water.Autoclave at 121C for 15 minutes.Coolthe autoclaved solution to about 50C,and combine with 500mL of 2X CMRL and 100 mL of sterile horse serum8(bothingredients also warmed to 50C).5.4 Aseptically dispense medium in 5 mL amounts in petri1This 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 1531 93.Last previous edition E 1531 93.2The boldface numbers in parenthesis refer to the list of references at the end ofthis standard.3Annual Book of ASTM Standards,Vol 11.05.4Available from Life Technologies,Gaithersburg,MD.5Available from Research Organics,Cleveland,OH.6Available from BBL Microbiology Systems,Cockeysville,MD.7Available from SeaPlaque agarose,FMC Bioproducts,Rockland,MA.8Available from Whittaker Bioproducts,Walkersville,MD.1Copyright ASTM,100 Barr Harbor Drive,West Conshohocken,PA 19428-2959,United States.dishes and allow to solidify.Store at 4C and prevent drying.The DM-1 plates have a shelf-life of eight weeks.6.Quality Control6.1 It is important that solid medium used for isolation havehigh plating efficiency;therefore,quality control should in-clude titration of mycoplasma suspensions onto the solidmedium to determine the highest dilution to produce colonies.6.2 Test mycoplasma strains should not be adapted toartificial media.Instead,cell culture grown mycoplasma strainsshould be used.Test strains should include:M.hyorhinis,BTS-7;M.orale,CH 19299;M.pirum,70159;M.arginini,G230;M.fermentans,PG-18;Mycoplasma infected BHK-21cell cultures are stored at 70C.6.3 Prepare test batches of DM-1 medium wit