ASTM_E_1536_-_00.pdf

Designation:E 1536 00Standard Practice forDetection of Mycoplasma Contamination of Bovine Serumby the Large Volume Method1This standard is issued under the fixed designation E 1536;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 in serum by direct microbiologicalculture.1.2 This practice does not cover procedures used for detec-tion of mycoplasma in cell cultures.1.3 This practice does not cover indirect methods fordetection of mycoplasma contamination.1.4 This practice does not cover methods for identificationof mycoplasma cultures.1.5 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 1532 Practice for the Detection of Mycoplasma Contami-nation of Cell Cultures by the Use of the BisbenzamideDNA-Binding Fluorochrome2E 1533 Practice for Indirect Detection of Mycoplasma inCell Culture by 48-6-Diamidino-2-2 Phenylindole(DAPI)Staining23.Terminology3.1 Definitions:3.1.1 direct mycoplasma detection,ndemonstration ofcharacteristic colonial growth on axenic agar medium.3.1.2 large volume testing,nusing a large volume inocu-lum in an enrichment culture.3.1.3 mycoplasma(Mollicute),nsmallest prokaryotes ca-pable of self replication.4.Significance and Use4.1 Mycoplasmas of bovine origin are prevalent contami-nants of cell cultures.Contamination can be detected by thelarge volume method.3,44.2 Heat inactivated serum need not be tested for mycoplas-mas.Heating serum to 56C for 30 minutes will kill myco-plasmas.4.3 Mycoplasmas may be present in any particular lot ofserum but may not be detected because of inadequate samplesize;thus,negative test results do not provide absolute assur-ance that the test serum is free of mycoplasmas.5.Liquid Medium Preparation5.1 Add 105-g mycoplasma broth base,5-g glucose,5-garginine,and 20 mL of a 0.5%solution of phenol red to 4080mL of distilled water.Mix to dissolve ingredients.5.2 Dispense medium,in 400-mL amounts into 500-mLscrew-capped bottles.5.3 Autoclave.5.4 Sterile refrigerated medium is stable for four months.6.Quality Control6.1 Prior to testing large volumes of bovine serum,checksterility and ability of liquid medium to support mycoplasmagrowth.6.2 Strains used to test for growth support:M.arginini,G230,M.bovis,Donetta;A.laidlawii,PG8.6.3 For quality control,a portion of the base liquid mediumis supplemented with 20%of newborn calf serum.This batchof serum must be extensively tested to ensure that it is free ofmycoplasma contamination and it should be in sufficientquantity to last for an extended period of time.Challengemycoplasma strains for the quality control test should bediluted so that approximately 100 colony-forming units arecontained in the inoculum.7.Test Procedure7.1 The sample is 100 mL of uninactivated bovine or equineserum.Multiple samples will increase the probability ofmycoplasma detection.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 June 2000.Originallypublished as E 1536 93.Last previous edition E 1536 93.2Annual Book of ASTM Standards,Vol 11.05.3Barile,M.F.,and Kern,J.,“Isolation of Mycoplasma arginini from Commer-cial Bovine Sera and Its Implication in Contaminated Cell Cultures,”Proceeding ofthe Society for Experimental Biology and Medicine,138,1971,pp.432437.4Del Giudice,R.A.,Tully,J.G.,“Isolation of Mycoplasmas from Cell Culturesby Axenic Cultivation Techniques,”Molecular and Diagnostic Procedures inMycoplasmology,Joseph G.Tully and Schmuel Razin,Eds.,Academic Press,1996,Vol II,pp.411418.1Copyright ASTM,100 Barr Harbor Drive,West Conshohocken,PA 19428-2959,United States.7.2 Inoculate one 100-mL sample of fetal bovine serum into400 mL of medium,and incubate for 21 days at 37C.7.3 After incubation for 5,10,and 21 days,0.1 mL issubcultured to each of two agar plates(see Practice E 1531).Incubate one plate anaerobically and one plate aerobically.Examine all plates after incubation for 5 and 14 days.7.4 Serum is considered contaminated if typical myco-plasma colonies grow on the agar medium.8.Keywords8.1 mycoplasma;serumThe American Society for Testing and Materials takes no position respecting the validity of any patent rights asserted in connectionwith any i