Phosphate-bufferedsaline(PBS)(0.01MNa2HPO4,0.15MNaCl,pH7.4) AdjustthepHto7.4with1NNaOHor1MHCl. DubelccoModifiedEagleMedium(CompleteMedia) with4.5g/lglucose) 1.Add50mlofNCTC-135mediumtoabottleofunopenedDMEMmedia.Mixandtransfer50mltoabottlelabelled"WashingMedia." 2.Add100mloffetalbovineserum,5mlantibioticand5mlfungizonetothe500mlbottleofDMEMmedia.Mix,labelthecontainer"CompleteMedia",andstoreintherefrigerator. WashingMedia (Note:Washingmediaisusuallyaquiredbyremovingmediabeforetheadditionoffetalcalfserum.Fetalcalfserumisexpensiveandnecessaryforcellgrowth.) 70%ethylalcohol Disinfectingsolution(forcleaning) Disinfectingsolution(forfillingwaterpan) HATmedia (Note:someformulationsare50xandshouldbeusedattherateof1ml/50mlmedia) HTmedia (NOTE:someformulationsare50xandshouldbeusedattherateof1ml/50mlmedia)sodiumphosphatedibasic(Na2HPO4) 0.87g sodiumphosphatemonobasic(NaH2PO4xH2O) 0.54g sodiumchloride 8.5g HPLCwater 1liter DMEM(1xliquidtissueculturemedium 70% 500ml NCTC-135medium 10% 50ml Fetalbovineserum 20% 100ml Antibiotic/antimycotic 1% 5ml Fungizone 1% 5ml DMEMmedia,unopened 500ml NCTC-135media 50ml 95%ethylalcohol 370ml Distilledwater 130ml RoccallII 5ml HPLCwater 1liter RoccallII 7.5ml HPLCwater 4liter HATsupplement,500x 0.1ml DMEMCompletemedia 50ml HTsupplement,500x 0.1ml DMEMCompletemedia 50ml
