1.Forrichmedia,weighoutappropriateingredientsandplaceintoaflask.Addwateruntilappropriatevolume.Useaflaskatleast2timeslargerthanthemediavolume.2.Forminimalmedium,makeseparate2XAgarand2XSaltflasks.Forexample,for1literofEplates,make500mlsof2XEsalts,and500mlsof2Xagar(1.5gper500mls).Useatleastoneflasklargeenoughtomixthemediatogetherafterautoclaving.3.Capwithapolypropylenebeakerandautoclavesmallbatches20min;autoclavelargevolumes30-40min.Alsoautoclavea1Literflaskforpouringlargevolumeofmedia.4.Coolto55Conabenchorinawaterbath(37Cinitialtemp).Forminimalmedia,combinethe2Xagarwiththe2Xsaltatthispoint.Iftheyarecombinedtooearly,theagarwillcarmelizeinthehighsaltsolution.5.Addantibioticsandotheradditionsifnecessary.6.Forlargevolumes,transferaportiontothe1literPouringFlask.Flamethetopofthemediatoremovethebubbles.7.Poureachplateto1/2volume,about25-30ml.Thiswillyieldabout35platesperliterofmedia.Ifyouaregetting25platesorfewer,theplatesaretoothick.Ifyouaregettingmorethan40plates,theyaretoothin.Thinkplatesdryoutquicklyandaredifficulttouse.Thickplateswastemediaandarehardtoprint.8.Pourthebottomplateofastackfirst,holdinguptheremainderofthestackwithyourfreehand.Captheplateandpourthenextoneuntilthestackof20iscompleted.Labelthetopplateofeachstack.8.Allowplatestocooluntilsolid.Thecoolerthemediaisuponpouring,thefastertheywillsolidify.9.Transferplatestothe37Cincubatorandincubateovernightuntildry.Fresh,wetplatesdonotabsorbliquidswellandatoowettouseforreplicaplating.10.Transferstacksof20intoplasticsleeves,tapeshut,andstoreat4C.

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