JeffLawrence(M05)1.SterilizeVelvetSquareswithwidthsatleast50%longerthanthediameterofapetriplate.2.Streakstrainstobeprintedforsinglecolonies.3.PrepareaprintingMasterPlate.PlaceaGridSheetunderthebaseofasterileplate.Typically,thegridscontain24or50squres,butgridbetween9and121squareshavebeenused.Useagridsystemoftheappropriatesizeforthenumberofstrainstobeprinted.4.Pickasinglecolonyofeachstrainandhatchan"X"shapeinthemiddleofagridsquare.Notewhichgridcontainswhatstrain.Make"X"shapesrathersmallsincetheytendtogetlargeronsubsequentlyprintedplates.Includecontrolstrainsonthelastfewgrid-squares.5.GrowtheMasterPlateovernight.6.Preparemediaforprinting.Eachplateshouldbedriedsothatthevelvetisnotexcessivelywetafterprinting.7.PlaceaSterileVelvetfaceuponaprintcolumnandfixthesideswithacollar.Printthemasterplateontothevelvetsurfacepressinglightlybutevenlytotransferallpatchestothevelvet.8.Removethemasterplateandimmediatelycoverthevelvetwiththefirstreplicaplate.Notetheorientationsofallplatesothatthelayoutofthestrainsisknown.Transfercellstothereplicaplatebypressinglightlyandevenlyagainstthevelvet.9.Repeatforallreplicas.TwotoSixreplicascaneasilybemadefromasinglemaster.Uptofifteenreplicashavebeensuccessfullymadewithouttoomuchpatch-spreADIng.10.Toreducecell-debristransfer,printtoaminimal-mediareplicafirst,thendiscardit.Tovastlyreducecell-debristransfer,makeaminimalmediareplicate,thenimmediatelyusethatreplicateasamaster.Printthenewmastertoafreshvelvetandprintasusual.11.Alwaysprintlasttoaplateonwhichallstrainswillgrow,likeLB.Thisensuresthatallstrainswere,infact,transferedtothevelvet.12.Toavoidnutrientcarry-over,printtonutrient-deficientmediafirst,thenprinttonutrient-richmedia.Thiswillpreventcontaminationofthevelvetwithtracenutrients.