厌氧培养基配方

Dehalococcoidesmedia(YangandMcCarty,1998,EST,3951-3597)StocksMaketracemetalsstocksolutions(0.5L),autoclaveandstoreinfridgeMakemineralstocksolutions(0.5L),autoclaveandinfridgeMakevitaminstocksolutions(0.5L),filtersterilizeandstoreinfridgeMakeresazurinstocksolution(2g/L),autoclave,storeinfridgeMakesodiumsulfidestock(1.25gNa2Sor3.95gNa2S.9H2OperL)Finalmedia(use2Lbottle)Makebasalmedium(below),makeupto1LwaterPurgewithN2/CO2(80/20%)for40minutesTaketoglovebox,distributetoserumbottles(~50mLtoeach)AutoclaveTaketogloveboxbelowAddfiltersterilizedsodiumsulfide(200uLper50mLsol.)leaveovernight(shouldbeclear)belowSodiumbenzoate1.3mM0.086Sodiumpropionate0.6mM0.125Sodiumbenzoate1.3mM0.086Sodiumpropionate0.6mM0.125gKHPO240.5NaCO231.5Yeastextract0.02BasalMedium(1L)BenzoateorpropionateTOC\o"1-5"\h\zResazurinstock(2g/L),finalconc.Of1mg/L 0.5mlTracemetalsstock 5mLMineralstock 20mLVitaminsstock 1mL1.TraceMetalsStock(X200)gperLgper0.5LAl2(SO4)3.18H2O0.040.02CoCl2.6H2O0.20.1CuCl2.2H2O0.020.01FeCl2.4H2O10.5H3BO30.120.06MnCl2.4H2O10.5Na2MoO4.2H2O0.020.01Na2SeO40.020.01Na2WO4.2H2O0.020.01NiCl2.6H2O0.020.01

ZnCl20.020.01ZnCl20.020.015mLoftracemetalstockto1Lmedia2.MineralsStockSolution(X50)gperLgper0.5LCaCl.2HO2221KCl52.5KHPO2452.5MgCl2.6H2O52.5NaCl4020NH4Cl50253.VitaminStockSolution(X1000)gperLgper0.5LBiotin0.020.01Cyanocolalamine(VitaminB12)0.050.025Folicacid0.020.01Nicotinicacid0.050.025p-aminobenzoicacid0.050.025Pantothenicacis0.050.025Pyridoxine0.10.05Riboflavin0.050.025Thiamine0.050.025Thiocticacid(lipoicacid)0.050.0251mLofvitaminstockto1LmediaRezasurinStockSolution(50mL)Resazurin(2g/L)0.1g add0.5mLto1LmediaResazurin(2g/L)SodiumSulfideStock(50mL)Donotautoclave,storeinglovebox,filtersterilewhenisaddedtomediaNa2S.9H2O 3.95gperLMakeonly50mL(use0.198gNa2S.9H2O)Add200uLto50mLmediaFinalconcofNa2Sinmediashouldbe5mg/LDehalococcoides介质(杨和McCarty,—九九八年EST,39513597)储备液配制微量金属储备液(0.5L)高压灭菌后放在冰箱中存储配制矿物质储备液(0.5L)高压灭菌后放在冰箱中存储配制维生素储备液(0.5L)过滤并灭菌后放在冰箱中存储4•配制resazurin储备液(2g/L)高压灭菌后放在冰箱中存储5•配制硫化钠化储备液(1.25gNaSor3.95gNaS.9HO每L)最终介质（用2L瓶）配制基础介质（下有），再补1L水用N/CO（80/20%）净化40分钟22&在手套箱（就是只能把手伸进去的，透明的，超净的那个东西），分配到血清瓶（50ml每个）高压釜高温灭菌在手套箱中添加5中的储备液到血清瓶中（200uL每50ml溶液）放置一夜（shouldbeclear没看懂，翻译不出来）基础介质(lL)gTOC\o"1-5"\h\zKHPO 0.524NaCO 1.523酵母提取物 0.02苯甲酸或丙酸下面有苯甲酸钠1.3mM0.086丙酸钠0.6mM0.125Resazurin储备液(2g/L)，最终浓度稀释到lmg/L【原文finalcone.Oflmg/L】 0.5ml微量金属储备液5ml矿物质储备液20ml维生素储备液1ml1•微量金属储备液（X200）g/Lg/0.5LAl(SO).l8HO24320.040.02CoCl.6HO220.20.lCuCl.2HO220.020.0lFeCl.4HO22l0.5HBO330.l20.06MnCl.4HO22l0.5NaMoO.2HO2420.020.0lNa2SeO40.020.0lNaWO.2HO2420.020.0lNiCl.6HO220.020.0lZnCl20.020.0l1L介质加5mL微量金属储备液2.矿物质储备液（X50）g/Lg/0.5LCaCl.2HO222lKCl52.5KHPO2452.5MgCl2.6H2O52.5NaCl4020NHCl450253.维生素储备液（X1000）

g/Lg/0.5L维生素H0.020.01维生素B120.050.025叶酸（维生素B）0.020.01烟碱酸0.050.025对氨基苯甲酸0.050.025泛酸0.050.025维生素B60.10.05核黄素0.050.025维生素B10.050.025硫辛酸0.050.0251L介质加1ml维生素储备液Rezasurin储备液(50ml)Resazurin(2g/L)0.