土壤PLFA--测定方法..docVIP

土壤PLFA测定方法 一、原理：Phospholipid fatty acids (PLFAs) are exclusively present in the membranes of all living cells. The PLFA profile of the soil microbial community reflects both species composition and relative species abundance. Phospholipids decompose rapidly on cell death (by hydrolysis of the phosphate group by cellular enzymes) therefore it is assured that only the living cells are studied. PLFA profiles are determined using a modification of the method described by Frostegard et al (1991), as based on the method described by Bligh and Dyer (1952) and White et al (1979). N.B.（注意事项） Avoid excessive exposure to light throughout this method, especially from fluorescent lights, as light degrades phospholipids. (易被光解，故后续操作用锡纸包裹) Glassware should be ion and organic free. Dirty glassware should be immersed in hot water with phosphate detergent (Decon) and rinsed deionised water. The dry glassware can then be either wrapped in aluminum foil and heated at 450oC for 4 hrs or alternatively rinsed in methanol then chloroform and dried. Alternatively use new sterile glass media bottles throughout. The needles of the sample concentrator should be washed in methanol before use. (氮吹仪针头，用前用正己烷擦拭) Fatty Acid Methyyl Esters (FAMEs) can be quantified by using nonadecanoic acid as an internal standard（定量用内标）. A FAME standard supplied by Supelco is used for quality assurance of the GC run. 附表：试验用药品、仪器清单。 准备 ——First Stage (0.5 day) 二、提取1、试剂 0.15 mol/L柠檬酸缓冲液：31.52g柠檬酸溶解于1L去离子水中，用NaOH调pH到4.0； Bligh-Dyer土壤提取液：氯仿：甲醇：柠檬酸缓冲液=1：2：0.8 (体积比), Add 0.005 % w/v (50 mg l-1) butylated hydroxy toluene () as an anti-oxidant，使保存时间增长。 2、步骤 取5.0g 鲜土，冷冻干燥于50ml特氟龙管加入15ml Bligh-Dyer土壤提取液超声30分钟，振荡30分钟（180rpm），离心（3800 rpm）10分钟用Pasteur pipette将上清液转移至50ml干净的玻璃管中（试管需提前用锡纸包好）再取10 ml Bligh-Dyer土壤提取液于特氟龙管中，超声20分钟，振荡10分钟，3800rpm离心10分钟，用Pasteur pipette将上清液转移至试管中（两次上清液混合）在上清液混合的试管中加ml柠檬酸缓冲液和4ml氯仿，涡旋振荡10秒锡纸封口4℃冰箱中静置过夜用长滴管吸出下层液体至10mL螺口管中，用氮气吹干（in a water bath set at 37oC to prevent the break down of unsaturated