No.1 扫描电镜样品制备方法
样品在2.5%的戊二醛溶液中4℃固定过夜，然后按下列步骤处理样品：✧倒掉固定液，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min；
✧用1%的锇酸溶液固定样品1-2h；
✧倒掉固定液，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min；
✧用梯度浓度（包括30%,50%，70%，80%，90%和95%五种浓度）的乙
醇溶液对样品进行脱水处理，每种浓度处理15min，再用100%的乙醇处理两次，每次20 min。
✧用乙醇与醋酸异戊酯的混合液（V/V=1/1）处理样品30min，再用纯醋酸
异戊酯处理样品1-2h。
✧临界点干燥。
✧镀膜，观察。
处理好的样品在Hitachi TM-1000型扫描电镜中观察。
1.Double fixation: The specimen was first fixed with
2.5% glutaraldehyde in
phosphate buffer (pH7.0) for more than 4hours; washed three times in the phosphate buffer; then postfixed with 1% OsO4 in phosphate buffer (pH7.0) for 1hour and washed three times in the phosphate buffer.
2.Dehydration: The specimen was first dehydrated by a graded series of
ethanol (30%,50%, 70%, 80%, 90%, 95% and 100%) for about 15 to 20 minutes at each step, transferred to the mixture of alcohol and iso-amyl acetate (v:v=1:1) for about 30 minutes, then transferred to pure iso-amyl acetate for about 1hour. In the end, the specimen was dehydrated in Hitachi Model HCP-2 critical point dryer with liquid CO2.
3.Coating and observation: The dehydrated specimen was coated with
gold-palladium and observed in Philips Model TM-1000 SEM.
No.2 Negative staining of bacterium
The bacterium suspension was stained by 1 to 2%solution of phosphotungstic acid (PTA) in a pH range of 6.5 to 7.0 for 15 to 30 seconds. Then, the bacterium was observed in TEM of Model JEM1230.
No.3透射电镜样品制备方法
样品在2.5%的戊二醛溶液中4℃固定过夜，然后按下列步骤处理样品：✧倒掉固定液，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min；
✧用1%的锇酸溶液固定样品1-2h；
✧倒掉固定液，用0.1M，pH7.0的磷酸缓冲液漂洗样品三次，每次15min；
✧用梯度浓度（包括30%,50%，70%，80%，90%和95%五种浓度）的乙
醇溶液对样品进行脱水处理，每种浓度处理15min，再用100%的乙醇
处理一次，每次20min；最后过度到纯丙酮处理20min。
✧用包埋剂与丙酮的混合液（V/V=1/1）处理样品1h；
✧用包埋剂与丙酮的混合液（V/V=3/1）处理样品3h；
✧纯包埋剂处理样品过夜；
将经过渗透处理的样品包埋起来，70℃加热过夜，即得到包埋好的样品。
样品在Reichert超薄切片机中切片，获得70-90nm的切片，该切片经柠檬酸铅溶液和醋酸双氧铀50%乙醇饱和溶液各染色15min，即可在Hitachi H-7650型透射电镜中观察。
1.Double fixation: The specimen was first fixed with
2.5% glutaraldehyde in phosphate buffer (pH7.0) for more than 4hours; washed three times in the phosphate buffer; then postfixed with 1% OsO4 in phosphate buffer (pH7.0) for 1hour and washed three times in the phosphate buffer.
2.Dehydration: The specimen was first dehydrated by a graded series of ethanol (30%,50%, 70%, 80%, 90%, 95% and 100%) for about 15 to 20 minutes at each step, transferred to absolute acetone for 20 minutes.
3. Infiltration: The specimen was placed in 1:1 mixture of absolute acetone and the final Spurr resin mixture for 1hour at room temperature, then transferred to 1:3 mixture of absolute acetone and the final resin mixture for 3hours and to final Spurr resin mixture for overnight.
4.Embedding and ultrathin sectioning: Specimen was placed in capsules
contained embedding medium and heated at 70℃for about 9hours. The specimen sections were stained by uranyl acetate and alkaline lead citrate for
15 minutes respectively and observed in TEM of Model H-7650.
直接观察的扫描样品
1、样品粘附在样品台上，在Eiko IB5型离子溅射仪中喷镀4-5min。
2、样品在日本Hitachi TM-1000型扫描电镜中观察。
The specimen was coated with gold-palladium in Eiko Model IB5 ion coater for 4-5min and then observed in Hitachi Model TM-1000 SEM.。