生物显微技术实验

生物显微技术实验 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 备注 生物显微技术 学时:32学时，学分:1.5，适用专业:生物工程 一、实验目的与任务 要求学生掌握制片的原理和主要方法，培养学生应用显微技术解决科研、 生产问题的能力。提高学生动手、实践和运用现代科学仪器的能力等综合素质。 二、生物制片的一般原理及方法 植物制片技术是植物显微技术课的一个重要组成部分，是研究植物细胞 学、解剖学、胚胎学的必要技术基础。在有关植物形态建成、植物杂交育种、 作物病虫害防治、药用植物的培育和鉴别以及林木材性鉴定等多方面的研究和 教学工作中，都需要应用显微制片技术，由于各种作物器官的性质差异以及研 究目的的不同，就需要不同的制片方法。 三、植物制片分类 (一)按制片方法可分为二大类: 1、切片法 (1)徒手切片法 指手拿刀片把植物新鲜材料切成薄片，所作的切片通常不经 染色或经简单染色后，制成临时的水装片用于观察。方法简便，要求设备简单， 但徒手切片技术要求熟练，方可切出符合要求的薄片。 (2)石蜡切片法 以石蜡作包埋剂。用旋转切片机将材料切成薄片(一般为8, 12 μm，可以切成连续的蜡带)，经一系列处理制成永存片。便于进行器官的 三维重构。切片厚度比较好掌握。此法多用手摇切片机切片。 (3)半薄切片法 方法步骤与石蜡制片相似，但包埋剂为塑料或环氧树脂，采 用玻璃刀或钢刀，切片厚度为1,2 μm，不能切成连续的蜡带。 (4)冰冻切片法 将待切材料快速冷冻，或固定以后再冷冻，用冰冻切片机切 片，用于做原位杂交、组织化学等。 (5)滑走切片机切片法 是利用滑走切片机切新鲜的或保存的材料，亦可以切 经由石蜡制片法包埋的材料。 将材料夹持于夹持物中(如石蜡、火棉胶、胡 萝卜等，木材软化后可直接切片)，并固定于滑走切片机夹物台座上进行切片， 此法切出的切片厚薄均匀、结构完整，适用于一些比较坚硬的材料。 (6)超薄切片法 用超薄切片机制作超薄切片，切片厚度为500Å 左右，供电 子显微镜观察用，技术要求更严格。 the oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used for other processes 备注 2、非切片法 (1)离析法 用一些化学药品或酶等使细胞间的胞间层溶解，细胞彼此分 离，从而得到单个、完整细胞的方法，便于研究细胞的立体结构。该方法 适用于木材导管、根尖、茎尖及叶表皮细胞及木材、药材的研究。 (2)压片法 经化学药剂处理植物的器官或组织并用手工机械地使植物细 胞分离，涂抹或压在载玻片上，使细胞成一薄层，便于进行观察的一种制 片方法。如染色体压片法。 (3)整体封藏法 将完整细小的组织块进行处理，封藏为永存片，如叶表 皮整体制片、团藻、水绵、黑根霉等装片。 (4)整体透明法 用透明剂将较小的材料整体透明，经透明以后可以在显 微镜下观察其内部结构。 (5)胚囊酶法分离技术 对植物的胚珠进行酶解，将胚珠内部的完整胚囊 分离出来，用于研究胚囊的发育、或者进一步分离出卵细胞。 (二)按是否能长期保存来分，可分为二类: (1) 临时制片法:制片用于临时观察，不需要长期保存(如临时装片、 徒手切片、冰冻切片等)。 (2)永存片制片法:制片可以长期保存(如石蜡切片、半薄切片、超薄切 片等)。 四、教学基本要求 1、要求重点掌握的技术: 石蜡制片技术(包括:取材、固定、脱水、包埋、切片、展片、烤片、染 色、透明与封藏); 2、要求掌握的一般技术: (1) 植物其它切片制片方法(徒手切片法); (2) 非切片制片技术(压片法、涂片法、整体透明法、酶解法等) (3) 摄影及照相机的使用方法; on or cable tray cannot be used for other processesnnecticonstruction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground colengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the cable tion is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify t a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparanot puand design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does hematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume al scsection number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electric verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on thend to eal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction ained the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great d. Combg in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the thirdr pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable layinthe oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling wate1 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 备 注 实验一 生物制片样品的选取与固定技术 (2 h) 一、实验目的要求 了解生物制片样品的选材与固定技术，为石蜡切片做准备。 二、实验原理 1、选材与切割:根据不同制片目的和制片方法选取合适的材料;材料要求新鲜， 无病虫害;先作徒手切片或剥离检查，决定适宜的材料立即固定处理;材料 大小要适当，如根的直径在5 mm 以内的，可切取5-10 mm 长的小段，直径 在5 mm 以上者可纵分割成1,2 或1,4，横切厚度约3-5 mm;叶的切割可切 成2-5 mm，若叶较宽可切10 mm 的长条，叶面切取的部位，以研究者的目的 灵活选取。切取材料必须使用新刀片，刀口和叶面垂直迅速切割下来;花、 果的切割:花药和雌蕊切割，花芽和幼穗的切割，须剥去外部鳞片及叶鞘和 其它部分，切去多余部分再放入固定液，有的花序则需去掉外方苞片，一般 小的花不经切割可投入固定掖，大花果才进行分割处理。 2、杀生(杀死):在制片过程中，将拟制片的材料(细胞或组织块)迅速杀死， 使组织块尽量保持原生活时状态，这种处理通称杀生。杀生时常用一种或多 种化学药品处理。一般以渗透力较强的药剂作杀生剂效果比较好。 3、固定:在杀生处理的过程中，将器官、组织、细胞按原来的形态保存下来， 并为后续制片保持固有形态，这样才达到固定的要求。杀生与固定关系密切， 通常二者兼有作用。 4、保存:组织块经杀生固定后，能较长时间保存下来，经久保存下来的组织块， 仍能制片，如酒精、醋酸，福尔马林液保存时间较长;而铬酸类固定液，常 须转换70 %酒精液后保存，才可较长时间保存。 三、实验材料与试剂 材料:南瓜茎或禾木植物的茎 试剂: 1、F.A.A 固定液(福尔马林,冰醋酸,酒精) (1)福尔马林(38 %甲醛) 5 mL(2)冰醋酸 5 mL (3)70 %酒精 90 mL 2、F.P.A 固定液(福尔马林,丙酸,酒精)制片效果有时较F.A.A 好。 (1)福尔马林(38 %甲醛) 5 mL (2)丙酸 5 mL (3)70 %酒精 90 mL 3、Canoys Fluid (卡诺氏固定液) (1)纯酒精(或95 ,酒精) 15 mL (2)冰醋酸 5 mL re affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shounclosuhe time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil levr other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectrild be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use e2 四、实验操作步骤 1、取样:用锋利刀片将南瓜茎切成小段，用作横切的切成0.8 cm长，用作纵切 的切成1 cm长，并切去相对两侧少许，最好选择髓腔较大的样品纵切为两半， 髓腔较小的样品不用纵切; 2、固定:用FAA固定24 h，抽气，除去材料中的气体，并保存于固定液中; 3、软化:先经70 %、50 %、30 %酒精及降至蒸馏水(每次瓶装1/3试剂)分别处 理2-4 h后，置于15 %氢氟酸中软化10-15 d备用。氢氟酸可将硅化物软化去 除南瓜茎表面的硅化物颗粒，便于制片。 on or cable tray cannot be used for other processesnnecticonstruction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground colengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the cable tion is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify t a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparanot puand design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does hematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume al scsection number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electric verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on thend to eal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction ained the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great d. Combg in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the thirdr pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable layinthe oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling wate3 思 思考作业: 考 1、 生物制片如何选择材料, 题 2、杀生和固定有何意义, 与 参 参考文献: 考 1、王灶安(植物显微技术(北京:农业出版社，1992 文 2、李和平(植物显微技术(华中农业大学，2006电子版 献 3、李正理(植物组织制片学(北京:北京大学出版社，1996 re affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shounclosuhe time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil levr other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectrild be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use e2 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 备 注 实验二 植物显微化学制片 (2 h) 一、实验目的要求 掌握植物徒手切片的制作技术与方法。利用临时装片进行显微化学鉴定，掌握常用植物显微化学实验的原理与方法。 二、实验原理 植物显微化学:将材料以徒手切片方法制成薄片，应用化学反应或染色与化学反应相结合的方法，将组织或细胞内的某种化学成分(淀粉粒、蛋白质、脂肪、果胶质、纤维素及木质素等)在组织切片内予以显示。用于研究和测定植物的器官、组织及细胞中内含物的存在、化学性质、含量和分布，简便迅速的方法，是野外调查和室内鉴定的极好方法。 三、实验材料与试剂 材料:土豆 试剂:碘-碘化钾溶液: (1)碘化钾 2 g;(2) 碘 1 g;(3)蒸馏水 300 mL 配法:先将碘化钾加热溶于5 mL 蒸馏水中，然后加入碘，待其溶化后再将溶液稀释至300 mL。 四、实验操作步骤 1、制作土豆临时装片:徒手切片法制作 (1)将材料切成长约2,3厘米的小段，削平切面。 (2)左手的三个指头拿住材料，使之固定不动摇。为防止刀伤，材料上端应超 出手指2,3 mm，不可高出过多，否则切片时材料容易动摇，也不容易切薄。 (3)右手大拇指和食指捏住刀片的右下角(刀片要非常锋利，双面刀片必须是 新用的)，刀口向内，并与材料切面平行，切片前先将材料和刀口上蘸些水， 使之切时滑润。 lengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the cable tion is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify t a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparanot puand design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does hematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume al scsection number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electric verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on thend to eal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction ained the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great d. Combg in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the thirdr pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable layinthe oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling wateon or cable tray cannot be used for other processesnnecticonstruction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground co3 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 (4)切的方法，以刀口自外侧左前方向内侧右后方拉切，这样可以边切边看清 切片的进展情况，同时切起来也比较顺手。切片时只用臂力而不要用腕力及 握刀指关节的力量，左手保持不动，不要两手同时拉动，两手不要紧靠身体 或压在桌子上，并且动作要敏捷，材料要一次切下，切忌中途停顿或推前拖 后作“拉锯”式切割。关键是要切得薄而平。如此连续切片，切下数片后，用 湿毛笔将切片轻轻移入培养皿的清水中备用。 (5)在切片过程中刀口和材料要不断蘸水，以保持刀口锋利和避免材料失水变 形。所切的材料和刀片一定要保持水平方向，不要切斜，如果切斜，即使很 薄，但由于细胞切面偏斜，同样影响观察。 (6)切下足够多的切片后，挑选薄而平的切片做成临时装片供镜检。挑选切片 时，不一定要材料切得很完整，只要切得很薄就行，有时只要有一小部分就 可以看清其结构了。因此切下的切片不是每一片都适用，也不是只有全面切 得薄的才能用，要根据需要进行选择，一次可多选几片置于载玻片上，制成 临时装片，通过镜检再进一步选择理想的材料用以观察。 2、染色:I-KI溶液染色，取一滴配好的溶液滴在切片材料上染色30 S，水洗; 2 3、观察:盖上盖玻片置显微镜下观察，可见细胞内含许多被染成蓝色的卵圆形 或椭圆形颗粒，即为淀粉粒。于高倍镜下观察，可见马铃薯淀粉粒依脐点和 轮纹不同有单粒、复粒和半复粒三种类型。 (1)单粒淀粉(图A):每粒淀粉有一个脐点，围绕脐点有许多同心环，即轮纹。 (2)复粒淀粉(图B-D):每粒淀粉有二个或二个以上的脐点和各自的轮纹，而 无共同的轮纹层。 (3)半复粒淀粉(图E):每粒淀粉具有二个或二个以上的脐点和各自少数的轮 纹，还有共同的轮纹层。 思考作业: 思 1、碘液染色时间稍长(30 s以上)出现黄色为何物质, 考 2、绘制马铃薯三种类型的淀粉粒图，并引线注明。 作 业 参考文献: 与 1、王灶安(植物显微技术(北京:农业出版社，1992 参 考 2、李和平(植物显微技术(华中农业大学，2006电子版 文 3、李正理(植物组织制片学(北京:北京大学出版社，1996 献 r other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectrild be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use ere affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shounclosuhe time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil lev4 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 备 实验三 生物样品的非切片制片法,木材解离制片 注 一、实验目的要求 掌握生物样品的非切片制片法,木材解离制片原理与方法，并观察木质部各组成细胞的形态结构。 二、实验原理 不经过切片而是以机械或药物将一部分细胞(或组织)或各个细胞分开的制片法称非切片制片，如用机械法分离叶表皮制片;用药物解离根尖、茎尖、木材等。 三、实验材料与试剂 材料:木材 试剂:浓硝酸 四、实验操作步骤 1、解离 将被检查的木质部切成火柴梗粗细，长约1-2 cm 的小段。解离方法常 有两种，任意选择。 (1) 将切好的材料置于试管或烧杯中，加浓硝酸使材料浸没，再加入数小粒氯化 钾，水浴(或酒精灯)上加热至有气泡发生并使材料变白为止(为了安全需在 排气柜内进行)。此法解离效果较好。 (2) 将切好的材料放于培养皿中，加入过氧化氢一冰醋酸液(以过氧化氢1 份+ 冰醋酸1份配制)密闭后，放置25-30 ?温箱(台)中，解离1,2 小时，待 解离适当转入下一步。该法解离效果欠佳。 2、水洗:用尼龙纱网滤去酸液，再换水浸泡数次，尽量除去酸液。 3(保存:用玻璃棒捣散组织，转入离心管中进行离心处理，或静置沉淀后，吸 去上清液，另加入50 %酒精保存备用。 4(染色:取少量木质部解离组织，置1 %番红液染5 min左右。 5(水洗:水洗数次去浮色。(至少3次) 6(粘片:用玻棒蘸取含有木质部细胞的水液，均匀涂布载玻片上，转置36 ?温箱中烘干。 7(封片:二甲苯过一遍，加拿大树胶封片(可不做)。 8、观察:解离细胞呈深红色。 on or cable tray cannot be used for other processesnnecticonstruction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground colengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the cable tion is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify t a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparanot puand design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does hematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume al scsection number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electric verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on thend to eal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction ained the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great d. Combg in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the thirdr pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable layinthe oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling wate5 (茎)缘孔纹导管 阶纹导管 (茎)网纹导管 思 考 作 业 与 参 考 木质部发达的螺纹导管 木质部发达的缘孔纹、螺纹导管 文 献 思考题与作业: 绘图并描述导管细胞的形态。 单孔导管 参考文献 1、王灶安(植物显微技术(北京:农业出版社，1992 2、李和平(植物显微技术(华中农业大学，2006电子版 3、李正理(植物组织制片学(北京:北京大学出版社，1996 r other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectrild be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use ere affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shounclosuhe time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil lev6 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 备 注 实验四 石蜡法制片——样品的脱水、透明与浸蜡(8 h) 一、实验目的要求 掌握石蜡制片中脱水、透明和包埋的主要原理与方法。了解各种透明剂的特点，了解实验中出现问题的原因。 二、实验原理 1、脱水:用的脱水剂去除组织内的水分，并使其组织细胞变硬;从而使溶解的石蜡顺利进入细胞内，以便进行石蜡切片。常用脱水剂有:乙醇(酒精)、二氧六环、正丁醇、叔丁醇、丙酮等药剂。 2、透明:是制片过程中的基础工作，材料经各级酒精脱水以后，要经过常用的有机溶剂进行透明，这样便于包埋剂(石蜡)及封藏剂(加拿大树胶)进入和溶合。只有当组织材料全为透明剂所占，并在显微镜下观察呈透明状态时，这种材料才符合制片要求，故透明是制片的重要环节。常用的透明剂有氯仿，二甲苯等。 3、浸蜡:在装有材料的纯氯仿瓶中，分次逐步加入纯石蜡蜡片，以石蜡透入细胞组织内的全过程，称为浸蜡。浸蜡完全的材料，组织块显得十分透明。 三、实验材料与试剂 材料:氢氟酸处理的南瓜茎 试剂: 1、苯胺番红酸性染料溶液 (1)苯胺蓝 1g (2)85 %或95 %酒精 100 mL 2、无水及不同浓度的酒精溶液 3、纯氯仿 4、石蜡:软材料用54,56 ?熔点的石蜡，若切5 μm 以下的厚度或炎热夏季 切片时，宜用58,60 ?的石蜡。石蜡商标上须注明为“生物组织切片用蜡” 字样，选用时还必须检查石蜡细致度、无气泡、无灰尘、无透明点痕、切 片时不断裂、不碎等优点。工业用蜡不能使用。 新石蜡和用后已切碎的石蜡，都必须经过先熔化，然后用3,4 层桑 皮纸，折叠成漏斗形，进行过滤除去渣滓。 50 %石蜡:一半石蜡和一半二甲苯配制而成; 70 %石蜡:70 %石蜡与30 %二甲苯配成; on or cable tray cannot be used for other processesnnecticonstruction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground colengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the cable tion is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify t a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparanot puand design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does hematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume al scsection number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electric verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on thend to eal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction ained the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great d. Combg in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the thirdr pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable layinthe oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling wate7 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 四、实验操作步骤 1、整染:将氢氟酸处理(13 d)后的南瓜茎取出，用流水冲洗20 h，蒸馏水浸 泡洗涤3次，每次2 h，然后经苯胺番红整染48 h。 2、多级酒精脱水:经30 %、50 %、70 %、85 %、95 %及无水酒精处理各2 h。 3、多级氯仿透明:经1/5、2/5、3/5、4/5及纯氯仿通明各2 h。 4、浸蜡:经50 %、70 %及纯蜡A、B置恒温箱中浸蜡各2 h。包埋时作横切的样 品竖放，作纵切的样品卧放。 附步骤及过程表 思考题与作业 思 脱水、透明对材料的浸蜡与包埋有何影响, 考 作 业 参考文献 与 参 1、王灶安(植物显微技术(北京:农业出版社，1992 考 2、李和平(植物显微技术(华中农业大学，2006电子版 文 献 3、李正理(植物组织制片学(北京:北京大学出版社，1996 r other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectrild be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use ere affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shounclosuhe time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil lev8 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 实验四 脱水、透明与浸蜡步骤及过程表 天 数 时 间 步 骤 第一天 实验课内 取流水冲尽酸液(自来水冲20 h)，换蒸馏水冲6 h，再用 苯胺番红整染48 h。 第三天 7:30 30 % 9:40 50 % 11:40 70 % 酒精完全浸没材料超过1,2 cm 15:40 85 % 19:40 95 % 第四天 7:30 无水酒精 9:40 无水酒精 最好3次，最后1次划格子，共划5格 11:40 1/5 氯仿 15:40 2/5 氯仿 换软木塞，每次吸出一格，加入一格纯氯仿 19:40 3/5 氯仿 第五天 7:30 4/5氯仿 同上 9:40 纯氯仿 全部吸出后，再加纯氯仿 11:40 纯氯仿再换1次纯氯仿 15:40 碎蜡 第一次加少量碎蜡 19:40 碎蜡 第二次加溶液体积的20 % 36? 恒温 第六天 7:30 50 %蜡 40,42 ? 9:40 70 %蜡 48,50 ? 11:40 纯蜡A 再换橡胶塞 15:40 纯蜡B 56,58 ? 19:40 纯蜡C 63 ?石蜡溶化为止 第七天 课内完成 包埋 tion is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify t a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparanot puand design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does hematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume al scsection number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electric verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on thend to eal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction ained the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great d. Combg in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the thirdr pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable layinthe oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling wateon or cable tray cannot be used for other processesnnecticonstruction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground colengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the cable 9 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 附 一、几种常用的脱水剂及脱水方法 录 1、乙醇(酒精) (ethyl alcohol) 乙醇是常用的脱水剂，但因易使组织收缩 及发硬，故不是理想的脱水剂。在配制脱水剂时，用95 %酒精配制为15 %、30 %、 50 %、70 %、85 %等各种浓度的酒精。由低浓度到高浓度按级过渡脱水过程。 一般材料从30 %酒精开始，细胞学常以15 %酒精开始，有时用与固定液同浓度 的酒精开始脱水。低浓度中不能停留时间太久，否则材料易吸水膨胀，高浓度 (在95 %无水酒精内)中也不能停留时间过长，否则材料易脆化收缩。为脱水 彻底，在无水酒精中应两次，如果材料转入二甲苯中，有乳白悬浮液出现时， 说明脱水不彻底，应退回无水酒精中继续脱水，在脱水进行中，应避免在无水 酒精内过夜处理。 2、二氧六环(dioxan) (CH)4O 又名二氧杂环己烷、氧化二乙烯。为22 无色液体，易挥发燃烧，有毒性化合物，不适宜学生实验用，因其毒气是无臭 的，吸入后，毒性有积累作用，故开瓶后须十分小心，勿使毒气扩散出。二氧 六环与水及酒精、油类以任何比例混合，其优点:?兼有脱水和透明作用，不 会使组织硬化和收缩，在脱水时，以逐级二氧六环脱水至纯二氧六环后进入石 蜡，但其比重大于石蜡，故在进入石蜡前，通过一次氯仿处理驱赶尽二氧六环 后再转入浸蜡为好。?固定液中含有苦味酸的，不必多冲洗即可转入二氧六环 中。?大的材料在浸入二氧六环前，先浸入苯胺油，其结果更好。?重铬酸钾 在二氧六环中不溶解，可在材料中成黄色的染色剂。?浸入冷的二氧六环中， 放于冰冻切片机的冻盘上，可冰冻切片。 3、正丁醇(normal butyl alcohol) 此剂可与水、酒精、二甲苯等剂混合， 也可单独或与酒精配级混合使用，是常用的脱水剂。为黄色液体，易挥发，吸 入后有人会产生头痛，有人则无反应。其优点:不会使组织收缩或变硬，能溶 解石蜡，可不经透明剂直接浸蜡，脱水时，用正丁醇和酒精配成一定比例，逐 级处理至纯正丁醇后，再转移至正丁醇与石蜡等量 混合液中，最后移入包埋蜡杯中，完成包埋工作。 r other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectrild be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use ere affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shounclosuhe time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil lev10 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 附 4、叔丁醇(Tertiary butyl alcohol) (简称TBA) 是较正丁醇更好的一种录 脱水剂，可单独用或与酒精混合用;其优点:为无毒性、不会使组织收缩和变 脆，还可不经过透明剂，其比重轻，易从石蜡中除去，故可简化脱水、透明、 浸蜡等步骤;因其价格较贵，不宜一般实验用。熔点为25?。具体脱水级别与 配法如下(单位:mL): 浓度级别 1 2 3 4 5 6 7 8 9 10 11 95 %酒精 5 11 18 30 40 50 50 40 25 0 0 蒸馏水 95 89 83 70 50 30 15 5 0 0 0 TBA(叔丁醇) 0 0 0 0 10 20 35 55 75 100 100 相当于酒精(%) 5 11 18 30 50 70 85 95 100 100 100 1) 材料经固定水冲洗后，先由5 %浓度酒精开始脱水至50 %酒精(每级经1 h)。 2)到第六级时转入欲配制的TBA 级别(此时处理时间为一夜)。 3)7 级至9 级又为1 h。 4)10-11 级为12-24 h;从11 级即可加碎蜡进行透蜡处理。 5、丙酮(Acetone) 可代替酒精作脱水剂，脱水能力强于酒精，但对组织 收缩力较大，能使蛋白质沉淀，组织硬化。与水、醚、酒精、氯仿、苯等能任 意混合，但不能溶解树脂、石蜡，所以仍需经过透明剂处理后才能包埋。 以上脱水剂各有其优缺点，但使用最多的仍为酒精。 6、甘油(Glycerin) 是藻类、菌类、高等植物的原丝体、原叶体等良好 的脱水剂。使用时较少收缩现象，甘油脱水前，必须冲洗干净固定液，以免影 响包埋、染色等处理。脱水级差从5 %开始，到纯甘油。如发现水仍未脱尽，临 时以正丁醇补充脱水一次。 二、几种常用的透明剂及透明方法 透明剂都是石蜡的溶剂，不能与水相混。现介绍以下透明剂。 1、二甲苯(xylol) 为较其他透明剂价格便宜，应用普遍的一种无色透明液体， 能与酒精混合，也可溶解石蜡，并能与树胶混合成封藏剂，但不溶于水， on or cable tray cannot be used for other processesnnecticonstruction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground colengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the cable tion is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify t a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparanot puand design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does hematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume al scsection number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electric verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on thend to eal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction ained the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great d. Combg in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the thirdr pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable layinthe oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling wate11 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 附 透明力强、速度快;其缺点是:使组织收缩变脆，故在二甲苯中不宜停留录 时间过长，同时必须在材料完全脱水后方可使用二甲苯，若有极少量水分在组 织内存在，就会出现乳雾状现象，通常在无水酒精与二甲苯之间，增添1,2 无 水酒精和1,2 二甲苯的混合液，以便脱水彻底又可使材料减少收缩。装二甲苯 的器具盖，要随手关闭，以免挥发污染室间空气。 2、氯仿(chloroform) 为常用的透明剂，能与酒精混合，溶解石蜡，在石 蜡制片中，浸蜡前的透明多采用氯仿，本编者习惯使用此剂。其优点是:?组 织在氯仿中浸久虽有收缩，但不强烈;?易挥发，如浸蜡时渗入组织中的氯仿 经蜡温易驱赶出去。不足之处是:渗透力稍弱(如比二甲苯、苯透明慢)，可延长 透明时间便于工作安排，另外，氯仿能退色，一般染色后的切片，不以氯仿处 理。一般用三级氯仿透明，若材料柔嫩可以增加到五级透明处理。 3、冬青油(wintergreen oil) 即水杨酸甲酯(Methyl salicylate)，又称冬 绿油。可作整体制片的透明剂，效果较好，对维管系统的制作透明也很理想， 但其渗透力很慢，且具毒性，用时要小心。 4、丁香油(clove oil) 为切片染色后封固前最好的透明剂;有的染料，如 固绿、桔红G 可以丁香油配成饱和溶液，有作染色、分色、透明三步合并应用 效果。另能溶于酒精及氯仿，可用95,酒精混合使用，而二甲苯必须用纯酒精 混合;丁香油的透明力比二甲苯效果好。其缺点是易使组织变脆，且经丁香油 透明的切片，需经二甲苯复处理，染色的部分才鲜明，封片后干燥时间较二甲 苯长。 5、香柏油(cedar oil) 为无色或绿色的芳香挥发性油，极毒，用时必须小 心。纯香柏油常用于油镜使用上，使用此油作透明剂，不易使材料收缩变硬， 作用慢，一般透明时间在10 h以上，且不易为石蜡代替。如用石蜡制片，仍须通 过二甲苯一次，以除尽香柏油才利于石蜡透入组织内。 6、苯(benzene) 苯的透明作用与二甲苯相似，用法相同，且收缩较少，但易 挥发、爆炸，吸入苯后会引起中毒，尽量少用。 7、甲苯(toluene) 甲苯的性能与二甲苯相同，可作二甲苯的代用品;其透明力慢， 组织浸置甲苯中10 h以上也不变脆。 r other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectrild be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use ere affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shounclosuhe time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil lev12 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 备 注 实验五 细胞组织分离制片法 (4 h) 一、实验目的要求 掌握组织分离制片原理与方法，并观察表皮细胞的形状及气孔的构造。学习数码显微照相技术。 二、实验原理 以机械或药物将一部分细胞(或组织)或各个细胞分开的制片法，如用机械法分离叶表皮制片。 三、实验材料与试剂 器材:生物显微镜、滴瓶、镊子、大方滤纸、培养皿、毛笔、染色缸、载玻片、剪刀、 材料:蚕豆叶 试剂:1 %番红水溶液;1 %龙胆紫;无水乙醇;盐酸;二甲苯;FAA固定液 四、实验操作步骤 1、取样:采新鲜无病斑的蚕豆叶或鸭陌草叶，剪去两端及两侧的叶缘一小部分。 2、固定:用FAA固定材料24 h并保存其中。 3、取表皮:用镊子将已固定的蚕豆叶下表皮轻轻撕下一块，置培养皿的水中，然后用纸(薄道林纸或普通纸张)捞起叶表皮，使其平展于纸上，再用剪刀将纸和表皮剪成3×3 mm的小块，将此小块转移至另一洁净的培养皿水中，待纸与表皮分离，镊出纸块，留表皮于培养皿中。 4、制片:在载玻片上加1滴蒸馏水，用毛笔挑取一块表皮至水滴中展平，倒去水，待表皮干燥。 5、染色:1 %番红水溶液(或1 %龙胆紫)染色1-5 min。 6、水洗:用蒸馏水洗数次，去浮色。 7、分色:在低倍镜下观察，用盐酸分色(去除浮色)至适度(番红染色可深点)。 8、水洗:自来水洗数次，洗净酸液，再用蒸馏水过一遍，终止分色。 9、封片:经无水酒精脱水，3次二甲苯透明，用棉布擦干净。 10、封藏:在材料上滴一滴二甲苯，再将加拿大树胶滴于其上，用载玻片沿着胶慢慢压下封片。 11、观察:在显微镜下观察，找到目标视野。 12、拍照:将数码相机调到微距离拍照档，在自动拍照状态下，照下显微镜中的目标图象。 on or cable tray cannot be used for other processesnnecticonstruction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground colengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the cable tion is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify t a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparanot puand design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does hematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume al scsection number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electric verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on thend to eal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction ained the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great d. Combg in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the thirdr pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable layinthe oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling wate13 叶片表皮的 双子叶植物 气孔分布 思 考 作 业 与 参 思考题与作业 考 文 献 1、每人制作一张永久片; 2、将观察结果绘图，并表明各部位的名称。 参考文献 1、王灶安(植物显微技术(北京:农业出版社，1992 2、李和平(植物显微技术(华中农业大学，2006电子版 3、李正理(植物组织制片学(北京:北京大学出版社，1996 r other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectrild be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use ere affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shounclosuhe time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil lev14 备 注 实验六 石蜡法制片——包埋 (2 h) 一、实验目的要求 掌握石蜡制片中包埋的主要原理与方法。了解各种透明剂的特点，了解实验中出现问题的原因。 二、实验原理 包埋:浸蜡完全的组织和熔解的石蜡，倒入蜡块盒，随之快速冷却定形成蜡块的过程。常用的包埋剂有:石蜡和火棉胶。 三、实验材料与试剂 材料:浸蜡的南瓜茎 试剂:纯蜡A、B、C 四、实验操作步骤 1、将电炉调节使蜡熔化达到 63-64 ?，保持其温度，若温度太高，即将蜡锅移出电炉，转入下步骤。 2、从温箱中取出纯蜡C 杯，置于金属温台上。 3、将纸片浸水润湿，放于玻璃块上，平放桌上(或水泥台上)，再放一个包埋 纸盒于湿纸上。用蜡勺取熔解石蜡转入包埋纸盒内，再从蜡杯C 中取出材料， 随之转入有溶蜡的纸盒中，并将样品整齐排放纸盒内。此时较敏捷地用镊尖 拨动材料，彼此间留一定距离，这样才便于切割成小蜡块安放切片机上切削。 4、当上述手续完毕时，盒内的蜡面渐渐呈薄膜凝固面，此时可用手拿起玻璃片， 先平放于脸盆水中，当进入脸盆后，玻璃较重便自行与纸片离开，纸盒也随 着漂浮水面，这时不要搅动水或翻动蜡块，以免蜡块内的熔蜡流出，造成蜡 块厚薄不均匀或蜡块内材料移动。 5、使用过的玻璃片可以反复使用。 (1)已冷却凝固的蜡盒，可从水中捞出，盒底向上，纸干后剥去盒纸，放入已 编号的纸袋中备用。 (2)包埋完毕后的用品，要及时整理清顺，集中存放下次再用。 he time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil levr other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectrild be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use ere affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shounclosu0 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 思考题与作业 思 材料包埋注意事项, 考 作 业 与 参考文献 参 1、王灶安(植物显微技术(北京:农业出版社，1992 考 文 2、李和平(植物显微技术(华中农业大学，2006电子版 献 3、李正理(植物组织制片学(北京:北京大学出版社，1996 on or cable tray cannot be used for other processesnnecticonstruction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground colengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the cable tion is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify t a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparanot puand design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does hematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume al scsection number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electric verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on thend to eal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction ained the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great d. Combg in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the thirdr pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable layinthe oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling wate1 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 附 三、包埋剂 录 1、石蜡 是石油中提炼出来的多种碳氢化合物，生物制片所用的石蜡为专用石蜡。石蜡产品按熔点度而分规格。熔点高的较硬，低熔点的则软。硬石蜡对切成蜡带不利，太软的则易使组织材料蜡带皱缩，展片时带来困难。 选用石蜡的原则:夏天室温高，应采用熔点高的(如58-60 ?)，冬天室温低，应采用熔点低的;如材料硬或拟切薄的切片应选用硬的熔点高的，厚的切片则采用低的。常用回收的石蜡较新蜡要好用，但要过滤(以桑皮四层叠成漏斗形进行过滤后再用)。 2、火棉胶 为易着火的一种硝化纤维，但不易爆炸。常用于材料质地坚硬、易断折或过软的组织块，用火棉胶包埋效果良好。不足处，价格高，操作时间久不能制作连续切片。常用浓度为2 %、4 %、6 %、8 %、10 %数种。购买火棉胶常见有透明固体和液体两类规格(能溶于丁香油、纯酒精、丙酮及乙醚中，上述浓度可按需要配制) he time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil levr other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectrild be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use ere affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shounclosu2 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 备 (5)开启切片机总开关，试摇切片机手轮(顺时针方向摇动)，左手持毛笔，注 实验七 石蜡法制片——切片、粘片及展片(5 h) 每摇动一圈，就切下一个蜡片，第一片常贴在刀刃上，当第二片切下时，第 一片与第二片的边缘即连在一起，连续摇动手轮，连续蜡带就形成。当蜡带一、实验目的要求 了解切片机的使用方法，掌握切片、粘片及展片技术。 连成一定长度后，用左手的毛笔将蜡带轻轻抬起(尽量避免蜡带与刀面接二、实验原理 触)，边摇边移动蜡带，待蜡带切下长达20-30 cm 时，停止摇手轮，用右 手执另一毛笔顺刀刃轻轻取下蜡带，两毛笔托着整个蜡带平放于切片机左侧1、切片:将包埋好的材料利用切片机(回转式切片机或滑走切片机)切成薄片 (蜡带)。 的蜡带盒内。使蜡带靠刀的光滑面向下，无光泽面向上;在粘片时，蜡带在 2、粘片与展片:是将切下的蜡带分割成适宜的片段，用粘贴剂(蛋清甘油或明载玻片上放置，也按光面向下(即紧贴玻璃面)的位置贴放。 (6)用手持扩大镜先逐一检查蜡带，选出合适的材料，以圆刃解剖刀切断蜡带，胶甘油)贴于载玻片上的过程。粘片时必须注意蜡带伸展平整，故粘片与展 片都是在载玻片上同时完成的。 保存在蜡带盒内以备粘片。 三、实验材料与试剂 (7)锁紧切片机总开关，进一步镜检切下蜡带是否适用。台木上材料不符合要 求时，剩下的蜡块不必再切，另换上材料继续切片，这样可保护切片刀刃锋器材:轮转式切片机、生物组织摊烤片机、展片台、台木、载玻片、毛笔、解剖 刀、镊子、放大镜、蜡铲 利。 试剂:蛋清甘油注意:在进行切片操作时，要耐心细致，不要对着蜡带讲话，以免气体 吹走蜡带，切片过程要随手注意关锁总开关钮，以防止手轮下降伤害人的手四、实验操作步骤 1、分割:将桌上垫一层纸，然后放置一小木板，将石蜡块分割成小蜡块。每个指和损坏材料或刀刃。 小样品四周留3-5 mm 的蜡边进行整修，每个蜡边与材料尽量呈平行线，上窄4、粘片及展片:每片载玻片粘一片蜡片，展片后在36?温箱中烘干。 (1)将展片台通电，调节恒温约35?左右; 下宽，横切片厚15 μm，纵切片厚20 μm，削好的样品，装入已编号的纸袋中(2)备用。蘸滴极少量蛋清甘油于载玻片的中央，以 洗净的右手无名指来回涂沫均匀。2、固着:每蘸一次蛋清甘油，可涂用烫蜡铲将小蜡块紧紧地4-5 张载玻片固着在台木上，; 再进一步修整呈上窄下宽的(3)将已涂蛋清甘油的载玻片平放于展片台上，并加数滴蒸馏水于载玻片上(水倾斜面。有些坚硬的材料在切片前，需将材料切面微露出(没有蜡包被着)， 呈均匀散开。若载玻片上蘸水为聚集状，说明玻片不干净，必须另调换清洁将露出的面朝向有水的培养皿中浸泡半天到一天再安装到切片机上切削，这 玻片)。如用明胶甘油作粘贴剂，则改用样能使组织软化，切面达到光滑不碎裂、无刀痕。在夏季切片时，常用小冰3 %-4 %福尔马林液作伸展蜡带用。 (4)将已选择的小蜡片，用解剖刀的刀尖蘸水(或用镊子)将蜡片转移至有水块在蜡块面上冰冻几秒钟后，再进行切片效果较好。 3、切片:先选择好刀片。的载片中部，当蜡片受热完全伸展看不到有任何皱纹时为止。在展片过程中， (1要随时注意观察伸展情况，并用解剖刀(或镊子)拨正材料正确的位置，另)装紧蜡块固定锁紧扳手，并调节好角度与距离; 外要注意温度，不使温度过高而溶化蜡片，或造成蜡片散乱，影响染色及封(2)安装好切片刀，当倾角为15?左右时，用固定螺旋扭紧; (3藏)先松开刀架固定钮移动刀架，同时从侧面观看，当切片刀刀刃与台木上的; 蜡面较平行且接近时进行固定，最后重新检查其它固定螺旋是否紧固。 (4)调整切片厚度控制盘的刻度，一般切片厚度约在8-12 μm 间。 t a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparanot puand design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does hematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume al scsection number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electric verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on thend to eal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction ained the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great d. Combg in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the thirdr pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable layinthe oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling wateon or cable tray cannot be used for other processesnnecticonstruction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground colengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the cable tion is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify 3 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 (5)从展片台上取下已展片的载玻片，略为倾斜使多余水分流入培养皿中，同 时对材料的位置作最后调整定位;材料无破损、内容较好、伸展平直、无任 何纤维丝和渣滓的切片为适合标准的，否则丢弃。 (6)将选中的切片继续在展片台上烘烤，使蜡片中心、周围水分都除去，材料 紧贴载玻片上，如材料干后有透明质感，表明粘、展片工作较好，如有粉质 状白色蜡片，说明材料与载玻片未贴紧(可能有水汽及空隙引起)，这为后 续处理(染色、脱水、透明，封藏)带来很多麻烦，因此，必须在酒精灯火 焰上较迅速地来回烘烤，直至材料发白处变成透明状而蜡不熔化为止。 (7)在粘片、展片过程中，不断将展片台上的合格切片，转移至铝制摊片盘中， 然后再转放至40 ?温箱中(中部或上层)继续烘烤3 d 左右方可制片，在放 入温箱中的摊片盘，要交叉叠放，这样放的数量多，不占温箱内体积，又可 避免磨损切片。 思 思考题与作业 考 1、切片过程易出现何种问题,如何解决, 作 业 2、粘片中蛋清甘油的加量对制片有何影响? 与 参考文献 参 考 1、王灶安(植物显微技术(北京:农业出版社，1992 文 2、李和平(植物显微技术(华中农业大学，2006电子版 献 3、李正理(植物组织制片学(北京:北京大学出版社，1996 4、龚志锦, 詹熔洲(病理组织制片和染色技术(上海:上海科学技术出版社， 1994。 nclosuhe time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil levr other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectrild be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use ere affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shou4 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 一、切片刀的选择与准备工作 注 每种切片机一般附有1-2 把切片刀，若需添购可向当地仪器公司购买适合意 的类型。 事 如双平面刀:刀两面平直，是常用的一类，若用于回转切片机刀较窄而短，项 用于滑行切片机则刀宽且长。使用时刀口与石蜡块倾角约为15?。 平凹面刀:刀的一面平直，另一面内凹，凹度深的适用于滑行切片机，作火棉胶切片，凹度浅的适用于回转切片机，作石蜡切片，刀口与石蜡块倾角为4?-6?。 双凹面刀:一般少用，常为回转切片机上使用。 磨刀:有机器磨刀和手工磨刀两种方法。目前国内生产有全自动磨刀机和半自动两种。上海产品属全自动型，使用尚普遍，操作也方便。 手工磨刀法步骤如下: 1、磨刀石与润滑剂选择。磨刀石(天然石)以平滑不含杂质的青石为常用，人工合成磨刀石，价格较高，市场不宜采购，为了节约资金，充分利用资源，还是采购天然青石为宜。 磨刀前，将青石上的尘埃用布拭净，如为新购回的青石，需用两块青石，面对面相互研磨，这样使两块石面磨平后即可使用。磨刀用的润滑剂，可用液体石蜡代替，向医药公司购置。 2、将切片刀装上刀臂与刀柄，再用吸管滴数滴液体石蜡于青石面上，后将刀刃朝向青石前方，左手紧握刀臂套多余的一端，右手握刀的手柄，在磨刀时，两臂膀及手腕应放松，使刀刃、刀臂下侧都自然地紧贴青石表面滑行研磨，研磨中双手都应加压力，两手用力要均匀。 3、左侧刀刃在前，右侧刀刃在后，慢慢推动切片刀刃在青石上全部磨完，紧接着右手持刀柄，左手持刀臂同时翻动，将刀刃向上翻(刀臂不离开青石面)，刀刃转向内方轻轻放于右面朝胸部研磨。如此向前向后转动来回研磨，直至刀刃锋利为止，接着进行鐴刀。切片刀磨后，必须用牛皮制的鐴刀皮鐴刀，这样经鐴刀后的刀刃锋利，切下的蜡带光滑无刀痕形成。鐴刀皮是用宽牛皮带，两端钉在平整的方形木棒上，便于双手持刀进行鐴刀。鐴刀时，刀刃向后拖鐴(来回都相同)，千万不可向前。 4、刀磨了一定时间后，须在低倍显微镜下检查刀刃是否有缺口，若呈一直线即算磨好。切片刀用后或磨后都须用布蘸二甲苯拭净液体石蜡及尘浆，如磨后不马上用，还必须涂黄油或液体石蜡于刀刃及刀面、刀背，以免生锈，最后将刀放回刀盒放置安全处保存。 磨刀、鐴刀、切片、收刀等诸环节，都要特别细心，既不要损伤刀刃，也不要用手或其它脏物拭刀的任何部分，还需注意安全，免手指受伤。 tion is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify t a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparanot puand design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does hematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume al scsection number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electric verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on thend to eal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction ained the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great d. Combg in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the thirdr pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable layinthe oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling wateon or cable tray cannot be used for other processesnnecticonstruction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground colengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the cable 5 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 注 二、切片过程易出现的问题及解决办法 意 1、切片纵裂或具纵条纹刀有缺口、鐴刀时间太短、组织软化不够等原因，事 都会发生此类现象，可移动刀刃位置，进一步鐴刀及重新取下台木，将蜡块倒项 放在有水的培养皿中浸泡半天，继续软化材料。 2、蜡带弯曲是蜡块的上下边缘不平行引起，如将蜡块长边修整为平行的， 即可纠正弯曲现象。 3、切片有横纹及厚薄、大小不匀刀夹或物夹松紧不均匀，刀倾角过大(调 小到1?,2?)，刀口用二甲苯拭去石蜡屑;大小不匀与调节系统出故障有关， 应考虑维修切片机等问题。 4、材料破碎，甚至整个材料由蜡中脱离形成空洞蜡带此种现像是脱水不彻 底、浸蜡不完全，因而使材料和蜡之间不融合，形成空洞蜡片，这种材料无法 补救，必须重新处理、制作。 5、切片卷起形成圆筒是刀刃太钝并附着不洁杂物、刀的倾角太大，切片厚 度太厚等原因所引起的，须鐴刀，调整刀的倾角，或用少量二甲苯拭净刀刃等 措施。 石蜡切片全过程中，每一环节都会影响切片质量，初学者必须细致操作， 及时发现问题予以解决，决不能因其中某步骤出现问题失去信心。 r other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectrild be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use ere affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shounclosuhe time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil lev6 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 附 四、粘贴剂 录 1、明胶粘贴剂(又称Haup[粘贴剂] 此剂除粘贴蜡带外，还能用于花粉及单细胞藻类的粘贴剂，其配法如下: 甲液:明胶(动物胶) l g; 蒸馏水 100 mL;甘油 15 mL;酚(石炭酸)结晶 2 g; 乙液:甲醛 4 mL; 蒸馏水 100 mL 配制时将明胶溶于蒸馏水中，并转入保温器具内使明胶溶解(温度控制在35-36 ?)，然后再加入甘油与石炭酸，边加边搅拌促其尽快溶解混合，再过滤于细口瓶中备用。 2、火棉胶粘贴剂 适用于较厚的组织材料，如木材或种子切片，先用明胶粘贴剂粘贴稍干后，再滴上火棉胶溶液(浓度为1-2 ,火棉胶溶液;即将火棉胶溶于纯酒精与乙醚各半的混合液中)，然后把贴好的切片完全烘干，以免染色时材料脱落。染色时可用石炭酸1 份、二甲苯4 份混合液来清除石蜡，然后转入纯酒精中脱水、染色等步骤。 3、粘贴剂(蛋清甘油)及其配制法:将鸡蛋一端打开一个小孔，只让蛋白慢慢流入烧杯内。用玻璃棒(清洁的)充分调拌成泡沫状，然后用粗滤纸或双层纱布过滤至量筒中，经一昼夜(或更长)能滤出透明的蛋白液。再加入等量甘油及少量防腐剂(如樟脑精)振荡使其完全混合后即可应用，不用时需放低温干燥处(如冰箱中)保存，可长期使用。 在使用中应注意以下几点: (1)教学上使用，应先分装成小指形管中，每瓶在瓶塞上附一细竹棒(粗细与 火柴棒相当)，作蘸取蛋清甘油用。 (2)配制此剂，不要在高温天气配制，否则过滤时间拖长，会使蛋清腐败变质。 (3)蘸一次蛋清甘油，可涂抹4-5 张载玻片，涂的太厚(多)会影响制片质量。 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 on or cable tray cannot be used for other processesnnecticonstruction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground colengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the cable tion is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify t a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparanot puand design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does hematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume al scsection number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electric verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on thend to eal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction ained the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great d. Combg in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the thirdr pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable layinthe oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling wate7 备 实验八 石蜡法制片——脱蜡、染色及封片 (5 h) 注 一、实验目的要求 了解脱蜡、复染、封片的方法，学习染色的知识。 二、实验原理 1、脱蜡:将切片中的石蜡脱去。 2、染色:制片染色的原理，归纳起来可以有物理上的和化学上的两方面，这两方面各自都可以说明染色中的某一问题，但对有些现象仍不能作出合理的解释，还有待于进一步探讨。 (1)染色的物理作用 主张物理作用的观点认为染色现象可用物理学上的理论来解释。物理作用包括以下三种。 A 毛细作用或渗透作用 认为被染物质是有孔隙的，染料可以因为渗透作用而进入组织的内部。 B 吸收作用或溶液作用 组织吸收染液后进行牢固的结合，所染颜色与溶液的颜色相同，而与干燥的染料颜色不同。例如品红在干燥时为绿色，其溶液为红色，所染植物组织也是红色。 C 吸附作用 当组织浸于染液内，染液中分散的色素粒子的分子引力作用，使色素粒子被组织吸附，而呈现颜色。 (2)染色的化学作用 主张化学作用的观点认为，植物细胞内的不同化学成分，对染料有不同的亲和力，因此细胞具有选择性的着色能力，使细胞的不同成分染成不同颜色。细胞中的酸性物质，易被碱性染料所染，因为它对碱性染料的亲和力很强。细胞内的碱性物质，易被酸性染料所染，即对酸性染料的亲和力强。 此外，染液的pH 值对染色有很大关系，一般碱性物质如细胞质在低pH(酸性)的染液内易着色，酸性物质如细胞核在高pH(偏碱)的染液内易着色。 染色的机制是很复杂的，上述两种染色理论，都不能全面的说明问题，目前多数学者认为可能是由于物理作用与化学作用综合作用的结果。 3、封藏:选用适合在显微镜下观察的较高折射率(加拿大树胶为n=1.524)作封藏剂，将完成制片全过程的材料用封藏剂封固以便长期保存，可获得透明和显示组织详细结构的制片，供分析、照相、研究用。 r other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectrild be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use ere affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shounclosuhe time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil lev8 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 染一、染料的分类 备 三、实验材料与试剂 料 注 植物制片中使用的染料种类较多，染料的分类方法较复杂，根据不同的角器材:染缸、滴瓶、镊子、载玻片、盖玻片 度可以将其进行不同的分类。 材料:石蜡粘片 (一)根据染料的来源划分 试剂:无水乙醇、95 %乙醇、二甲苯、树胶、苯胺固绿染色液 1、天然染料 由植物或动物中提取，如苏木精、洋红、地衣红等。天然染料四、实验操作步骤 虽然种类不多，但在生物染色中普遍使用。 1、脱蜡:将两个装有二甲苯的脱蜡缸，有“脱蜡”、“透明”标签的一面朝向工作 2、人工染料 由人工合成。最初的人工染料是由苯胺制成的，称为苯胺染料。者。将待脱蜡的载玻片有蜡带的一面，朝向标签方向，一张张小心插入脱蜡目前许多人工合成染料与苯胺并无关系，也不是苯胺的衍生物。但人工合成染料缸的齿槽中，待30 min(在夏季38-40?温箱内可减少到15-20 min即可)，当均可由煤焦油中的一种或数种物质制成，故可统称为煤焦油染料。大多数染料属蜡片中材料的周围蜡溶去后，将经脱蜡后的载玻片转移至透明缸中5-10 min。 于此类，如番红、固绿、碱性品红等。 2、复染:先分别经无水酒精、95 %酒精处理4-5 s，再用苯胺固绿染色5 min，(二)根据染料的化学性质划分 然后经95 %酒精、1/2无水酒精，1/2二甲苯、二甲苯(2次)分别处理4-5 s。 1、碱性染料 它是一种色碱的盐，通常为氯化物，也有磷酸盐或醋酸盐。它镜检观察染色效果，若不行，按操作步骤原路返回重新染色。 含有碱性助色团氨基或二甲氨基，主要有色部分为阳离子。如苏木精、碱性品红、3、封片: 番红等。 (1)在桌上放一张白纸，将载玻片由二甲苯中取出后，以清洁布块(选用无纤 2、酸性染料 是一种色酸的盐，通常是钠盐、钾盐或铵盐，它含有酸性助色维短绒的布块为宜)迅速擦去材料以外的二甲苯，平置纸上，当材料上二甲团如羟基羧基，主要有色部分为阴离子。如固绿、伊红、桔红G、酸性品红等。 苯尚未全挥发时，滴一滴树胶于材料上。 3、中性染料 或称复合染料，它们的阳离子和阴离子都有颜色。如中性红等。 (2)用右手执镊子夹住盖玻片右侧，将它放在树胶的左边，左手以食指抵住盖(三)根据染色的性能而分 玻片的左边，右手将镊子逐渐下降，待盖玻片接触树胶后再轻放抽出镊子， 1、核染料 能与细胞核中的染色质发生作用的染料，包括天然染料的苏木精、这时树胶就均匀展开布满盖玻片，材料同时被覆盖。如果胶量过少，或盖盖洋红及人工染料的番红、甲苯胺蓝、甲基绿、碱性品红等。 片太快，就会在盖玻片下留有气泡，影响观察效果。 2、细胞质染料 能与细胞质发生作用的染料，如伊红Y、固绿、桔红G、苯(3)如果滴的树胶量太少，可在盖玻片有空隙的地方，再滴注少量树胶，使其胺蓝、酸性品红等。 慢慢浸满盖玻片空间，如树胶过多，溢出盖玻片外，不必马上拭除，待树胶 3、细胞壁染料 能与细胞壁发生作用的染料，如番红、苯胺蓝等。 干燥后用刀片刮去多余树胶，再用纱布蘸二甲苯擦拭干净即可。最后贴上标 4、组织化学染料 如苏丹?、溴酚蓝、碱性品红、苯胺蓝黑等。 签，全部制片过程完成。 5、荧光染料 应用含荧光色团的物质作为染料，使之与细胞中和该物质有特思考题与作业 思 殊亲和力的物质相结合，即可在紫外线或短光波的照射下被激发出荧光，这类染1、每人制作2张永久片; 考 料称为荧光染料(fluorescen dye)专用的荧光染料如H33258、DAPI、FITC、水2、复染时应注意什么, 作 溶性苯胺蓝、荧光增白剂、荧光素二醋酸酯、金胺O 等(详细介绍见荧光显微业 与 术内容)。 参考文献 参 6、活体染料 植物中可用作细胞活体染色的染料，如中性红和许多荧光染料考 1、王灶安(植物显微技术(北京:农业出版社，1992 文 等。 2、李和平(植物显微技术(华中农业大学，2006电子版 献 construction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground colengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the cable tion is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify t a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparanot puand design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does hematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume al scsection number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electric verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on thend to eal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction ained the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great d. Combg in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the thirdr pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable layinthe oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling wateon or cable tray cannot be used for other processesnnecti9 3、李正理(植物组织制片学(北京:北京大学出版社，1996 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 染二、几种常用染料的性质及配制 料 (一)苏木精(haemaloxylin) 是制片技术中的最主要染料之一。苏木精是从热带植苏木中提取的淡黄色或棕黄色结晶，溶于酒精，甘油及热水中。苏木精不能直接染色，必须经氧化成为氧化苏木精后才能应用。氧化苏木精脱掉两个氢原子成为苏木红，这个氧化过程叫做成熟。一般采用在空气中自然氧化，如急用可加入强氧化剂以加速氧化，可随配随用。 配制苏木精常用的氧化剂有高锰酸钾、氧化汞、碘酸钠及过氧化氢等。 苏木精很少单独使用，因为它虽经氧化成为苏木红，但是与组织之间的亲和力还是很小，如加上金属盐的媒染剂(硫酸铝铵、硫酸铁铵、硫酸铝钾)以后，可大大增强染色的效果。 苏木精染色后，在分色过程中，因处理的情况而异，颜色会有改变，经酸性溶液(如盐酸酒精)分色后呈现红色，水洗后仍可回复蓝色，碱性溶液(如氨水)分色后为蓝色，经水洗后呈蓝黑色。 苏木精广泛应用于细胞学，胚胎学的制片工作中。常用的配方有以下几种。 1、爱氏(Ehrlich’s)苏木精染色液(苏木精原液) 配方: 苏木精 2 g ;冰醋酸 10 mL;甘油 100 mL;95 %酒精 100 mL; 蒸馏水 100 mL;硫酸铝钾 5 g 配制步骤: (1)将苏木精溶于少量酒精中，加冰醋酸后搅拌，加速其溶解。 (2)加入甘油及其余酒精。 (3)研碎钾矾，溶于水中并加温。 (4)将加温的钾矾溶液一滴滴地加入染色剂中，并不断搅动。 (5)瓶口用双层纱布包扎，放于通风处，并经常摇动，直至颜色变为紫红时即可使用，成熟时间约需2-4 周以致数月之久，若加0.2 g 碘酸钠即可立即成熟。己成熟的原液，须用瓶塞密封，置低温暗处长期保存。 此染色液主要用于细胞核的染色。用于整体染色效果甚好。使用时以原液1 份加入 50 %酒精与冰醋酸等量混合液1 份或2 份。 2、德氏(Delafield’s)苏木精染色液配方: 甲液:苏木精4 g;95 %酒精25 mL;硫酸铝铵饱和水溶液400 mL 乙液:甘油100 mL;甲醇100 mL r other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectrild be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use ere affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shounclosuhe time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil lev10 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 配制步骤: (1)将苏木精溶于95 %酒精中。 (2)将上液慢慢滴入硫酸铝铵的饱和水溶液，并用玻棒不断搅动。 (3)用双层纱布紧扎瓶口，放在光线充足处氧化，约需1 周。 (4)将上液过滤，再加入甘油及甲醇。 (5)继续氧化直至颜色变为蓝紫色为止，再过滤密封备用。 此染色液与爱氏苏木精染色液用途相似，染色作用更强，使用时以蒸馏水3-5 份稀释之。分色液用0.5 %或1 %盐酸酒精(0.5 mL 或1 mL盐酸与99 mL 的70 %酒精混合而成)。 3、海氏(Heidenhain′s)苏木精染色液 又称铁矾苏木精染色液 配方: 甲液(媒染剂):硫酸铁铵(铁矾)2-4 g、蒸馏水100 mL 必须保持新鲜，最好临用前不久配制。 乙液(染色剂):苏木精0.5 g、95 %酒精l0 mL、蒸馏水100 mL 乙液配制步骤: (1)将苏木精溶于酒精中，瓶口用双层纱布包扎，使其充分氧化(4 周左右)。 (2)加入蒸馏水，塞紧瓶口，置冰箱中可长期保存。 切片需先经甲液(4 %)媒染，并充分水洗后才能以乙液染色，染色后又经水 稍洗再用另一瓶甲液(2 %)分色至适度。铁矾苏木精染液为细胞学上染细胞 核内染色质最好的染色剂，但甲液与乙液在任何情况下决不能混合，切勿疏 忽。 (二)番红(safranin O) 为组织学上应用最广泛的一种染料，尤其对于高等植物中木质化细胞及角质化组织的染色不可缺少，对蛋白质、染色质、染色体、孢子、花粉外壁都可染成鲜艳的红色，并可与固绿、苯胺蓝等作二重染色，与结晶紫、桔红G 作三重染色。番红为碱性染料，溶于水、酒精。既可整染，又可片染、滴染。常规的配方有以下几种。 1、番红水溶液:番 红 1 g;蒸馏水 100 mL 2、番红酒精溶液:番 红 1 g;50 %(或95 %)酒精 100 mL 3、苯胺番红染色液: 番 红 5 g;95 %酒精 50 mL;蒸馏水 450 mL;苯 胺 25 mL 配后摇匀，用前须过滤。番红染液为我们用于整染及滴染的重要染色液，节 省时间、药品与用具，效果极好。 lengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the cable tion is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify t a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparanot puand design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does hematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume al scsection number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electric verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on thend to eal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction ained the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great d. Combg in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the thirdr pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable layinthe oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling wateon or cable tray cannot be used for other processesnnecticonstruction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground co11 (三)固绿(fast green) 与苏木精、番红一并称为植物组织学上三大染料，应用也很广泛，通常是与番红作二重染色，或再加入桔红G 作三重染色，再加入结晶紫作四重染色。固绿见光不易褪色，能将纤维素壁、细胞质染为鲜艳绿色。 固绿为酸性染料，在水中溶解度较低，易溶于酒精。固绿着色很快，故染色时间要很好掌握。常用的配方有以下几种。 1、固绿酒精溶液:固 绿 0.5 g;95 %酒精 100 mL 2、苯胺固绿染色液:固 绿 1 g;95 %酒精 40 mL;苯 胺 10 mL 边配边搅，配后摇匀，用前过滤。后者与苯胺番红染色液配合使用于滴染，即使是初学者也易掌握，效果很好，已得到广泛应用。 (四)碱性品红(basic fuchsin) 是强有力的核染色剂，可作为组织化学试剂使用。如在孚尔根反应中用于检验去氧核糖核酸，在高碘酸—席夫反应中用以检验多糖物质，在一般植物组织学制片中较少使用。 1、孚尔根(Feulgen)反应 (1)水解液:1N 盐酸:盐酸(比重1.18) 82.5 mL;蒸馏水 917.5 mL (2)席夫试剂(Schiffs reagent 脱色品红) 碱性品红 0.5 g;蒸馏水 l00 mL;1N 盐酸 10 mL;偏重亚硫酸钠(钾) 1-3 g 配制步骤: ?将碱性品红缓缓加入煮沸离火的蒸馏水中，再继续煮5 min，并随时搅拌。 ?待冷却到50 ?即过滤到有玻璃塞的棕色试剂瓶中。 ?加入1 N 盐酸。 ?冷却到25 ?即加入偏重亚硫酸钠(钾)，盖紧玻璃塞，置暗处过夜。第二天即为脱色品红，过滤后，盖紧并包以黑纸在低温或冰箱中贮存。此液必须清彻透明，无色无沉淀，如颜色变红，可再加入少许偏重亚硫酸钠(钾)，使其转为无色再用。 2、石炭酸,品红染色液 又称卡宝染色液(Carbol fuchsin) A 液:取3 g 碱性品红溶于100mL 70 %酒精中(可长期保存)。 B 液:取10 mL A 液加入90 mL 5 %的石炭酸酚水溶液中(2 周内使用)。 C 液:取 55 mL B 液加6 mL 冰醋酸及6 mL 福尔马林。 D 液:取C 液20-30 mL 加70-80 mL 45 %冰醋酸和1.8 g 山梨醇。 配后为淡品红色，立即使用染色较淡，放置2 周后，染色力明显增强，放置时间越久，染色效果越好。C 液可用于组织培养中核及核分裂的染色，此液中有较多甲醛，不能使组织软化。D 液则可普遍用于组织的压片染色，效果很好。 re affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shounclosuhe time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil levr other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectrild be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use e12 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 染三、染色时应注意的事项 料 1、应根据观察目的，样品的结构性质来选择适当的染色方法。 2、材料从某一溶液中进入染色液时，这两种溶液的浓度必须相同。 3、酸性染料着色速度快于碱性染料，故二重染色时，碱性染料先染，酸性染 料后染。 4、染色宁可深一点，因染色后在水洗和脱水过程中颜色会变浅，如果使用分色剂，染色更要偏深。 5、使用分色剂时，要在低倍显微镜下随时观察，分色适度时即彻底洗净，否则以后还会退色，并影响下一步染色。 6、染色的时间不是一成不变的，书上所说的时间仅供参考，还须根据染色剂的性质、配方、切片的厚薄、材料的结构在使用时灵活掌握。 7、发色团 产生颜色的化学结构式“醌式环”称为发色团(chromophore) (或称呈色基团、色基)，是使化合物带有色彩性质的部分，无发色团的有机化合物是没有颜色的，如苏木精分子结构里无发色团，必须经过氧化作用使成为氧化苏木素才有颜色。 8、助色团 要使一种物质成为染料除应含发色团外，还需要有一种能使化合物发生电离作用的辅助原子团(酸碱性基团)称为助色团(auxochrome) (或称作用基团);最主要的是氨基、羧基、羟基、磺酸基、甲氨基、二甲氨基。助色团的作用在于使染料分成带电荷的离子，以便加强与被染物质组织的亲和力。 on or cable tray cannot be used for other processesnnecticonstruction, not allowed to use electricity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground colengths and set aside. Bridge should be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the cable tion is completed, later in each enclosure affixed on both sides of each side of the cable you want to lay accurately verify t a cable one cable. Finally, at the time of inventory, prepared to take the same path the cables together. Inventory preparanot puand design inventory check cable to cable with and without omission, laying of the cable in order to ensure the process does hematics, in accordance with the principle of accounting the cable path cable volume, the number of cores used, total volume al scsection number. At the time of preparation of the inventory of cable, first of all, the professional carefully study electric verify cable length, specifications, familiar with the cable channel and the device location and bracket cable bracket on thend to eal of control and signal cables. So you should carefully review, checks for missing physical cable, ready for construction ained the engineering field to find fault, adding omitted items. Because the unit used computer DCS control and adds a great d. Combg in front of the cable construction, design, supervision and construction units to deliver the design, and drawing the thirdr pipe fittings, valve parts before installation to check ... 4.2 measures to eliminate quality defects of cables cable layinthe oil level to the normal oil level. (2) for the secondary prevention of the leakage of cooling wate13 长江大学生命科学学院备课用纸 长江大学生命科学学院备课用纸 附 五、封藏剂 录 常用的封藏剂有加拿大树胶(canada balsam)、油派胶(euparal)、达马树胶(dammar balsam)、甘油胶冻(glycerin jelly)等，我们常采用加拿大树胶及南京林业大学生产的冷杉胶两种。树胶要以专制树胶瓶盛装。 1、加拿大树胶:是从北美一种冷杉(Abies balsamea)植物体中取得的树脂，可溶于二甲苯、苯、氯仿、叔丁醇等溶剂中，通常以溶于二甲苯中最常用。市售有已配制的中性加拿大树胶。 在配制此树胶时，应掌握其浓度，如以玻璃棒蘸沾树胶，玻棒在末端呈小滴慢慢滴下，而不成丝状为适度。加拿大胶的熔点为61?，但配制时不能加热，加热后会变为深褐色和酸化而影响制片效果。如已酸化，胶中可放一些纯大理石(为碳酸钙)，碳酸钙为弱碱性，有中和作用。加拿大树胶的折光率(1.547)与玻璃的(1.51)很相近，故透明度较佳。 2、油派胶:是由樟脑,水扬酸苯脂(camsal)(等量混合)、山达脂( sandarac)、桉树油(eucalyptol)、三聚乙醛(paraldehyde)混合而成(其比例为1?4?2:1)。其折光率(1.48)稍低于加拿大树胶。材料可在95 %酒精脱水取出后，不经透明剂即可用此种封藏剂封藏。在容易收缩的材料和染色体压片过程中，使用油派胶封片能取得好的制片效果。 3、无水封藏剂 达马树胶(dammar balsam)，为松柏科的柳安(Shorea wiesneri)分泌的树脂，能溶于二甲苯、苯、氯仿和酒精中。 其配法:溶解25 g 达马树胶于250 mL 氯仿和250 mL 二甲苯中，过滤，蒸发到 l00 mL 时即可使用。或溶于二甲苯中，过滤成二甲苯达马树脂。 2、含水封藏剂 用含水封藏剂封藏的标本，其保存的时间不及无水封藏剂时间长。 (1)乳酚甘油 此封藏剂适用于整体封藏，如藻、菌、原叶体及其他小材料的封藏都可适用。配方为: 酚 1份; 乳 酸 1 份; 甘 油 1 份或2 份;蒸馏水 1 份 (2)甘油胶冻(glycerin jelly) 下面介绍两种配制方法 A 明胶(gelatin) 5 g(1 份);蒸馏水 30 mL(6 份);甘油 35 mL(7 份);酚(石炭酸) (溶解于10 滴水中)0.5 g; 每l00 mL 加l g。 先将明胶溶解于35?温水中，然后将其他药剂再加入，待完全溶化(此时尚有余温)，接着用粗滤纸过滤至培养皿中，待冻结后划成小块，贮藏备用(引自郑国昌生物显微技术)。 B 明胶 l0 g;蒸馏水 30 mL;甘油 30 mL;酚(石炭酸) l g 将四种药剂加热(混合后)至70-80 ?，装入大口瓶中密闭，后置50 ?温箱中48 h，再加热到80 ?并用纱布(消毒过)连续过滤两次即可。 此剂在室温下为固态。使用时，先转置60 ?水浴锅中液化，取一滴于载玻片上，加入材料(菌丝或子实体等)，迅速加盖玻片，便封藏久存(引汤华光 ld be combined, correcting errors, avoid straight, no cable bridge scaffolding used in the construction, not allowed to use ere affixed on both sides of each side of the cable you want to lay accurately verify cable lengths and set aside. Bridge shounclosuhe time of inventory, prepared to take the same path the cables together. Inventory preparation is completed, later in each ee with and without omission, laying of the cable in order to ensure the process does not put a cable one cable. Finally, at to cablrinciple of accounting the cable path cable volume, the number of cores used, total volume and design inventory check cable tion of the inventory of cable, first of all, the professional carefully study electrical schematics, in accordance with the pparat, familiar with the cable channel and the device location and bracket cable bracket on the section number. At the time of preshould carefully review, checks for missing physical cable, ready for construction and to verify cable length, specifications o youfault, adding omitted items. Because the unit used computer DCS control and adds a great deal of control and signal cables. S sign, supervision and construction units to deliver the design, and drawing the third. Combined the engineering field to findon, dee installation to check ... 4.2 measures to eliminate quality defects of cables cable laying in front of the cable constructiel to the normal oil level. (2) for the secondary prevention of the leakage of cooling water pipe fittings, valve parts beforthe oil levr other processescity (fire) cutting, fix cable tray, cable tray is not allowed as a welding ground connection or cable tray cannot be used folectri14 等改良剂)。