第1章 组织学实验方法
Chapter 1 Histological experimental method

（一）如何正确使用显微镜观察组织标本

1．对照图片，结合实物，熟悉显微镜的构件。

2．将显微镜轻轻地移至观察者的前方，使镜座的后缘距实验台边缘大约10 cm。

3．旋开显微镜电源开关，并使光线亮度适当。

4．旋转物镜转换器，将10倍物镜旋至镜筒下方，对准载物台中央的孔。

5．打开聚光镜孔径光阑，可见光线经聚光器→载物台中央的孔→10倍物镜→目镜。

6．注意身体坐姿，睁开双眼，把观察注意力集中于显微镜的视场内，可见两个不完全重合的视场光斑，双手推移两目镜，使两光斑合二为一。

7．按照实习指导和切片标本目录，从标本盒内取出要观察的标本，进行肉眼观察，初步了解该标本的大小、形状和染色等。

8．将载玻片放在载物台上，盖玻片朝上，标签位右侧，用切片夹夹好。旋转推进器移动旋钮，将载玻片上的组织标本移至物镜下方。调节电源开关，将视野亮度调至适中。在右手旋转推进器移动旋钮移动切片的同时，左手慢慢地旋转粗调旋钮和微调旋钮聚焦（先旋粗调旋钮，后旋微调旋钮）。因推进器位置在不同显微镜可能不同，左右手分工可能互换。

9．显微观察标本常规是从低倍放大开始，这样视场大，有利于对标本进行全面观察。使用低倍物镜时，观察者左手转动粗调旋钮，使载物台缓慢上升或下降，同时，观察者用双眼从目镜中观察视场，当见到组织标本像轮廓时，转动微调旋钮直至组织标本像清晰。

10．当要进一步放大观察时，则直接旋转物镜转换器，将更高倍物镜（如4倍转成10倍，或10倍转成40倍）旋至镜筒下方。此时，只需转动微调旋钮调焦（必要时升降聚光器或调节聚光镜光阑的孔径），便可获得更清晰的进一步放大的标本像。在不同切片和不同放大倍数下观察时，应注意随时调节亮度，使之最适宜。

11．移动切片观察时，要注意片中组织标本与镜像方位完全相反。注意二维平面标本像与其三维立体图像的关系。

12．观察完一张切片后，应回忆总结一下在该片中辨认了几种细胞、组织、结构或该器官的特征性结构，每片如此积累，可以大大提高阅片能力和学习效率。

（二）如何观察和理解切片物像

观察切片的步骤 应养成由肉眼到低倍，再到高倍，系统观察标本的习惯。先要了解标本的取材部位、制片方法、切片方位和染色方法，再从宏观到微观，由浅入深逐步观察组织、细胞的微细结构。在掌握组织、细胞光镜结构的基础上，可结合观察某些细胞或结构的电镜照片，并联系机能融会贯通深入理解。

正确理解切片的立体形象 人体结构极为复杂，就同一个器官或细胞来说，所切的部位不同或者所切的方向不同，则切片所显示的物像就不相同。

我们将一个煮熟的鸡蛋示意为一个细胞，通过不同方向和部位所作的各种切面，则可得到不同的物像，如Fig.1-2所示。若对呈辐射状排列的细胞群体作各种切面，其各种物像如Fig.1-3所示。若对呈管状的器官作各种切面，其形状如Fig.1-4、Fig.1-5所示；若对呈束状的器官作各种切面，其形状如Fig.1-6所示。

切片中的人为现象 在切片中出现的一些人为假象，并非组织结构，应予鉴别：①刀痕：因切片刀锋有缺口造成组织标本纵行刀痕。②裂纹：组织透明、浸蜡的时间过长，组织脆硬，切片时可引起组织裂开，呈不规则裂纹。在制片过程中，由于组织或细胞各部分结构的收缩不一致，或贴片时水温过高，也可导致某些人为裂隙。③皱褶：贴片时组织未充分展平。④气泡：封片时将少许空气封入切片树胶中。⑤异留物：如染色时残留的染料沉渣等。

I. How to use a microscope to examine the slides

Step 1. Locate each part of your microscope according to the above image.

Step 2. Check the glass slides in the box and inform the teacher if there is any one broken or missing.

Step 3. Procedure for examination of the slides using the microscope

A. Gently move the microscope towards you，keeping the distance of about 10 cm between the rim of microscope base and your bench edge.

B. Turn on the power switch，make sure that your light source is functioning，and ensure that the lens nosepiece is rotated properly and the 10X objective lens is fixed.

C. Hold the slide up and examine it first with the naked eye to get an overview of the tissue size，shape and placement on the slide.

D. Put the slide on the stage and fasten it with the stage clip.

E. Look at the stage from the side and turn the coarse focus knob so that the stage goes upward. Move it as close as possible to the objective lens，without touching it！

F. Now，look through the eyepiece，meanwhile turn the coarse knob so that the stage moves away from the objective lens. Continue until the image comes into focus. Then use the fine knob for fine focusing.

G. Move the interesting image in the center of the field of view，then change to the 40X objective lens for further observation.

II. How to examine and understand microscopic images

Tips for examination:

1．Observe the section with naked eyes first for orientation and then at the lower magnification，and increasingly higher magnifications.

2．Be familiar with the sampling site and staining method，which is helpful in understanding the structure.

3．Electron micrograph available in the lab is valuable in connecting the morphology with function of some cells and tissues.

Transition from 3D to 2D:

When viewing a histological sample or specimen，you have to bear in mind that the tissue has been reduced from being three-dimensional to two-dimensional. You are no longer viewing a full specimen or even the exterior of a specimen，but rather a sliver of tissue taken from a specimen.

The following diagrams demonstrate various sectional shapes of a tissue sample cut from different orientations.

Artifacts:

Be aware that each step of tissue preparation introduces artifacts by altering or distorting the natural appearance of cells. Artifacts in slides may result from improper fixation and dehydration，paraffin infiltration，and poor microtome sectioning.

1．Bubbles：The air is sucked in under the coverslip when the mounting media is too thin or dries off.

2．Black precipitates：Formalin-heme pigment forms when the formalin buffer is exhausted and the tissue becomes acidic.

3．Ripples and wrinkles：They can be introduced during cutting and handling of sections when the tissue section stretches unevenly around structures of differing consistencies.

4．Scratches and “chatter”：Scratches are caused by flaws or dirt on the cutting edge, and appear as straight slashes or ragged tears across the specimen.“Chatter” is the visible record of knife vibration. The process of slicing sometimes induces vibrations in the knife edge，which then cause variations in thickness in the section. These appear as narrow parallel bands，usually evenly spaced，across a tissue specimen.