Name: microscope slide prep and staining
Uploaded: 2023-04-30T11:59:49.000Z
Duration: 1 min 3 s
Channel: JoVE

大多数生物组织样本较厚且不透明，在显微镜下观察前必须经过处理。
组织首先用固定液处理，以杀死细胞并固定细胞组分，从而维持其结构。
固定后的样品经过浓度递增的酒精梯度处理，以使样品脱水。
为了防止脆弱的样品受损，需将其包埋在石蜡等疏水性材料中，并用切片机切成薄片。
将切好的薄片转移至载玻片上，加入几滴有机溶剂以去除附着的石蜡。
接下来，对切片进行染色，并加入封片剂，盖上盖玻片。封片剂的折射率应与盖玻片和载玻片相同，以获得最佳的图像分辨率。
样本现已准备好，可在显微镜下进行观察。

固定和切片

Fixation and Sectioning

使用光学显微镜观察标本有两种基本的制备方法：湿封片和固定标本。

最简单的制备类型是湿封片，即将样本置于载玻片上的一滴液体中。可以使用滴管将液体样本直接滴在载玻片上。可以将固体标本（例如皮肤刮片）放置在载玻片上，然后添加一滴液体来制备湿封片。有时液体只是水，但通常会添加染色剂以增强对比度。将液体添加到载玻片后，将盖玻片放在顶部，样本就可以放在显微镜下检查了。

制备光学显微镜标本的第二种方法是固定。样品的“固定”是指将细胞附着到载玻片上的过程。固定通常通过加热（热固定）或对样本进行化学处理来实现。除了将标本固定在载玻片上之外，固定还可以杀死标本中的微生物，停止其运动和新陈代谢，同时保留其细胞成分的完整性以供观察。

热固定样品时，将样本的薄层铺在载玻片上（称为涂片），然后在热源上短暂加热载玻片。对于组织标本来说，化学固定剂通常比加热更好。乙酸、乙醇、甲醇、甲醛（福尔马林）和戊二醛等化学试剂可以使蛋白质变性、停止生化反应并稳定组织样本中的细胞结构。

除了固定之外，在光学显微镜下检查样本之前，染色几乎总是用于对样本的某些特征进行着色。染色剂或染料由带有正离子和负离子的盐组成。根据染料的类型，正离子或负离子可能是发色团（有色离子）； 另一个无色离子称为反离子。如果发色团是带正电的离子，则染色剂被归类为碱性染料； 如果发色团是带负电的离子，则该染色剂被认为是酸性染料。

根据染料和所观察样本的化学性质选择用于染色的染料，这决定了染料如何与样本相互作用。在大多数情况下，最好使用正染色剂，即一种被观察的细胞或生物体吸收的染料，为感兴趣的物体添加颜色，使其在背景中脱颖而出。然而，在某些情况下，使用被背景吸收但不被样本中的细胞或生物体吸收的负染色剂是有利的。负染色在彩色背景下产生生物体的轮廓或剪影。

由于细胞通常具有带负电荷的细胞壁，因此碱性染料中的正发色团往往会粘附在细胞壁上，从而使其呈正染色。因此，碱性染料如碱性品红、结晶紫、孔雀石绿、亚甲蓝和番红通常用作正染色剂。另一方面，酸性染料中带负电的发色团被带负电的细胞壁排斥，使它们成为负染色剂。常用的酸性染料包括酸性品红、曙红和玫瑰红。

一些染色技术仅在样品上使用一种染料； 而另一些则需要不止一种染料。在简单染色中，使用单一染料来强调样本中的特定结构。简单的染色通常会使样品中的所有生物体呈现相同的颜色，即使样品包含多种类型的生物体。相反，差异染色根据生物体与多种染色剂的相互作用来区分生物体。换句话说，差异染色样品中的两种生物体可能会呈现不同的颜色。临床中常用的鉴别染色技术包括革兰氏染色、抗酸染色、内生孢子染色、鞭毛染色和荚膜染色。

用于在光学显微镜下观察标本的两种基本制备方法是湿封片和固定标本。  
最简单的制备类型……

Education

教育

JoVE Core

Cell Biology

细胞生物学

Unit 1

第一单元

Visualizing Cells, Tissues, and Molecules

细胞、组织与分子的可视化

KEY TERMS AND CONCEPTS

关键术语与概念

microscope slide prep and staining

Microscope slide preparation includes wet mounts, fixation, and staining. These steps help make specimens easier to see under a light microscope. They also help preserve cells and highlight important structures.

A wet mount is the simplest slide preparation. The specimen is placed in a drop of liquid on the slide. A liquid sample can be added directly with a dropper, and a solid sample, such as a skin scraping, can be placed on the slide before the liquid is added. The liquid is sometimes water, but stains are often mixed in to improve contrast. A coverslip is placed on top, and the sample is then ready for viewing.

The other main method is fixation, which means attaching cells to the slide. Fixation can be done with heat or with chemicals. Heat fixing uses a thin smear of the specimen that is briefly heated over a heat source. This method also kills microorganisms, stops movement and metabolism, and helps preserve cell structures for observation. Chemical fixatives are often better for tissue samples. Common chemical agents include acetic acid, ethanol, methanol, formaldehyde, and glutaraldehyde. These chemicals denature proteins, stop biochemical reactions, and stabilize cell structures.

Staining is usually used along with fixation to add color before light microscopy. Stains, or dyes, are salts with a positive ion and a negative ion. The colored ion is called the chromophore, and the uncolored ion is the counterion. If the chromophore is positive, the dye is a basic dye. If the chromophore is negative, the dye is an acidic dye. Dyes are chosen based on the chemical properties of both the dye and the specimen.

Most of the time, a positive stain is preferred because it is absorbed by the cells or organisms being observed. This makes the objects of interest stand out from the background. In some cases, a negative stain works better. A negative stain colors the background but not the cells, so the organisms appear as outlines or silhouettes. Because cell walls are usually negatively charged, basic dyes tend to stick to them and act as positive stains. Examples include basic fuchsin, crystal violet, malachite green, methylene blue, and safranin. Acidic dyes have negatively charged chromophores and are repelled by negatively charged cell walls. Common acidic dyes include acid fuchsin, eosin, and rose bengal.

Some staining methods use one dye, while others use more than one. Simple staining uses a single dye to make certain structures easier to see. It usually makes all organisms in a sample appear the same color, even when more than one type is present. Differential staining uses multiple stains to tell organisms apart. In a differentially stained sample, two organisms may appear different colors. Common differential stains used in clinical settings include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining.

Microscope Slide Prep and Staining

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key terms and concepts

Learn how wet mounts, fixation, and staining prepare specimens for light microscopy, including positive and negative stains and simple versus differential staining.

Microscope Slide Prep and Staining | JoVE K12

cell biology

JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

Microscope Slide Prep and Staining

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