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

La plupart des échantillons de tissus biologiques sont épais et opaques et doivent être traités avant d’être observés au microscope.
Les tissus sont d’abord traités à l’aide d’une solution de fixation qui tue les cellules et immobilise les composants cellulaires pour maintenir leur structure.
Les échantillons fixés sont passés à travers un gradient croissant d’alcool, ce qui déshydrate l’échantillon.
Pour éviter que les échantillons fragiles ne soient endommagés, ils sont noyés dans un matériau hydrofuge comme la cire de paraffine et coupés en fines sections à l’aide d’un microtome.
Les lames minces sont transférées sur des lames, où quelques gouttes de solvants organiques sont ajoutées pour éliminer la cire adhérente.
Ensuite, les sections sont teintées et un support de montage est ajouté, avec une lamelle sur le dessus. Le support de montage doit avoir le même indice de réfraction que la lamelle et la lame pour obtenir la meilleure résolution d’image.
L’échantillon est maintenant prêt à être visualisé au microscope.

fixation et découpage

Fixation and Sectioning

Deux types de préparation de base sont utilisés pour visualiser des échantillons avec un microscope optique : les supports humides et les échantillons fixes.

Le type de préparation le plus simple est la préparation humide, dans laquelle l'échantillon est placé dans une goutte de liquide sur la lame. Un échantillon liquide peut être déposé directement sur la lame à l’aide d’un compte-gouttes. Les échantillons solides, tels que ceux provenant d'un grattage cutané, peuvent être placés sur la lame avant d'ajouter une goutte de liquide pour préparer le support humide. Parfois, le liquide est simplement de l’eau, mais des taches sont souvent ajoutées pour améliorer le contraste. Une fois le liquide ajouté à la lame, une lamelle est placée dessus et l’échantillon est prêt à être examiné au microscope.

La deuxième méthode de préparation des échantillons pour la microscopie optique est la fixation. La « fixation » d’un échantillon fait référence au processus de fixation de cellules sur une lame. La fixation est souvent obtenue par chauffage (fixation thermique) ou par traitement chimique de l'échantillon. En plus de fixer l'échantillon à la lame, la fixation tue également les micro-organismes présents dans l'échantillon, arrêtant leur mouvement et leur métabolisme tout en préservant l'intégrité de leurs composants cellulaires pour l'observation.

Pour fixer à chaud un échantillon, une fine couche de l’échantillon est étalée sur la lame (appelée frottis), puis la lame est brièvement chauffée sur une source de chaleur. Les fixateurs chimiques sont souvent préférables à la chaleur pour les échantillons de tissus. Les agents chimiques tels que l'acide acétique, l'éthanol, le méthanol, le formaldéhyde (formol) et le glutaraldéhyde peuvent dénaturer les protéines, arrêter les réactions biochimiques et stabiliser les structures cellulaires des échantillons de tissus.

En plus de la fixation, la coloration est presque toujours appliquée pour colorer certaines caractéristiques d'un spécimen avant de l'examiner au microscope optique. Les taches, ou colorants, contiennent des sels constitués d'un ion positif et d'un ion négatif. Selon le type de colorant, l'ion positif ou négatif peut être le chromophore (l'ion coloré) ; et l’autre ion incolore est appelé le contre-ion. Si le chromophore est l’ion chargé positivement, la tache est classée comme colorant basique ; si l'ion négatif est le chromophore, la tache est considérée comme un colorant acide.

Les colorants sont sélectionnés pour la coloration en fonction des propriétés chimiques du colorant et de l'échantillon observé, qui déterminent la manière dont le colorant interagira avec l'échantillon. Dans la plupart des cas, il est préférable d'utiliser une coloration positive, un colorant qui sera absorbé par les cellules ou les organismes observés, ajoutant de la couleur aux objets d'intérêt pour les faire ressortir sur le fond. Cependant, il existe des scénarios dans lesquels il est avantageux d’utiliser une coloration négative absorbée par le fond mais pas par les cellules ou les organismes de l’échantillon. La coloration négative produit un contour ou une silhouette des organismes sur un fond coloré.

Étant donné que les cellules ont généralement des parois cellulaires chargées négativement, les chromophores positifs des colorants basiques ont tendance à adhérer aux parois cellulaires, ce qui en fait des colorants positifs. Ainsi, les colorants basiques tels que la fuchsine basique, le violet cristal, le vert malachite, le bleu de méthylène et la safranine servent généralement de colorants positifs. D’un autre côté, les chromophores chargés négativement dans les colorants acides sont repoussés par les parois cellulaires chargées négativement, ce qui en fait des taches négatives. Les colorants acides couramment utilisés comprennent la fuchsine acide, l'éosine et le rose Bengale.

Certaines techniques de coloration impliquent l’application d’un seul colorant sur l’échantillon ; d'autres nécessitent plus d'un colorant. Lors d'une coloration simple, un seul colorant est utilisé pour mettre en valeur des structures particulières dans l'échantillon. Une simple coloration fera généralement apparaître tous les organismes d’un échantillon de la même couleur, même si l’échantillon contient plus d’un type d’organisme. En revanche, la coloration différentielle distingue les organismes en fonction de leurs interactions avec plusieurs taches. En d’autres termes, deux organismes dans un échantillon coloré différemment peuvent apparaître de couleurs différentes. Les techniques de coloration différentielle couramment utilisées en milieu clinique comprennent la coloration de Gram, la coloration acido-résistante, la coloration des endospores, la coloration des flagelles et la coloration des capsules.

This text is adapted from <a href="https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens">Openstax, Microbiology 2e, Section 2.4: Staining microscopic specimens</a>

Fixation et Découpage

Deux types de préparation de base sont utilisés pour visualiser les échantillons avec un microscope optique : les montures humides et les échantillons fixes.
Le type de préparation le plus simple ...

Education

Enseignement

JoVE Core

Cell Biology

Biologie cellulaire

Unit 1

Unité 1

Visualizing Cells, Tissues, and Molecules

Visualisation des cellules, des tissus et des molécules

KEY TERMS AND CONCEPTS

TERMES ET CONCEPTS CLÉS

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

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Microscope Slide Prep and Staining

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