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

La mayoría de las muestras de tejido biológico son gruesas y opacas y deben procesarse antes de ser observadas bajo un microscopio.
Los tejidos se tratan primero con una solución de fijación que mata las células e inmoviliza los componentes celulares para mantener su estructura.
Las muestras fijas se pasan a través de un gradiente creciente de alcohol, que deshidrata la muestra.
Para evitar que las muestras frágiles se dañen, se incrustan en un material repelente al agua como la cera de parafina y se cortan en secciones delgadas con un micrótomo.
Las secciones delgadas se transfieren a portaobjetos, donde se agregan unas gotas de solventes orgánicos para eliminar la cera adherida.
A continuación, se tiñen las secciones y se añade un medio de montaje, con un cubreobjetos en la parte superior. El medio de montaje debe tener el mismo índice de refracción que el cubreobjetos y la corredera para obtener la mejor resolución de imagen.
La muestra ya está lista para su visualización al microscopio.

fijación y seccionamiento

Fixation and Sectioning

Se utilizan dos tipos básicos de preparación para visualizar muestras con un microscopio óptico: preparaciones húmedas y muestras fijas.

El tipo de preparación más simple es el montaje húmedo, en el que la muestra se coloca en una gota de líquido sobre el portaobjetos. Se puede depositar una muestra líquida directamente en el portaobjetos utilizando un gotero. Se pueden colocar muestras sólidas, como raspados de piel, en el portaobjetos antes de agregar una gota de líquido para preparar la preparación húmeda. A veces el líquido es simplemente agua, pero a menudo se añaden tintes para mejorar el contraste. Una vez que se ha agregado el líquido al portaobjetos, se coloca un cubreobjetos encima y la muestra está lista para examinarla bajo el microscopio.

El segundo método de preparación de muestras para microscopía óptica es la fijación. La "fijación" de una muestra se refiere al proceso de unir células a un portaobjetos. La fijación se logra a menudo calentando (fijación térmica) o tratando químicamente la muestra. Además de fijar la muestra al portaobjetos, la fijación también mata los microorganismos de la muestra, deteniendo su movimiento y metabolismo y preservando al mismo tiempo la integridad de sus componentes celulares para la observación.

Para fijar térmicamente una muestra, se extiende una capa delgada de la muestra sobre el portaobjetos (llamada frotis) y entonces el portaobjetos se calienta brevemente sobre una fuente de calor. Los fijadores químicos suelen ser preferibles al calor para las muestras de tejido. Agentes químicos como el ácido acético, el etanol, el metanol, el formaldehído (formalina) y el glutaraldehído pueden desnaturalizar proteínas, detener reacciones bioquímicas y estabilizar las estructuras celulares en muestras de tejido.

Además de la fijación, la tinción casi siempre se aplica para colorear ciertas características de una muestra antes de examinarla con un microscopio óptico. Los tintes o tintes contienen sales formadas por un ión positivo y un ión negativo. Dependiendo del tipo de tinte, el ion positivo o negativo puede ser el cromóforo (el ion coloreado); y el otro ion incoloro se llama contraión. Si el cromóforo es el ion cargado positivamente, el tinte se clasifica como tinte básico; si el ion negativo es el cromóforo, el tinte se considera un tinte ácido.

Los tintes se seleccionan para teñir en función de las propiedades químicas del tinte y de la muestra que se observa, que determinan cómo interactuará el tinte con la muestra. En la mayoría de los casos, es preferible utilizar un tinte positivo, un tinte que será absorbido por las células u organismos observados, añadiendo color a los objetos de interés para que destaquen sobre el fondo. Sin embargo, hay situaciones en las que resulta ventajoso utilizar una tinción negativa absorbida por el fondo pero no por las células u organismos de la muestra. La tinción negativa produce un contorno o silueta de los organismos sobre un fondo colorido.

Debido a que las células suelen tener paredes celulares cargadas negativamente, los cromóforos positivos en los tintes básicos tienden a adherirse a las paredes celulares, convirtiéndolas en tinciones positivas. Por tanto, los colorantes básicos como la fucsina básica, el violeta cristal, el verde de malaquita, el azul de metileno y la safranina suelen servir como colorantes positivos. Por otro lado, los cromóforos cargados negativamente en los tintes ácidos son repelidos por las paredes celulares cargadas negativamente, lo que las convierte en tinciones negativas. Los tintes ácidos de uso común incluyen la fucsina ácida, la eosina y la rosa de bengala.

Algunas técnicas de tinción implican la aplicación de un solo tinte a la muestra; otros requieren más de un tinte. En la tinción simple, se utiliza un único tinte para resaltar estructuras particulares de la muestra. Una simple tinción generalmente hará que todos los organismos de una muestra parezcan del mismo color, incluso si la muestra contiene más de un tipo de organismo. Por el contrario, la tinción diferencial distingue organismos en función de sus interacciones con múltiples tinciones. En otras palabras, dos organismos en una muestra teñida de manera diferente pueden aparecer con colores diferentes. Las técnicas de tinción diferencial comúnmente utilizadas en entornos clínicos incluyen tinción de Gram, tinción acidorresistente, tinción de endosporas, tinción de flagelos y tinción de cápsulas.

Fijación y Seccionamiento

Se utilizan dos tipos básicos de preparación para visualizar muestras con un microscopio óptico: monturas húmedas y muestras fijas.
El tipo de preparación más simple ...

Education

Educación

JoVE Core

Cell Biology

Biología Celular

Unit 1

Unidad 1

Visualizing Cells, Tissues, and Molecules

Visualización de células, tejidos y moléculas

KEY TERMS AND CONCEPTS

TÉRMINOS Y CONCEPTOS CLAVE

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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fixation and sectioning

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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

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JoVE Core Biología Celular ilustra procesos celulares complejos mediante completos vídeos animados fáciles de entender. Esta colección se centra en los componentes esenciales de las células y su funcionamiento, incluyendo los orgánulos y las membranas, el transporte celular y el metabolismo energético celular. Todo este material se complementa con los vídeos de científicos en acción, que muestran la aplicación de la biología celular en experimentos reales de investigación.

Microscope Slide Prep and Staining

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