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

A maioria das amostras de tecido biológico é espessa e opaca e deve ser processada antes de ser observada ao microscópio.
Os tecidos são tratados primeiro com uma solução de fixação que mata as células e imobiliza os componentes celulares para manter sua estrutura.
As amostras fixas são passadas através de um gradiente crescente de álcool, que desidrata a amostra.
Para evitar que as amostras frágeis sejam danificadas, elas são incorporadas em um material repelente de água, como cera de parafina, e cortadas em seções finas com um micrótomo.
As seções finas são transferidas para lâminas, onde algumas gotas de solventes orgânicos são adicionadas para remover a cera aderente.
Em seguida, as seções são manchadas e um meio de montagem é adicionado, com uma lamínula na parte superior. O meio de montagem deve ter o mesmo índice de refração que a lamínula e a lâmina para obter a melhor resolução de imagem.
A amostra agora está pronta para visualização ao microscópio.

fixação e seccionamento

Fixation and Sectioning

Dois tipos básicos de preparação são usados para visualizar amostras com um microscópio óptico: montagens úmidas e amostras fixas. O tipo de preparo mais simples é a montagem úmida, no qual a amostra é colocada em uma gota de líquido sobre a lâmina. Uma amostra líquida pode ser depositada diretamente na lâmina usando um conta-gotas. Amostras sólidas, como raspagens de pele, podem ser colocadas na lâmina antes de adicionar uma gota de líquido para preparar a montagem úmida. Às vezes o líquido é simplesmente água, mas muitas vezes são adicionadas manchas para aumentar o contraste. Depois que o líquido for adicionado à lâmina, uma lamela é colocada por cima e a amostra está pronta para exame ao microscópio. O segundo método de preparação de amostras para microscopia óptica é a fixação. A “fixação” de uma amostra refere-se ao processo de fixação de células a uma lâmina. A fixação geralmente é obtida por aquecimento (fixação por calor) ou tratamento químico da amostra. Além de fixar a amostra à lâmina, a fixação também mata os microrganismos da amostra, interrompendo seu movimento e metabolismo, preservando a integridade de seus componentes celulares para observação. Para fixar uma amostra a quente, uma fina camada da amostra é espalhada na lâmina (chamada de esfregaço) e a lâmina é então brevemente aquecida sobre uma fonte de calor. Os fixadores químicos são frequentemente preferíveis ao calor para amostras de tecido. Agentes químicos como ácido acético, etanol, metanol, formaldeído (formalina) e glutaraldeído podem desnaturar proteínas, interromper reações bioquímicas e estabilizar estruturas celulares em amostras de tecido. Além da fixação, a coloração é quase sempre aplicada para colorir certas características de uma amostra antes de examiná-la ao microscópio óptico. As manchas ou corantes contêm sais compostos por um íon positivo e um íon negativo. Dependendo do tipo de corante, o íon positivo ou negativo pode ser o cromóforo (o íon colorido); e o outro íon incolor é chamado de contra-íon. Se o cromóforo for um íon carregado positivamente, a mancha é classificada como corante básico; se o íon negativo for o cromóforo, a mancha é considerada um corante ácido. Os corantes são selecionados para coloração com base nas propriedades químicas do corante e da amostra que está sendo observada, que determinam como o corante irá interagir com a amostra. Na maioria dos casos, é preferível utilizar um corante positivo, um corante que será absorvido pelas células ou organismos observados, acrescentando cor aos objetos de interesse para destacá-los do fundo. No entanto, existem cenários em que é vantajoso utilizar uma coloração negativa absorvida pelo fundo, mas não pelas células ou organismos da amostra. A coloração negativa produz um contorno, ou silhueta, dos organismos contra um fundo colorido. Como as células normalmente têm paredes celulares carregadas negativamente, os cromóforos positivos nos corantes básicos tendem a aderir às paredes celulares, tornando-os corantes positivos. Assim, corantes básicos como fucsina básica, violeta cristal, verde malaquita, azul de metileno e safranina normalmente servem como corantes positivos. Por outro lado, os cromóforos carregados negativamente em corantes ácidos são repelidos pelas paredes celulares carregadas negativamente, tornando-os corantes negativos. Os corantes ácidos comumente usados incluem fucsina ácida, eosina e rosa bengala. Algumas técnicas de coloração envolvem a aplicação de apenas um corante na amostra; outras requerem mais de um corante. Na coloração simples, um único corante é usado para enfatizar estruturas específicas da amostra. Uma coloração simples geralmente fará com que todos os organismos em uma amostra tenham a mesma cor, mesmo que a amostra contenha mais de um tipo de organismo. Em contraste, a coloração diferencial distingue os organismos com base nas suas interações com múltiplas manchas. Em outras palavras, dois organismos em uma amostra corada diferencialmente podem ter cores diferentes. As técnicas de coloração diferencial comumente usadas em ambientes clínicos incluem coloração de Gram, coloração ácido-resistente, coloração de endósporos, coloração de flagelos e coloração de cápsula. Este texto é adaptado de <a href="https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens">Openstax, Microbiology 2e, Seção 2.4: </a><a href="https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens">Coloração de amostras microscópicas</a>

Fixação e Seccionamento

Dois tipos básicos de preparação são usados para visualizar amostras com um microscópio de luz: montagens úmidas e amostras fixas.
O tipo mais simples de preparação ...

Education

JoVE Core

Cell Biology

Biologia Celular

Unit 1

Unidade 1

Visualizing Cells, Tissues, and Molecules

Visualizando Células, Tecidos e Moléculas

KEY TERMS AND CONCEPTS

TERMOS-CHAVE E CONCEITOS

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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Optical microscopy uses light and lenses to make tiny biological samples visible. Antonie van Leeuwenhoek built the first compound optical microscope in the 17th century to view blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister developed an essentially modern light microscope. During the 20th century, microscopes gained better magnification and resolution. A sample is placed on a glass slide and clipped onto the stage, which is the flat platform of the microscope. The...

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fixation and sectioning

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...

immunofluorescence microscopy

Fluorescent Antibody Staining in Cells

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Antibody Staining in Cells and Tissues

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Confocal Microscopy for Sharp Cell Images

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Fluorescent Methods for Protein Movement

Protein movement in living cells can be measured with fluorescence-based methods such as FRAP, FRET, and PET. These techniques help scientists track how proteins move, interact, and change distance inside the cell. Fluorescent recovery after photobleaching, or FRAP, measures protein movement rates within the cell. In this method, a small region is exposed to a strong laser beam. The beam permanently bleaches fluorophore-tagged proteins in that area. Over time, bleached proteins diffuse away...

total internal reflection fluorescence microscopy

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Surface-Only Imaging with TIRF Microscopy

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Electron microscopy uses electrons instead of visible light to make images with much higher detail than a light microscope. Visible light limits the resolution of light microscopes, which usually magnify up to 1000X, and sometimes up to 1500X. Electrons have a much shorter wavelength, about 0.005 nm, so they can produce resolution as fine as 0.05 nm. An electron microscope can also magnify images up to 2,000,000X and reveal subcellular structures and some molecular structures, including single...

scanning electron microscopy

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A scanning electron microscope (SEM) reveals the surface details of a sample with an electron beam. The beam scans across the sample in a two-dimensional pattern. SEM can image areas from about 1 centimeter down to 5 micrometers wide. SEM is useful for studying bacteria, viruses, tissues, and larger samples such as insects. Conventional SEM can produce magnification from 20X to 30,000X. It also offers spatial resolution of about 50 to 100 nanometers. The process starts when the electron gun...

transmission electron microscopy

How TEM Reveals Tiny Structures

Transmission electron microscopy uses electrons to reveal very small structures inside a specimen. The technique grew from the first electron microscope prototype developed in 1931 by physicist Ernst Ruska. He built on the idea that magnetic fields can direct an electron beam, just as lenses direct light in an optical microscope. In a transmission electron microscope, a hot tungsten element produces electrons. An electron gun then accelerates them with a potential difference, giving the beam...

preparation of samples for electron microscopy

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Electron Microscopy Sample Prep Methods

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Immunogold Labeling in Electron Microscopy

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Cryo-EM for Native Biomolecule Imaging

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3D Imaging with Electron Microscopy

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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 - Biologia Celular ilustra processos celulares complexos em vídeos de animação fáceis de entender e, ao mesmo tempo, abrangentes. Esta coleção foca nos componentes e funções essenciais das células, incluindo organelas e membranas, transporte celular e energia celular. É complementado por vídeos de Cientistas em ação que demonstram a aplicação da biologia celular em experimentos de pesquisa do mundo real.

Microscope Slide Prep and Staining

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