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

La maggior parte dei campioni di tessuto biologico sono spessi e opachi e devono essere elaborati prima di essere osservati al microscopio.
I tessuti vengono prima trattati utilizzando una soluzione fissante che uccide le cellule e immobilizza i componenti cellulari per mantenere la loro struttura.
I campioni fissati vengono fatti passare attraverso un gradiente crescente di alcol, che disidrata il campione.
Per evitare che i fragili campioni vengano danneggiati, vengono incorporati in un materiale idrorepellente come la cera di paraffina e tagliati in sezioni sottili con un microtomo.
Le sezioni sottili vengono trasferite su vetrini, dove vengono aggiunte alcune gocce di solventi organici per rimuovere la cera aderente.
Successivamente, le sezioni vengono macchiate e viene aggiunto un supporto di montaggio, con un vetrino coprioggetti sulla parte superiore. Il supporto di montaggio deve avere lo stesso indice di rifrazione del vetrino coprioggetti e del vetrino per ottenere la migliore risoluzione dell'immagine.
Il campione è ora pronto per la visualizzazione al microscopio.

fissazione e sezionamento

Fixation and Sectioning

Per visualizzare i campioni con un microscopio ottico vengono utilizzati due tipi fondamentali di preparazione: supporti bagnati e campioni fissi.

Il tipo più semplice di preparazione è il montaggio bagnato, in cui il campione viene posto in una goccia di liquido sul vetrino. Un campione liquido può essere depositato direttamente sul vetrino utilizzando un contagocce. I campioni solidi, come quelli ottenuti mediante raschiamento cutaneo, possono essere posizionati sul vetrino prima di aggiungere una goccia di liquido per preparare il supporto bagnato. A volte il liquido è semplicemente acqua, ma spesso vengono aggiunte macchie per migliorare il contrasto. Una volta aggiunto il liquido al vetrino, viene posizionato sopra un vetrino coprioggetto e il campione è pronto per l'esame al microscopio.

Il secondo metodo per preparare i campioni per la microscopia ottica è la fissazione. Il “fissaggio” di un campione si riferisce al processo di fissaggio delle cellule a un vetrino. La fissazione viene spesso ottenuta riscaldando (fissaggio termico) o trattando chimicamente il campione. Oltre a fissare il campione sul vetrino, il fissaggio uccide anche i microrganismi presenti nel campione, arrestandone il movimento e il metabolismo e preservando l'integrità dei loro componenti cellulari per l'osservazione.

Per fissare a caldo un campione, uno strato sottile del campione viene steso sul vetrino (chiamato striscio) e il vetrino viene quindi riscaldato brevemente su una fonte di calore. I fissativi chimici sono spesso preferibili al riscaldamento per i campioni di tessuto. Agenti chimici come acido acetico, etanolo, metanolo, formaldeide (formalina) e glutaraldeide possono denaturare le proteine, arrestare le reazioni biochimiche e stabilizzare le strutture cellulari nei campioni di tessuto.

Oltre alla fissazione, la colorazione viene quasi sempre applicata per colorare alcune caratteristiche di un campione prima di esaminarlo al microscopio ottico. Le macchie, o coloranti, contengono sali costituiti da uno ione positivo e uno ione negativo. A seconda del tipo di colorante, lo ione positivo o negativo può essere il cromoforo (lo ione colorato); e l'altro ione incolore è chiamato controione. Se il cromoforo è lo ione carico positivamente, la macchia è classificata come colorante basico; se lo ione negativo è il cromoforo, la macchia è considerata un colorante acido.

I coloranti vengono selezionati per la colorazione in base alle proprietà chimiche del colorante e del campione osservato, che determinano il modo in cui il colorante interagirà con il campione. Nella maggior parte dei casi, è preferibile utilizzare una colorazione positiva, un colorante che verrà assorbito dalle cellule o dagli organismi osservati, aggiungendo colore agli oggetti di interesse per farli risaltare sullo sfondo. Tuttavia, esistono scenari in cui è vantaggioso utilizzare una colorazione negativa assorbita dallo sfondo ma non dalle cellule o dagli organismi presenti nel campione. La colorazione negativa produce un contorno o una sagoma degli organismi su uno sfondo colorato.

Poiché le cellule hanno tipicamente pareti cellulari caricate negativamente, i cromofori positivi nei coloranti basici tendono ad attaccarsi alle pareti cellulari, rendendole macchie positive. Pertanto, i coloranti basici come la fucsina basica, il cristal violetto, il verde malachite, il blu di metilene e la safranina servono tipicamente come coloranti positivi. D'altra parte, i cromofori caricati negativamente nei coloranti acidi vengono respinti dalle pareti cellulari caricate negativamente, rendendole macchie negative. I coloranti acidi comunemente usati includono fucsina acida, eosina e rosa bengala.

Alcune tecniche di colorazione prevedono l'applicazione di un solo colorante al campione; altri richiedono più di un colorante. Nella colorazione semplice, viene utilizzato un singolo colorante per enfatizzare particolari strutture nel campione. Una semplice colorazione generalmente farà apparire tutti gli organismi presenti in un campione dello stesso colore, anche se il campione contiene più di un tipo di organismo. Al contrario, la colorazione differenziale distingue gli organismi in base alle loro interazioni con più macchie. In altre parole, due organismi in un campione colorato in modo differenziale possono apparire di colori diversi. Le tecniche di colorazione differenziale comunemente utilizzate in ambito clinico comprendono la colorazione di Gram, la colorazione acido-resistente, la colorazione dell'endospora, la colorazione dei flagelli e la colorazione della capsula.

Questo testo è adattato da <a href="https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens">Openstax, Microbiology 2e, Section 2.4: Staining microscopic specimens</a>

Fissazione e sezionamento

Per visualizzare i campioni con un microscopio ottico vengono utilizzati due tipi fondamentali di preparazione: montature umide e campioni fissi.
Il tipo di preparazione più semplice ...

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Cell Biology

Biologia cellulare

Unit 1

Unità 1

Visualizing Cells, Tissues, and Molecules

Visualizzazione di cellule, tessuti e molecole

KEY TERMS AND CONCEPTS

TERMINI E CONCETTI CHIAVE

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

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Microscope Slide Prep and Staining

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