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

Most biological tissue samples are thick and opaque and must be processed before being observed under a microscope.
The tissues are first treated using a fixing solution that kills the cells and immobilizes the cellular components to maintain their structure.
The fixed samples are passed through an increasing gradient of alcohol, which dehydrates the sample.
To prevent the fragile samples from being damaged, they are embedded in a water-repelling material like paraffin wax and sliced into thin sections with a microtome.
The thin sections are transferred to slides, where a few drops of organic solvents are added to remove the adhering wax.
Next, the sections are stained, and a mounting medium is added, with a coverslip on top. The mounting medium should have the same refractive index as the coverslip and the slide to obtain the best image resolution.
The sample is now ready for visualization under a microscope.

fixatie en sectie

Fixation and Sectioning

Er worden twee basistypen preparatie gebruikt om specimens met een lichtmicroscoop te visualiseren: natte montages en vaste specimens.

De eenvoudigste voorbereiding is de natte montage, waarbij het preparaat in een druppel vloeistof op het objectglaasje wordt geplaatst. Een vloeibaar monster kan met behulp van een druppelaar rechtstreeks op het objectglaasje worden aangebracht. Vaste monsters, zoals huidafkrabsels, kunnen op het objectglaasje worden geplaatst voordat een druppel vloeistof wordt toegevoegd om de natte montage voor te bereiden. Soms is de vloeistof gewoon water, maar vaak worden er vlekken toegevoegd om het contrast te vergroten. Nadat de vloeistof aan het objectglaasje is toegevoegd, wordt er een dekglaasje op geplaatst en is het monster klaar voor onderzoek onder de microscoop.

De tweede methode voor het voorbereiden van monsters voor lichtmicroscopie is fixatie. Het ‘fixeren’ van een monster verwijst naar het proces waarbij cellen aan een objectglaasje worden bevestigd. Fixatie wordt vaak bereikt door het preparaat te verwarmen (warmtefixatie) of chemisch te behandelen. Naast het bevestigen van het monster aan het objectglaasje, doodt fixatie ook de micro-organismen in het monster, waardoor hun beweging en metabolisme wordt gestopt, terwijl de integriteit van hun cellulaire componenten voor observatie behouden blijft.

Om een monster met hitte te fixeren, wordt een dunne laag van het monster op het objectglaasje uitgespreid (een uitstrijkje genoemd) en het objectglaasje wordt vervolgens kortstondig verwarmd boven een warmtebron. Bij weefselspecimens verdienen chemische fixeermiddelen vaak de voorkeur boven verhitting. Chemische middelen zoals azijnzuur, ethanol, methanol, formaldehyde (formaline) en glutaaraldehyde kunnen eiwitten denatureren, biochemische reacties stoppen en celstructuren in weefselmonsters stabiliseren.

Naast fixatie wordt kleuring vrijwel altijd toegepast om bepaalde kenmerken van een preparaat te kleuren voordat het onder een lichtmicroscoop wordt onderzocht. Vlekken of kleurstoffen bevatten zouten die bestaan uit een positief ion en een negatief ion. Afhankelijk van het type kleurstof kan het positieve of het negatieve ion de chromofoor (het gekleurde ion) zijn; en het andere ongekleurde ion wordt het tegenion genoemd. Als de chromofoor het positief geladen ion is, wordt de vlek geclassificeerd als een basische kleurstof; als het negatieve ion de chromofoor is, wordt de vlek als een zure kleurstof beschouwd.

Kleurstoffen worden geselecteerd voor kleuring op basis van de chemische eigenschappen van de kleurstof en het monster dat wordt waargenomen, die bepalen hoe de kleurstof met het monster zal interageren. In de meeste gevallen verdient het de voorkeur om een positieve kleurstof te gebruiken, een kleurstof die wordt geabsorbeerd door de cellen of organismen die worden geobserveerd, waardoor kleur wordt toegevoegd aan interessante objecten, zodat ze opvallen tegen de achtergrond. Er zijn echter scenario's waarin het voordelig is om een negatieve kleur te gebruiken die door de achtergrond wordt geabsorbeerd, maar niet door de cellen of organismen in het monster. Negatieve kleuring produceert een omtrek of silhouet van de organismen tegen een kleurrijke achtergrond.

Omdat cellen doorgaans negatief geladen celwanden hebben, hebben de positieve chromoforen in basische kleurstoffen de neiging aan de celwanden te blijven kleven, waardoor ze positieve vlekken worden. Basische kleurstoffen zoals basische fuchsine, kristalviolet, malachietgroen, methyleenblauw en safranine dienen dus doorgaans als positieve vlekken. Aan de andere kant worden de negatief geladen chromoforen in zure kleurstoffen afgestoten door negatief geladen celwanden, waardoor ze negatieve vlekken worden. Veelgebruikte zure kleurstoffen zijn onder meer zure fuchsine, eosine en Bengaalse roos.

Bij sommige kleurtechnieken wordt slechts één kleurstof op het monster aangebracht; anderen hebben meer dan één kleurstof nodig. Bij eenvoudige kleuring wordt een enkele kleurstof gebruikt om bepaalde structuren in het monster te benadrukken. Een eenvoudige kleuring zorgt er over het algemeen voor dat alle organismen in een monster dezelfde kleur hebben, zelfs als het monster meer dan één type organisme bevat. Differentiële kleuring daarentegen onderscheidt organismen op basis van hun interacties met meerdere vlekken. Met andere woorden: twee organismen in een verschillend gekleurd monster kunnen verschillende kleuren hebben. Differentiële kleuringstechnieken die vaak in klinische omgevingen worden gebruikt, zijn onder meer gramkleuring, zuurvaste kleuring, endosporekleuring, flagellakleuring en capsulekleuring.

Fixatie en sectie

Two basic types of preparation are used to visualize specimens with a light microscope: wet mounts and fixed specimens.
The simplest type of preparation ...

Education

JoVE Core

Cell Biology

Celbiologie

Unit 1

Eenheid 1

Visualizing Cells, Tissues, and Molecules

Visualisatie van cellen, weefsels en moleculen

KEY TERMS AND CONCEPTS

BELANGRIJKE TERMEN EN CONCEPTEN

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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