What is the process of preparing the specimen on the slide for microscopy?

If you’re involved in biological research, chances are at some stage you’ve submitted tissue specimens to a histology lab. Somehow they magically produced beautiful slides for you – each containing thin sections of your specimens, ready for microscopic evaluation.

Have you ever wondered how the histology technician does this histology slide preparation?  Read on for the five important stages in histology slide production.

The Five Steps of Histology Slide Preparation

1. Tissue fixation

Slide preparation begins with the fixation of your tissue specimen. This is a crucial step in tissue preparation, and its purpose is to prevent tissue autolysis and putrefaction. For best results, your biological tissue samples should be transferred into fixative immediately after collection.

Although there are many types of fixative, most specimens are fixed in 10% neutral buffered formalin. The optimum formalin-to-specimen volume ratio should be at least 10:1 (e.g., 10ml of formalin per 1 cm3 of tissue). This will allow most tissues to become adequately fixed within 24-48 hours. Formalin containers should be capped and leak-proof, and labeled correctly.

2. Specimen Transfer to Cassettes

After fixation, specimens are trimmed using a scalpel to enable them to fit into an appropriately labeled tissue cassette. Specimens should not be so big that they fill the cassette – they are trimmed so as not to touch the edges. Additionally, they must not be too thick (ideally they should be less than 4 mm), otherwise, they risk being “waffled” when the cassette lid is closed. The filled tissue cassettes are then stored in formalin until processing begins.

3. Tissue Processing

Processing tissues into thin microscopic sections is usually done using a paraffin block, as follows:

1. Dehydration, which involves immersing your specimen in increasing concentrations of alcohol to remove the water and formalin from the tissue.
2. Clearing, in which an organic solvent such as xylene is used to remove the alcohol and allow infiltration with paraffin wax.
3. Embedding, where specimens are infiltrated with the embedding agent – usually paraffin wax. The tissue becomes surrounded by a large block of molten paraffin wax, creating what is now referred to as the “block”.  Once the block solidifies, it provides a support matrix that allows very thin sectioning.

4. Sectioning

Your tissue specimen is now ready to be cut into sections that can be placed on a slide.

1. Wax is removed from the surface of the block to expose the tissue.
2. Blocks are chilled on a refrigerated plate or ice tray for 10 minutes before sectioning.
3. A microtome is used to slice extremely thin tissue sections off the block in the form of a ribbon.

The microtome can be pre-set to cut at different thicknesses, but most tissues are cut at around 5 µm. You can discover more ways to slice tissue sections here.

Once cut, the tissue ribbons are carefully transferred to a warm water bath. Here they are allowed to float on the surface, and can then be scooped up onto a slide placed under the water level. Charged slides work best for this process – they improve tissue adhesion to the glass, and help to reduce the chance of sections washing off the slide during staining.

Slides should be clearly labeled, and then allowed to dry upright at 37oC for a few hours to gently melt the excess paraffin wax, leaving the tissue section intact.

5. Staining

Most cells are transparent and appear almost colorless when unstained. Histochemical stains (typically hematoxylin and eosin) are therefore used to provide contrast to tissue sections, making tissue structures more visible and easier to evaluate.  Following staining, a coverslip is mounted over the tissue specimen on the slide, using optical grade glue, to help protect the specimen.

Histology Slide Preparation Wrapped Up

So as you can see, histology slide preparation is no breeze – it’s quite an intricate work of art!  Although you may want to learn how to do this to help cut costs in the lab, I’d advise you to think twice about this, and instead send the specimens to a histology laboratory for this purpose – especially if tissue evaluation is an important part of your study. You’ll save yourself a lot of stress and time by leaving this job to an experienced histology technician, and your slides will be much easier to evaluate.

While histology is great for lower resolution imaging of whole tissues, it is limiting if you want to investigate subcellular structures for example when studying changes in brain tumors. In this case, a more detailed and high-powered technique such as brain cancer electron microscopy is required.

If histology has enough resolution for your needs, do you prefer to make your own slides, or send tissues to a histology lab for processing? Leave a comment below.

Want to know more about histology? Visit the Bitesize Bio Histology Hub for tips and tricks for all your histology experiments.

Further Reading

If you found this article useful, you might want to check out some of our related articles below:

Originally published April 4, 2012. Reviewed and updated on November 19, 2020.

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Sectioning is performed using microtomy or cryotomy. Sectioning is an important step for the preparation of slides as it ensures a proper observation of the sample by microscopy.

– Paraffin-embedded samples are cut by cross section, using a microtome, into thin slices of 5 micrometers. Glass slides are covered with a solution that contains an additive in order to keep the section attached onto the slide. Slides are then placed onto a hotplate in order to ensure a uniform spreading of the sample. Once the slides are heated, the residual liquid is removed by hand. The slides are then dried at room temperature.

– Frozen samples are cut using a cryostat. The frozen sections are then placed on a glass slide for storage at -80°C.

Bioalternatives evaluates the suitable fixation and sectioning conditions according to your sample and to your study.

The choice of these preparation conditions is crucial in order to minimize the artifacts. Paraffin embedding is favored for preserving tissues; freezing is more suitable for preserving DNA and RNA and for the labeling of water-soluble elements or of those sensitive to the fixation medium.

Before you start building your slides, make sure you have everything you will need, including slides, cover slips, droppers or pipets and any chemicals or stains you plan to use.

You will be using two main types of slides, 1) the common flat glass slide, and 2) the depression or well slides. Well slides have a small well, or indentation, in the center to hold a drop of water or liquid substance. They are more expensive and usually used without a cover slip.

Standard slides can be either plastic or glass and are 1 x 3 inches (25 x 75 mm) in size and 1 to 1.2 mm thick.

Wet slides will use a cover slip or cover glass, a very thin square piece of glass (or plastic) that is placed over the sample drop. Without the cover in place, surface tension would cause the droplet to bunch up in a dome. The cover breaks this tension, flattening the sample and allowing very close inspection with minimal focusing. The cover also serves to protect the objective lens from interfering with the sample drop.

MOUNTS

There are four common ways to mount a microscope slide as described below:

Dry Mount

In a dry mount, the specimen is placed directly on the slide. A cover slip may be used to keep the specimen in place and to help protect the objective lens. Dry mounts are suitable for specimens such as samples of pollen, hair, feathers or plant materials.

Wet Mount

In a wet mount, a drop of water is used to suspend the specimen between the slide and cover slip. Place a sample on the slide. Using a pipette, place a drop of water on the specimen. Then place on edge of the cover slip over the sample and carefully lower the cover slip into place using a toothpick or equivalent. This method will help prevent air bubbles from being trapped under the cover slip.

Your objective is to have sufficient water to fill the space between cover slip and slide. If there is too much water, the cover slip will slide around. Take a piece of paper towel and hold it close to one edge of the cover slip. This will draw out some water. If too dry, add a drop of water beside the cover slip. Practice this until you get used to it.

Wet mounts are suitable for studying water-bound organisms such as paramecium or bodily fluids such as saliva, blood and urine.

Section Mount

In a section mount, an extremely thin cross-section of a specimen is used. Using a microtome, cut a thin slice of your selected specimen such as an onion, and carefully set it on your slide. Then follow the instructions for a dry or wet mount. A stain can often be applied directly to the specimen before covering with a cover slip.

Section mounts are suitable for useful for a wide variety of samples such as fruit, vegetables and other solids that can be cut into small slices.

Smear

A smear is made by carefully smearing a thin layer of the specimen across a slide and then applying a cover slip. Typically, a smear should be allowed to air dry before applying a stain. 

STAINS

Stains are used to help identify different types of cells using light microscopes. They give the image more contrast and allow cells to be classified according to their shape (morphology). By using a variety of different stains, you can selectively stain different areas such as a cell wall, nucleus, or the entire cell. Stains can also help differentiate between living or dead cells. 

Stains tend to be grouped as neutral, acidic or basic, depending upon their chemical makeup and will attract or repel different organisms accordingly. For example, scientists and health professionals use Methylene Blue, a slightly alkaline stain, to reveal the presence of deoxyribonucleic acid, more commonly known as DNA.

Stain Types

Iodine is one of the more commonly available stains and is used to identify starch in a variety of samples. It will stain carbohydrates in plants and animal specimens brown or blue-black. Glycogen will show as red.

Methylene Blue is an alkaline stain useful in identifying acidic cell nuclei and DNA in animal, bacteria or blood samples. It’s also useful in aquariums to prevent the spread of fungal infections in fish. See more details >

Eosin Y is an acidic stain which stains pink for alkaline cells (cytoplasm, for example). It colors red for blood cells, cytoplasm and cell membranes. Eosin's most important medical uses are in blood and bone-marrow testing, including the PAP smear. See more details >

Gram's Stain is one of the most frequently used processes in identifying bacteria – used daily in hospitals. It is a primary test that quickly and cost effectively divides bacteria into one of two types: Gram positive or Gram negative.  See more details >

STAINING STEPS

1. Prepare a wet mount slide.
2. Collect a drop of stain with an eye dropper or pipette.
3. Put a drop of stain on an outer edge of your cover slide.
4. Place a piece of napkin or paper towel against the opposite side of your cover slip, right up against the edge. This will help draw the stain under the cover and across the specimen.
5. You may need to add another drop to ensure complete coverage.
6. The slide is now ready for viewing.