Preparing Microscope Slides
Microscopy is only as good as the slide preparation underlying it. This lesson covers the techniques required by OCR module 2.1.1 (d): preparing temporary and permanent mounts, the use of common stains, and the reasons why each stain is chosen for a particular cell component. This is a practical-focused topic that is assessed in both written papers and PAG (Practical Activity Group) tasks.
Key Terms:
- Mounting — placing a specimen on a slide beneath a cover slip in a suitable medium.
- Fixation — preserving a specimen and preventing decay using chemicals such as formaldehyde.
- Sectioning — cutting very thin slices of embedded tissue using a microtome.
- Staining — applying a dye to selectively colour structures and increase contrast.
Why Prepare Slides at All?
Most biological specimens are transparent and colourless. Under a light microscope, an unstained cell can look almost featureless, because there is little contrast between the cytoplasm and organelles. By staining, sectioning, and mounting correctly, we produce a slide that:
- Has sufficient contrast between structures of interest.
- Can be focused upon with the objective lens (specimens must be thin enough to transmit light).
- Is preserved against drying or decomposition during observation.
- Is protected from physical damage and lens contact by the cover slip.
Temporary Mounts (Wet Mounts)
A temporary mount (often called a wet mount) is the simplest and fastest slide preparation, suitable for demonstrations and observation of living material.
Procedure
- Clean a glass slide with ethanol and a lint-free cloth to remove grease and dust.
- Using a pipette, place a small drop of water (or appropriate buffer) in the centre of the slide.
- Place a thin section of the specimen into the water. For epidermal peels (e.g., onion epidermis), use fine forceps; for pond water samples, use a dropping pipette.
- Add one drop of stain (if used) beside the specimen.
- Using a mounted needle or forceps, lower a cover slip onto the drop at an angle of around 45°, slowly, so that the liquid spreads under the cover slip and air bubbles are excluded.
- Blot away any excess liquid around the cover slip with filter paper.
Typical Specimens
- Onion epidermis (cells of the bulb scale): a classic introduction to plant cells. Cells appear as rectangular units with visible nuclei, cell walls, and large vacuoles when stained with iodine.
- Human cheek cells (buccal smear): scrapings from the inside of the cheek. Stain with methylene blue to reveal nuclei.
- Pond water: living protists, algae, and microscopic invertebrates can be observed swimming.
- Moss leaves: to see chloroplasts.
Advantages and Disadvantages of Temporary Mounts
| Advantages | Disadvantages |
|---|
| Quick and cheap | Dry out within minutes, so not permanent |
| Allow observation of living cells | Only thin specimens can be used |
| Minimal equipment required | Difficult to preserve |
| Dynamic processes (e.g., cytoplasmic streaming) visible | Risk of air bubbles spoiling the image |
Permanent Mounts
For long-term study and teaching collections, permanent mounts are prepared. These can last for decades if stored correctly. The process is more involved and uses harsher chemicals.
Procedure Outline
- Fixation — the specimen is preserved in a chemical such as formalin (formaldehyde solution) to halt decomposition and denature enzymes.
- Dehydration — the water is removed by passing the specimen through a graded series of alcohols (50%, 70%, 90%, 100% ethanol).
- Clearing — the alcohol is replaced with a clearing agent such as xylene, which is miscible with both alcohol and the embedding wax.
- Embedding — the specimen is infiltrated with molten paraffin wax, which sets hard on cooling. This supports the tissue so it can be sliced very thinly.
- Sectioning — a microtome is used to cut sections typically 5–10 µm thick. For TEM, an ultramicrotome cuts sections 50–100 nm thick.
- Mounting and staining — sections are mounted on slides, de-waxed, rehydrated, and stained.
- Cover slipping — a drop of mountant (e.g., Canada balsam or DPX resin) is added, a cover slip is lowered, and the slide is allowed to set.
Why Permanent Mounts Are Useful
- Preserve a specimen for repeated observation.
- Allow the use of stains and counterstains that cannot be used on living material.
- Permit very thin sectioning that is impossible on fresh tissue.
- Provide standardised reference specimens for teaching and research.
Staining: Principles and Common Stains
Stains bind to specific chemical components within a cell and selectively colour them. This increases contrast and enables structures to be identified.