Preparing Microscope Slides

Spec Mapping — OCR H420 Module 2.1.1 — Cell structure, content statements covering the preparation of temporary and permanent mounts of biological specimens for microscopy, including the use of stains to enhance contrast (refer to the official OCR H420 specification document for exact wording). This is the most heavily practical-assessed topic in the whole module; PAG 1 sits squarely on top of it.

Microscopy is only as good as the slide preparation underlying it. This lesson covers the techniques required by OCR module 2.1.1: 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.

The history matters. Camillo Golgi discovered the eponymous Golgi apparatus in 1898 by developing a silver-impregnation stain that randomly blackened a small fraction of cells, revealing their internal cisternae against an unstained background. Christian Gram developed the Gram stain in 1884 to distinguish bacterial species by the structure of their cell walls — still used 140 years later in every hospital microbiology laboratory. The selectivity of a stain is, in every case, a chemical story: a dye that binds to one biomolecule and not another, exploiting electrostatic, hydrogen-bond, or hydrophobic interactions.

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.

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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:

1. Has sufficient contrast between structures of interest.
2. Can be focused upon with the objective lens (specimens must be thin enough to transmit light).
3. Is preserved against drying or decomposition during observation.
4. Is protected from physical damage and lens contact by the cover slip.

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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

1. Clean a glass slide with ethanol and a lint-free cloth to remove grease and dust.
2. Using a pipette, place a small drop of water (or appropriate buffer) in the centre of the slide.
3. 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.
4. Add one drop of stain (if used) beside the specimen.
5. 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.
6. 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

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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

1. Fixation — the specimen is preserved in a chemical such as formalin (formaldehyde solution) to halt decomposition and denature enzymes.
2. Dehydration — the water is removed by passing the specimen through a graded series of alcohols (50%, 70%, 90%, 100% ethanol).
3. Clearing — the alcohol is replaced with a clearing agent such as xylene, which is miscible with both alcohol and the embedding wax.
4. 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.
5. Sectioning — a microtome is used to cut sections typically 5–10 µm thick. For TEM, an ultramicrotome cuts sections 50–100 nm thick.
6. Mounting and staining — sections are mounted on slides, de-waxed, rehydrated, and stained.
7. 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.

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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.

• Basic (cationic) stains are positively charged and bind to negatively charged components, notably nucleic acids (DNA and RNA). Examples: haematoxylin, methylene blue, toluidine blue.
• Acidic (anionic) stains are negatively charged and bind to positively charged components, particularly some proteins in the cytoplasm. Examples: eosin, acid fuchsin.
• Differential stains (counterstains) use combinations of dyes so different cell components appear in contrasting colours (e.g., haematoxylin and eosin — "H&E" — is the classic histological counterstain).

Common Stains You Must Know

Stain	What it stains	Colour produced	Typical use
Haematoxylin	Nucleic acids (DNA, RNA)	Blue/purple	General histology; nuclei in animal tissues. Often used with eosin.
Eosin	Cytoplasmic proteins	Pink/red	Counter-stain with haematoxylin (H&E).
Methylene blue	Nucleic acids and acidic structures	Blue	Cheek cells, bacteria, to show nuclei.
Iodine (in KI solution)	Starch grains (and cell walls slightly)	Blue-black on starch; cells appear yellow-brown	Plant cells, especially onion epidermis; amyloplasts in potato.
Acetic orcein (also written aceto-orcein)	Chromosomes (DNA)	Red/purple	Root tip squashes for mitosis — stains condensed chromosomes vividly.
Gram stain (crystal violet + safranin)	Peptidoglycan in bacterial cell walls	Purple (Gram +) or pink (Gram −)	Bacterial identification.

How Staining Affects What You See

Without stain, a cheek cell under a light microscope appears as a pale disc with only the faintest outline. Adding a drop of methylene blue causes the nucleus to become a clearly visible dark blue blob in the centre. The same principle applies to onion cells with iodine, where nuclei and cell walls become distinct.

Exam Tip: Questions often give you an unstained cell image and ask which stain you would use. Identify what structure is being highlighted (nucleus, starch, chromosomes) and pick the appropriate stain from the table above. Always justify by stating which biomolecule the stain binds to.

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Safety considerations and laboratory technique

Each stain or fixative carries specific risks that examiners expect candidates to identify in PAG-style questions:

• Formalin (formaldehyde solution): carcinogenic; use in a fume hood, wear gloves, avoid inhalation.
• Xylene: flammable, neurotoxic on chronic exposure; use in a well-ventilated area.
• Methylene blue, iodine, acetic orcein: skin/eye irritants; wear gloves and safety glasses.
• HCl (1 mol dm⁻³): corrosive; warm dilute HCl is used in mitosis preparation — eye protection essential.
• Lead and uranium salts (used in TEM heavy-metal staining): toxic and radioactive; handled only in specialist facilities, not in school laboratories.
• Glass slides and cover slips: sharp edges; handle by the long edges, dispose in a sharps bin if broken.
• Open flames and heating: some staining protocols require gentle warming (e.g. acetic orcein on a root squash). Use a water bath or hot plate rather than a Bunsen flame to avoid boiling and stain decomposition.

A typical PAG 1 risk assessment requires you to identify the hazard, the route of exposure, and the control measure for each chemical used. CPAC criteria reward candidates who can articulate the rationale for each control — not merely listing them, but explaining why each is appropriate. For example, "wear gloves to prevent skin contact with formalin, because formalin is a probable human carcinogen and chronic skin exposure has been associated with allergic contact dermatitis."

A Practical: Root Tip Squash Using Acetic Orcein

This is a classic PAG used to observe mitosis. The steps are:

1. Place the root tip (e.g., garlic or onion) in 1 mol dm⁻³ hydrochloric acid at 60 °C for 5 minutes to break down the middle lamella and separate cells.
2. Rinse in water.
3. Cut off the terminal 2 mm of the root (the meristem, where cells are actively dividing).
4. Place on a slide with a drop of acetic orcein stain, and warm gently (without boiling) to aid stain uptake.
5. Lower a cover slip and press down firmly with a pencil eraser or gloved thumb to squash the tissue into a single layer of cells.
6. Observe under ×400; chromosomes appear as dark red/purple bodies. Count how many cells are in each stage of mitosis to calculate a mitotic index.

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The chemistry of staining: cationic vs anionic dyes

Stain selectivity is a story of charge complementarity. Most biological macromolecules carry a characteristic charge at physiological pH:

• Nucleic acids (DNA, RNA) carry strongly negatively charged phosphate backbones, attracting basic (cationic) dyes such as methylene blue, haematoxylin, and toluidine blue.
• Cytoplasmic proteins with basic residues (lysine, arginine) tend to carry net positive charge, attracting acidic (anionic) dyes such as eosin and acid fuchsin.
• Polysaccharides are typically uncharged but contain many hydroxyl groups; iodine binds amylose by inserting into its helical structure, producing the characteristic blue-black colour reaction used in food tests.

The chemical logic of the Gram stain is particularly elegant:

1. All bacteria are first stained with crystal violet, a positively charged dye that enters all cells.
2. Iodine is added, forming a crystal violet–iodine complex inside the cells.
3. The slide is decolourised with alcohol or acetone, which disrupts cell membranes.
4. Gram-positive bacteria retain the dye because their thick peptidoglycan layer is mechanically robust and traps the complex.
5. Gram-negative bacteria lose the dye through their thin peptidoglycan layer once their outer membrane has been disrupted by the alcohol.
6. A counterstain (safranin) colours the now-decolourised Gram-negative bacteria pink.

The result: Gram-positive bacteria appear purple, Gram-negative pink — a 140-year-old clinical workhorse that still guides antibiotic prescribing in hospitals worldwide.

A second PAG-style practical: stained onion epidermis

Onion epidermis is the simplest plant-cell preparation in PAG 1. The procedure:

1. Cut a small section of onion bulb (~5 mm × 5 mm) and gently peel a single layer of transparent epidermal cells from the inner concave surface of a fleshy scale leaf.
2. Place the peel on a clean slide with a drop of water.
3. Add one drop of iodine in potassium iodide (KI) solution beside the specimen.
4. Lower a cover slip at ~45° to exclude air bubbles.
5. Blot away excess liquid with filter paper.
6. Observe under ×100 (low power) then ×400 (high power).