🔬 Big idea: You cannot see a cell wall, a nucleus or a xylem vessel in an untreated piece of tissue — the material is thick and almost colourless. Sectioning makes light pass through it; staining makes the structures differ from one another. Every step exists to solve one of those two problems.
Summary — What This Topic Covers
Why sections must be thin, and how to cut them
What the common stains bind to and what colour they give
Preparing a wet mount and a squash without trapping air
Using a graticule and stage micrometer to measure what you see
Calculating magnification and actual size
1. Sectioning — Why Thin Matters
A light microscope works by shining light through the specimen. Tissue more than a few cells thick blocks the light and gives a dark, muddled image with several layers of cells superimposed.
Cut with a sharp blade in one smooth stroke — sawing tears the tissue
Support soft tissue between two pieces of pith, or in a rolled leaf, so it does not squash
Take the thinnest section you can and select the best one under the microscope — you are expected to cut several
Keep the section moist throughout, or the cells shrink and distort
Analogy
Holding a whole apple up to a lamp shows nothing. Cut a slice thin enough and the light comes through, and you can finally see how it is arranged inside.
2. Stains — What Binds to What
Stains work because different cell components have different chemistry, so a dye binds to some and not others. Learn the pairing, not just the colour.
STAIN BINDS TO RESULT
─────────────────────────────────────────────────────────────────
Iodine in KI starch blue-black
Methylene blue nucleic acids / nuclei blue
Acetic orcein chromosomes dark red
Toluidine blue O nuclei and cell walls blue / purple
Phloroglucinol + HCl lignin red
Sudan III / IV lipids red
Key Term
A differential stain colours different structures differently, so they can be told apart. This is why a stained section shows a nucleus clearly while an unstained one does not.
Common mistake
Saying a stain "makes it easier to see". Say what it binds to and what colour that structure becomes — that is where the mark is.
3. Wet Mounts and Squashes
The standard mount, in order:
1. place a drop of water (or stain) on a clean slide
2. add the section, flat, using forceps or a mounted needle
3. lower the coverslip SLOWLY from one edge, using a needle
4. draw off excess liquid with filter paper from the opposite edge
Why lower it slowly
Dropping the coverslip flat traps air bubbles. Under the microscope these are round, very dark-edged and often mistaken for cells — they are the most common thing students misidentify.
A squash (used for root tip mitosis) adds a step: after staining and gentle warming in acid, press down firmly on the coverslip through filter paper. This spreads the cells into a single layer so individual chromosomes can be seen.
The distinction examiners test
Press vertically, never sideways. Sideways movement rolls the coverslip and breaks the chromosomes apart, destroying the very thing you are trying to see.
4. Measuring with an Eyepiece Graticule
Key Terms
An eyepiece graticule is a scale in the eyepiece. Its divisions are arbitrary — they mean nothing until calibrated. A stage micrometer is a slide with a scale of known length, used to calibrate it.
CALIBRATION
stage micrometer: 100 divisions = 1 mm, so 1 division = 10 µm
at ×400, 50 eyepiece divisions line up with 20 stage divisions
20 stage divisions = 20 × 10 = 200 µm
1 eyepiece division = 200 ÷ 50 = 4 µm
a cell spanning 12 eyepiece divisions = 12 × 4 = 48 µm
What mark schemes look for
The calibration must be redone for every objective lens. Change from ×10 to ×40 and one eyepiece division no longer means the same distance — this is the single most examined point about graticules.
5. Magnification and Actual Size
The formula
magnification = image size ÷ actual size
Rearranged: actual size = image size ÷ magnification
A drawn cell measures 45 mm across; the magnification is ×1500.
actual size = 45 ÷ 1500 = 0.03 mm
= 0.03 × 1000 = 30 µm
1 mm = 1000 µm 1 µm = 1000 nm
Easy marks
Convert to the same units before dividing, and state the unit in the answer. Most lost marks here are unit conversions, not the arithmetic.
Practice Questions
Practice Question 1
Explain why a section of plant tissue must be very thin, and describe how you would obtain one.
Practice Question 2
A student prepares a wet mount and sees several perfectly round, very dark-edged circles. Suggest what these are and how they arose.
Practice Question 3
An eyepiece graticule is calibrated at ×100 so that one division equals 10 µm. The student then switches to the ×400 objective. Explain why the calibration must be repeated.
Practice Question 4
A cell measures 36 mm on a photomicrograph taken at ×900. Calculate its actual size in micrometres.
What to Memorise
Thin section → light passes throughIodine → starch → blue-blackMethylene blue → nucleiAcetic orcein → chromosomesLower the coverslip slowly → no bubblesSquash presses cells into one layerRecalibrate the graticule for every objectivemag = image ÷ actual1 mm = 1000 µm
Concepts Checklist
Exam Tips
What mark schemes look for
For a stain, name the structure it binds to and the colour it produces. "Makes it easier to see" is not an answer.
The trap
Graticule calibration is specific to one objective lens. Changing magnification without recalibrating is the classic error the question is looking for.
Easy marks
Magnification calculations are pure arithmetic — the marks lost are nearly always unit conversions. Convert first, divide second, label the answer.
Worth remembering
If asked to identify a round dark-edged circle on a slide, air bubble is far more often the intended answer than any cell structure.