Counting Cells: the Haemocytometer & Flow Cytometry
Two ways to count and analyse cells. A haemocytometer is a precision counting chamber you read under a microscope — count, take a mean, and convert to a concentration with careful unit conversions. Flow cytometry streams single cells past a laser to sort them by size, granularity and fluorescence — used in blood analysis and to analyse the cell cycle by DNA content. Both are made interactive below.
The haemocytometer method and calculations apply to all exam boards. Flow cytometry is a specific requirement of OCR Biology B (2.1.1 e, f).
Counting cells with a haemocytometer
A haemocytometer is a thick glass slide with a laser-etched grid and a coverslip held a fixed 0.1 mm above it, so a known volume of liquid sits over each square. Count the cells in the four large corner squares, take a mean, and — because you know the volume — convert to a concentration. The interactive below demonstrates the counting rule; the next section builds the full calculation. (Relevant to all exam boards; OCR B specification 2.1.1(e): determining erythrocyte counts and converting to a concentration.)
Triple lines: on a real haemocytometer each large square is bordered by three closely-spaced lines; the middle one is the true boundary. North-West rule: count a cell if it touches the top or left boundary; ignore any cell touching the bottom or right boundary. This stops the same cell being counted twice between adjacent squares.
The calculation — step by step
Enter your counts and dilution and watch the concentration build, with every unit conversion shown. This mirrors the standard worked example: 80 cells in 4 squares, diluted 1 in 10, gives 2,000,000 cells cm⁻³ (2 × 10⁶).
Highlighted: the 4 corner squares counted for white blood cells / total cells.
Each large corner square is 1 mm × 1 mm × 0.1 mm = 0.1 mm³. Used for white-blood-cell / total-cell counts. You count 4 of them and take a mean.
The dilution factor is how many times the original sample was diluted before loading (e.g. a 1 in 10 dilution → factor 10). Blood is normally diluted because there are far too many cells to count undiluted.
Volume of one corner square
The volume is width × height × depth. The square’s width and height come from the etched grid, and the depth is the fixed 0.1 mm gap between the grid and the coverslip. Multiplying all three gives the volume of liquid sitting over that square — which is what lets you turn a count into a concentration:
Cells per mm³
= 20 ÷ 0.1 = 200 cells mm⁻³
| Step | What you do | Worked example |
|---|---|---|
| 1 | Count cells in the 4 corner squares (North-West rule) | 80 cells |
| 2 | Mean per square = total ÷ 4 | 80 ÷ 4 = 20 |
| 3 | Volume of one square = 1 × 1 × 0.1 | 0.1 mm³ |
| 4 | Cells per mm³ = mean ÷ volume | 20 ÷ 0.1 = 200 |
| 5 | × dilution factor (diluted 1 in 10) | 200 × 10 = 2000 |
| 6 | Cells per cm³ = × 1000 (1 cm³ = 1000 mm³) | 2000 × 1000 = 2,000,000 |
Flow cytometry (OCR Biology B)
A haemocytometer counts cells; flow cytometry counts and characterises thousands of cells per second. Cells are forced into a single-file stream by hydrodynamic focusing and passed one at a time through a laser. Detectors then measure how each cell scatters and emits light. (OCR B specification 2.1.1(f): the principles and use of flow cytometry in blood analysis, including fluorescent labels.)
| Measurement | What the detector sees | What it tells you |
|---|---|---|
| Forward scatter (FSC) | Light scattered forwards, in line with the laser | Cell size |
| Side scatter (SSC) | Light scattered at 90° to the laser | Internal complexity / granularity |
| Fluorescence | Light re-emitted by a bound fluorescent label | Cell type (antibody markers) or DNA content (DNA stain) |
Plotting side scatter against forward scatter separates the white blood cells of a blood sample into distinct clusters — small, simple lymphocytes; larger monocytes; and granular neutrophils / granulocytes. Adding fluorescent antibody markers then identifies specific cell types precisely, which is how flow cytometry is used to analyse blood.
You need the principles and use of flow cytometry, and the role of fluorescent labels. OCR B states that details of the different lasers are not required — focus on FSC = size, SSC = granularity, and fluorescence = cell type or DNA content.
Sorting blood cells by scatter — animated
Here is flow cytometry doing blood analysis. Each white blood cell passes the laser and is plotted by its forward scatter (size) and side scatter (granularity). Watch the three populations separate into clusters: small, agranular lymphocytes; medium monocytes; and large, granular neutrophils / granulocytes. Press play.
Analysing the cell cycle by DNA content — animated
Flow cytometry can tell you which stage of the cell cycle each cell is in — by measuring how much DNA it contains. Here is the idea, built up step by step, then animated.
• G1 (before copying) has 2C of DNA, so it glows the least brightly.
• S phase (copying in progress) is somewhere between 2C and 4C — each cell has copied a different fraction of its DNA so far, so these cells cover a range of medium brightnesses.
• G2 / M (finished copying) has 4C — double the DNA — so it glows twice as brightly as G1.
Why are there two peaks? Because cells cluster at two set brightness values. Lots of cells are sitting in G1 (all at exactly 2C), so they pile up at the same brightness into a tall peak on the left. Another group have finished copying and are in G2/M (all at exactly 4C, twice as bright), so they pile up into a second peak on the right, at double the DNA.
And the S-phase region isn’t “low fluorescence” — it’s a low number of cells. S-phase cells are actually at medium brightness, but each one has copied a slightly different amount of DNA, so instead of stacking up at one value they are smeared across every brightness from 2C to 4C. Spread thinly like that, only a few land at any single value — so the graph is low and flat there, even though the cells are perfectly bright. Low on the graph means “few cells,” not “dim.”
Press Play and watch it build: each dot is one cell crossing the laser, dropping into the histogram at its brightness. The two peaks appear as the cells accumulate.
Test yourself
Three sets. Work each out first, then check.
Score: 0 / 0