BenchMasteryMolecular biology techniques & virtual lab

Cell counting & viability

Manual counting in a ruled chamber under a 10× objective. Cheap and transparent, but slow and operator-dependent.

cells/mL · % viableSimulator readyCore simulatorEngine: Cell & microbial culture and growth
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Hemocytometer

Manual counting in a ruled chamber under a 10× objective. Cheap and transparent, but slow and operator-dependent.

In this lesson · about 1 min read
  1. Counting cells in a known volume
  2. Dye exclusion
  3. Where counts go wrong
  4. Key terms
  5. When to use it
  6. Sub-techniques

1Counting cells in a known volume

A hemocytometer is a thick glass slide with a precisely etched grid. When the coverslip sits on its ridges, the chamber is exactly 0.1 mm deep, so each 1 × 1 mm large square holds 0.1 µL (10⁻⁴ mL). Count the cells in several large squares, average them, and multiply by 10⁴ and by any dilution to get cells per mL.

cells/mL

Density

Mean cells per large square × 10⁴ × dilution factor.

% viable

Viability

Unstained cells ÷ all cells × 100. Healthy log-phase cultures are usually above 90%.

±1/√N

Precision

Counting is a Poisson process: 100 cells gives ~10% error, 400 cells ~5%.

2Dye exclusion

Trypan blue is a large charged dye that can't cross an intact membrane. Live cells stay bright and refractile; cells whose membranes have failed take it up and look blue. The dye itself is toxic, so live cells begin to stain after a few minutes: count within 3–5 minutes of mixing.

3Where counts go wrong

  • Cells settle and clump within minutes: mix gently right before taking the sample.
  • Overfilling lifts the coverslip and makes the chamber deeper than 0.1 mm, inflating the count.
  • Cells on the grid lines must follow a rule (count top and left, skip bottom and right) or edge cells get counted twice.
  • Too many cells per square (over ~100) and you miss some; too few and the count is noisy.

Key terms

Practise these as flashcards →
Hemocytometer

Counting chamber with a ruled grid and fixed depth (Neubauer improved: 0.1 mm).

Large square

1 × 1 mm square; at 0.1 mm depth it holds 0.1 µL.

Dilution factor

Every dilution applied before counting, including the dye (1:1 trypan blue = ×2).

Viability

Percentage of cells with intact membranes (unstained).

Confluence

Fraction of the dish surface covered by adherent cells; estimated by eye, not counted.

When to use it

  • Before every seeding, transfection or freezing step, so each well or vial gets a known number of live cells.
  • To follow growth and decide when to passage.
  • To check a thawed or treated culture's health.

Compared with alternatives

Viability & cytotoxicity →

Plate assays (MTT, CellTiter-Glo) measure viability of whole wells after a treatment rather than counting single cells.

Flow cytometry & FACS →

Counts and classifies thousands of cells with viability dyes and markers; overkill for routine counts.

Aseptic technique & passaging →

Counting is the step that tells you how to split.

CFU counting →

The microbiology equivalent: count colonies from plated dilutions.

Sub-techniques

Choosing one here switches the whole page, including the protocol and the lab, to it.

Manual counting in a ruled chamber under a 10× objective. Cheap and transparent, but slow and operator-dependent.