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Same lesson, with the figures in motion.

Cell Counting with a Microscope (Neubauer-improved)

Count exactly how many cells you have (cells/mL) and how many are alive (viability %) — so you know precisely how much yeast to pitch. Uses a Neubauer-improved counting chamber and methylene blue. Works for both a slurry and a finished starter.

Materials

  • Neubauer-improved counting chamber (haemocytometer) + its coverslip
  • Microscope (100–400×)
  • Precision pipette (micropipette)
  • Test-tube rack with at least 3 tubes (for the dilution steps)
  • Distilled or boiled, cooled water (diluent)
  • Methylene blue solution (0.01–0.1%)
  • Tally counter
  • Lint-free wipes + sanitiser
  • Yeast sample (homogenised slurry or starter)

Before you start

  1. Clean the counting chamber and coverslip with a lint-free wipe — no fingerprints or dust on the grid.
  2. Homogenise the yeast sample well (swirl/shake) so the cells are evenly distributed — otherwise the count is off.
Asepsis isn't critical here (you're not culturing the sample), but clean tools and tubes give a cleaner count.

Step 1 — Dilute the sample

  1. Dilute in clean tubes from the rack. Thick slurry: 1:100 (1 part sample + 99 parts water). Thin starter: maybe 1:10.
  2. Aim for the central large square to hold ~100–200 cells (~4–8 per medium square) — too dense is unreadable, too sparse is statistically shaky.
  3. Note your dilution factor (e.g. 1:100 → factor 100). You need it for the calculation.
Pipette accurately — the dilution is the biggest source of error in the whole method.

Step 2 — Stain (viability)

  1. Add a drop of methylene blue to the diluted sample and mix gently. Wait 1–2 min.
  2. Live cells push the dye out and look clear; dead cells take it up and turn blue.
  3. Viability vs vitality — read more →
Don't wait too long — after a while even live cells take up the dye and viability is underestimated.

Step 3 — Load the chamber

  1. Place the coverslip over the grid. Touch the pipette tip to the edge of the coverslip and let capillary action draw in ~10 µL. Don't overfill — liquid in the channels, not flooded.
  2. Let it sit ~3 min so the cells settle into the focal plane.

Step 4 — Count

  1. Focus on the central large square (1 mm², 25 medium squares) at 100–400×.
  2. Boundary rule: count cells touching the square's top and left lines (green ✓), but NOT those touching the bottom and right (red ✗) — so you don't double-count. ✓✓✗✗ ✓✗
  3. Buds: a bud counts as a separate cell if it's ≥ half the mother's size, otherwise as one cell.
  4. Click live (clear) and dead (blue) separately. Aim for ≥100 cells total for good statistics.

Step 5 — Calculate

  1. Cells/mL = cells in the central large square × 10,000 × dilution factor. (E.g. 120 cells at 1:100 → 120 × 10,000 × 100 = 1.2 × 10⁸ = 120 million cells/mL.)
  2. Viability % = live / (live + dead) × 100.
  3. Total cells = cells/mL × sample volume (mL).
  4. Counted only some of the 25 medium squares? Scale: cells/mL = (cells / medium squares counted) × 25 × 10,000 × dilution.
  5. Let the Pitch Calculator do it → under Measured (Haemocytometer) — enter live, dead, squares counted and dilution, and cells/mL and viability fill themselves in. It does step 6 as well — it compares against your target and tells you how many mL to pitch.

Step 6 — Compare to your pitch

  1. Look up your target cell count in the Pitch Calculator (batch volume × OG × pitch rate) and compare with your total.
  2. Account for viability — pitch by live cells (total cells × viability %).
Methylene blue overestimates viability, most when it's low – below about 90% (classic home method). Methylene violet is more accurate but harder to find.
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