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SOP — Yeast Cell Count & Viability

FUERST WIACEK Version 1.5 | Lab — yeast analysis | Frequency: after each yeast harvest, before pitching

Purpose

Cell count and viability analysis determines the concentration and health of harvested yeast before pitching. Results are used to calculate the correct pitch volume for the wort being produced.

PPE Required

  • Gloves
  • Safety goggles

Equipment

  • 2 × dry measuring cups or beakers (minimum 1000 ml)
  • 2 × EPI (Eppendorf-style pipette tube)
  • Big pipette — set to 800
  • Small pipette with fresh needle
  • Haemocytometer and cover slips
  • Microscope with power supply
  • Weighing scales (accurate to 1 g)
  • Microfibre cloth and paper towels
  • 2 × cell counter (clicker) — one for live, one for dead
  • Yeast Calculation spreadsheet
  • Methylene blue dye at 1:10 dilution
  • Water (~900 ml)
  • Alcohol (for cleaning the haemocytometer)
  • Clean, dry spoon

Key Principles

Use clean, dry sample glasses and EPIs to perform the dilutions. Always gently but thoroughly stir each sample glass with a clean, dry spoon before drawing from it. When moving a sample to the next EPI, draw from the bottom of the glass or EPI upward through the sample — this gives the most representative selection.

Process

1. Pull Yeast Sample

Pull a yeast sample of at least 100 g from the yeast brink as per the Yeast Harvest SOP. The sample should be taken at approximately 50% fill from the T-piece connection.

Avoid diluting the sample or allowing any sanitiser or other liquid to enter it.

De-gas and homogenise the slurry by pouring it back and forth between two cups a few times.

2. Prepare Lab

Set the big pipette to 800. Use it to fill one EPI with a single shot of water.

Take the small pipette with a fresh needle. Use it to fill a shot of 1:10 Methylene Blue solution into the same EPI.

3. Make a 1:10 Dilution

  1. Fill a measuring cup with 100 g of yeast slurry sample.
  2. Top up to 1000 ml with water (you add about 900 ml).
  3. Mix well by filling it from one cup to the other a couple of times.

4. Make a 1:100 Dilution with Methylene Blue

  1. Take the small pipette. Use it to fill a shot of well mixed yeast slurry sample from the 1:10 dilution you prepared in step 3 into the same EPI prepared in step 2.
  2. Close the EPI and shake well.
  3. Allow the methylene blue and yeast to sit undisturbed for at least 5 minutes — the dye needs time to be metabolised before counting. Viable cells will metabolise the dye; dead cells cannot.

5. Load the Haemocytometer

  1. Prepare the clean, dry haemocytometer and cover slip.
  2. Gently stir the 1:100 sample.
  3. Place a single drop into one chamber of the haemocytometer. Allow capillary action to pull the sample under the cover slip.
  4. Avoid flooding the moats.

6. Count Cells

Keep the microscope light as low as possible while still being able to see clearly. Excess light dries out and kills the yeast.

Count cells in 5 squares: the 4 corner squares and the centre square of the grid. Within each of these, count all 25 smaller squares. Repeat the same on the other side of the haemocytometer.

Use 2 counters (clickers) — one for living cells, one for dead cells.

Grid Line Counting Rules

Count these 5 squares — in order 1, 2, 5, 4, 3

1 2 3 4 5

Line counting rule (zoom of one square)

✓ ✓ ✓ ✓ ✓ × × count all inside area Count: interior + left + bottom lines Do NOT count: top + right lines
**DO COUNT** cells touching the **left** or **bottom** lines of the square (green). **DO NOT COUNT** cells touching the **top** or **right** lines of the square (red). Count **live** and **dead** cells separately.

Viability — Live vs Dead

Cells are counted as viable (alive) if they have metabolised some or all of the methylene blue dye. Only fully dark blue cells that are not budding are counted as dead.

Deep blue
Dead — count with clicker, add 1 tally to dead count
Light blue translucent
Alive — has metabolised some dye. Count as healthy cell.
Clear with blue rim
Alive — has metabolised some dye. Count as healthy cell.
Clear
Alive — fully metabolised dye. Count as healthy cell.

Budding Cells — What to Count

Budding cells must be at least ½ the size of the mother cell to be counted as a separate cell.

Live (blue) budding cells:

Dark blue + dark blue daughter ≥ ½ size → count both with clicker
Dark blue + daughter < ½ size → count ONLY mother with clicker. Small cell does NOT count.
Dark blue mother + clear daughter ≥ ½ size → daughter is large enough, count both with clicker
Cluster of budding dark blue cells — count 4 viable cells with clicker. Small cells do NOT count.

Dead (clear) budding cells:

Single clear cell → count with clicker
Budding, daughter ≥ ½ size → count both with clicker
Daughter < ½ size → count ONLY mother. Do not count small cell.
Cluster — highlighted cell < ½ size of mother. Count 4 cells with clicker (small cell does NOT count).

7. Final Product Count (Packaged Beer)

When counting yeast in packaged beer (not a harvest), the cell density is much lower. A different procedure applies:

  • No dilution — do not dilute the sample with water.
  • Mix the sample 1:1 with 1:10 Methylene Blue solution only.
  • Allow to sit for at least 5 minutes as normal.
  • Count all 25 squares on the haemocytometer (not just 5).
  • Apply the same line counting rule: count cells touching left and bottom lines, exclude top and right lines.

Note

Minimum representative count: Aim for a total of at least 150 cells across all 25 squares for a statistically reliable result. Below 100 total cells the margin of error is too large to trust the count. If you count all 25 squares and the total is below 100, count a second chamber and average the two results. If cell counts are consistently very low and you are concerned about the accuracy, discuss with Lukasz.

8. Calculate and Communicate

  1. Enter counts into the Yeast Calculation spreadsheet. When counting for a yeast pitch you count both sides of the haemocytometer, so your totals are doubled — divide each count by 2 before entering it into the sheet (only if you counted both sides).
  2. The spreadsheet calculates the required pitch volume based on cell count, viability, and wort volume.
  3. Communicate the required amount of yeast to the cellar team.

9. Clean Up

  1. Dump and rinse all sample glasses and spoons with distilled water.
  2. Rinse pipettes thoroughly.
  3. Rinse the haemocytometer and cover slip with alcohol, then wipe dry with the microfibre cloth.
  4. Tidy the counter and wipe down the counter and sink.
  5. Turn off and unplug the microscope. Place a clean, dry cloth over it as a dust cover.

Checklist

  • [ ] Yeast sample pulled — at least 100 g, de-gassed and homogenised
  • [ ] All equipment clean and dry before starting
  • [ ] Big pipette set to 800 — EPI filled with water
  • [ ] Small pipette with fresh needle — 1:10 Methylene Blue added to EPI
  • [ ] 1:10 dilution prepared — 100 g slurry topped to 1000 ml with water, mixed
  • [ ] 1:100 dilution made — slurry shot added to EPI, closed, shaken
  • [ ] EPI left undisturbed for at least 5 minutes
  • [ ] Haemocytometer loaded — no flooding of moats
  • [ ] 5 squares counted (centre + 4 corners), both sides — live and dead on separate clickers
  • [ ] Grid line rules applied correctly (no top 3 / right 3)
  • [ ] Budding cell rules applied — daughter ≥ ½ mother size to count
  • [ ] Results entered into Yeast Calculation spreadsheet
  • [ ] Pitch volume calculated and communicated to cellar team
  • [ ] Equipment cleaned, microscope covered and unplugged

Troubleshooting

Issue Likely Cause Action
Cells drying out under microscope Light too bright Reduce microscope light intensity. Work faster between loading and counting.
Haemocytometer flooded / moats filled Too much sample applied Clean and dry the haemocytometer. Reload with a smaller drop.
All cells staining deep blue Old or stressed yeast, or incorrect dye dilution Check dye dilution. Assess yeast health with team before pitching.
Count inconsistent between chambers Uneven sample distribution Clean and reload the haemocytometer. Re-mix the sample before reloading.