Cell Counting by Weight SOP

This method determines yeast slurry cell density by diluting a sample using mass rather than volume. It also includes a methylene blue viability stain, and uses EDTA in place of phosphoric acid to de-flocculate saison and lager yeasts (EDTA is compatible with methylene blue staining).

1. Obtain a sample from a full yeast brink of homogenous yeast slurry - it doesn't need to be degassed at this stage.
2. Collect 3 clean volumetric flasks.
3. Fill each flask with 90 grams of water. For lager or saison yeast, use a 0.5% EDTA solution for the first dilution (add 0.45 grams of EDTA to a flask, then top up to 90 grams with water), and plain water for the remaining two.
4. Add 10 grams of yeast slurry to the first flask, cap, and shake vigorously to combine (10x dilution).
5. Take 10 grams from the first flask and add to the second flask, cap, and invert several times to mix (100x dilution).
6. Take 10 grams from the second flask and add to the third flask, cap, and invert several times to mix (1000x dilution).
7. Take 10 grams from the third flask into a 50 mL flat-bottomed test tube, add 10 grams of 0.01% methylene blue solution, cap, and invert several times to mix (1:1, 2000x dilution).
8. Load the hemocytometer and let it sit for 2-5 minutes - healthy cells need at least 2 minutes to fully metabolize the methylene blue, but may start dying (giving false negatives) after 5 minutes, since methylene blue is toxic.

1. Count all cells, including blue-stained ones, and record the total.
2. Now count only the blue cells, excluding:
   • Blue cells that are budding (budding yeast won't metabolize methylene blue).
   • Lightly stained cells, or cells that are only blue on the cell wall - only count cells that are dark blue all the way through.
3. Calculate percent viable cells as: 1 minus (blue cells / total cells).

If your viability number looks unusually low, double-check you're only counting fully dark-blue cells before assuming a real problem with the yeast.