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​Yeast 4. Microscopic Yeast Cell Counting​​​​​​​​​

This is the fundamental method for determining how many yeast cells is in one milliliter of sample, whether it be a yeast slurry, yeast propagation, or fermenter sample. It utilizes a tool called a hemocytometer that makes counting cells much easier due to its gridded pattern. Because of the accurate volume above the counting area, we can determine the number of yeast cells in that area of an appropriately diluted sample and extrapolate to a 1mL volume through the method calculations. This is an important value for determining pitch rate and monitoring fermentations. There are many opportunities for error therefore each step must be performed precisely and consistently

Figure 1 - Click to Enlarge

​The hemocytometer is a device that uses a counting chamber with a specific volume in order to calculate the number of cells in one milliliter of a yeast sample.

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Figure 2 - Click to Enlarge

​The volume of liquid directly over the ruled area of the counting chamber where yeast cells will be counted, depicted by the red square, is typically 0.0001cm³ = 1 x 10^-4 cm³ = 0.0001mL or 1 x 10^-4​mL. This will need to be recalculated if the dimensions of the chamber are different from what is expressed in the method. The final calculations must therefore be adjusted as well.

Green Chemistry

Green Action	Principle	Benefit
Use EDTA as the deflocculating agent over sulfuric acid	Safer Solvents	Prevents technici​ans from using a corrosive and dangerous chemical.
	​Prevent Accidents
Use glass volumetric pipettes for reagents transfers.	Prevent Waste	Washing glass volumetric pipettes between each use reduces the need to single-use plastics.
Collect waste for proper disposal.		Working with local management for reagent disposal prevents release of hazardous materials to the environment.

​Cleaning the Counting Chamber

• If the counting chamber is dirty, the volume of the counting area will be inaccurate, which is the source of erroneous cell counts

• ​​Use the table below for guidance on choosing what dilution rate is needed for your sample and how to prepare the dilution. The table is not to be used as a hard and fast rule, but a general starting point. The number of cells on the hemocytometer counting grid must follow the counting rules outlined in the method. If it does not, then you will have to adjust the dilution accordingly and prepare again.

Counting

• It does not matter which line counting rules you apply so long as they stay consistent within one laboratory.
• One counting square is surrounded with a three-lined boundary. The counting rules apply to the inside line.
• Although not validated by the ASBC Technical Committee, a quicker alternative method is commonly used within the brewing industry. Rather than counting all of the cells in the entire ruled area of 25 squares, count the four corners and center square, a total of 5 out of 25 counting squares. A multiplication of 5 must be applied to the final calculation. Counts from both sides of the slide should agree within 10%. Standardized counting technique must be used. It is advised to perform an in-house validation if this alternative method is used to determine variability and number of total counts needed to reach a statistically valid result.

Calculations

• The 10⁴ factor within the equation represents how many times the volume of the ruled counting area goes into one milliliter in order to get end results in units of cells/mL.

​A Big Thanks To...

Rob Christiansen, Liz Nagle, Krystin Norman, Joe Palausky, Robert Fulwiler, Katie Fromuth​