# Using a Hemocytometer

The Hemocytometer is a tool for estimating the concentration of cells in a sample. It has a chamber of 0.1mm deep and a grid of precisely measured squares (See Figure). Because we know the depth of the chamber, counting the number of cells in a given area of the grid allows you to estimate the number of cells per unit volume. (for instance, a 1 mm^{2} area with a depth of 0.1mm has a volume of 1×10^{-4} mls)

**STEP 1: The sample**

- You need a sample of yeast slurry to test. 10mls is sufficient but you can take more if it is easier.
- Take a representative sample!!!! In statistics, a representative sample is one that fairly represents the population it was taken from.
- The two biggest obstacles to representative sampling of yeast in the brewery are gas and settling. Below are two possible solutions to these obstacles.
- Yeast harvested from beer can have a huge amount of gas incorporated. Many samples are more than 50% air! This will throw off your estimates and make pitch rates inconsistent. Either de-gas the entire batch by harvesting into a brink with cold yeast dilution liquor or de-gas before counting (see below)
- Yeast will settle quickly in brinks, jars, tubes etc. If you don’t re-suspend well your sample may be much more concentrated or dilute than the actual population. Re-suspend well before sampling! It’s worth noting– it is virtually impossible to re-suspend a 15-30 gallon stainless brink that has settled for more than a day. You are better off taking a small sample for enumeration immediately after filling.

**STEP 2: Dilute sample for counting and staining**

Yeast needs to be at approximately 500,000 cells/ml to count on the Hemocytometer (more on this below).

Since most slurry harvested from conical fermenters is approximately 1-2 billion cells/ml you need to dilute. Dilute your slurry 1:2,500 fold in H2O to reach approximately 500,000 cells/ml.

If needed, de-gas the yeast while diluting it 2,500x. Start by adding 5ml H2O to a 15ml tube. Add slurry until the volume reaches 10mls. (No matter what fraction of the slurry was gas you now know you’ve added 5mls of liquid volume!). Re-suspend well by gently inverting several times.

If you de-gassed as above, add 80µl of the above to 99ml H2O, otherwise add 40µl to 99ml H20 (now your yeast is diluted 2500 fold). Note: 1µl = .001 mls i.e. 40µl = 0.04 mls. If you do not have the equipment to work with small volumes don’t sweat it! Just do serial dilutions i.e. you could add 1ml yeast to 99ml of H20, then take 4ml of this dilution and add it to 96ml H20 to reach the same 2,500x final dilution

Add 1ml of 0.1% (W/V) Methylene Blue to the 99mls from above (final 10ppm).

Re-suspend well by gently inverting several times and let sit 1-2 minutes before loading Hemocytometer

**STEP 3: Load the Hemocytometer and count**

Load the Hemocytometer by touching the pipette tip to the injection site. Eject app. 10µl of sample and let it fill the chamber by capillary action.

Count two of the 9 large squares on the grid– choose the upper left and lower right.

Here’s how to count (see figure below)

- Start in the upper left most small square
- Count all the yeast (blue and clear) touching the left and upper line
- Next count all the yeast from top to bottom, left to right NOT touching the lower and right boundary (shown in red)
- Move to the next square to the right and repeat.
- Count buds as a separate cell when they are >1/2 diameter of the mother
- Once you are finished, return and count all the blue yeast (yeast that stain blue are likely not viable)

**STEP 4: Determine the total cell concentration, % Viability and concentration of viable yeast**

** **Calculate total concentration in Cells/ml using the equation below

**Total Cells/ml (TC) = (total cells from 2 squares/2) x 2,500 x 10,000**

*where TC= total cells per ml, 2,500 is the dilution factor and 10,000 accounts for the Hemocytometer chamber volume.*

Calculate % Viability using the equation below

**% Viability (%V) = 100 – ((Blue cells x 100)/Total Cells)**

*where %V = % viable yeast*

** **Calculate the concentration of viable yeast using the equation below

**Viable cells/ml (VC) = TC x (%V/100)**

* where VC = viable cells per ml, TC = Total cells per ml and %V = % viable yeast*

**STEP 5: Use results from STEP 4) to determine optimal pitch rate**

Calculate pitch rate in gallons using the following equation. (Exchange 0.75 for 1.0 for wort over 17 Plato)

**Gallons of slurry to pitch = (0.75 x 1 x 10 ^{6 }x Deg Plato x 117,348 x BBLs Wort/VC)/3,785.4**

*where 0.75 x 1 x 10^{6 }x Deg Plato = the Fix pitch rate for a standard ale (substitute 1 for ales above 17 plato and 1.5 for lagers), 117,348 = mls per BBL, BBLs Wort = # BBLs of wort you are pitching to, VC = viable cells per ml and 3,785.4 = mls per gallon*

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