Cell Counting With A Hemocytometer

Thanks to Mad Dog and Lib I have a brand spanking new binocular compound microscope with a mechanical stage! One of the main things I will be using this for is doing accurate cell counts so I know what my pitching rate and viability is for every batch. I also scored two hemocytometers for about $25 and some methylene blue for staining and checking viability for another $12.

Even though my schooling is science lab heavy, I had yet to use a hemocytometer, so I used the tutorial here for a quick rundown on the calculations and protocols. A hemocytometer has two small wells (0.10mm deep each), along with a grid etched into the glass in which you count the cells. You count only a few squares, usually five, then take the average and then extrapolate the count to the size of your total volume (usually a starter for homebrewers, 1-2 liters). It will become more clear with my example.

Hemocytometer diagram

The picture to the right shows a diagram of the entire hemocytometer. For my sample I drew off a few milliliters of a fermenting beer. I swirled up the sample real well before every step to make sure the yeast was evenly distributed in solution (homogeneous). Next, I took 1 ml of the sample and diluted it with 10 ml of DI water. From this, I used a pipette to draw a small amount of the newly diluted sample and put the sample in the counting chamber. To do this, you must first cover the chambers with a cover slip, then place the pipette tip on the edge of the counting chamber. You don't even need to squeeze the sample in, or else the wells will overflow, it should go in easily through capillary action.

Below is an image of the hemocytometer grid. For yeast, you only need to use the large middle square out of the 9 large squares (since yeast a so small). Inside of the large middle square, you count 5 of the 25 smaller squares. I counted the corners and the very middle one, which is the norm.

Grid specifications

One thing to note is that cells on the border of each square are not always counted. The counter chooses two sides of the square to include in the count. For my count, I chose to not include cells that were on the right side and bottom borders of the square, and include those on the top and left borders. Also, those cells which are budding are usually only counted as one. I did read in Yeast, however, that brewers usually count a budding cell as two cells if the daughter cell is at least half the size of the mother.


After I finished counting the squares, I averaged my count per square, which ended up at 10 cells/square.

Next, I found the volume of a single square:

Volume = (W)(H)(D) = (0.25 mm)(0.25 mm)(0.10 mm) = 0.00625 mm³

Next, I found the dilution factor from diluting my sample:

Dilution Factor = (Final Volume)/(Sample Volume) = (10 ml)/(1 ml) = 10 Dilution Factor.

Next the cell density:

Cell Density = [(Average cells per square)(Dilution Factor)]/(Volume of square)
                   = [(10 cells)(10)]/(0.00625 mm³)
                   = 16,000 cells/mm³

The sample I drew was from 5 gallons of fermenting beer, which I thought I definitely under pitched in a rush from a top cropping to get an IPA done for a competition.

5 gallons = 19,000 ml.

There are 1,000 mm³  in 1 ml, so that yields (1.60 x 10^7 cells/ml)(19,000 ml) = 3.04 x 10^11

or 304 billion cells.

Note worthy is that my 1.060 OG IPA = 15 degrees plato needed 15 million cells/ml, and I had 16 million cells/ml, not bad for eye balling.

Below is my sample on the counting grid. This is exactly what you want; a sample evenly distributed across the grid, representative of the population.

Picture of the counting grid taken with my phone, I'm surprised at how well it turned out