Wednesday, January 15, 2014

Yeast Cell Counting and Viabilty: Hemocytometer

Cell counting is one of the most important skills for a brewer. It allows a brewer to be able to pitch the correct number of cells into their wort. It also ensures that the cells are healthy and ready to make beer.

The most basic way for a brewer to count cells is to use a hemocytometer. This is a tool that was developed in the 1800's for counting blood cells. It just so happens that blood cells and yeast cells are about the same size. For this reason a hemocytometer can also be used for counting yeast cells.

The hemocytometer itself is a rather ingenious invention. It is simply a grid on a slide that holds a known volume of liquid. The volume inside of any one of the 9 large squares on the grid contains exactly one 10000th of a mL of liquid. To obtain  the total cells in 1 mL of liquid you can count all the cells in that 10000th of a mL and multiply by 10,000 to come up with the cells in 1 mL of liquid.

In combination with counting cells they can also be stained for viability. Viability is how many cells in the population are alive. The number does not tell you how healthy the cells are, (there is another method called a Vitality Test that can be used to tell you how healthy the cells are) only if they are alive or dead. To stain the cells you can use a solution of 2% Methylene Blue, which is a dye that enters into all the yeast cells. The yeast cells that are still alive have an active enzyme that will break down the dye. Yeast cells that are dead have no active enzyme to break down the dye and soon accumulate it inside. This causes the living cells to appear unstained and the dead cells to stain a dark blue/black color. By counting dead and live cells a percentage of living cells can be obtained, this number is the viability of the culture.

Here is how both a cell count and a viability count are done.

Loading the Hemocytometer
Photo 14:12, 13 November 2008 (UTC) by Jacopo Werthe
 
  1. Take your yeast slurry or whatever sample you want to count.
  2. Mix the sample up very well, if the yeast are heavily concentrated you will need to dilute them in water (you can not count the cells if there are too many on the slide, aim for between 80-200 cells in the 5 counting squares).
  3. Take the diluted sample and add a few drops of methylene blue until the solution turns a medium blue color.
  4. Add about 15 uL of the sample to the hemocytometer slide using the sample loading notch.
  5. Count all of the cells in the boxes that are highlighted below (the 5 4x4 boxes that are located inside the large center square). Note that each of the boxes is rimmed by a triple line. Cells that are on the upper and right triple line should not be counted (they are considered out of bounds). Cells that are on the lower and left triple line should be counted as in bounds. If a cell is budding count it as two cells if the bud is greater than or equal in size to the mother cell. Count the budding cell as one cell if the bud is less than half the size of the mother cell.
  6. Take the number of cells you counted, multiply by 5 (because you counted only 5 of the 25 squares in the area), multiply by 10,000 (because your volume counted was a 10000th of a mL) and multiply by your dilution factor. If you diluted 1:20 and counted 89 cells the math would look like this...
    • 89 x 5 x 10,000 x 20 = 89,000,000 or 8.9E7 cells/mL

    The 5 cells to be counted are highlighted in blue.
    Photo from http://dictybase.org/techniques/media/dicty_growth.html

  7.  To determine the viability count the number of living and dead cells in all 5 highlighted squares, then divide the living cells by the total number of counted cell. If you counted 86 living and 3 dead the math would look like this... 
    • 86/(86+3) = .966 or 96.6% viability.





Stained cells.



 Now that you know how many cells you have in your slurry you can calculate how much volume of your yeast slurry to add to your fermenter. I will be adding a post on how much yeast to add to your wort and will post a link here.

    1 comment:

    1. Perhaps using HemocyTap would make things slightly easier. Keep up with the good work!

      ReplyDelete