Back from a friend’s party where you tried homemade beer? And now you want to try yourself right? I see. Well, here we will (only) go through the details of getting the so-called “pitching rate” (or initial cell density) using a hemocytometer. Try not to be drunk when you do this. I am serious – you’re using glass material after all! For more info on the basic materials you will need to count yeast cells, check our cell counting starter kit post and our beer making supplies post.
1. Taking a representative sample of yeast
If you want to count cells it’s certainly because you already started a culture. So go get 1mL for your count. Make sure you mix properly before taking the sample, it should be as homogeneous as possible.
2. Adding viability dyes
If you’re interested in the healthiness of your yeast, you should consider adding a viability dye. A viability dye (such as methylene blue) will penetrate cells that are dead but will be excluded (that’s the PhD word) from live ones – so you can count both populations separately and know the percent of live over total cells.
It is common to dilute directly in a methylene blue solution (0.1%) but you can also add one drop of the original solution as long as the volume of cells you have is quantitatively bigger (for example, it won’t make a difference in terms of dilution to add 1 drop to 10mL – i.e. you can do it). See step 3 for the dilution part.
You should wait a few minutes for the dye to react. Methylene blue is oxidized in live cells but remains unchanged in dead cells. Be aware that viabilities under 90% might not be accurate due to the metabolic origin of the reaction. Check the viabiliy dyes post for alternatives.
3. Diluting the sample
This step is quite subjective. You will get better with time, but in the beginning it’s just trial and error.
The idea behind this is that you don’t want to have too many cells in the small space where you are going to count (they would overlap and you would get a headache trying to figure out which is which), but you need to have enough (at least 100) for your count to be significant. Let’s say you decide to dilute your 1mL in 9mL (if you haven’t done it yet with your methylene blue solution). You should do steps 4&5 to check that there are at least 100 cells in the central big square, but they are countable. If you decided to go for the methylene blue dilution in step 2, then that’s your diluting solution. Otherwise, do it with distilled water.
4. Preparing the hemocytometer and adding the sampleTo prepare your hemocytometer, make sure that the chambers and the coverslip are clean (use water or ethanol, and a soft tissue). Place the coverslip on the hemocytometer, on top of the two coverslip supports on the sides of the overfill chambers.
Mix your sample well and pipette a small amount into both chambers, avoiding to push the fluid too hard; it should go in almost on its own by capillarity.
5. Counting the yeast in the central square of the hemocytometerYeast cells have an average size of 5-10μm. In comparison to the size of a hemocytometer square (1mm), they are 100-200 times smaller. So you should count the smaller squares in this case (i.e., the ones in the central square, which measure 25 x0.2mm). They also have helper lines that divide each of them into 16 even smaller squares.
Count 5 of the 0.2mm squares, evenly distributed throughout the central square (commonly, the four corners and the central one). The rules are the same as for the other uses of the hemocytometer: establish beforehand two sides (lines) that you are not going to count (bottom and right for example) and keep those consistently for all the 0.2mm squares. Count the live and the dead cells and record your counts somewhere (or use this awesome app for iPhone/Android).
An important remark however is that yeast duplicates by budding (creating a bump that becomes bigger and finally results in a second, independent cell). It is common practice to count cells that haven’t divided yet (even if they have the bump) as one, not two. This is because there are different levels of “buddyness”, so your choice for counting one or two would be biased.
6. Practice the counting
There’s this great site that generates hemocytometer squares for you – so you can practice the counting before getting hands on!
7. Performing the calculations
Perform the calculations as in hemocytometer calculation, except that you have to multiply by 10000*25 = 250000 (because there are 25 small squares in a 1mm wide square) to get the cell density. There’s a couple of tools that will make your life SO MUCH easier when doing hemocytometer calculations: