For any microbial cell whether it is fungal or bacterial, the best and most accurate method for  counting  is TVC. Make serial dilutions of the said culture and go for a pour plate or spread plate method (In case of bacteria use nutrient agar  and in case of fungi use potato dextrose agar). if your fungus is sporulating you can also go for Spore counting using Neubar's chamber or haemocytometer. 

Dear Kunal

If your yeast cells grow in suspension liquid culture, I recommend first calculating a growth curve by setting up cultures (in triplicate, ideally) and counting the cell number on a hemacytometer at regular intervals (for our slow-growing fungus, we take a time point about every 12-24h over 3-4 days). If you have a spectrophotometer and can measure the optical density at the same time, you can calculate how many yeast cells/OD unit.

To use the hemacytometer, we make known dilutions and count the yeast cells. For experiments, since frequently there will be budding yeast in your count, you will need to decide whether you count every cell as an individual (which we do when we count for multiplicity of infection for in vitro work, i.e., the number of yeast per mammalian cell in the Petri dish) or every mother-daughter group as one colony-forming unit (CFU), which we do for inoculum preparation during infections and for plating counts.   You should also plate your serial dilutions onto appropriate media for your fungus and count the colonies to confirm viability and plating efficiency.  Plate in duplicate for better count accuracy. 

From infected tissues, we plate serial dilutions of organ homogenate onto appropriate media in duplicate (Brain Heart Infusion plates work well for yeast recovery from mammalian tissues) and count colonies (CFU) per organ.

Since hemacytometer counts do not allow you to distinguish between live and dead cells in the culture, if you need to differentiate between viability and plating efficiency, you can also carry out simple live:dead staining (e.g., Janus green exclusion dye) on a different microscope slide to estimate the percentage of dead cells in your culture. This percentage will change during the growth curve of the culture - with a greater percentage of dead/dying cells present in stationary phase. To avoid having many dead cells in our inoculum etc, we only use our cells during logarithmic growth (measured by OD and time of growth after inoculation calculated from our growth curve- see above).

You can count your purified spores on the hemacytometer the same way as above and should look at percentage viability by plating.

Best of luck!

Davina

Counting yeast with a hemocytometer

Posted on April 9, 2013 by biolabprotocols • 1 comment

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!

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.

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.

yeast dead cells

yeast dead cells (Photo credit: Wikipedia)

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.

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.

Preparing the hemocytometer and adding the sample

To 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.

Counting the yeast in the central square of the hemocytometer

Yeast 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.

hemocytometer squares yeast

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).

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.

Practice the counting

There’s this great site that generates hemocytometer squares for you – so you can practice the counting before going to the lab!

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 densit

For total microbial count whether fungal or actinomycetes or bacterial, the best method for  counting in petriplate by preparing serial dilutions of the said culture by spread plate method mostly...