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I just got started with yeast cell counting and was surprised to observe yeast cells that are at different z axis depths at 400x. That is, by adjusting the focus (not the x/y stage) I can make some cells go into view as others go out (there is some overlap in some cases but not always).

Is this normal? It means that I will have to count at probably 2 different depths which I haven't seen anyone else talk about. I had assumed everything would be visible at a single depth.

It is possible I did something wrong like not properly seating the cover slide. My filling technique, based on some videos I watched, was to breathe on the cover slide and the hemocytometer, place the cover slide on and slide it around a bit, and then touch the dropper to the edge of the slide to fill the chamber (I did not observe any obvious overflow/overfill).

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No, you only count what lay on the X,Y plane. The volume factor is based one cell depth.

Cell stacking is usually not an issue, just dilute your slurry with pure water and adjust your volume factor accordingly.

  • "The volume factor is based one cell depth" I'm not exactly sure you mean by this, but if the poster is using a standard hemocytometer (a fair assumption, I think) the height of the cover slide above the counting grid, and therefore the depth of liquid, will be exactly 0.1 mm (deep enough to stack maybe 10-20 average-sized yeast cells atop one another). The volume "factor" is purposely independent of cell size, allowing the chamber to be used for many different types of cells. – Franklin P Combs Feb 27 '19 at 2:29
  • @FranklinPCombs basically his sample isn't diluted enough to allow the cells to settle or break flocculation. bitesizebio.com/13687/… – Evil Zymurgist Feb 27 '19 at 2:53
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'Is this normal?'

It's common, but (as you suspect) it's not ideal for counting.

I've see this a lot and, for the most part, find it's usually just a matter of leaving the slide to sit for a few minutes to let the cells settle before proceeding.

Another issue can be how well-mixed your sample is. I've seen strains that need a lot of shaking (once in suspension) to cause the clumps of yeast to separate enough to be counted. Here's a picture I took recently of a particularly troublesome one (NB the distinct layering of cells):

enter image description here

If you continue to see this happen try agitating the sample more before loading the slide. The ASBC's method recommends the suspension be "mixed for a minimum of 5 min using [a] magnetic stirrer before filling the hemocytometer."

Any yeast that is chain-forming (e.g. many hefeweizen strains) may cause issues as well, since the strong cell-to-cell bonding can result in a distinct three-dimensional structure of the chain that can't be easily disrupted.

It is also possible your dilution is not high enough (as EvilZymurgist suggests). I generally feel comfortable with a dilution that leaves you with ~5-10 cells in each of the smallest counting squares.

'It is possible I did something wrong'

Unless your slide or cover is dirty, or you are seeing gas bubbles (either air or CO2) in or above the sample, I don't think it would be an issue of your technique, but that's hard to know for sure.

The bottom line is that you should only have to count cells in a single plane. Otherwise, something probably isn't quite right.

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