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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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  • $\begingroup$ Did you wait for the cells to settle or did you look immediately after loading the volume? $\endgroup$ – Cell Feb 5 at 1:54
  • $\begingroup$ I looked immediately but I also looked over different time periods--probably up to 10 minutes... and did not notice any change in stacking. I did however notice that the yeast cells start dying off over time with the stain creeping into formerly live cells--so I'm not sure I'd want to wait very long either. $\endgroup$ – Clark Updike Feb 5 at 14:07
  • $\begingroup$ That sounds very odd. Yeasts are very hardy and in my experience I can dilute them 1:100 in sterile water and let them sit for an hour on the bench but I never observe a significant drop in colony forming units, let alone observe them dying under the microscope. $\endgroup$ – Cell Feb 6 at 0:02
  • $\begingroup$ I should mention the yeast were not that healthy--I was just using an old sample to test the technique--that may explain why they seemed to die quickly. I did counting more recently on a healthy sample and did not have any issues (and there was only negligible Z axis stacking for a couple budding cells). I'm guessing I didn't properly seat the cover when I had the problem originally. $\endgroup$ – Clark Updike Feb 11 at 12:47

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