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).