Why an unequal distribution of cells on the hemocytometer counting chamber usually occurs?

Answer 1

The reasons are almost TNTC (Too Numerous To Count, which is somewhat of an inside joke in Microbiology. It's the notation used when there are too many colonies to count on a culture plate) A Hemocytometer counter is a special kind of microscope slide that is used to count cells in cell culture microbiological, clinical, biotechnological and other fields. It has an etched grid, raised sides, and a cover. The grid has variable markings that are used to calculate different areas, and a fixed depth, so the volume of the different portions can be calculated and the volume of the entire chamber is constant. Liquid with cells suspended inside are added to the space with the grid, the analyst looks through the microscope, and using the grid markings, counts how many cells are in a specific area. Knowing the area, volume and number of cells, the analyst can calculate how many cells are in his original sample. Now, to the matter of unequal distribution of cells in the counting chamber. Before loading the chamber, the sample has to be prepared. Skimping on any of the preparation steps can cause clumping of cells. Is the sample well mixed? Is is free of contaminants? If its an adherent culture, are the cells well separated? What dilution did you use and did you mix the cells well enough? when you pipetted the sample to load the chamber did you pipette up and down enough times to prevent clumping? When you filled the chamber did you disturb the coverslip during uptake? Are the cells sticky and need some kind of surfactant you didn't know about to be counted at all? And I haven't even started on dyes, age, shape size, drying, film distortions and just plain sullenness on the part of your cells that day (don't laugh if you don't work with cultures, all of them need to be beaten into submission now an then). There are no hard and fast reasons for anything in the biological sciences, or any science for that matter, just best guess the seems to work almost all of the time. That's why we talk about theories, facts keep showing up that don't fit in with what everyone accepts and we all gotta change what we accept.

Answer 2

Unequal distribution of cells on a hemocytometer counting chamber can occur due to uneven mixing of the cell suspension, improper filling of the chamber leading to unequal distribution, or differences in cell settling rates based on size or density. Additionally, factors like user error or inadequate microscope focusing can also contribute to an uneven distribution of cells.