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Counting with a Hemocytometer

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This process describes how to count cells with a Hemocytometer Slide.

Reagents and Materials

  • Trypan Blue
  • Cell Solution
  • Hemocytometer Slide
  • Counter
  • Microscope

Things to Note before starting

Why use this?

The hemocytometer is very good at calculating cell counts for low concentration solutions. If the cell count is expected to be low (below 1e6/mL), using a hemocytometer might be more accurate.

Estimated Timing

Counting a hemocytometer slides takes ~5 minutes per sample.

Procedure

  1. Aliquot 10uL of Cell Solution into a 96 well plate.
  2. Add 10uL of Trypan Blue into the same well. Mix by pipetting up and down.
  3. Add 10uL of the mixture into the hemocytometer Slide.
  4. Place slide underneath the microscope. The slide should have a grid that looks like this:

![[Pasted image 20230708174329.png]]

  1. There are 4 big squares in the corners of this grid. Count1 the number of alive cells 2 present in the upper right square and the lower left square. 3
  2. Multiply that number by 1e4 to calculate the number of cells / mL in that sample.

Next Steps

After confirming the cell count with the hemocytometer, you can proceed with your cell culture, experiment, or preparing samples for genomic submission.

Appendix

Footnotes

  1. If a cell sits on the boundary of the square, make a call to either include it or not include it. The more important thing is that you are consistent when the edge case occurs again. ↩

  2. Live cells will appear white. Dead cells will appear blue. ↩

  3. When I say upper right square and lower left square, I mean the 4 x 4 grid in the upper right and lower left. ↩