# Determine concentration of yeast

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Jump to navigationJump to search## haemocytometer

A haemocytometer or counting chamber is a fancy microscopy slide with a grid etched into it, with the depth exactly defined.

Using the dimensions of the grid multiplied by the depth gives a known volume in which cells can be counted, thus making it possible to get to a concentration.

### Prepare counting chamber

- carefully clean the chamber with a kim wipe and ethanol (this is a precision instrument with fine engraving we don't want to damage)
- wetten the cover slip support on either side with water ever so slightly
- push the thick coverslip onto the support bars to get rainbow refraction rings

### Prepare cells

- make sure the sample is in a homogeneous suspension
- add a dye if necessary
- if the density is too high, consider making a dilution into Saline
- take 10µl suspension and pipette directly next to the cover slip on the counting surface
- wait till the capillary forces draw the sample in
- you can repeat your measurement on the bottom chamber, or add a different sample if precision is not that important
- let the cells settle for a few minutes

### Count

- Focus on the grid lines and try to get the cells into view
- depending on the density, 10X or 20X objectives should give a good magnification
- count cells in enough squares of the appropriate size
- when counting, ignore cells that are touching two of the sides of the square (bottom and right), but keep the ones on the other two sides (top and left)

### Calculate

Make sure you know which counting chamber you are using, as we have two different ones:

- Calculate the volume you have counted the cells in.
- Depth is 0.1mm
- Height and width depend on the size of the square you counted (see figure)
- Multiply the depth x height x width (all in mm) to get the volume in µl per square, and multiply by the number of squares.

- Divide the number of cells by the volume counted to get a concentration per µl counted
- Correct for the dilution if needed