Determine concentration of yeast
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
- 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
- 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)
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