Determination of cell density

Rince and dry the counting chamber (Neubauer improved, Vel, Leuven, Belgium)

Place a microscope cover glass on top of the chamber

Carefully fill the chamber with about 10 µl of an appropriately dilluted cell suspension (OD₆₀₀ = 0,1 - 0,2). Let the cells settle onto the haemocytometer grid for a fewminutes.

Examine the counting chamber with the light microscope set at 40 X 10 magnification

Count the number of cells in at least five (diagonal) squares of 0,2 x 0,2 mm (see picture)
To be statistically correct, at least 100 cells have to be counted.
Saccharomyces cerevisiae divides by budding from a mother cell. Count budded cells as a single cells. Count cells with equal bud sizes as two cells there is evidence of additional buds forming on eithercell. Some strains form clumps of cells which reduce plating efficiency. A single clump of cells will only give rise to one colony on a plate, which may complicate further analysis.

Calculations :
The number of cells in 1 ml of an undiluted cell suspension =
the mean number of cells per square of 0,2 x 0,2 mm X dillution factor X 2,5.10⁵

By measuring OD₆₀₀

Swich on the Novaspec^®II Visible Spectrophotometer (Pharmacia LBK Biotechnology, Uppsala, Sweden) about 1/2 hour before measurements
Set wavelength at 600 nm

Zero the spectrophotometer (reference is sterile medium used to grow the cultures)
Measure the OD₆₀₀

• The relationship between cell number and OD is strain specific !
  Keep in mind that these data were calculated for yeast strain FY1679