Estimating yeast percentage cell disruption
We provide detailed instructions on how to use our cooled bead beater, including recommended ratios of cells/buffer/beads, which are vital when using any cell-breaking equipment. Furthermore we include test data for a variety of commonly used organisms, such as Saccharomyces cerevisiae, Pichia fermentans and Kluyveromyces marxianus. However, we understand that most labs will wish to develop their own procedures, so we have included some notes below on cell counting to estimate percentage lysis.
When optimizing extraction protocols for yeast/fungus in unicellular form, an essential first step is to use a hemacytometer and microscope to do a quick estimation of cell lysis. Using a phase contrast filter (e.g. a λ/4 PH2 filter), intact cells appear bright, while disrupted cells appear as dark “ghosts” (see Figure 1 below).
Unfortunately, simply assaying for your target molecule without microscopic examination can yield confusing results. For example, if you assay enzyme activity without microscopic examination and get disappointing activity in the clarified fraction, it is impossible to know whether:
(a) the cells were lysed but the enzyme activity was poor prior to cell disruption
(b) the cells simply did not lyse
(c) cells were lysed, enzyme activity was good prior to lysis but the enzyme is stuck to cell debris
(d) other possibilities
Once you have used a hemacytometer to verify that the cells have been broken, other protocol adjustments (culture conditions, buffer) can then made to maximize extraction of target molecules.
As estimating percentage cell breakage with a hemacytometer is a “rough and ready” procedure compared to precise estimation of total cell count, it is not necessary to invest in a top quality hemacytometer, a plastic disposable version will suffice.
Figure 1. Partially disrupted Saccharomyces cerevisiae at 400x magnification using a phase contrast filter (a λ/4 PH2 filter and a Leica DM750 microscope). Intact cells appear bright, while disrupted cells are dark. These images represent exactly what you should see when using this filter, no adjustments have been made to this image. If you do not see a similar gray background with bright and dark cells, please contact the manufacturer of your microscope for advice on the correct settings needed.