Yeast lysis with glass beads and a vortexer
In order to effectively disrupt 1 g of yeast in a 15ml centrifuge tube, combine;
1 g Wet weight of yeast, washed 3x in buffer of choice (see note 1 below on buffer molarity and also see the section on defining wet weight before and after freezing. Cell pellets shrink by about 40% after freezing, so these notes are important for good results)
2 g Glass beads, 0.5 mm diameter
0.25 ml Buffer of your choice (if using frozen and resuspended cells)
0.75 ml Buffer of your choice (if using unfrozen, freshly harvested cells and resuspended cells)
When viewed through the walls of the tube, it should appear to be about 66% beads. Agitate on a vortexer at maximum speed for 5 minutes. A temperature rise of about 20C will generally result. If temperature is a concern, agitate for 1 minute, place tube on ice for 1 minute and repeat five times.
50% cell lysis as verified by counting under a microscope can be expected. We strongly recommend cell counting using a microscope and a hemacytometer for verification, other approaches, particularly in the early stages of a project, can yield misleading results. We have provided a protocol and images on estimating cell disruption in this manner (yeast counting). After lysis verification, clarify the yeast lysate by centrifugation at 16,000 x g for 10 to 20 minutes and remove the supernatant, which will contain soluble target molecules.
Optional: Some of these soluble target molecules will remain in the pellet, recovery can be increased by a secondary washing step. This involves adding 2 ml of buffer, re-suspending the pellet by vortexing and then re-centrifuging.
Optional: If the target molecules are known to insoluble, or you suspect that they are attached to cell debris, we suggest you confine detergent use to this secondary washing step. The rationale for this is that use of detergent during disruption on the vortex can lead to foaming, which can fill the headspace above the liquid and prevent the formation of a vortex inside the tube. Detergent use during this secondary washing step may solubilize target molecules, which may then be recovered in the supernatant.
Note 1. The buffer composition is largely up to the individual researcher, but we advise using a high molarity buffer (i.e. with a high buffering capacity), to compensate for the low volume of buffer employed, and the relatively high volume of cell contents that will be released.
Problems associated with disruption of yeast, fungus or algae using glass beads
The old approach of manually holding a tube of beads and cells on a vortexer is somewhat effective for smallsamples of yeast. Minute samples (e.g. 0.1 g) for DNA analysis can be disrupted in microcentrifuge tubes, shaken on a vortexer, this generally yields good results, as (a) the amount of DNA needed is usually not very large and (b) the narrow diameter of the microcentrifuge tube helps create a vigorous vortex.
Slightly larger scale cell disruption (up to 1 g wet weight) for protein work in 15 ml centrifuge tubes is possible using a vortexer, if temperature control and labor are not key concerns. However, it is our experience that attempting to scale up this approach by using bigger samples in 50 ml centrifuge tubes yields very poor results. The wider diameter of the 50 ml tubes produces much slower agitation and therefore slower cell disruption, while simply adding bigger samples to 15 ml tubes fails as the essential vortex will not form.