Yeast Cell Disruption Methods
Yeasts are more difficult to lyse with enzymes and detergents than bacteria. Accordingly, bead beating is often used to crack open yeasts for both nucleic acid isolation and enzyme assays. Bead beating can be performed using a Pulsing Vortex Mixer, HT Mini homogenizer, GenoGrinder, or HT Homogenizer. Unfortunately, vortexing samples mixed with glass beads on basic laboratory vortexers is not that effective for disrupting cells.
The most effective bead size for Saccharomyces is 400 µm silica beads. However smaller yeasts, such as Pichia, are best homogenized with 200 µm zirconium beads. Samples containing very low numbers of cells should be disrupted using low binding beads so that analytes remain in the lysate instead of adsorbing to the beads.
Protocol - General Parameters
Bead beating yeasts can be performed in microfuge tubes, deep well plates, 4 ml vials, 15 ml vials, or in customized 125 ml jars for batch processing. Generally the container should not be more than half full of beads and bacterial sample. Of that, 1/3 should be beads and 2/3 sample. Thus, in a 2 ml microfuge tube, roughly 350 µl should be beads and 650 µl sample. It is important that the beads and yeast have head space to move in during beating, thus no detergents should be added during processing.
Depending upon the application, yeasts can be homogenized in culture medium or centrifuged and resuspended in a suitable homogenization/lysis buffer. Prepare cells and add to pre-filled microfuge tubes or other tubes or plates containing beads. Tubes and plates should be "loaded" with beads before the sample is added. A small scoop which is approximately 300 µl can be used to load tubes quickly. Plates can be loaded using a scoop and small funnel to load individual wells. Custom tube and plate fillers are also available to load racks of tubes and plates more rapidly.
Cap the tubes/plates with closures that do not leak. For screw cap microfuge tubs, be certain to use caps with "O" rings. For plates, tight fitting polypropylene strip caps are recommended. With strip caps, each well is sealed with an individual cap. Silicone press on mats have been found not to work well with high throughput bead beating as they tend to leak and cause cross contamination.
Individual tubes can be processed in a Pulsing Vortex Mixer or HT Mini homogenizer effectively. For full racks of tubes or plates, then a high throughput homogenizer is needed. Both the Geno/Grinder and HT Homogenizer are very effective at homogenizing large number of yeast samples. For a basic homogenization procedure, place the samples in the homogenizer and process for 2 minutes on high speed. Generally yeasts are more resilient than bacteria so processing may be longer.
If a microscope is available, examine the sample under 20X or 40X. Disrupted yeast will appear as dark "ghost" cells while intact yeasts are refractile (i.e., bright). If the proportion of disrupted yeasts is low, continue the processing and check again microscopically.
Optimizing the homogenization process requires beating the sample for short durations and then assessing the degree of disruption at each time point. The release of the enzyme lactate dehydrogenase from disrupted cells can be successfully used to measure homogenization efficiency in many different biological samples (see link for protocol). For yeasts, prepare at least six microfuge tubes with cells and beads (ideally it would best to run this test in triplicate, thus 18 tubes would be better if space allows in the homogenizer). Start by removing a tube before processing and labeling it 0 min. Run the homogenizer for 3 minutes, stop and remove a sample(s) and label. Restart the homogenizer and repeat the process removing samples at 6, 9, 12, and 15 minutes. Label all tubes accordingly.
Pellet the yeast cells and debris by centrifugation. Test the supernatant for lactate dehydrogenase (LDH) activity using a commercially available test kit or standard LDH assay protocol (link). The tube with the highest enzyme activity or where the activity peaks with the least processing represents the optimal processing time. Note that LDH is only one of many possible cellular enzymes that can be used for assessing cell disruption.