Yeast DNA, mRNA and intracellular proteins are oftentimes difficult to extract intact from cells by traditional enzymatic methods. Lysing enzymes are often crude preparations containing nucleases and proteases that will attack not only the cell wall, but also the molecules of interest. Furthermore, protoplasts generated from enzymatic digestion usually require lysis with detergents that will also denature many proteins to inactivity. Therefore, mechanical cracking/fracturing of the yeast cell is often required to liberate the molecules.
Mechanical disruption of yeasts has traditionally been accomplished by using either a press or bead mill (i.e., bead beater). In both approaches, samples are processed individually. For experiments where large numbers of yeast clones must be examined in a high throughput screening environment, individual processing is a major bottleneck and impractical. Consequently, a method is needed that combines mechanical disruption of cells in a high throughput format. The Geno/Grinder®, a bead mill originally designed to smash seeds in deep well plates, can be used to disrupt yeast in a microwell plate format.
Saccharomyces cerevisiae was cultured overnight in YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 30°C with agitation (150 rpm). Using a sterile 96-well microtiter plate, 100 µl of culture broth was added to each well along with 250 mg of acid washed glass beads (OPS Diagnostics). A range of bead sizes was used including 100 micron silica, 200 micron zirconium, and 400 micron silica. Culture broth without glass beads was also prepared as a negative control. The plate was sealed with a rubber-sealing mat and locked into the Geno/Grinder. The yeast cells were disrupted for 5 and 10 minutes at 1500 rpm. Following disruption, the yeast cultures were examined by phase contrast microscopy and photographed.
The efficiency of yeast disruption is dependent upon the size of the beads and the duration of the grinding. Figures 1-3 demonstrate the efficiency of cell disruption with 400 micron silica beads at 5 and 10 minutes. Smaller beads were less effective in cracking cells with the 100 micron beads being particularly ineffective. It is concluded that 400 micron silica beads are most suited for disrupting Saccharomyces.
The design of the Geno/Grinder is also believed to be a factor in the disruption process. Standard bead mills adapted to microwell plates are modeled after "paint shakers" and move the plates in a "figure eight" motion. This motion is not believed to lead to uniform cell disruption. The Geno/Grinder is designed to effectively disrupt cellular materials by oscillating the plate vertically. This motion allows glass beads to impact the cells more directly than standard mills where beads impact the well walls in addition to the cells.
|Figure 1. Negative control yeast without glass beads. The yeast remained intact after processing for 10 min. in the GenoGrinder.||Figure 2. Yeast disrupted for 5 min. with 400 micron silica beads. Cracked yeast appear as dark "ghosts" while intact yeast continue to refract light and appear as bright.||Figure 3. Yeast disrupted for 10 min. with 400 micron silica beads. Most yeast have been effectively disrupted as is demonstrated by cell ghosts and cellular debris. Some intact yeast remain.|