Method for rupturing large quantities of microorganisms.

Many procedures have been described (Umbreit et al., 1957) for the mechanical rupturing of yeast and bacterial cells. These techniques vary from simple hand grinding with abrasives to the use of extrusion or high speed shaking devices. These techniques, although generally satisfactory when dealing with small quantities of cells, are unsuitable for breaking larger quantities of cells required in many investigations. To provide a more suitable method for grinding larger quantities of cells, an attempt was undertaken to rupture cells in a cell-glass bead slurry under conditions where high shearing forces could be obtained. The Eppenbach3 laboratory colloid mill Model QV-6 was chosen for this purpose. This colloid mill has a rotor 2 in. in diameter and operates at 8000 rpm. The clearance between the rotor and stator can be varied from 0.0005 to 0.125 in. The homogenizing zone and the lower portion of the feed reservoir are jacketed for cooling purposes and a bypass line provided for recirculating the discharged slurry back to the feed reservoir. Runs were made with 300 to 600 ml batches of heavy cell suspensions to which were added Superbrite4 glass beads. The beads used were 120 to 130 ,u in diameter except for a single run with Bakers' yeast where I to 20 ,u beads were tried. Permanent antifreeze at -25 C was circulated through the jacket to maintain the slurry temperature at 15 to 20 C. Lower temperatures could not be maintained without material freezing on the interior surfaces of the mill; however, this problem could readily be solved by increasing the heat transfer area and raising the coolant temperature. Two representative organisms were chosen to survey the conditions for optimal cell breakage: one was Escherichia coli and the other a semiconstitutive 3-glucosidase hybrid yeast strain (Saccharomyces fragilis 610 X Saccharomyces dobzhanskii 1974) furnished by Dr. Wickerham. The influence of bead concentration, mill setting, and time on cell breakage is summarized in table 1. Protein and enzyme assays showed that the