Monitoring cell lysis
The MiniBeadbeater will disrupt over 90% of the cells in about 2-5 minutes of operation. The homogenization process involves cell “cracking” action rather than high shear. After homogenization, cell membranes may mistakenly appear to be intact when viewed under a microscope (sometimes called "ghosts"). Therefore, to monitor the time course of cell breakage, rely on assay methods that measure intracellular constituents (e.g., OD260, enzyme activity, protein staining, PAGE).
As the beads collide in the vial, the homogenate will warm about 10 degrees per minute of MBB operation. To minimize sample heating start with ice-cold vials, beads and buffer and operate the MBB for one minute. Then turn off the MBB, remove the vial and chill it in crushed ice and water for one minute. Cycle thus, beadbeat/cooling, until homogenization is complete. Note that operating the MBB in a cold room does not help keep product cool. Also note that the above temperature control is usually not necessary when isolating proteins in denaturing media such as SDS solution or nucleic acid extraction media containing phenol/chloroform, guanidinium-SCN, or that provided in a commercial extraction kit.
The correct size beads are 0.1 mm dia. for bacteria, 0.5 mm dia. for yeast and fungi, and 1.0 mm dia. or 2.5 mm dia. for chopped-up plant or animal tissue**. While glass bead media is usually adequate, denser bead media may be needed for tough materials. For bead media information: http://www.biospec.com/category/beads/.
** Tissue samples sizes over a few tens of milligrams should be prechopped into pieces less than 1 mm in cross-section. If your sample is already harvested and frozen, do not thaw. This is especially important if you are isolating nucleic acids. Rather, Cryopulverize the sample. For information on this method see http://www.biospec.com/product/40/biopulverizer/.
The 2 ml vials used in the MBB are available from Biospec Products as well as other suppliers. All current cell disrupters on the market use the same 2 ml microvials.
Typical Operating Conditions
Fill the screw-cap vial at least 1/2 full with beads (exact measurement of beads to be added is not necessary. A 1/2 fill by eye is okay) and the rest of the volume with buffer and biomaterial. The maximum biomass concentration during homogenization is 400 mg (wet weight). The vial should be filled to the top to exclude as much air as possible when the vial cap is screwed on. Note that dry beads can entrap air, especially when using 0.1 mm dia. beads. You may have to invert the vial to wet the beads and then top-up the vial with more media.
Insert the vial securely in the arm assembly. Close the safety cover. Operate the MiniBeadbeater for a total of three minutes by selecting a speed (the rabbit!) of 4800 rpm (48 on display) and a time (the clock!) of 180 seconds (18 on display). At the end of a continous 3-minute run, the temperature of the homogenate will be about 25 deg C. For cooling considerations during beadbeating, see comments above.
Beads quickly settle to the bottom of the vial by gravity. With beads 0.5 mm dia. or larger, simply plunge the micropipette tip through the beads, to the bottom of the vial and suck out the homogenate.