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. 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 “ON”. Then chill the vial in crushed ice and water for one minute. Cycle thus until homogenization is complete. Operating the MBB in a cold room does not keep product cool. The above temperature control is usually not necessary when isolating proteins in SDS solution or nucleic acids in aggressive extraction media containing phenol/chloroform, guanidinium-SCN, or a commercial kit-extraction solution.
The correct size beads are 0.1 mm dia. for bacteria, 0.5 mm dia. for yeast, 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 are available for tough materials. For more 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 and other suppliers. Other branded beadmill cell disrupters use the same vials.
Typical Operating Conditions
Fill the screw-cap vial at least 1/2 full (1/2-3/4 is okay) with beads and the rest of the volume with buffer and biomaterial. 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 several times 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 BeadBeater 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, see comments above.
The 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 liquid.