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 the 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 diameter for bacteria, 0.5 mm diameter for yeast and fungi, and 1.0 or 2.5 mm diameter 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: https://www.biospec.com/instructions/bead-guide-lines.
** Tissue sample sizes over a few tens of milligrams should be prechopped into pieces less than 1 mm in cross-section. If the sample has already been frozen, do not thaw it in order to prechop it - - this is especially important if isolating nucleic acids. Rather, Cryopulverize the sample. For information on this method see https://www.biospec.com/products/biopulverizer?taxon_id=28.
Two ml screw-cap, microvials used with the MiniBeadbeater are available from Biospec Products.
Typical Operating Conditions
Wet Milling. Fill the screw-cap vial at least 1/2 full with beads (exact measurement of beads to be added is not necessary...1/2 fill by eye is okay). Add up to 400 mg (wet weight) of prechopped biomaterial and fill the rest of the vial with extraction media. The microvial should be filled almost to the top with extraction media, thus excluding as much air as possible when the microvial cap is screwed on. This minimizes foaming during beadbeating. Note: When using 0.1 mm dia. beads, entrapped air may still remain entrapped in the beads. Invert the vial to wet the beads and then top-up the vial with a little more media.
Insert the microvial securely in the arm assembly and close the safety cover. Operate the MiniBeadbeater for a total of three minutes by selecting a speed (the rabbit!) of 4800 rpm (48 on the display) and a time (the clock!) of 180 seconds (18 on display). At the end of a continuous 3-minute run, the temperature of the homogenate will be about 25 deg C. (See above if temperature control is a concern). Beads quickly settle to the bottom of the microvial by gravity. When using 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.
Dry grinding and Cryogrinding with the MiniBeadbeater-1. For this application bead media will be either chrome steel or zirconia. Depending on the size of the beads use 1 to 5 beads in the microvial: Five 2.3 mm diameter beads, three 3.2 mm diameter beads, or a single 6.3 mm diameter chrome steel bead. Ordinary microvials can crack during beadbeating with chrome-steel beads. Use BioSpec's "XXTuff" reinforced polypropylene microvials or BioSpec's Stainless Steel microvials with silicone rubber caps.