BeadBeater

This is not a detailed protocol for specific applications. It is a collection of notes that may be of interest to users of the BeadBeater.

Operating Notes
Monitoring cell lysis.
The BeadBeater will disrupt over 90% of the cells in about 2-5 minutes of operation. The homogenization procedure involves cell 'cracking' action rather than high shear. After homogenization, cell membranes may still 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).

If the goal is to isolate intact intracelluar organelles, use the same size beads as suggested for cell disruption. To maximize the yield of intact organelles, homogenize for a shorter period of time to get about 70% maximum cell disruption. A homogenization time study of 1, 2, 3, and 5 minutes would be instructive.

Temperature control.
The homogenate will be warm after three minutes of 'BeadBeating'. When isolating proteins, membranes or organelles, cooling
may be necessary. When isolating nucleic acids in aggressive extraction media containing phenol/chloroform, guanidinium salts, and/or detergents, temperature control is usually not necessary. The easiest way to minimize heating is by operating the BB, with the ice water jacket installed, for one minute and then let the homogenate sit for one minute, cycling thus until homogenization is complete. Also, consider replacing the clear polycarbonate chamber with a stainless steel chamber for better heat transfer to the ice water. For an even more stringent cooling technique see Methods in Enzymology, Vol.182, p.162-164.

Bead size selection.
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 most commonly used, denser bead media is available for tough materials.

Typical Operating Conditions
1. Fill the large, clear container or chamber 1/2 full with ice-cold beads and the rest of the volume with cells suspended in cold extraction media. Using the standard large chamber, that would be about 200 ml of beads and 200 ml of buffer containing 1-80 g wet weight of cells. Homogenizing cells at lower concentrations is okay but, in the interest of efficient down-stream purification, it may be better to keep cell concentrations high by using a Small Chamber designed to process 50 ml of cell suspension.

2. Place the rotor assembly on top of the container, making sure the gray rubber gasket is in between the top lip of the container and the rotor assembly. Leave as little air as possible in the container. Holding the container and rotor assembly on one hand and the ice-water jacket (held up-side-down) in the other hand, screw them together. If temperature control is not a concern, use the Metal Ring (it looks like a thick Mason jar lid ring) to seal the filled chamber instead of the ice water jacket.

3. Invert the whole assembly, fill the ice water jacket with crushed ice and water and place the assembly on the BeadBeater motor. The bead-chain attached to the side of the motor is only used to hold down the Large Container when it is used with the Metal Ring.

4. Run the BeadBeater for about three minutes (5 min. maximum).

5. The homogenate can be recovered by simply decanting. To recover the entire product, one can either pour the homogenate, beads and all, into a Buchner funnel containing filter paper, and suction the homogenate out of the beads or one can attach a glass tube with a sintered glass tip (commonly used to aerate cultures) to a side arm flask and suck out the homogenate directly from the chamber.

Cleaning
In most cases, cleaning new glass or ceramic bead media is unnecessary. The only contaminate - carbon black - is so inert that its presence in your prep has no effect and is removed upon centrifugation or filtration in the steps that usually follow cell disruption.

After use, wash beads with water and then detergent. Rinse the beads repeatedly with DI or RO water and dry them overnight at 50 deg C in a shallow glass or stainless steel tray. Properly cleaned beads will flow freely. If some of the beads cake on the sides of the drying container, repeat the cleaning process. Beads can be autoclaved or baked, if desired. If you want beads free of all nucleic acids or nucleases, soak the beads for 5 minutes in a 1:10 dilution of ordinary household bleach solution in place of the detergent wash. Beads can be reused about ten times.

Do not let a 'dirty' chamber sit around. Residual cell homogenate is corrosive and will lead to jamming and leaking of the chamber. Hand wash the plastic rotor assembly and chamber promptly. Then, disassemble the rotor/shaft unit for further cleaning and remove it from the black plastic bushing unit.

To do so, begin by holding the white Teflon rotor with your fingers so that it cannot rotate and invert the chamber and unscrew the black rubber clutch (the six toothed engaging wheel which mates to a similar clutch on the motor shaft). The shaft has a left-handed screw thread, so push on the slanted side of the teeth on the clutch (i.e. turn clockwise).

Remove the gray fiber washer. When reassembling the chamber, this washer must be positioned completely over the shaft of the rotor/shaft assembly before screwing on the clutch. Now lift the rotor/shaft assembly out of the bushing unit. Clean everything with detergent, rinse well with water, blot dry and reassemble. It is only necessary to 'finger-tighten' the black rubber clutch.

This rotor assembly cleaning ensures removal of any abrasive glass fragments and other contaminants that might have worked their way into the shaft and bushing area

About every tenth cleaning, put a light coat of silicone grease on the threads of the shaft which engage the clutch and lubricate the bronze bushing in the center of the black plastic bushing unit with a single drop of mineral oil. Mineral oil is inert and will not contaminate your biopreparation.

Things Not To Do
• Do Not fill the polycarbonate chamber more than ¾ full with glass or ceramic beads. Sample heating will be excessive and the motor may burn out. On the other hand, the chamber must be at least ½ full of beads in order to get good cell disruption.
• Do Not use beads larger than 2.5 mm diameter with the Large Chamber nor larger than 1mm diameter with the 15 ml or 50 ml Small Chamber. Steel beads cannot be used because they are too heavy to be agitated.
• Do Not use larger vessels (Mason jars, etc.) with the BeadBeater. These containers do not achieve good homogenization and, if made of glass, may break and cause injury.
• Do Not use flammable solvents in the chamber. The polycarbonate plastics in the chamber may be attacked. Furthermore, sparks from the BeadBeater brush-motor might ignite leaking solvent or fumes.