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 (see Beads). Beadbeating is a very effective for this purpose. 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 closed chamber (accessory, #60801) for much 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. Additional information on bead media is located at Beads.
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 polycarbonate 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 low concentrations is okay but, in the interest of efficient down-stream purification, it may be better to keep cell extract concentrations high by using a Small Chamber (accessory, #110803) designed to process 15 or 50 ml of cell suspension. Lay the large black rubber gasket on the the top lip of the filled chamber and gently lower the rotor assembly into the filled, clear plastic chamber. It is important to leave as little air as possible in the filled chamber. Holding the chamber/gasket/rotor assembly in one hand and the ice-water jacket (held up-side-down) in the other hand, firmly screw them together. If temperature control is not a concern, use the plastic, threaded Ring (it looks like the outer ring a thick Mason jar lid) in place of the ice-water jacket to seal the filled chamber.
2) Invert the whole assembly, fill the ice water jacket with crushed ice and water and place the assembly on the BeadBeater motor base. The bead-chain attached to the side of the motor base is only used to hold down the filled Large Chamber when it is sealed with the plastic, threaded Ring.
3) Run the BeadBeater for about three minutes (5 min. maximum). For cooling considerations, see Temperature Control comments above.
4) 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.
Wash the 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 be 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.
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. BioSpec Products has extensive experience with the use of continuous bead-mills capable of processing multi-liter quantities of cell suspension. We would be happy to share our experience with you.
- 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.
Cleaning BeadBeater Chambers
There is a method for thorough cleaning of BeadBeater chambers. It only takes a minute more than simply washing out the intact chamber but will assure that chamber components last longer. The rotor/shaft part of the BB homogenization chamber is temporarily removed from its black plastic bushing unit. By doing so, you will remove any abrasive glass fragments that might have worked their way into the shaft and bushing area:
- Hold the white Teflon rotor with your fingers so that it cannot rotate. 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). Hopefully the clutch will come off without much effort. If this enhanced cleaning procedure is not routinely done shortly after receiving a 'new' chamber, it soon becomes difficult to unscrew the clutch.
- Remove the gray fiber washer and pull the rotor/shaft assembly out of the top part of the black plastic bushing unit.
- Clean everything with detergent, rinse well with water, blot dry.
- Reassemble: Insert the rotor/shaft assembly into the top of the bushing unit. Pushing on the rotor to hold it in place, turn everything up-side-down and position the gray fiber washer completely over the shaft of the rotor/shaft assembly before screwing on the clutch. It is only necessary to 'finger-tighten' the black rubber clutch. 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.