For Cell Disruption we suggest:
- When working with Bacteria use the 0.1mm diameter glass beads.
- When working with Yeast/Fungi use the 0.5mm diameter glass beads.
- When working with Most Tissue use the 1.0mm diameter glass beads or zirconia/silica beads.
- When working with Skin or 'soft' plant material use a 2.0mm diameter zirconia beads.
- When working with tough or fibrous tissue use the same sized beads (see above) but choose a more dense bead material. For example, researchers prefer 0.1mm zirconia-silica beads for disruption of spores or 2.3 mm chrome-steel beads for extraction of tough fibrous plant material like monocotyledon leaves. Some users have had good results using the MiniBeadbeater in a 'dry grinding' process - either at ambient or at liquid nitrogen temperatures. For example, a single seed can be pulverized into a fine powder in 30 seconds using one 6.3 mm diameter chrome steel bead in our 2 or 7 ml vials.
- Critique: Special and supposedly unique combinations of different sizes and types of beads in the same vial are mostly marketing hype from certain vendors of pre-filled microvials. There is little, if any, comparative documentation available to back up their claims of enhanced performance. With limited exception, measurable improvements in cell lysis derived from "magic" bead combinations are marginal.
- Glass has a density of 2.5 g/cc (most commonly used bead media for 'Beadbeating')
- Zirconia/Silica has a density of 3.7g/cc (50% more dense than glass - good for spores and most tissues)
- Silicon Carbide (sharp particle, not a bead) has a density of 3.2 g/cc (May work faster on tough tissue samples because the particles have sharp cutting edges. Their utility is still under investigation, but see Brein's comments below)
- Garnet (an iron-aluminum silicate, sharp particle) has a density of 4.1 g/cc. Like SiC particles, it may accelerate lysis of tough tissue due to its sharp edges. However, garnet particles are easily fragmented during beadbeating. Facile separation of the final grinding media from the homogenate can be problematic. Nevertheless, this very fragementation during beadbeating may be useful when homogenizing tissue, fecal or soil samples containing bacteria because you end up with a useful size mix of grinding media that can both rapidly disperse the sample and also disrupt the microorganism. For this specialized application, start off with the vial containing 1 or 2 mm sized grinding particles.
- Zirconia has a density of 5.5g/cc (100% more dense than glass - good for tough tissue). Chemically inert and resistant to fragmentation.
- Steel has a density of 7.9g/cc . (Used mostly for grinding leaves and seeds). Stainless steel beads are expensive but reusable. Only 1-3 steel beads are added to the microvial. Chrome steel beads are a recommended substitute for s.s. beads. They are 10X cheaper - cheap enough to be use once and thrown away. Thus, no cleaning or cross-contamination concerns.
- Tungsten Carbide has a density of 14.9g/cc. While very dense, this bead is generally not used for biopreps - it leaves prep dirty.
Other uses for beads:
- Quick and easy plating of yeast and bacteria. Add a few sterile 6.3mm diameter glass beads to the plate to evenly spread yeast and bacteria inoculums. No need to remove them afterward.
- Increase the surface area for tissue culture growth by packing roller bottles, tubes or vials with 6.3 mm diameter glass beads. In this application, the beads should be packed tightly to prevent movement of the beads during rolling or shaking. And, when growing cells in non-agitated culture flasks, confluence is reached faster and cell density enhanced by adding a layer of 0.1 mm diameter beads (see Growth of Three Established Cell Lines on Glass Microcarriers by James Carani, et. al., Biotechnology and Bioengineering, Vol.25, p.1359-1372 (1983).
- Create a bio-reactor by packing a column with glass beads and inoculate with select surface-adhering micro-organisms or cells.
- Keep dialysis tubes vertical during dialysis by adding a few large beads before sealing.
Nanotechnology. Grinding of hard or friable particles to a sub-micron size. A short review "High-tech Ball Mills Spur Nanotechnology Advances" contains practical information for bead-mill grinding of nanoparticles using a Retsch E-max™ bead mill. The MiniBeadbeater series of grinders extends this application to even smaller amounts (<0.5g) of precious materials.
- Research Opportunity. Biospec is seeking cooperative interaction with a research lab that routinely works with tissue cultures. We want to explore a new application of our MiniBeadbeater shakers - the dispersion of viable single cells from animal tissue. Successful development of a superior method for producing single cells would be well received in the bioscience research community. Contact Tim Hopkins at email@example.com.
Feed Back, Santhosh Chelian of UC Davis. "Fresh rice leaves or tree needles were powdered or pulverized with a MiniBeadbeater using several 2.5 mm or 3.2 mm chrome steel beads in BioSpec's "XXTuff" stainless steel microvials equipped with silicone rubber caps. The capped vials contents were immersed half way into liq. N2 and and then quickly inserted into a MBB. No liq. N2 is added into the vial. Pulverize for one-half minute. Refreeze and repeat for another 1/2 min, if necessary. Forty to sixty mg of plant material was used in each vial. After cryo-pulverizing the above tissue, DNA or RNA extraction solution was added to the vial and beads and the mixture was beadbeat for an additional 1-3 minutes at room temperature. Nucleic acids were recovered in high yield."
BIOSPEC NOTES: Large steel beads used for dry milling at liq N2 temperatures in our MBB machines blast right through common plastic microvials! Glass or ceramic beads are okay. BioSpec has developed a new, much stronger 2 ml microvial called "XXTuff". This "XXTuff" polypropylene microvial or our Stainless steel microvial should be used when dry milling with steel beads; For MiniBeadbeater-96 users, this same cryogenic protocol works well using 12 strips of 1.2 ml micro dilution tubes (96 well format, USA Scientific, #1212-8000). Also, consider using the MBB-96 solid aluminum vial holder (Cat. No. 702ALU) chilled to liq. N2 temperatures to maintain the microvial's ultralow temperature.
If very high molecular weight DNA is desired, cryo-pulverize the tissue using the above beadbeater method or with a BioPulverizer followed by gentle extraction with DNA extraction solution without further physical homogenization.
Freeze-dried leaf tissue or dry seeds can also be dry ground. In this case, cryo-temperatures are not needed during grinding. Use two 6.3 mm diameter glass beads or a single 6.3 mm chrome steel bead per vial and grind for 15 - 60 seconds. When dry grinding with steel beads BioSpec's "XXTuff" polypropylene vials or BioSpec's Stainless Steel vials with silicone rubber caps should be used.
Preliminary information suggests that plant and animal tissue can be disrupted more quickly with sharp-edged material rather than smooth beads. In this case, it may not be necessary to prechop larger sized tissue into smaller pieces before 'beadbeating'. If you want to check this out, we now stock three sizes of sharp particles, made of Silicon carbide or Garnet. See report below...
Feedback, James Brien of Health & Science, University in Portland, OR. Brien reports that several whole organs from mice, cut into 3-4 pieces, were completely homogenized using 1 mm diameter SiC sharp particles. His objective was to measure virus load using a plaque assay. He found that the usual prechopping of the tissue to 1mm cross-section pieces was not necessary. Using a dye permeability test, he estimated that better than 99% of the cells were lysed after 2 minutes of shaking in a Mini-BeadBeater-96. Lysis in both 2 ml and 7 ml vials was tested. One half of the vial volume was filled with the sharp particles. Controls using similar loads of 1 mm diameter spherical glass beads gave poorer results.
BIOSPEC NOTE: A minor 'downside' on use of sharp particles is the particles abrade during beadbeating and the raw homogenate will have a yellow-gray appearance due to the colloidal-sized grinding particles suspended in the homogenate. Both SiC and Garnet are considered essentially chemically inert and can be separated from the homogenate by the usual down-stream steps of filtration, centrifugation, adsorption or precipitation.
- Coating beads is a waste of time. The coating will quickly be removed during shaking.
- Most bead media is reusable. Wash with lab detergent (not acid!) and rinse well (see bead cleaning link, above).
Most beads are sold in one pound bottles with easy-to-use pour spouts.
Our listed bead sizes given are median values. Because beads are sorted by sieves, the actual bead sizes will range between +/-10% of the listed value.
We also offer PRELOADED beads in 2ml screw cap vials. Because of the many possible combinations of bead size, bead composition and bead load, we do this on a custom basis. Let us know what you need at firstname.lastname@example.org and we will give you a price quote.