Beads (Guide lines)
For Cell Disruption we suggest:
- When wet bead milling Bacteria use the 0.1mm diameter glass beads.
- When wet bead milling Yeast/Fungi use the 0.5mm diameter glass beads or zirconia/silica beads.
- When wet bead milling Soft Tissue (e.g, liver, brain, muscle) use 1.0mm diameter glass beads or zirconia/silica beads. When wet beadmilling milling tissue, a sample size over a few tens of milligrams should first be prechopped into pieces less than 1 mm in cross section before beadbeating in initiated.
- When wet milling Connective tissue, Skin or 'non-woody" Plant material use a 2.0mm diameter zirconia beads.
- When working with especially tough or fibrous tissue use the same sized beads suggested above but choose a more dense bead material. For example, use 0.1mm zirconia-silica beads for disruption of spores or three 2.3 mm chrome-steel beads for extraction of tough fibrous plant material like monocotyledon leaves.
The MiniBeadbeater can be used in a 'dry grinding' mode - either at ambient or at liquid nitrogen temperatures. For example, a single seed can be pulverized into a fine powder in 30 seconds using three 3.2 mm diameter or one 6.3 mm diameter chrome steel bead in the vial. Hair can be powdered at liq N2 temperatures using similiar sized steel beads. Dry grinding with steel beads requires vials made of special, extra-durable polypropylene (see XXTuff microvials) or stainless steel.
How many beads are in a one pound bottle?
6.23 mm diameter ~430
3.2 mm diameter ~3300
2.3 mm diameter ~7900
Glass Beads: Multiply by 3.2; Zirconia-Silica Beads: Multiply by 2.1; Zirconia Beads: Multiply by 1.4
TECH ADVICE re. Bead Combinations: Some vendors sell pre-filled microvials containing suposedly unique combinations of different sizes and types of beads in the same vial. This is mostly marketing hype. Comparative documentation is not available to back up claims of uniqueness or enhanced performance. With few exceptions, measurable improvements in cell lysis derived from "magic" bead combinations are marginal. Save money by adhering to the guidelines above and loading vials yourself...a process that takes less than a couple of seconds per vial. Some Do-It-Yourself bead loaders are described here. And, if you need to load hundreds of microvials or micro-plate wells, check out our stable of Commercial Bead Loaders. If you would rather have somebody else do the bead loading for you, BioSpec can do that for you. Contact us for a quote.
- 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 fragmentation 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 microorganisms. 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.
- Chrome-Steel and Stainless Steel has a density of 7.9g/cc . Used mostly for dry-grinding leaves and seeds. Only 1-3 steel beads are added to the microvial. Relative to chrome-steel, stainless steel beads are expensive - but reusable. In most cases, chrome-steel beads are a recommended substitute for s.s. beads. They are 10X cheaper...indeed, cheap enough to be "disposible". The Advantage: No cleaning or cross-contamination concerns when processing multiple samples. One Caution: When using chrome-steel beads in an aqueous media, get them out of the lysate promptly to avoid rust contamination. A small rare-earth magnet makes that chore easy. Ask for one when ordering a bottle of chrome-steel beads.
- Tungsten Carbide has a density of 14.9 g/cc. While very dense, this bead is generally not used for biopreps because it leaves prep "dirty". A hard centrigugation will clarify the homogneate but that is time consuming and other media usually do just as a good a job.
Other Uses for Beads
- Quick and easy plating of yeast and bacteria. Add a dozen or so sterile 6.3mm diameter glass beads to the solid plated media. Shake the plate (or a stack of plates) using a sideways motion to evenly distribute the added liquid suspension of yeast and bacteria over the surface of the plate. While many chose not to remove the beads after mixing, they can be poured off after the inoculum has permeated the medium gel by inverting the plate and spilling the beads out of the plate lid.
- 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 glass beads before sealing.
- Nanotechnology. Grinding of hard or friable particles to 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 the application to smaller amounts (<0.5g) of precious material.
Lens for a Smartphone microscope attachment. See our glass beads page here.
Replace water or ice in commercial lab baths with glass or aluminum beads. No wet tubes to wipe, no tubes tipping over, no need for racks, floats or weights and, in the case of aluminum beads, good thermal transfer. See here for more details.
Improved yields of viable single cells from animal tissue while being incubated with matrix-digesting enzymes. Biospec would like to work with a research lab that routinely harvests single cells from tissue for subsequent culture. We woud like to explore a new application for our beads. See developement proposal.
Faster QuEchERS technique. Make this popular protcol for extraction and analysis of small organics (cannabinoids, pesticides, herbicides, pharmaceuticals) from plants, animal tissue and foods even faster. Halve the time for sample hydration and homogenization by including a few 2 - 6 mm diameter glass beads in the extraction tube and vortex or, better yet, beadbeat.
Feedback: Santhosh Chelian of UC Davis. "Fresh rice leaves or tree needles were powdered or pulverized with a MiniBeadbeater using several 2.3 mm or 3.2 mm chrome steel beads in BioSpec's 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."
Due to the very high shaking energies of BioSpec's minibeadbeaters, dry milling with large steel beads can crack or even escape common plastic microvials! This problem is especially true at cryo-temperatures. BioSpec has developed new, stronger 2 ml microvials for use with steel beads..."XXTuff" polypropylene microvials and Stainless steel microvials. And for MiniBeadbeater-96 users, we have laboratory tested and found Porvair deep well microplates to have superior resistance steel bead damage. Also available is the MBB-96 solid aluminum vial holder. Pre-chilled to liq. N2 temperatures, this vial holder rigorously maintains a 2 ml microvial's ultralow temperature during beadbeating.
If very high molecular weight DNA is desired, cryo-pulverize tissue using the above dry-milling method (or use a BioPulverizer) followed by passive extraction with DNA extraction solution. Minimize physical agitation during the cell lysis step.
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 to be used for grinding 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.
Listed bead sizes are median (average) values. Because beads are sorted by sieves, bead size varies between +/-10% of a listed value.
We also offer PRELOADED beads in 2ml screw cap vials. There are too many possible combinations of bead size, bead composition and bead load to consider stocking all prefilled vials, so 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.