Bead Selection Guidelines
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 bead milling tissue, a sample size over a few tens of milligrams should first be pre-chopped into pieces less than 1 mm in cross-section before beadbeating.
- When wet milling Tough Tissue (e.g., Connective tissue, Skin, or 'non-woody" Plant material), pre-chop or cryo-pulverize first and use 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 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 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 when embrittled at liq N2 temperatures using similar-sized steel beads. Note that Dry grinding with steel beads requires special, reinforced polypropylene vials (see XXTuff microvials and stainless steel microvials).
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 which supposedly contain unique combinations of different sizes and types of beads in the same vial. This is mostly marketing hype and comparative data to back up their claims of uniqueness or enhanced performance are not available. 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 a couple of seconds per vial. BioSpec has a "Do-It-Yourself" bead loaders described here. And, if you need to load hundreds of microvials or micro-plates, check out our stable of custom-designed Bead Loaders. If you would rather purchase pre-loaded vials, tubes, or microplates, BioSpec Products does that too.
- Glass beads have a density of 2.5 g/cc and is the most common bead media used for 'Beadbeating'.
- Zirconia/Silica beads have a density of 3.7g/cc (50% denser than glass - good for spores and most tissues).
- Silicon Carbide sharp particles (not a bead) have a density of 3.2 g/cc (Thet may work faster on tough tissue samples because the particles have sharp cutting edges. Their superiority over less expensive Zirconia/Silica beads is under investigation. But see Brein's comments below.
- Garnet (an iron-aluminum silicate, sharp particle) has a density of 4.1 g/cc. Like dense, sharp-edged SiC particles, it accelerates the lysis of tough tissue. Unlike SiC sharp particles, garnet particles fragment during beadbeating. This property can be useful when homogenizing soft tissue or fecal and soil samples containing bacteria. No mix of bead sizes will be needed. Starting with microvials half filled with 2 mm Garnet particles, the sample is rapidly dispersed. At the same time, the beadbeating has produced, in situ, much smaller Garnet fragments which can efficiently disrupt the microorganisms.
- Zirconia has a density of 5.5g/cc (100% denser than glass) - This ceramic bead, used for tough tissue, is chemically inert and resistant to fragmentation.
- Chrome-Steel and Stainless Steel have a density of 7.9g/cc. These heavy beads are most often used for dry-grinding leaves and seeds. Depending on the size of the bead, 1 to 5 steel beads are placed special reinforced 2 ml polypropylene microvials or 2 ml stainless steel microvials. Ordinary screw-cap polypropylene microvials can crack or leak when used with steel beads. Chrome-steel beads often are a good substitute for stainless steel beads. While stainless steel beads are often selected because of their corrosion resistance, chrome-steel beads are inexpensive...indeed, they are cheap enough to be used as "disposable", thus eliminating cleaning and cross-contamination concerns. One Caution: Eventually, chrome-steel beads will rust in contact with aqueous media. They should be promptly removed from the cell lysate. A small magnet provided by BioSpec when ordering chrome-steel beads makes that chore easy.
- Tungsten Carbide has a density of 14.9 g/cc. While very dense, this bead is generally not used for biopreparations because it leaves the homogenate looking "dirty". High g-force centrifugation will clarify the homogenate but that is time-consuming. The other dense media listed above can usually do just as 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 nutrient 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. The beads can be poured off after the inoculum has penetrated into the media gel by inverting the plate and tipping the beads out of the 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 tightly packed to prevent the movement of the beads during rolling or shaking. Similarly, when growing cells in non-agitated culture flasks, a 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 inoculating it with select surface-adhering micro-organisms or cells.
- Keep dialysis tubes vertical during dialysis by adding a few large glass beads or marble 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 bead mills extend this application to processing smaller amounts (<0.5g) of precious material.
- Lens for a Smartphone microscope attachment. See the glass beads page.
- Replace water in commercial lab baths with aluminum beads. No wet tubes to wipe, no tubes tipping over, no need for racks, floats, or weights. BioSpec Products "Tiny Teardrops" aluminum beads provide superior thermal transfer to the tubes. For details see Tiny Teardrops Aluminum Thermal Beads.
- Improved yields of viable single cells derived from animal tissue incubated with matrix-digesting enzymes. Explore a novel application of plastic beads. Replace tedious "manual trituration" of tissue fragments being lysed by digestion of the tissue's extracellular matrix with appropriate enzymes. See a Development Proposal.
- Faster QuEchERS technique. Make this popular protocol for extraction and analysis of small organics (cannabinoids, pesticides, herbicides, pharmaceuticals) from plants, animal tissue, and foods faster. Halve the time for sample hydration and homogenization by including a few 2 -3 mm diameter glass beads in the extraction tube and vortex. Or, better yet, increase throughput by homogenizing multiple extraction tubes in a MiniBeadbeater.
Feedback from 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 halfway into liq. N2 and then quickly inserted into a MiniBeaterbeater. No liq. N2 is added to the vial. Pulverize for a 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 microvials with 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 Product's series of Minibeadbeaters, dry milling with large steel beads can crack (or even escape from) common polypropylene microvials. This problem is exacerbated at cryo-temperatures. To get around this, BioSpec stocks two kinds of 2 ml microvials specifically designed for use with steel beads: "XXTuff" reinforced polypropylene microvials and Stainless steel microvials with silicone stoppers. For MiniBeadbeater-96 users, we also stock reinforced Porvair deep well microplates. Also available is a MBB-96 solid aluminum vial holder. When it is pre-chilled to liq. N2 temperatures, this vial holder will maintain ultralow temperatures during the beadbeating process.
For the extraction of very high molecular weight DNA, tissue samples can be cryo-pulverized to a fine powder by either the above dry-milling method or a BioPulverizer. Then follow by very gently rocking or swirling the powdered sample in a DNA extraction solution using a speed-adustable shaker like BioSpec Products' BioRotator-6. Minimize shearing is essential during this extraction step...no lab vortexing, no stirring rods, no stir bars, no repetitive pipetting.
Freeze-dried leaf tissue or dry seeds can also be dry ground. In this case, cryo-temperatures will not be needed during grinding. Use three 2.3 mm diameter glass beads or a single 2.3 mm chrome steel bead per vial and beadbeat for 15 - 60 seconds. When dry grinding with chrome-steel beads BioSpec's "XXTuff" reinforced polypropylene microvials or Stainless Steel microvials with silicone rubber caps must be used.
Recent information suggests that plant and animal tissue can be disrupted with sharp-edged particles more quickly than smooth beads. BioSpec Products stocks sharp particles of Silicon carbide or Garnet in three sizes.
Feedback: James Brien of Health & Science, University in Portland, OR. Brien reported that several whole organs from mice, cut into 3-4 pieces, was 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 pre-chopping 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. In both cases, one-half of the vial volume was filled with sharp particles. Controls using similar loads of 1 mm diameter spherical glass beads gave poorer results.
BIOSPEC NOTE: A minor 'downside' on the use of sharp particles is that particles abrade during beadbeating and the cell homogenate will have a yellow-gray color. This off-color is colloidal-sized grinding particles suspended in the homogenate. Both SiC and Garnet are essentially chemically inert and the colloidal particles can be separated from the homogenate downstream by filtration, centrifugation, adsorption, or precipitation.
Additional Bead Information
- For faster filling of microvials and microplates with beads, see Commercial Bead Loaders and Do-It-Yourself bead-loading tools.
- Coating beads to be used for grinding is a waste of time. The coating will quickly be removed by abrasion during shaking.
- Most bead media is reusable. Wash used beads with lab detergent and rinse well. For more on this see How to clean your beads.
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.
BioSpec Products offers PRELOADED beads in 2ml screw cap vials or 96 well microplates. There are too many possible combinations of bead size, bead composition, and bead load to stock prefilled microvials or microplates. We do this on a custom basis with a quick turnaround. Tell us what you need at firstname.lastname@example.org and we will give you a price quote.