For Cell Disruption, we suggest:

Size

• 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 prechopped into pieces less than 1 mm in cross-section before beadbeating is initiated.
• When wet milling Connective tissue, Skin, or ‘non-woody” Plant material, 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 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 similar-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?

Chrome-steel Beads:

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 documentation is not available to back up their 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.

Density

• 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% denser 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 dense, sharp-edged SiC particles, it accelerates the lysis of tough tissue. However, unlike SiC sharp particles, garnet particles are easily fragmented during beadbeating. This fragmentation during the beadbeating process can be useful when homogenizing tissue, fecal or soil samples containing bacteria. Starting with a vial containing only large Garnet beads initially needed to rapidly disperse the sample, the beadbeating process produces in situ much smaller Garnet fragments needed to efficiently disrupt the microorganisms. For this specialized application, start off with a vial containing 2 mm grinding particles.
• Zirconia has a density of 5.5g/cc (100% denser than glass – good for tough tissue). Chemically inert and resistant to fragmentation.
• Chrome-Steel and Stainless Steel have a density of 7.9g/cc. These beads are used mostly for dry-grinding leaves and seeds. Only 1-3 steel beads are added to the microvial. Stainless steel beads are essential corrosion resistant. In most cases, chrome-steel beads are a good substitute for s.s. beads. They are 10X cheaper…indeed, cheap enough to be “disposable”. Thus, cleaning or cross-contamination concerns are eliminated when processing multiple samples. One Caution: When using chrome-steel beads in an aqueous media, promptly remove them from the lysate 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 biopreparations because it leaves the homogenate looking “dirty”. High-force centrifugation will clarify the homogenate but that is time-consuming and other dense media listed above 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 the movement of the beads during rolling or shaking. And, 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 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 extend this application to smaller amounts (<0.5g) of precious material.
• Lens for a Smartphone microscope attachment. See our glass beads page here.
• 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, and aluminum beads offer an excellent thermal transfer. See here for details.
• Improved yields of viable single cells derived from animal tissue incubated with matrix-digesting enzymes. Explore a novel application of beads to replace “manual trituration” of tissue fragments being digested with appropriate enzymes.
• 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, grind the sample in one of our MiniBeadbeaters.

Applications

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 halfway into liq. N2 and then quickly inserted into a MBB. 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 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:

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 to a fine powder using the above dry-milling method or use a BioPulverizer followed by passive extraction with the DNA extraction solution. Importantly, minimize physical agitation during the extraction step…no vortexing, no stirring rods, no stir bars…just slow tube rocking.

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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.

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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 pre-chop 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 the 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 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 were tested. 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 the particles abrade during beadbeating and the crude homogenate will have a yellow-gray color due to 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.

Additional Information

• MSDS on glass or ceramic beads
• How to clean your beads
• For faster filling of vials and microplates with beads, see Bead Dispensers. See also Do-It-Yourself bead loading tools.
• 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)

Ordering Information

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 info@stratech.co.uk and we will give you a price quote.

Operating Instructions for the BioPulverizer

General

The BioPulverizer reduces tissue frozen in liquid nitrogen or dry-ice to a course powder. The method is especially useful for tough tissue such as skin, cartilage, and cornea. Cryo-fracturing with the BioPulverizer is a preliminary step for cell disruption. Final disruption of the cells is accomplished using mechanical disrupters and/or aggressive extraction solutions.

Operating Instructions

1) The mortar is consists of two parts… an inner sleeve and an outer s.s. cup. The inner sleeve of the mortar nests in the outer cup. It is the hole in the inner sleeve that receives the tissue sample. Place the mortar and pestle of the BioPulverizer (BP) in an insulated container and thoroughly chill it with liquid nitrogen or, second best, crushed dry-ice. Stainless steel is relatively slow to cool down and later, slow to warm up. Slowly add liq N2 to the stainless steel parts until liq N2 no longer flashes off vapor and some liquid lingers on the stainless steel.

2) Add pre-frozen (-80 deg C or lower) tissue to the well of the mortar. Fresh tissue sticks to a cold BP on contact and cannot be inserted into the well.

3) Remove the BP from the insulated container and place it on the lab bench. There will be ample time to work with the chilled BP. Insert the pre-chilled pestle into the well of the mortar, patterned end down, and deliver one to three sharp blows to the pestle using the provided dead-blow hammer. Rotate the pestle slightly between blows.

4) Remove the pestle and the inner sleeve of the motar, thus providing easy access to the powdered sample.

IMPORTANT! Powdered tissue has a “dry” appearance and, with the exception of highly fatty tissue, can be poured into a test tube or plastic weighboat containing extraction solution. Pulverized tissue thaws quickly when exposed to room temperature, so work quickly or you will have a sticky mess.

The BioSqueezer is used to quickly freeze animal and human tissue samples.  Tissue is compressed into a thin disc under a metal clamp head that is pre-cooled in liquid nitrogen or dry ice.  Freezing occurs in a few milliseconds, thus giving a snapshot of metabolic events in the cells.  The hard frozen disc is easily fragmented or ground without thawing for subsequent extraction of labile nucleic acids, proteins and intracellular metabolite products.

Operation of the BioSqeezer:

A small beaker, styrofoam cup or similiar vessel is placed on the platform and the metal clamp is lowered.  Liquid N2 is slowly and carefully poured into the vessel until the boiling action of the liqN2 subsides.  This cooling process takes about a minute and, once cooled to the desired temperature, the stainless or aluminum metal clamp head will remain adequately cold for several minutes.  The vessel is removed and the sample, by itself or already in a poly-bag, is placed on the platform and the knob quickly lowered and clamped.  The clamping step must be done rapidly or the sample will freeze uncompressed.
It may be necessary to adjust the BioSqeezer using ‘practice’ tissue.  The optimal clamp setting can vary with the softness and size of the tissue sample.  Clamping force and the degree of squeezing on the tissue can be adjusted by changing the position of the metal clamp head with the wing nut, black plastic nut and the stainless steel hex bolt.
The thin waffer of tissue can be manually fragmented inside the poly bag while still frozen.  Presenting the tissue in small, thin pieces is ideal for quick homogenization and extraction or for storage in appropriate stabilizing solutions such as RNAlater or other excipients.

Instructions on Cryo-Cup Grinder

The Cryo-Cup Grinder is a mortar and pestle specifically designed to pulverize and powder soft plant and animal tissue samples at liquid nitrogen temperatures. It consists of a double-wall insulating cup in which is embedded a stainless steel, semi-circular bowl or mortar. Up to 5 grams of issue is placed in the pre-cooled mortar and ground to a fine powder with a special pestle made of non-heat conductive plastic. Two different sized pestles are provided for a variety of applications.

Selecting the correct size pestle: For samples over 1 gram start with the smaller polypropylene ball (1 3/8 inch diameter). Use the larger ball (1 7/8 inch diameter) with smaller samples. The larger ball gives a finer grind.

Cooling the Cryo-Cup Grinder

Using a Dewer (Thermos) flask as the container, draw about a cup of liquid from a liq N2 tank. Slowly pour liq N2 into the stainless steel cup. At first, liq N2 will quickly evaporate off the cup’s surface. Continue to slowly add liq N2 until a small amount of liq N2 remains in the stainless steel cup. The Cryo-Cup Grinder (CCG) is now cold enough to proceed with grinding. There is no need to have a puddle of liq N2 in the CCG. The cup should retain its cold temperature long enough to complete the grinding. Further addition of liq N2 may be needed if you are working up multiple samples.

Because the pestle (the black ball plus black handle) is made of inert plastic, it is a poor heat conductor. That is the intent of the design. The pestle will not transfer heat to the sample during grinding. Do not try to thoroughly pre-cool the pestle. At the most, cool its surface briefly with liq N2 – this is good enough. Deep cooling of the pestle will cause stress cracks and eventual failure of the pestle ball.

There is an exception to this advice. If you have purchased an accessory solid stainless steel ball, it should be thoroughly cooled before use. A stainless steel ball on the pestle is only needed if you are processing many samples and/or the grinding process goes on for an extended time.

Grinding process

The tissue sample MUST BE HARD FROZEN in liq N2 before attempting to grind. Begin with a downward, circular motion of the pestle inside the cup. Sometimes it will be necessary to gently hammer the sample with the pestle to break it up into smaller sized pieces so that the circular motion of the pestle can finish the grinding. Work quickly in transferring the ground sample. Once removed from the stainless steel cup surface it can quickly thaw.

Cleaning the Cryo-Cup Grinder

The stainless steel insert of the mortar can be removed by turning the cup upside down and taping the cup against a table top. Prompt washing and drying of all parts is recommended. Despite the name, stainless steel can be attacked by biomaterial. Washed parts can be autoclaved, if desired.

The DUELING SYRINGES Soft Tissue Disperser is a low shear method used to recover intact, viable cells or as the initial step in the isolation of high quality intracellular organelles. The disperser consist of a pair of 1 ml syringes connected together via their Luer connections. One of the syringes contains an embedded 500 micron stainless steel screen. Tissue is loaded into the syringe and forced back and forth through the screen by alternate manual pushing of syringe plungers. Compared to the classic syringe-needle-syringe method of tissue dispersion, the DUELING SYRINGES method is faster and more gentle in processing small samples of soft tissue. Less pushing effort is required and the number of multiple passes from one syringe to the other is less.

Operating Instructions

Two syringes are used in this device. Up to 100 mg of soft tissue (e.g., muscle, liver, brain) is inserted into the “sample syringe” – the one containing a stainless steel screen embedded in the barrel, a clear rubber pad attached to the end of the plunger and an attached white plastic female-female connector.

1) Remove the “sample syringe” plunger and insert the tissue sample into the barrel.

2) Reinserted the plunger and lightly push the plunger down the barrel of the syringe, thus excluding most of the air inside the syringe.

3) Fill the “receiving syringe” with 0.3 to 1 ml of dispersion media and connect the two syringes together with via the white, female-female Luer connector.

4) Start by pushing all the dispersion media into the “sample syringe”. Then push everything back into the “receiving syringe”. If pushing resistance builds up, reverse flow direction. Continue to push back and forth 4-6 times until all that is left inside the “sample syringe” is a small amount of off-white colored connective material. Depending on the type of tissue, passage through the screen may need to be terminated when pushing resistance increases to an unacceptable level.

5) Remove the “receiving syringe” from the Luer connector and dispense the cell suspension for plating or other processing.

Cleaning connective material from the screen is difficult and uncertain. The used syringe pair is intended to be a disposable. Reordering information: BioSpec Products, Dueling Syringes Tissue Disperser, Prod. No. 116.

The Lever Tissue Press is a modified garlic press enhanced by addition of a silicone-rubber gasket to the bottom of the lever press piston and the inclusion of an extra “can” to facilitate processing of multiple samples.

Special App – Disaggregation of tissue for primary culture:  Isolation of cells from solid tissue samples commonly involves digestion of extracellular matrix with proteolytic enzymes.  The first step in most protocols call for manually chopping the tissue into little pieces with a scissors or scalpel in order to increase access of the digestive enzymes to tissue surfaces.  The Lever Tissue Press eliminates this tedious manual chopping and delivers a significant improvement by producing much smaller tissue pieces than chopping.  Incubation time needed for enzyme-assisted tissue disaggregation is significantly reduced and diffusion of nutrients and gasses to the tissue mass is increased, thus optimizing cell viability during the incubation period.

For an excellent review of Enzymatic Disaggregation of Tissue Samples see http://www.worthington-biochem.com/tissuedissociation/default.html .

Operating Instructions:

Retract the piston of the Lever Tissue Press from its stainless steel can.  Add 1 to 10 g of fresh tissue to the cavity of the stainless steel can and swing the piston into position.  Grip the levers together, thus forcing the piston down the press can or barrel.  The dispersed tissue protruding from the bottom of the can is removed with the swipe of a single edge razor blade or scalpel.

Note: Tissue must be “soft”, relatively non-fibrous and have high water content.  Acceptable examples are muscle, brain, liver, plant fruits, some plant roots and tubers.  If the tissue is fatty or partially fibrous trim off the tough or fatty parts before pressing.  Significant fibrous content in tissue samples interferes with the passage of the tissue through the press and lowers the yield of dispersed cell material.  Unacceptable tissues are skin, hide, connective tissue, woody or fibrous plant stems, many dry seeds and tissues.  Rehydration of dried samples may make them amenable.

Operating Instructions

Partially screw out the piston of the TissuePress to make room for the tissue. Unscrew the brown ‘cap’ and remove the stainless steel wire screen. Place your sample into the cavity of the TissuePress.  If the tissue is partially fibrous or covered with membrane, trim out these tough parts and/or precut the sample into small pieces. Connective or fibrous tissue can interfere with the passage of soft tissue through the press.

Generally, the 2 mm array of holes molded into the cap is used. If you chose to use the 500 micron stainless steel wire screen, center it on top of the clear TissuePress cylinder before screwing the brown ‘cap’ onto the clear cylinder section.

Crank down the piston to extrude the tissue. It will exit the press having a paste-like texture. Quit when it becomes difficult to turn the piston or it bottoms out. Do not attempt to use tools to extrude more tissue. It will break the TissuePress. Depending on the content of connective or fibrous material in the tissue, the yield of dispersed tissue will vary from 20-95%.

Clean the TissuePress promptly after use. Unscrew the piston from the cylinder and unscrew the cap. Remove tissue debris and clean all parts with a non-abrasive cleaning solution. Removal of tissue debris from the stainless steel screen can be difficult.  An overnight soak of the screen in 1 N NaOH followed by scrubbing will help.

Operating Instructions

General

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. Click here for more details.

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.

Cleaning

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:

1. 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.
2. Remove the gray fiber washer and pull the rotor/shaft assembly out of the top part of the black plastic bushing unit.
3. Clean everything with detergent, rinse well with water, blot dry.
4. 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.

Monitoring cell lysis

The MiniBeadbeater will disrupt over 90% of the cells in about 2-5 minutes of operation. The homogenization process involves cell “cracking” action rather than high shear. After homogenization, cell membranes may mistakenly appear to be intact when viewed under a microscope (sometimes called “ghosts”). 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).

Temperature control

As the beads collide in the vial, the homogenate will warm about 10 degrees per minute of MBB operation. To minimize sample heating start with ice-cold vials, beads and buffer and operate the MBB for one minute. Then turn off the MBB, remove the vial and chill it in crushed ice and water for one minute. Cycle thus, beadbeat/cooling, until homogenization is complete. Note that operating the MBB in a cold room does not help keep product cool. Also note that the above temperature control is usually not necessary when isolating proteins in denaturing media such as SDS solution or nucleic acid extraction media containing phenol/chloroform, guanidinium-SCN, or that provided in a commercial extraction kit.

Bead selection

The correct size beads are 0.1 mm dia. for bacteria, 0.5 mm dia. for yeast and fungi, and 1.0 mm dia. or 2.5 mm dia. for chopped-up plant or animal tissue**. While glass bead media is usually adequate, denser bead media may be needed for tough materials. Click here for more details.
** Tissue samples sizes over a few tens of milligrams should be prechopped into pieces less than 1 mm in cross-section. If your sample is already harvested and frozen, do not thaw. This is especially important if you are isolating nucleic acids. Rather, Cryopulverize the sample.

Vial selection

The 2 ml vials used in the MBB are available from Biospec Products as well as other suppliers. All current cell disrupters on the market use the same 2 ml microvials.

Typical Operating Conditions

Fill the screw-cap vial at least 1/2 full with beads (exact measurement of beads to be added is not necessary. A 1/2 fill by eye is okay) and the rest of the volume with buffer and biomaterial. The maximum biomass concentration during homogenization is 400 mg (wet weight). The vial should be filled to the top to exclude as much air as possible when the vial cap is screwed on. Note that dry beads can entrap air, especially when using 0.1 mm dia. beads. You may have to invert the vial to wet the beads and then top-up the vial with more media.

Insert the vial securely in the arm assembly. Close the safety cover. Operate the MiniBeadbeater for a total of three minutes by selecting a speed (the rabbit!) of 4800 rpm (48 on display) and a time (the clock!) of 180 seconds (18 on display). At the end of a continuous 3-minute run, the temperature of the homogenate will be about 25 deg C. For cooling considerations during beadbeating, see comments above.

Beads quickly settle to the bottom of the vial by gravity. With beads 0.5 mm dia. or larger, simply plunge the micropipette tip through the beads, to the bottom of the vial and suck out the homogenate.

Preparation of Sample

Use 0.1 mm beads for bacteria, 0.5 mm beads for yeast, fungi and tissue culture cells and 1.0 mm or 2.5 mm beads for fresh plant and animal tissue. If you have solid tissue, pre-chop it into pieces having approximate 1mm cross-section with a single-edge razor blade or fragment the tissue at liquid nitrogen temperatures with a BioPulverizer (available from BioSpec Products). Up to 400 mg (wet wt) of biomaterial can be disrupted per ml of extraction media. In most applications, beads made of glass or zirconia-silica give excellent results. In special cases (grinding dry leaf material, wet grinding soaked seeds, disrupting skin or cartilage) beads made of denser material such as steel may be required. See beads here.

Fill the 2 ml screw-cap microtube one-half to two-thirds full with beads. Then add extraction media and cells, being sure to fill the microtube almost to the top. Exclude as much air from the microtube as possible. Use screw-cap microtubes with integral o-ring seals in order to eliminate aerosol formation during the homogenization. Be sure there are no beads on the threads of the microtubes when screwing down the cap.

(CAUTION – All snap-top microtubes aerosol. Nevertheless, it is possible to use snap-top microtubes in the MBB-8. Reverse the black cap of the MBB-8 so that its lip pushes into the chamber holder when screwed on.)

1) Insert one to eight microtubes into the chamber holder. Distribute symmetrically. Screw in the black plastic chamber cap, flat side down, until it is in contact with the tops of the microtube caps. Finally, screw on tight the white nylon wing nut.

2) Set the toggle switch on the control box to ‘Time’ and select the speed. A typical setting for cell disruption is 3 minutes at Homogenize. If you are working with heat-sensitive material, consider homogenizing for 1 minute, then cooling the vials in ice-water for 1 minute. Cycle for a total ‘On’ time of three minutes. Nucleic acid extraction does not need cooling.

3) Start the homogenization by pushing the white button in the middle of the timer dial. The timer dial resets itself automatically at the end of the run.

CAUTION – While the MBB-8 is running, changing the Timer settings will damage the timer. If you must change the time setting, press and hold down the white start button while changing the position of the timer dial.

Operate the MBB-8 with the black plastic hood over the chamber. This prevents the user from coming in contact with the shaker during operation and will help trap anything that might break free.

The noise level of the disrupter exceeds 85 dB. You may want to isolate the MBB-8 to a side room.  Do not isolate the MBB-8 in a cold room.  It will lead to premature motor failure and does not help to keep samples cold during homogenization.

Instructions for the Mini-BeadBeater-16

Preparation of Sample

Use 0.1 mm beads for bacteria, 0.5 mm beads for yeast, fungi and tissue culture cells and 1.0 mm or 2.5 mm beads for fresh plant and animal tissue. If you have solid tissue, pre-chop it into pieces less than 1 mm in cross-section with a single-edge razor blade**. Up to 400 mg (wet wt) of biomaterial can be disrupted per ml of extraction media. In most applications, beads made of glass or zirconia-silica give excellent results. In special cases (grinding dry leaf material, wet grinding soaked seeds, disrupting skin or cartilage) beads made of denser material such as zirconia or steel may be required. Click here for additional guidelines on selecting bead media.

** If your sample is already harvested and frozen, do not thaw. This is especially important if you are isolating nucleic acids. Rather, Cryopulverize the sample. For information on this method click here.

Fill the screw-cap vial at least 1/2 full (1/2-3/4 is okay) with beads. Then add extraction media and cells, being sure to fill the microtube almost to the top. Exclude as much air from the microtube as possible. Use screw-cap microtubes with integral o-ring seals in order to eliminate aerosol formation during the homogenization. Be sure there are no beads on the threads of the microtubes when screwing down the cap.

( CAUTION – snap-top microtubes release aerosol. Nevertheless, it is possible to use snap-top microtubes in the MBB-16. However, an accessory adapter ring will be required. Call for information)

Operating the Mini-BeadBeater

1) Load 1 to 16 microtubes into the clear, vial holder ring holder. Distribute them symmetrically as you would do with a centrifuge. If using less than 4 sample vials, insert ‘blank’ vials so that at least four vials are in the holder.

2) Place the loaded vial holder ring on the aluminum wiggle head (the later being attached to the motor). Rotate the loaded vial holder ring to a position where the hole in the vial holder ring is aligned with the anti-rotation pin sticking out of the wiggle head. Slide the vial holder ring down the pin and seat it on the wiggle head. Next, align the large, black plastic hold-down cap and slide it down to contact the tops of the microtubes.

3) Screw on and firmly hand tighten the aluminum knob. To do this, the black locking pin, which is part of the aluminum knob, must be in the lowered position. A slight lifting/twist action of the black locking pin keeps it either in the raised or lowered position. As you tightening the aluminum knob the pin it will engage with a ring of metal teeth on the black hold-down cap. You will hear clicking sounds as you continue to hand tighten the knob aluminum. Firmly hand tighten the aluminum knob until it is impossible to get an additional “click”. But, do not use a tool to tighten the aluminum knob.
IMPORTANT! The locking pin is an important safety feature. Do not proceed if you do not hear clicking sounds as you tighten down the hold-down cap. Remove the cap and visually test to see if the locking pin is in the down position.

4) Set the timer. A typical setting for cell disruption is 2-3 minutes. (Note: If you are working with heat-sensitive material, consider homogenizing for a shorter period, say 1 minute, then remove the vial holder with its vials and cooling the vials in ice-water for 1 minute. Cycle thus, for a total ‘On’ time of three minutes. No cooling is needed for nucleic acid extraction providing you are doing cell disruption in nucleic acid extraction media). The timer automatially resets to it last run time.

5) Start the machine by pressing the start/stop button. The timer automatically resets itself at the end of a run. For pre-April 2008 MBB-16 units having a different timer mechanism, press the white button in the center of the timer dial. Also for pre-April 2008 units: While the MBB-16 is running, changing the Timer setting can damage the timer).

6) To remove the vials and vial holding ring, first raise the locking pin in the up position (a slight lift and turn of the locking pin knob will keep it in the raised positon). Unscrew the aluminum knob, remove the black vial hold down cap and, finally the vials in their vial holding ring. The vial holding ring can be conviently be placed on a provided blue vial ring holder (alias: up-side-down tumbler).

Safety Concerns

Operate the MBB-16 with the black plastic hood over the chamber. This prevents the user from coming in contact with the shaker during operation and helps trap anything should it break free.

Maintenance

The external spring (for pre-April 2008 MBB-16 units) or the external O-ring will need to be replaced from time to time. See BioSpec for a repair kit should the spring or O-ring break. The change out is a simple 5 minute task and does not require opening the MBB.

MINIBEADBEATER-24 OPERATING INSTRUCTIONS

Preparation of Sample

Use 0.1 mm beads for bacteria, 0.5 mm beads for yeast, fungi and tissue culture cells and 1.0 mm or 2.5 mm beads for fresh plant and animal tissue. If you have solid tissue, pre-chop it into pieces less than 1 mm in cross-section with scalpel ort single-edge razor blade**. Up to 400 mg (wet wt) of biomaterial can be disrupted per ml of extraction media. In most applications, beads made of glass or zirconia-silica give excellent results. In special cases (grinding dry leaf material, wet grinding soaked seeds, disrupting skin or cartilage) beads made of denser material such as zirconia or steel may be required. Click here for additional guidelines on selecting bead media.

** If your sample is already harvested and frozen, do not thaw. This is especially important if you are isolating nucleic acids. Rather, Cryopulverize the sample. For information on this method click here **

Fill the screw-cap vial at least 1/2 full (1/2-3/4 is okay) with beads. Then add extraction media and cells, being sure to fill the microtube almost to the top. Exclude as much air from the microtube as possible. Use screw-cap microtubes with integral o-ring seals in order to eliminate aerosol formation during the homogenization. Be sure there are no beads on the threads of the microtubes when screwing down the cap.

Operating the Mini-BeadBeater

1. Load 1 to 24 microtubes into the clear, vial holder ring holder. Distribute them symmetrically as you would do with a centrifuge. If using less than 4 sample vials, insert vials so that at least four vials are in the holder.
2. Place the loaded vial holder ring on the aluminum wiggle head (the later being attached to the motor). Rotate the loaded vial holder ring to a position where the hole in the vial holder ring is aligned with the anti-rotation pin sticking out of the wiggle head. Slide the vial holder ring down the pin and seat it on the wiggle head. Next, align the large, black plastic hold-down cap and slide it down to contact the tops of the microtubes.
3. Screw on and firmly hand tighten the aluminum knob. To do this, the locking pin, which is part of the knob, must be in the down position. A slight twist of the pin keeps it in either a down or raised position. As you tighten the aluminum knob a clicking sound will be heard as the locking pin engages holes in the black hold-down cap.
   IMPORTANT! The locking pin is an important safety feature. Test that the pin has engaged into one of the ring of holes on the hold down cap. To do this, attempt to further tighten the knob. Do not proceed if the cap does not lock. Raise up the pin on the aluminum knob and repeat the tightening, locking and testing process. Failure to sufficiently tighten the aluminum knob or to engage the locking pin can result in rapid destruction of the central shaft of the aluminum wiggle head.
4. Set the timer. A typical setting for cell disruption is 2-3 minutes. (Note: If you are working with heat-sensitive material, consider homogenizing for a shorter period, say 1 minute, then remove the vial holder with its vials and cooling the vials in ice-water mix for 1 minute. Cycle thus, for a total runtime of three minutes. No cooling is needed for nucleic acid extraction providing you are doing cell disruption in nucleic acid extraction media).
5. Select the shaking speed with the slide knob on the top of the MBB-24. The speed can be varied from about 2000 oscl/min (2.0 on the speed label) to 3450 oscl/min. Speeds lower than the maximum are seldom used in cell disruption applications except in special circumstances. Converting low settings described in the literature for FastPrep 24 (MP Biomedicals) machines are problematic as their speed setting are defined in confusing units of M/sec. As a rule, operating a MBB-24 at its maximum 3450 rpm will deliver optimal cell disruption yields.
6. Start the machine by pressing the start/stop button. The timer resets itself automatically at the end of the run.
7. To remove the vials and vial holding ring, first raise the locking pin in the up position (a slight turn of the locking pin knob will keep it in the raised positon). Unscrew the aluminum knob, remove the black vial hold down cap and, finally the vials in their vial holding ring. The vial holding ring can be conviently be place on a provided blue vial ring holder (alias:up-side-down tumbler).

Safety Concerns

The MBB-24 will only operate with the black plastic hood closed over the shaking mechanism. This prevents the user from coming in contact with the shaker during operation and helps trap any material that might leak or break free.

Maintenance

Internal components of the MBB-24 have no servicable parts. A large sealed bearing inside the wiggle head can get hot during prolonged running times. While not a requirement, allowing the bearing to cool down between runs will increase its lifetime. Lubrication inside the sealed bearing is compromised at cold temperatures. Operation of the MBB-26 in a cold room is not recommended. Furthermore, cold air is not an effecient way to cool vials during the beadbeating process.

The large, black O-ring just below the aluminum wiggle head will need to be replaced from time to time. Without the O-ring the aluminum wiggle head will spin rather than shake. Should the O-ring break, go to BioSpec’s Parts and Accessories. Because this O-ring is located on the outside of the MBB-24, change-out is a simple five minute task.

Preparation of Sample

Use 0.1 mm beads for bacteria, 0.5 mm beads for yeast, fungi and tissue culture cells and 1.0 mm beads for plant and animal tissue. Up to 400 mg wet wt of biomass can be disrupted per ml of extraction media. If you have solid tissue, pre-chop it with a single-edge razor blade or scalpel.**
** Tissue samples sizes over a few tens of milligrams should be pre-chopped into pieces less than 1 mm in cross-section.  If your sample is already harvested and frozen, do not thaw.  This is especially important if you are isolating nucleic acids.  Rather, Cryopulverize the sample.  For information on this method click here.

Fill the screw-cap vial at least 1/2 full (1/2-3/4 is okay) with beads (BioSpec has a simple bead loader for 96-well microplates which makes loading a few plates easy and accurate, Cat. No. 702L. For loading numerous deep-well microplates, see Cat. No. 617L). Bead size and type should be matched to the biomaterial you are processing (see Beads for details). Then add your extraction media and cells, being sure to fill the microtubes or wells almost to the top. Exclude as much air from the microtubes or wells as is practical. We recommend using screw-cap microtubes with o-ring seals. All snap-top microtubes aerosol during shaking. Be sure there are no beads on the threads of the microtubes or on the lip area of the microplate(s) when sealing the microtube or microplate(s). If sealing microplates with a flexible mat or with adhesive film, firmly press the mat or film over the entire top surface of the microplate. BioSpec offers a manual mat roller which makes sealing easy and secure.

Some microplates and their matching mats or sealing films perform better than others when using the high energy MBB-96. BioSpec has tried different sealing techniques and microplates and mats made by several different manufacturers in an effort to find those that delivers the integrity needed to prevent sample leakage from well to well or, in the case of using steel bead media, rupture of the bottoms of the plate wells during the beadbeating process. To date, only deep well microplate products 219030 (2 ml) and sealed with EVA polymer sealing mats 219004, both made by Porvair Sciences (Wales, UK) and distributed by BioSpec Products, passed our rigorous leakage and breakage tests.

Operating the Mini-Beadbeater-96

1) Insert microtubes into the tube holder, if applicable. Distribute the tubes symmetrically. Place the rack of microtubes or microplate (if using 1 ml microplates you can stack two at a time) into the chamber holder, top side toward you. Screw down the two black plastic knobs until the top stainless steel retainer plate is in contact with the microtube caps or top of the microplate(s). Tighten knobs snug but not overly tight. Finally, screw down the white nylon wing nuts.

2) Turn on the ‘on-off’ switch on the front of the MBB-96 cabinet and select the cell disruption time (0-5 min). Typical cell disruption times are 3 minutes. If you are working with heat-sensitive material such as native proteins, consider pre-cooling your beads and sample, homogenizing for 1 minute, then removing the vials or plate and cooling in ice-water for 1 minute. Cycle for a total ‘on’ time of three minutes. Cooling is not need when extracting nucleic acids in nucleic acid isolation media.

Safety Concerns

You must operate the MBB-96 with the black plastic hood lowered over the chamber. This prevents the user from coming in contact with the shaker during operation and will trap anything should it break free. Do not by-pass the safety features of the lid.

Do not operate the MBB-96 in a cold room.  Special electronics in the unit will prevent the machine from starting.  Furthermore, cold air does not appreciably cool samples while beadbeating.

Contact Us for the complete Instruction Manual.

Instructions

IMPORTANT! REMOVE AND DISCARD TWO PACKING SCREWS FROM THE BOTTOM BEFORE OPERATION. SEE THE TWO ARROWS.

Note: The Mini-BeadBeater-Plus is a dual voltage instrument (120v or 230v) and comes pre-set to 230v. Select the appropriate voltage for your location using the switch at the back of the product. Failure to select the correct voltage could result in unrepairable damage. Damage resulting from user error is not covered under warranty.

The Mini-BeadBeater-Plus is a single sample, high energy bead mill designed to violently agitate microbial or tissue samples a sealed micro vial containing tiny glass, zirconia or steel beads. The Mini-BeadBeater-Plus features a single speed, brushless motor operating at 4500 rpm and two user programmable time settings (0-99 seconds). For complete product details click here.

Typical Operating Conditions:

Fill a 2.0 ml, screw-cap microvial at least 1/2 full (1/2-3/4 is okay) with beads. The rest of the microvial volume is filled with your sample and buffer. The maximum amount of biomass that can be homogenized is 400 mg wet weight. Tissue samples larger than a few 10’s of milligrams should be pre-chopped into pieces less than 1 mm in cross-section using a scalpel or single edge razor blade. Finally, the vial is filled to the top with buffer or extraction media. Try to exclude as much entrapped air as possible when the vial cap is screwed on. Note that dry beads, especially when using 0.1 mm diameter beads, can entrap air. You may have to invert the vial several times to wet the beads and then top-off the vial with a little more buffer.

Firmly insert the vial into the arm assembly, pointed end first. Close the blue safety cover. Select a time of 90 seconds on the MBB+ panel and start the MBB+. Repeat beadbeating for another 90 second (the MBB+ retains the last time setting used). This total beadbeating run time of 3 minutes disrupts over 90% of the tissue or cells. Shorter beadbeating times may be acceptable for sensitive analytical or PCR applications.

Homogenate can be recovered by simply decanting after the beads settle by gravity to the bottom of the vial. To maximize recovery of the entire product, one can either wash the beads with buffer or, when using beads 0.5 mm diameter or larger, plunge the micropipette tip through the settled beads to the bottom of the vial and aspirate the homogenate.

Bead Selection:

For comprehensive bead selection guidance and a complete list of lysis bead media click here.

Temperature Control:

At the end of the 90 second run, the temperature of the cell homogenate will be approximately 15 °C warmer. Cooling steps are necessary when isolating proteins, membranes, or organelles. When isolating nucleic acids in aggressive extraction media containing phenol/chloroform, guanidinium salts, and/or detergents, temperature control is not necessary. To minimize heating, begin with a pre-chilled vial, beads, and buffer and operate the MBB+ for one minute. Then let the homogenate rest for one minute in an ice water bath. Cycle thus until homogenization is complete. Operating the MBB+ in a cold room does cool the homogenate during the beadbeating process.

Monitoring Lysis:

The MBB+ will disrupt over 90% of the cells in 2 to 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).

Performance optimization of the SoniBeast™ for your particular sample is highly recommended. 

TIME STUDY:  

Conduct a beadbeating time study using control material.  Try each of the six time settings on the SB (2, 5, 10, 20, 40, and 60 sec).  For nucleic acid isolation, always beadbeat in an accepted nucleic acid isolation media.  Good nucleic acid yields should be found in the range of 5-20 seconds when using 0.5 ml locking-top PCR tubes and 1-3 minutes when using 2 ml screwcap microvials.

BEAD AND VIAL SELECTION:  

Use 0.1 mm zirconia-silica beads for bacteria, 0.5 mm zirconia-silica beads for yeast, fungi and tissue culture cells and 1.0 mm or 2.5 mm zirconia-silica beads for fresh plant and animal tissue.  In special cases such as grinding of dry leaf material, wet grinding of seeds, disrupting skin or cartilage, beads made of denser media such as zirconia or steel may be required.  Addition guidelines on selecting bead media is availble.

The Soni-Beast™ (SB) Beadbeater-type Cell Disrupter holds up to twelve 0.6 ml PCR tubes or three 2.0 ml microvials.  Always use locking-cap PCR tubes or screw-cap microtubes with integral o-ring seals in order to eliminate possible aerosol formation or even ejection of the tube during beadbeating.

SAMPLE SIZE:

Tissue sample sizes over a few tens of milligrams should be prechopped**.  Up to 150 mg (wet wt) of biomaterial can be disrupted per 0.6 ml vial containing beads and extraction media.  Up to 400 mg of biomaterial can be disrupted in 2 ml microvials.

** Note: Pre-chop solid tissue into pieces less than 1 mm in cross-section with a scalpel or single-edge razor blade. If your sample is large but has already been harvested and stored frozen, do not thaw.  Rather, cryopulverize the sample.  For information on cryopulverization click here. **

TEMPERATURE CONTROL:

In general, start beadbeating with ice cold media and sample.  During beadbeating the sample will be warmed.  Warming will not effect the quality and yield of nucleic acids isolated in nucleic acid media, nor proteins isolated in urea- or SDS detergent-based media.  However, if you are lysing cells to obtain native cellular components such as enzymes, antibodies, membranes, functional subcellular organelles or viable micro-organisms associated with tissue, feces or soil, close attention to heating during beadbeating is necessary.  For each 10 seconds of beadbeating in the SB using 0.6 ml tubes, temperature will increase approximately 10 degrees.  Heating in 2.0 ml tubes is slower (10 degrees per minute), but the cooler advantage is offset by longer times for complete cell disruption.  Heating is best controlled by beadbeating for several short periods interspersed with a minute of manual cooling of the vial and its contents in ice/water mixture.

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PROTOCOL

1. Fill the tube or vial at least 1/2 full with beads. A approximate delivery of beads by eye is adequate.  Then add up to 100 mg of cells and extraction media, being sure to fill the microtube almost to the top with media.  For best grinding results, try to exclude as much air in the sealed vial as possible.
2. Load 1 – 12 locking-cap PCR tubes or 1 – 6 screw-cap microtubes into the SB vial holder table. Distribute them symmetrically as you would do with a centrifuge.  Be sure to seat the tubes or vials all the way down into the rubber grommets.
3. Depress either the timer button (TIMED) or the manual button (PULSE).  A typical time for complete cell disruption is 5-30 seconds using 0.6 ml PCR tubes or 2-3 minutes using 2 ml screw-cap microvials.

General Procedures:

1. Chop or cut tissue into small pieces (approximately 1mm in length) using a safety razor blade or scissors. If your sample is already prefrozen and too large to be easily drawn up into the tip of the probe, you may need to cryopulverize the sample (see BioPulverizer.).
2. Place tissue in homogenizing vessel containing 3 to 10 volumes of homogenizing medium.
3. Lower the bottom one inch of the probe into tissue suspension, turn on the Tissue-Tearor, and operate for 5 seconds to 2 minutes using an up-and-down dabbing motion. Do not immerse the probe more than one-half way into the homogenization solution. Use lower speeds for subcellular particle preparation and the maximum motor speed for biochemical or macromolecule extractions. If desired, the homogenizing vessel can be placed in an ice bath during homogenization. The Tissue-Tearor can be stand-mounted using a tri-clamp and ring-stand. This is convenient when processing numerous samples.
4. To avoid splashing, turn off the Tissue-Tearor before withdrawing from the homogenizing vessel.
5. Place the TT in the plastic stand provided when not being used so that liquids can drain out. Do not place it on its side.

Cleaning:

Cleaning of the Tissue-Tearor is important. Many biological materials and extraction media are surprisingly corrosive, even if the probe is stainless steel. If the biomaterial gets into the upper part of the probe and into the motor, the lower motor bearing will be destroyed. Between repetitive samples, the unit can be superficially cleaned by briefly operating the TT in a vessel containing water or detergent. At the end of the day, unscrew the stainless steel stator (the outer part). This exposes the rotor for thorough cleaning. [CAUTION, never turn on the Tissue-Tearor with the stator removed – it will bend the rotor]. Wash in detergent, dry the parts and reassemble the stator. If necessary, both the entire probe (both stator and rotor) can be removed from the motor unit and autoclaved.

Maintenance:

The inert, fluorocarbon sleeve-bearing on the rotor shaft should be replaced from time to time. This is usually indicated by an observed increase in vibration and a noisy, buzzing sound during operation. Spare bearings have been included with the Tissue-Tearor along with the special wrench.

If you have replaced the fluorocarbon bearing and the unit is still noisy then you may have a bent rotor, or the armature and bearing assembly is rusted. Please contact us for further information and spare part numbers.

The  SpiralPestle™ microtube grinder is used to homogenize culture cells and small tissue samples (<100 mg wet weight).  It is also routinely used to disperse centrifugal pellets, precipitates, and other soft solids.  The SpiralPestle is compatible with both 1.5 ml conical and 0.6 ml conical microvials.

The unique screw-like pattern on the SpiralPestle’s surface actively draws 10-100 mg of soft plant or animal tissue suspended in 200 to 500 µl of extraction solution downward to the bottom of the 1.5 ml microvial where it is macerated and mechanically dispersed between the walls of the pestle and the microvial.  Tissue grinding is further enhanced by including of a pinch (~0.1 cc) of glass grinding beads.  While glass grinding beads are not needed for dispersion of pellets or precipitates, addition of SpiralPestle Grinding Beads is highly recommended for efficient cell lysis of tissue and microorganisms.  A small sample of the beads are included with your SP.

Traditional Pestle and Tube grinders are often operated by hand rotation. However, to achieve optimum homogenization efficiency, the SpiralPestle should be combined with a  BioVortexer™.  The BioVortexer is a low-cost, hand-held mixing motor drive turning at thousands of revolutions per minute.  With its greatly increased rotational speed and with the addition of grinding beads, soft tissue is completly homogenized in seconds.

Operating Instructions

Unlike traditional pestle and tube combinations, the SpiralPestle functions best with some clearance from the walls of the microtube.  This leaves space for the tissue (and beads) to be drawn downward to the bottom of the microtube by screw action of the rotating pestle.

If grinding by hand, add enough beads to fill the vial a little over the 0.1 ml mark (eye-ball accuracy is okay), add extraction media to the 0.5 ml mark and finally add the tissue or microbial sample.  Apply back and forth rotation to the pestle shaft and occasionally lift the pestle to re-capture tissue mass under the pestle.  For large tissue samples, grinding will be faster if the tissue is first prechopped into pieces less than 1 mm in cross section using a single edge razor blade or scalpel.

Using a BioVortexer to rotate the SpiralPestle makes things happen much faster.  Follow the hand-grinding protocol but be careful to avoid splashing.  It helps to not fill the microvial beyond the 1.0 ml mark and to grip the microvial by its hinged cap.  If the BioVortexer is turning too fast for your application, operate BioVortexer with only one battery, rather than two, in its handle, any questions please contact us here.

Ordering information:

• Product No. 1017:  Package of 100 SpiralPestles and 1.5 ml Microtubes
• Product No. 1017MC:  Prod. #1017 plus a BioVortexer stirrer (Prod. #1083)
• Product No. 1017GB:  SpiralPestle Grinding Beads for Pestle & Microtube grinders, 100 cc in bottle with pour spout