Practical aspects of mechanical cell disruption are discussed. Sources and approximate prices of equipment are given. A more extensive review of the subject by the author, Tim Hopkins, is available in ‘Purification and Analysis of Recombinant Proteins’, Seetharam and Sharma, editors, published by Marcel Dekker, Inc (New York)1991.
In bead milling, a large number of minute glass, ceramic or steel beads are vigorously agitated by shaking or stirring. Disruption occurs by the crushing action of the beads as they collide with the cells. Compared to ultrasonic and high-pressure methods of cell disruption wet bead milling is low in shearing force. By selecting the correct sized beads membranes are large and intracellular organelles can often be isolated intact. The method has been used for years to disrupt microorganisms. It is considered the method of choice for disruption for spores, yeast and fungi and works successfully with tough-to-disrupt cells like cyanobacteria, mycobacteria, spores and microalgae. More recently, bead mill homogenization has been applied to soil samples and to small samples of plant and animal tissue. If PCR techniques are to be used, this homogenization method is one of the few that totally avoids possible cross-contamination between samples because both vials and beads are disposable. The size of the beads used is important. Optimal size for bacteria and spores is 0.1 mm , 0.5 mm for yeast, mycelia, microalgae, and unicellular animal cells such as leucocytes or trypsinized tissue culture cells and 1.0 or 2.5 mm for tissues such as brain, muscle, leaves and skin. Speed of disruption is increased about fifty percent by using like-sized, heavier ceramic beads made of zirconia-silica or zirconia rather than glass (Hopkins, unpublished observations). Really tough tissue sometimes requires chrome-steel beads – which are 5 times more dense than glass beads. However, steel beads are too heavy to be used in rotor-type bead mills and, in some high energy shaking shaking-type bead mills, the steel beads will break through commonly available polyproplyene microvials. Specially designed, more durable polypropylene microvials (branded ‘XXTuff’) or microvials made of stainless steel are available (BioSpec Products). There are reports that non-bead, sharp edged particles made of silicon carbide, also do a good job of disrupting tissue. The loading of the beads should be at least 50% of the total liquid-biomass volume but can be up to 80%, provided adequate agitation of the bead slurry is still possible. Generally, the higher the volume ratio of beads to cell suspension, the faster the rate of cell disruption. After treatment, the beads settle by gravity in seconds and the cell extract is easily removed.
Microorganisms can be disrupted manually using the beadmill technique: To a cell suspension is added an equal volume of beads. Agitate the mixture at top speed on a vortex mixer for ten or more minutes. This hand-held method is tedious and the low mixing power of a vortexer makes it inefficient. Therefore, special high energy electromechanical agitators have been developed. Bead agitation by these devices is either by shaking or stirring.
Most shaking-type bead mills are restricted to sample sizes of 0.5 cc or less and use 2 ml polypropylene screw-cap microvials. Screw-caps are used because snap-top microvials can release aerosols of their contents during the high energy shaking process. These machines orient the vials either a vertical position or a slightly more efficient near-horizontal position. All machines shake the beads in the direction of the vial axis. While there are some noticeable performance differences between these machines, especially when disrupting tough cells or tissue, disruption generally takes about 1-3 minutes and yields are high. Heat generation can be a concern in shaking bead mills and effective cooling during shaking is difficult to achieve. Without cooling, the temperature increases about 10 degrees per minute of homogenization. Operation of the shaker in a cold-room does not help. A popular cooling strategy for shaking-type bead mills is to beadbeat for a short period of time, then remove the vial(s) from the shaker and cool the vial in an ice/water mixture for a minute, cycling thus for the full time duration required for disruption. Some shaking bead mills come with special cooling blocks to help ameliorate this problem. Temperature control is not a concern when disrupting cells or tissue in nucleic acid extraction media.
As mentioned earlier, while the above small volume cell disrupters were originally used mostly for the disruption of microorganisms, they have become very popular homogenisers for the extraction of 10 mg to 0.4 g of plant and animal tissue. This newer application is suitable for both soft tissue and tough or fibrous samples such as skin, tendon or leaves. Extraction yields of nucleic acids, viruses, receptor complexes and intracellular organelles are often superior to that of other methods. For nucleic acid isolation, consider disrupting the cells directly in the nucleic extraction solution (phenol, guanidinium SCN, etc). Nuclease concerns will be eliminated and yields enhanced. If PCR techniques are being used, shaking-type bead mills using disposable micro-vials and beads totally eliminate cross contamination concerns between samples. Selective homogenization is sometimes possible using different bead sizes or speeds of bead agitation. For example, it is possible to selectively disrupt only the epidermal layer of whole leaves or to obtain intact subcellular organelles by using larger beads, smaller charges of beads and/or shorter disruption times.
The first company to introduce lab-scale bead mills designed for cell disruption was BioSpec Products. It manufactures six models of high energy shaking machines. The Micro-MBB and MBB-1 hold a single 1 or 2 mL screw-cap microvial. Their other models process more than one vial at a time. The MBB-8 disrupts up to eight 2 ml samples or five 7ml samples, the MBB-16 can process up to sixteen 2 ml vials or eight 7 ml tubes and the newest one, the MBB-24, process up to 24 microvials. The MBB-96, a high through-put machine utilizing deep well microplates, can process up to 192 samples at a time. It can also process up to 48 microvials or twelve 7 ml tubes.
Commercial bead mill cell disrupters currently available are: Mini-BeadBeater (BioSpec Products, Bartlesville, OK), Retsch Mixer MM 301(F. Kurt Retsch GmbH, Haan, Germany), FastPrep (MP Biomedicals, Irvine, CA), Precellys-24 and Minilys (Bertin Technologies, France), 2010 Geno/Grinder (SPEX CertiPrep, Metuchen, NJ), MagNA Lyser (Roche Applied Science, Penzberg, Germany), Powerlyser-24 (MO BIO Laboratories, Carlsbad, CA), BeadRupter-24 (Omni International, Kennesaw, GA), TissueLyser (Qiagen Inc-USA, Valencia, CA), and Talboys H.T.H. (Troemner, Thorofare, NJ). Other shaking devices based on vortex-type mixing (‘Genie’ and ‘Bullet Blender’) deliver lower mixing energies and, therefore, take much longer shaking times to get good cell disruption.
Larger capacity laboratory bead mill cell disrupters agitate the beads with a rotor rather than by shaking. Equipped with efficient cooling jackets, larger sample volumes can be processed without overheating. By far the most widely used rotor-type bead mill is the BeadBeater (BioSpec Products, Bartlesville, OK). This laboratorysized unit will disrupt about 250 mL or, with smaller chamber attachments, 50 or 15 mL batches of cell suspension in 3-5 minutes. Cell concentrations as high as forty percent (wet wt) can be used. VirTis Company (Gardiner, NY) offers an attachment for its line of high speed rotary homogenisers which efficiently agitate glass beads in a special test-tube or fluted flask.
These homogenisers are well suited for homogenizing plant and animal tissue in liquid media volumes of 1 ml to a few liters. They generally outperform cutting-blade type Benders. Compared to a blender, foaming and aeration are minimized and smaller sample volumes are easily accommodated. The cellular material is drawn into the apparatus by suction created by a rotor sited inside the end a long static tube or probe (also called stator). The material centrifugally exits through slots or holes located on the tip of the stator. The product is repeatedly recycled, and because the rotor is turning at very high speeds, the tissue is reduced in size by a combination of liquid shear forces and scissor-like mechanical shearing occurring at the tip of the probe. The process is quite fast and, depending upon the toughness of the tissue sample, desired results are usually obtained in 5-60 seconds. For the recovery of intracellular organelles or receptor site complexes, shorter times and/or reduced rotor speeds are used. When using smaller sized rotor-stator probes the tissue sample must often be pre-chopped into pieces less than 1 mm in cross-section with a scalpel or razor blade prior to processing in order for the sample to be drawn inside the hole at the tip of the stator. If the sample has already been stored frozen, a cryopulverizer (a device that quickly powders tissue at liquid nitrogen temperatures – see below) can be used to break the tissue sample into small pieces without thawing. Some rotor stator manufacturers offer probes having a saw-like structure on the tip of the stator which helps break up samples initially too large to enter the probe. This feature helps but homogenization time is slower. Unlike many other types of mechanical cell disrupters, rotor-stators homogenisers generate essentually no heat during operation.
Most laboratory rotor-stator homogenisers are top driven with a compact, high speed electric motor which turns at 8,000 to 60,000 rpm. The size of the rotor-stator probe (also called the generator) can vary from the diameter of a drinking straw for 0.5-50 mL sample volumes to much larger units capable of handling 10 liters or more. There is an important relationship between rotor speed and stator diameter. In principle, the top rotor speed of the homogenizer should double for each halving of the rotor diameter. It is not rpm per se but the tip velocity of the rotor that is the important operating parameter. Ten to twenty meters per second (2000 to 4000 fpm) are acceptable tip speeds for tissue disruption. Unfortunately, some of the smaller-sized commercial rotor-stator homogenisers do not meet this standard. Other factors such as rotor-stator design (there are many), materials used in its construction and ease of cleaning are also important factors to consider in selecting a rotor-stator homogenizer. Some manufactures are BioSpec Products (Bartlesville, OK), Brinkmann Instruments (Westbury, NY), Charles Ross & Son Company (Hauppauge, NY), Craven Laboratories (Austin, TX), IKA Works (Cincinnati, OH), Omni International (Gainsville, VA), Pro Scientific (Monroe, CT), Silverson Machines (Bay Village, OH), and VirTis Company (Gardiner, NY).
Most laboratory sized homogenisers function properly only with liquid samples in the low to medium viscosity range (<10,000 cps). The speed and efficiency of homogenization is compromised by using too small a unit, and the volume range over which a given homogenizer rotor-stator size will function efficiently is only about ten fold. Foaming and aerosols can be a problem with rotor-stator homogenisers. Keeping the tip of the homogenizer well submerged in the media and the use of properly sized vessels helps with the first problem. Square shaped homogenization vessels give better results than round vessels and it is also beneficial to hold the immersed tip off center. Aerosols can be minimized, but not completely eliminated, by using properly covered vessels (VirTis, Brinkmann and Omni). Even though a number of the laboratory rotor-stator homogenisers use fully enclosed motors, none of them are explosion-proof. Therefore, due caution should be followed when using flammable organic solvents such as acetone, alcohol or chloroform by conducting the homogenization in a well ventilated hood.
Bottom-driven laboratory rotor-stator homogenisers are a new entry to the laboratory. The rotor-stator assembly is usually placed within a sealed chamber or container, fits blender motor bases and have working volumes of 100-1000 mL. They costs about $250 – $400 and are available from BioSpec Products (Bartlesville, OK) and Eberbach Corporation (Ann Arbor, MI).
Closely related homogenisers, called dispersers, are used for preparing large volumes of crude plant and animal aqueous extract. Operating like a household garbage disposal unit, the rotor-stator mechanism quickly homogenizes and liquefies kilogram quantities of biomass. The sample is suspended in one or more liters of media, loaded into a top reservoir and homogenized either in a continuous or batch mode.
Although less efficient than rotor-stator homogenisers and aeration, and foaming can be a problem, blade homogenisers (commonly called blenders) have been used for many years to produce fine brie and extracts from plant and animal tissue. Blenders cannot efficiently disrupt microorganisms. In this class of homogenizer a set of stainless steel cutting blades rotate at speeds of 6,000-50,000 rpm inside a glass, plastic or stainless steel container. The blades are either bottom- or top-driven. Some of the higher speed homogenisers can reduce tissue samples to a consistent particulate size with distributions as small as 4 microns, as determined by flow cytometric analysis. After blending, some plant tissue homogenates undergo enzymatic browning – a oxidation and cross-linking process which can complicate subsequent separation procedures. Enzymatic browning is minimized by carrying out the extraction in the absence of oxygen or in the presence of oxygen scavenging thiol compounds such as mercaptoethanol. Sometimes, addition of polyethylene imine, metal chelators, or certain detergents such as Triton X-100 or Tween 80 also help.
When using a blender, use caution when blending with flammable solvents such as alcohol or acetone or when homogenizing diseased tissues. Blenders use brush motors to achieve their high speeds and, therefore, spark during operation. Also, aerosols readily form while blending. Use a sealed blender container and operate it in a well ventilated hood. Blade homogenisers can process liquid sample sizes from 2 mL to one gallon. Accessories for blenders include cooling jackets for temperature control, closed containers to minimize aerosol formation and entrapment of air, special vessels made of stainless steel, semi-micro containers and even insulated vessels for use with cryogenic solvents (see Freeze fracturing). Manufactures of a scientific line of blenders include British Medical Enterprises (London, England), ESGE (Basel, Switzerland), Hamilton Beach Commercial (Washington, NC), Omni International (Waterbury, CT), Professional Diagnostic (Edmonton, Alberta Canada), The VirTis Company (Gardiner, NY) and Waring Products Division (New Hartford, CT). Accessory vessels for Hamilton-Beach brand blenders are manufactured by BioSpec Products (Bartlesville, OK) and for Waring brand blenders by Eberbach Corporation (Ann Arbor, MI).
Both microbial pastes and plant and animal tissue can be frozen in liquid nitrogen and then ground with a common mortar and pestle at the same low temperature. The hard frozen cells are fractured under the mortar because of their brittle nature. Also, at these low temperatures ice crystals may act as an abrasive. The end product of this process can range from very small pieces of tissue the size of grains of salt to preparations with most of the cells actually disrupted. With respect to the later, cryopulverization is a unique mechanical cell disruption method capable of delivering very high molecular weight DNA.
A ceramic motar and pestle precooled to liquid nitrogen temperatures is the classic cryopulverizer. BioSpec Products (Bartlesville, OK) makes a version of this tool specifically designed for cryopulverization called the Cryo-cup Grinder. There are additional devices used to cryopulverize tissue samples. Spectrum Medical Industries (Carson, CA) and BioSpec Products manufacture a freeze fracturing deviice called a Bessman tissue pulverizer or BioPulverizer. It fragments 10 mg to 10 g quantities of soft or fibrous tissue such as skin or cartilage to the size of grains of salt. This material is then easily and quickly homogenized by other cell disruption methods. Looking somewhat like a tablet press, this pulverizer consists of a hole machined into a stainless steel base into which fits a piston or rod. The base and piston are pre-cooled to liquid nitrogen temperatures. Ten mg to ten grams of hard frozen animal or plant tissue is placed in the hole. The piston is placed in the hole and given one or two sharp blows with a hammer. The resulting frozen, powder-like material can be further processed by Pestle and Tube, Bead Mill or Rotor-stator homogenisers. The BioPulverizer come in several sizes and cost $200 to $400. Other cryopulverizers available from BioSpec Products are: the MicroCryoCrusher, a hand operated press that is especially suited for cryopulverizing small samples of fresh bone or teeth and the CryogenicTissueGrinder, a high speed blade mill that cryopulverizes 0.5 – 10 grams of plant or animal tissue to a fine powder in the presence of dry ice.
Grinding biological material in a mortar or tube with fine sand, alumina or glass powder is roughly the equivalent of bead-milling (see above). The method works reasonably well with all types of biomass but is strictly small scale and is labor intensive. Cell pastes or solid mass with a minimum volume of buffer are mixed with 0.5-1 volume of grinding media and ground with a mortar and pestle. Disruption efficiency is poor if lower cell densities or smaller charges of grinding media are used. Also, glass powder has a high surface area and may adsorb significant amounts of charged biomolecules such as nucleic acids and proteins.
Are used to disrupt fresh animal tissue. While variations of the pestle and tube homogenizer have names like Potter, Potter-Elvehjem, Dounce, and Ten Broeck, as a group they consist of test-tubes made of glass, inert plastic or stainless steel into which is inserted a tight-fitting pestle (clearance about 0.1-0.2 mm) made of like materials. The walls of the test-tube and pestle can be smooth or have a ground finish. Most tissues must be cut or chopped into small pieces (~1 mm in cross-section) with scissors or a single-edge razor blade before being suspended in a 3-10 fold volume excess of medium in the test-tube. The pestle is manually worked to the bottom of the tube, thus tearing and fragmenting tissue as it is forced between the sides of the pestle and the wall of the tube. The grinding action occurs again as the pestle is withdrawn. Five to thirty repetitions of this low shear method homogenizes the tissue. Rotation of the pestle at about 500-1000 rpm with an electric motor while the test-tube is manually raised and lowered speeds up the process. While pestle and tube homogenization is simple and the equipment used is usually inexpensive, it is both labor intensive and, in the case of fragile glass homogenisers, potentially dangerous. Even so, this homogenizer continues to be popular because of its extremely gentle action. Often it is the method of choice for the preparation of small quantities of subcellular organelles from soft animal tissues such as brain or liver. Microorganisms cannot be disrupted with pestle homogenisers.
Commercially available glass or plastic pestle homogenisers with batch capacities of 0.1-50 mL generally cost $15-$100 and are available from many manufacturers including Ace Glass (Vineland, NJ), Bellco Glass (Vineland, NJ), BioSpec Products (Bartlesville, OK), Kontes (Vineland, NJ), Thomas Scientific (Swedesboro, NJ), Tri-R Instruments (Rockville Center, NY), Sage Products (Crystal Lake, IL) and Wheaton Industries (Milville, NJ). Disposable, plastic pestles which fit into microcentrifuge tubes are available from Kontes. They also offer a small, hand-held motor unit to drive the pestle. While precision stainless steel tissue grinders are more expensive ($200 – $250, BioSpec Products and Wheaton), they are efficiently cooled and tolerate vigorous homogenization without risk of breakage. A ‘Rolls-Royce’ homogenizer costing about $3000 has a variable speed motor, cooling jacket, and hand- operated lever to rise and lower the pestle (B. Braun Biotech Bethlehem, PA). A continuous pestle homogenizer is available from Yamato USA (Northbrook, IL). Grooves machined on the upper one-third of the pestle catch and guide tissue through the close tolerance region of the lower two-thirds of the cylinder pestle. The resultant homogenate exits from the bottom of the cylinder. Recycling is usually necessary.
The household meat grinder or mincer has been used for many years for the preparation of animal tissue extracts. Tissue is mechanically pressed through holes in a metal sieve plate while rotating blades slowly sweep across the face of the plate cutting the meat in 0.3-0.5 mm fragments. While it is not an effective way to disrupt cells per se , it is useful as a preliminary step for complete homogenization using other physical or chemical methods. Meat grinders cut flexible tissue like muscle better if the tissue is processed slightly frozen.
For small tissue samples, BioSpec Products (Bartlesville, OK) manufactures hand operated screw presses for the preparation of tissue extracts as does EDCO Scientific (Chapel Hill, NC). Capable of generating considerable force, sample sizes from 0.1 grams up to 50 grams of soft tissue are pushed through sieve plates having 0.5 to 3 mm holes, much like the action of a kitchen garlic press. Hard or fibrous tissues like tendon, skin, leaves and seeds will not pass through the press.
Are widely used to disrupt cells. These devices generate intense sonic pressure waves in liquid media. Under the right conditions, the pressure waves cause formation of microbubbles which grown and collapse violently. Called cavitation, the implosion generates a shock wave with enough energy to break cell membranes and even break covalent bonds.
Modern ultrasonic processors use piezoelectric generators made of lead zirconate titanate crystals. The vibrations are transmitted down a titanium metal horn or probe tuned to make the processor unit resonate at 15-25 kHz. The rated power output of ultrasonic processors vary from 10 to 375 Watts. What really counts is the power density at the probe tip. Higher output power is required to sustain good performance in large sized probes. For cell disruption, probe densities should be at least 100 W/cm2 and the larger the better for tip amplitude (typical range: 30-250 microns). Some manufacturers of ultrasonic disintegrators are Artek Systems (Farmington, NY), Branson Sonic Power Company (Danbury, CT), RIA Research Corp. (Hauppauge, NY), Sonic Systems (Newton, PA), Ultrasonic Power Corporation (Freeport, IL) and VirTis Company (Gardiner, NY).
Ultrasonic disintegrators generate considerable heat during processing. For this reason the sample should be kept ice cold. For microorganisms the addition of 0.1 – 0.5 mm diameter glass beads in a ratio of one volume beads to two volumes liquid is recommended, although this modification will eventually erode the sonicator tip. Tough tissues like skin or tendon should be macerated first in a tissue press, grinder or pulverized in liquid nitrogen (see details above). Use small vessels during ultrasonic treatment and place the probe tip deep enough in the sample to avoid foaming. Finally, one should be aware that free radicals can be generated during sonication and that these radicals react with most biomolecules. Damage by oxidative free radicals can be minimized by flushing the solution with nitrogen and/or including scavengers like cysteine, dithiothreitol or other -SH compounds in the media.
Other cell disruption apparatus or techniques not covered in this review are discussed by the author in the book, Purification and Analysis of Recombinant Proteins, Seetharam and Sharma, editors, published by Marcel Dekker, Inc., 1991. They include High-Pressure Homogenisers, Autolysis, Enzymatic lysis, Dehydration, Chemical lysis, Solvent lysis and Programmed self-destruction.