Breaking Up Isn't Hard To Do: A cacophony of sonicators, cell bombs, and grinders

Date: November 9, 1998Comparison of Disruptors With names such as cell crushers, grinders, disintegrators, and pulverizers dominating the field, the business of cell disruption is not for the faint of heart. Some of the functions of these machines are so twisted and diabolical that they would make the Marquis de Sade wince. These devices do the dirty work of the research world, ripping and tearing at the fabric of plants and animal tissues with extreme prejudice to both forms of life. Many scientists turn a blind eye to this unsavory business, leaving such duties to grad students. Behind the secrecy of closed doors, this little-acknowledged aspect of research has gone without comment until now. Finally, these machines and their creators will be revealed. There are primarily five methods for disrupting and homogenizing cell tissue. The most familiar is the common vertical blender that typically resides in most household kitchens. The vertical blender is not only the perfect appliance for margaritas and protein shakes, but it makes a nice puree out of almost any kind of animal or plant tissue. Whether you're tending bar for friends or grinding samples around the lab, the versatility of the vertical blender makes it a popular favorite. The rotor stator is probably the most widely used homogenizer because of its very large shearing effect. Invented in Germany and marketed by Brinkmann as the Polytron, the rotor stator turns a long shaft that is arrayed with some fierce-looking knives at the bottom. These knives shred material that is drawn up into the stator tip, which is ejected from small holes on the sides of the stator to be recycled over and over again in seconds. Dounce, or mortar and pestle, homogenizers are typically used for very small sample volumes of cellular tissue. The sample is placed in a test tube and agitated using a glass or Teflon pestle that creates friction against the glass wall of the tube. This is a manual procedure that is very hard to use. Another technique for liberating the cell of its contents is the so-called cell bomb method. This technique involves pressurizing cells in a chamber up to 25,000 psi and then rapidly decompressing the chamber. The resulting bubbles give the cells a severe case of the bends, and they consequently burst open because of enormous fatigue caused by the expanding bubbles. Sonic disruptors, or sonicators, are some of the most technologically sophisticated homogenizers to come along and are growing in popularity for many as the instrument of choice. The shearing effect produced by modern ultrasonic disruptors comes from the generation of intense ultrasonic waves in the region of 18 to 50 kHz. Though inaudible to the human ear at this level, waves of this frequency are capable of exerting pressures of more than 500 atmospheres and temperatures of 5,000šC. Every sonicator has the same basic configuration with some minor variations to accommodate sample volume and workspace limitations. An electrical signal generator amplifies and rectifies electric signals to 20,000 cycles, converting 50/60 Hz current to high frequency 20 kHz electrical energy. A piezoelectric transducer, which consists of stacks of PCT crystals µead zirconate), changes the electric current into mechanical oscillations. The mechanical vibrations are communicated to the sample by an amplification horn that is attached to the converter by a resonant length of titanium alloy metal. The probe/horn amplifies the sound waves into an intense beam for cutting and shearing. Most of the advances that have been made in ultrasonic devices have to do with microprocessors and the design of booster horns for special applications. In the following article particular attention will be paid to sonic disruptors and cell bombs as some of the most varied and specialized disruptors on the market. The basic design of many ultrasonic cell disruptors has not changed appreciably in over 50 years. Much like the shark, with its ideally streamlined body and formidable rows of serrated teeth, the homogenizer has reached an impasse. Its evolution can go no further, perfection having been achieved. Short of some major new discovery, manufacturers will be producing much the same product for years to come with only minor variations in design and materials, complemented by an array of attachments and accessories. Titanium casing, noise-reducing cabinets, and portability are just some of the features that have been added to give the sonicator a fresh appearance. The real test is whether the machine can deliver rapid homogenization of sample materials. About a half-dozen companies specialize in the production of sonicators for use as homogenizers, and each of these has an attentive audience. Misonix Sonicator XL2020 with Sonabox acoustic enclosure Misonix, based in Farmingdale, N. Y., makes four different sonicators, which, in combination with 12 booster horns and as many microprobes and extender tips, are adaptable to almost any laboratory application imaginable. The XL2020 series generator is intended for a variety of uses. As a biological sonicator, it is best suited for lysing bacteria, virus, and even pesky yeast cells for the extraction of protein, DNA and RNA, and other cellular components. In chemical experiments, an HW Half-Wave Extender is used in conjunction with a sonochemical flask to accelerate chemical reactions, making the XL2020 generator an important tool for cataloging reactions and providing greater yields. In its guise as an environmental sonicator, the XL2020 can be used to test soil and sediment samples and reduces the time for extraction from 4-18 hours to 8-10 minutes with the dual horn and stepped extender attachment. Additional uses for the XL2020 have been in dispensing dyes and inks as well as emulsifying and degassing creams and lotions. The XL2000 series is convertible with a combination of booster horns and HW extender tips made of titanium and sapphire for extended life and reduced cavitation. Also included are Rosett cooling cells for more efficient circulation and sample cooling. Misonix's most recent invention is the 431-T Tray Horn. The Tray Horn accessory sonicates- 384 and 96-well microtiter plates evenly, accelerating reactions for high-throughput screening and eliminating the loss of sample material. IKA-U 50 Ultrasonic Processor IKA, which is located in Wilmington, N.C., has two different sonicators to its credit, including both a land-locked version and a hand-held model for the scientist on the go. The portable U50 Control Ultrasonic Processor can be used in almost any environment, in or out of water. The instrument has a new frequency scanning system that ensures safe oscillation stimulation for the exchangeable sonotrodes and is radio-interference suppressed in order to protect the integrity of radio transmissions within the immediate vicinity. When combined with IKA's selection of sonotrodes, this instrument is capable |of delicate procedures such as cell homogenization or even more aggressive applications such as rock sample preparation. One of the ways companies are looking to improve their products is by consolidating many of the time-consuming and redundant procedures that researchers are forced to perform. IKA has a unique sonovessel that holds up to five test tubes at one time. The U-BB1 reduces the scientist's workload by dispersing acoustic radiation throughout the sonic bath. Individual test tubes can also be treated in the U-BB1 with the benefit that indirect irradiation provides by preventing sample contamination. Intense cavitation can cause material from the sonotrode tip to be dissolved into the solution, but, the indirect method eliminates this complication. Branson Ultrasonics has been making ultrasonic equipment from its headquarters in Danbury, Conn., since 1946, when it offered the Audigage as a nondestructive material thickness tester. In 1960, Branson acquired Sonic Energy Corporation, which made the Sonifier® cell disruptor. From that time forward, Branson Ultrasonics has focused on the transduction of sound for the purpose of biological and industrial research. Today's model 250/450 Digital Sonifier® Ultrasonic Cell Disruptor is the culmination of 50 years experience in producing ultrasonic equipment. The Sonifier can be adapted for emulsification, dispersion, and acceleration of chemical reactions and functions in five different system modes: Continuous, Timed, Pulse, Temperature, and Pulse/Pause. A digital readout gives parameter settings, and a serial interface permits operation from a remote terminal. One of the most unusual features of the instrument, however, is a setting that allows the user to choose from among five different languages during operation: English, German, Italian, French, and Spanish. No doubt, this was calculated as means to gain access to the European market for sonicators. Yet for the English-speaking researcher, this function gives scientists the opportunity for unlimited flights of fancy. One day you can pretend you're homogenizing cells on the French Riviera, and the next, Florence, Italy! Not that bench scientists need such diversions. Certainly the business of cell disruption is excitement enough for the world-weary researcher. The fast-paced world of ultrasonic disruption is not all about multilingual microprocessors and exotic destinations, however. One could well argue that the most powerful electrical supply and the most intelligent microprocessor would be useless if the converter and disruptor horns were inadequately designed for their intended purpose. That is why Branson has a great variety of disruptor horns for transmitting ultrasonic energy into solution with a whole range of amplitudes. Branson has three basic catagories of horns: an exponential horns for compressing powders and tissues, high-gain horns for higher amplitude disruption, and step horns threaded for microtip accessories. One of the company's most impressive designs is a high-intensity cup horn that processes samples in a sealed test tube or vessel, which permits the disruption of infectious or radioactive materials. Branson also makes a sealed-atmosphere treatment chamber for environment isolation and ice-bath cooling. This unit can be used in six different configurations with Branson's externally threaded disruptor horns. Tekmar-Dohrmann Cincinnati, Ohio manufacturer Tekmar-Dohrmann has been in the business of making laboratory instruments for over 35 years. In an effort to satisfy all of its customers' needs, Tekmar-Dohrmann has produced three instruments that prepare samples ultrasonically. The TSD-501 is a moderately priced alternative to some of the company's more sophisticated TSX models. It includes a variable power supply, a built-in timer/pulser, and dual output. For those who want something with a little more pizzazz, the TSX series has microprocessor control, menu-driven programming, storage memory, and digital display. The TMS series has been created for small-volume applications, and like its cousin the TSD model, it has a no-frills approach to cell disruption. Simplicity keeps the cost of these instruments low, but all of the necessary (and some of the not-so-necessary) elements are there. Though microprocessors can be complexified by the addition of microchips and storage capacity and amperage can be increased ad infinitum, the development of booster horns is still a matter of pure engineering. The design of new horns is a classic test of inventiveness and creativity. Adding to its selection of horns, Tekmar-Dohrmann has developed a brand new dual probe that allows the user to process two samples simultaneously. A multi-element horn is capable of holding up to four microtips for rapid sample processing. With all those extra tips, one might think the noise would be intolerable. That is why Tekmar-Dohrmann has introduced sound-abating enclosures that reduce noise levels by 43 dB. To give some idea of the reduction in noise this enclosure affords, 55 dB is the level of normal conversation. Used in combination with Tekmar-Dohrmann's sound abatement technology, sonication can be reduced to a whisper. Sonics & Materials Vibra Cell Sonics & Materials, located in Newtown, Conn., has been in the business of making ultrasonic instruments for 29 years and has the most extensive line of ultrasonic processors in the industry. A significant part of S&M's business relies on making ultrasonic welding systems that use vibratory energy to bond thermoplastic materials. The laboratory applications that S&M provides include both ultrasonic liquid processors for cell lysing, disaggregation, and homogenization and ultrasonic atomizers for atomization of low-viscosity liquids. The Vibra-Cell VCX series of ultrasonic processors is S&M's most advanced model to date. It can prepare sample volumes of 100 milliliters up to 15 liters per batch. At full capacity, the Vibra-Cell can process 300 liters per hour when used with a high-volume continuous-flow cell. Some of the most important advances that S&M has made in its ultrasonic equipment involve the development of new control circuitry. An automatic frequency control circuit eliminates the need for constant adjusting by compensating for changing amplitude and varying load conditions. Its proprietary Stall Prevention Circuitry also ensures continuous power supply. Best of all, Vibra-Cell power supplies and converters are covered by a full three-year unconditional warranty. TheVirTis Company, an SP Industries Company, provides ultrasonic processing power with its Virsonic 475 and Digital Ultrasonic Cell Disruptors. These units are overload/overheat protected and can be adjusted for variable output when horns and probes are changed. The Digital 600 is completely programmable, allowing run times, pulsing capability, and power settings to be entered into memory using the touch-sensitive membrane keypad. The Virsonic 100 Ultrasonic Cell Disruptor is suited for small samples of bacteria, viruses, and spores, as well as emulsification and dispersion of liquids. Biospec Products Mini-Bead Beater The development of the Bead Beater is a classic example of necessity and know-how coming together in one dynamic solution. It has been 20 years since Tim Hopkins, president of BioSpec Products, created the Bead Beater to aid him in his own research. The idea of using ceramic beads in combination with a high-speed rotor to finish cells has made his Bartlesville, Okla., company one of the major contributors to a field where change happens slowly but steadily. As the exclusive maker and distributor of the Bead Beater, BioSpec Products has captured a place in the market that requires a very high percentage of cell breakage. The Bead Beater method is especially effective for lysis of micro-organisms. Its smaller cousin, the Mini-Bead Beater, is well suited for PCR applications because it eliminates contamination from extraneous DNA, even the slightest traces of which will ruin an entire experiment. Homogenization inside disposable microcentrifuge vials guarantees that cross-contamination of samples will never occur. Bioreba Incorporated's Homex 6 features disposable sample containers and is unique in the industry. This homogenizer of large tissue samples such as stems, leaves, and roots has been used in genetic engineering, diagnostics, and pesticide production. The instrument is even capable of homogenizing harde specimens such as seeds and nuts. "People that use it are very committed," said Werner Martin, product manager for the Carrboro, N.C-based company. The technique of using disposable plastic bags in a circular grinder filled with roller bearings produces a clear extract that is easily removed without contamination of the instrument. People like the quick and easy process afforded by disposable bags because it eliminates time-consuming procedures involved in sterilizing instrumentation. Qiagen; Qiashredder The QIAshredder from Qiagen in Valencia, Calif., is designed for homogenization of cell lysates using a microcentrifuge. Its main function is to reduce viscosity and filter debris, breaking tissue and opening cells with a biopolymer shredding system. Qiagen recommends the QIAshredder as an adjunct to other product lines such as Omni's Shear Flow line of tissue homogenizers or Brinkmann's Polytron. The QIAshredder's disposable cell format is ideal for basic homogenization of mammalian cells and-like the Homex 6 and Mini-Bead Beater, prevents cross-contamination. Microfluidies M-110L Microfluidizer® Microfluidics' patented Microfluidizer Processor is one of the newest cell disruption techniques to become available for commercial use. The Microfluidizer bombards samples with an intense stream of fluid that disrupts cells at high pressure (20,000 to 40,000 psi). The design of the interaction chamber causes opposing streams to collide at the precise location of the sample, which results in a very significant shearing effect. Because the size of the machine can be scaled up or down without affecting the quality of disruption, the Microfluidizer can be used for laboratory or industrial applications. In fact, the device has been used for such varied purposes as mixing ice cream and rupturing Baker's yeast. One of the chronic problems with Dounce or Potter-Elvehjem homogenizers is that they tend to create a great deal of friction and subsequently heat the sample, denaturing delicate proteins. Cell bombs have the opposite effect when, during the decompression cycle, they cause the sample to expand, resulting in adiabatic cooling. The drawback to using this technique is that only small samples can be treated. The reason for this limitation is that pressurizing anything larger than a bread box up to pressures that normally reach 25,000 psi is dangerous. A leak or fracture in the instrument casing could cause an explosion. However, by reducing the size of the disruptor, accidents due to equipment failure become much less of a concern. Cell Scientific ML025K MicroDisruptor™ Cell Scientific, Champaign, Ill. manufacturer of cell disruption systems, makes the ML025K Micro Disruptor, which uses high pressure and decompression to disrupt cellular membranes. The rapid expansion of a cell as it passes from the pressurized chamber to the atmospheric chamber causes the cell membrane to rupture. Because there is no mechanical contact between the cell structures and a grinding medium, the integrity of nuclear material is upheld. As much as 99 percent of the sample volume can be disrupted to completion using this method on the first cycle. Although sample volumes remain small (0.05-2.0 ml), quick loading and an autoclavable assembly make rapid exchange of samples and consequently higher throughput a compensating factor. As an added precaution, the ML025K also comes with a full-length safety shield. Even though failure of the compression chamber is extremely unlikely, it's still nice to have some protection against flying particles in case something unpleasant should happen. Cell Scientific expects to release a new super-quiet system in early 1999. Parr Instrument Company in Moline, Ill., uses the nitro-gen decompression method, that is one of the most thoroughly tested methods of cell disruption. In this technique, nitrogen is dissolved into the cell under high pressure. When the pressure is released, the expanding bubbles of nitrogen rupture the cell membranes. The benefits of using the nitrogen decompression method are twofold. First, nitrogen dissolves into the entire sample homogenously, allowing uniform disruption of the entire sample. Second, because nitrogen is an inert gas, it insulates the sample material from oxidation that can degrade tissue. Studies cited by Parr Instrument Company show that when compared to Dounce tissue grinders or Potter-Elvehjem pestle and glass tube homogenizers, the nitrogen decompression technique achieves two to three times greater yield of polysomes and twice as much microsomal protein per gram of tissue Parr Instrument Company. has essentially two basic configurations available for the consumer. The 1-gallon general-purpose model 4635 can be used for large samples, and the 45 ml bomb model 4639 is designed for smaller applications. Larger bombs can also be purchased, but the 1-gallon 4635 model is recommended before moving up to the larger units. The bombs are made of stainless steel and come equipped with a pressure gauge and two valves for pressurizing and withdrawing homogenate. Glas-Col Apparatus BioNeb® Cell Disruption System Glas-Col of Terre Haute, Indiana, makes the BioNeb® Cell Disruption System that operates on one of three different gases: nitrogen, helium, or argon. The device shears large biomolecules at pressures of 250 psi. The BioNeb was originally designed to disrupt yeast cells and has proven successful on other hard-to-disrupt cells such as E. coli. BioNeb can be used in continuous-flow operation or recycling modes that disrupt greater than 90 percent of the homogenate after only one or two passes. This system is well suited for small samples that might be easily damaged by excessive heat. For those who were waiting for an in-depth discussion of rotor stators, or Potter-Elvehjem homogenizers that will have to be reserved for another day. There are just too many products and manufacturers to be included in a single article. Comparison of Disruptors Author Brent Johnson can be reached at bjohnson@the-scientist.com.

Breaking Up Isn't Hard To Do: A cacophony of sonicators, cell bombs, and grinders

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