Bead Beating Guide Contents and Introduction
Lindsay E. Gibbons, Halley C.G. Brangs, and David W. Burden
OPS Diagnostics LLC, P.O. Box 348, Lebanon, NJ 08833 USA
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Bead beating is an effective mechanical method used to disrupt a wide range of biological samples. At a minimum, bead beating is accomplished by rapidly agitating a sample with grinding media (beads or balls) in a bead beater (device that shakes the homogenization vessel). Bead beaters have been designed to homogenize samples in microwell plates, tubes, or vials with beads or balls that are made of glass (silica), ceramic (zirconium) or steel. Samples can be processed with or without buffer or solvent at either ambient or cryogenic temperatures.
Researchers often bead beat because it is a very efficient method to break down most samples. Unlike handheld rotor-stators, bead beaters do not directly contact the sample, thereby reducing cross-contamination when multiple samples are processed. Bead beating instruments vary in the number of samples that can be processed at a time, physical motion, and the speed at which the samples are agitated.
A variety of devices are used to bead beat samples. In all cases, the tube, vial or plate is shaken so that grinding media can impact and disrupt the sample. Vortexers, the simplest (and least effective) bead beater, work by swirling the sample and beads/ balls in a motion that promotes disruption of microorganisms. Other bead beaters, including dental amalgamators and shaking mills, oscillate tubes, often in a figure-eight motion, which allows for crushing and grinding of samples. High throughput homogenizers, which can process samples in deep well plates in addition to other formats, have a linear motion that focuses the kinetic energy of the grinding media on the sample instead of on the sides of the container. Extremely resilient samples, such as seeds, are most effectively homogenized via linear motion bead beaters. While vortexers are limited to processing microorganisms, other beat beaters have a wider range of sample processing capabilities (Table 1).
Single and multitube vortexers can be used to disrupt microorganisms, but are very ineffective at homogenizing solid tissues. Vortexers are a good choice when a limited number of samples require processing, or for laboratories on very tight budgets. Adding beads and a suspension of microorganisms to a snap cap tube and holding it on a vortex mixer for a minute will result in disrupted cells. However, this method is generally less than half the effectiveness of dedicated bead beaters. Some vortexers have been modified to function more like a bead beater. For instance, the Pulsing Vortex Mixer can process 12 samples in microcentrifuge tubes at a fraction of the cost of a dedicated bead beater. Though more expensive, a Multitube Vortexer can accommodate full racks of tubes or microwell plates, thus allowing higher throughput processing.
Table 1. Homogenizer Characteristics and Capacities. (click on table for larger image)
NOTES: The HT 24™ can run for 90 seconds continuously, but can cycle 10 times using the pulsing feature. When this feature is active, a pause (1-120 seconds) is inserted between the run times. Thus, total processing time can be up to 15 minutes with pauses of as little as 1 second. Initially, the GenoGrinder 2010 could be adapted to hold four deep well plates. The clamp assembly was later modified to hold up to six plates.
Multitube bead beaters like the HT Mini™ and the HT 24™ use a high speed oscillating motion to homogenize microorganisms and tissues samples. The HT Mini™, which is based on the design of a dental amalgamator, can effectively homogenize microorganisms and small tissue samples by rapidly shaking microcentrifuge tubes clamped in a small arm back and forth between 2800 and 4000 rpm. Microorganisms placed in tubes with grinding beads can be homogenized in as little as 30 seconds. The HT Mini™ has been designed to hold one to three 2 ml screw cap nonskirted disruption tubes. The HT 24™ can oscillate up to 24 samples in 0.5 ml or 2 ml disruption tubes in a figure-eight multidirectional motion at speeds between 2400 to 4200 rpm. Heat, which can be generated as a result of the high-speed agitation, can affect the integrity of many biomolecules. Consequently, the HT 24™ has a pulsing feature that can be programmed to operate in short bursts with interval rest periods, allowing heat to dissipate between cycles.
High throughput homogenizers are specifically designed for processing samples in deep well plates and vials, but they can also accommodate microcentrifuge tubes and small bottles, allowing for the processing of large samples. Organs that are too large to be processed in a dental amalgamator or multitube bead beaters can be homogenized in high throughput homogenizers. Although these instruments can be expensive, they are capable of efficiently processing hundreds to thousands of samples daily. Models include the HT Homogenizer™, 1600 Mini G™, and 2010 GenoGrinder®. These homogenizers have a range of capabilities, which is reflected in their cost.
All high throughput homogenizers can process samples in plates or vial sets in SBS (Society for Biomolecular Science) formats (e.g., 24 and 96 well). This format permits liquid handling robots to process samples downstream of homogenization. The HT Homogenizer™ can process a single deep well plate, a single rack of disruption tubes, up to twenty four (24) 4 ml vials, or five 15 ml vials at a time. The 1600 Mini G™ has twice the processing capability of the HT Homogenizer™, as well as the ability to process samples in tall centrifuge tubes (15 and 50 ml conical tubes) and cryogenically in a Cryoblock. The 2010 GenoGrinder® has all of the advanced features as the 1600 Mini G™ (i.e., cryogenic grinding, digital controls, and sleek design), but with much greater capacity. It can process up to six deep well plates, four vial sets, and multiple racks of disruption tubes at a time.
The term sample resiliency refers to the degree by which a sample is unscathed following bead beating. Some samples, such as thymus and liver, have very low resiliency and are relatively easy to homogenize by bead beating. Other samples, such as skin, fish scale, and nail, are very resilient and are homogenized with great difficulty. A few samples are totally resilient, like palm nuts, which remain virtually untouched when using even the largest, most dense grinding balls. Luckily, most samples are of moderate resiliency and can be effectively homogenized.
The key to effectively homogenizing samples is to find a balance between sample mass, vessel volume and grinding media size. With very soft samples, like liver, several hundred milligrams can be processed in a 2 ml disruption tube with a 5/32” grinding ball in a HT Mini™ or HT 24™. However, a 20 mg sample of rat skin, which is very durable, may require a 4 ml vial and a 3/8” grinding ball under cryogenic conditions using a GenoGrinder®. This highlights an important point: it is easy to purchase different grinding vessels and media, but much more difficult to switch homogenizers. While many laboratories have bead beaters that have been inherited, it is very important for researchers purchasing new homogenizers to consider the potential sample types that will be processed in the future (Table 2).
Table 2. Cross reference of sample type and bead beater capability. (click on table for larger image)