Many customers who are just starting to investigate laboratory homogenization techniques call us and ask what the difference is between Ultrasonic Homogenization and Mechanical Shear or Rotor/Stator Homogenization.  As we offer both products, it is fairly simple for us to meet the requirements of all the customers whether they need sonication or mechanical shear technology.  Although you may be able to get similar results from both ultrasonic and mechanical homogenizers, the way they create the energy to process the sample is different and can have distinctive effects on the sample and the end result.

Ultrasonic homogenization is created by power that is supplied by electrical energy that is transferred to a probe, usually titanium, where it is converted to mechanical energy.  This mechanical energy shows up as longitudinal vibrations at the tip of the probe.  Once the energy reaches the tip it causes microscopic vapor bubbles that implode and cause shock waves throughout the sample that cause the processing effect or what is called cavitation.

Ultrasonic probes are solid so they have no tearing or cutting capabilities however they are extremely effective on very small samples as the sample has no chance of hiding in a blade assembly.  Sonicators are more effective at processing bacteria and spores along with soil and sediment samples as they have more of an impact effect for breaking the hard walls of these samples. Ultrasonic Homogenizers are also very effective for DNA shearing.  However, they do generate more heat than Mechanical Shear Homogenizers so heat sensitive samples must sometimes be cooled.

Rotor/Stator Homogenization is driven by an electrical motor that is used to drive a long shaft with a rotor/stator (blade assembly) attached to the bottom of it.  This motor causes rotation of the blade assembly at very high speeds anywhere from 5,000 rpm’s all the way up to 75,000 rpm’s.  The blade (rotor) which is rotating pulls the sample into the processing area and then forces it through very sharp windows in the outer blade assembly (stator) to fully and continuously process the sample.

Mechanical shear homogenization is usually better suited to samples that need a tearing or cutting effect on them.  Examples of these samples include animal tissue and cells such as mouse organs, tumors, and muscle.  It is also very useful for making emulsions and combining powders and liquids along with other general lab applications.

Did you like this article? Stand by, our newest Ultrasonic article entitled “Getting Specific: Ultrasonic Homogenizer Applications” is coming soon!

Designed to Save Time, Improve Yield, and Eliminate Cross-Contamination

by Karl Jahn

Rotor–stator technology is a highly efficient method utilized by many laboratories to homogenize, disrupt, emulsify, and blend a broad range of samples, including tissue samples. As a result, rotor–stator homogenizers are used whenever possible for most laboratory homogenizing tasks. Sample processing times are generally very short, homogenization is fast and efficient, and sample temperature rise is minimized or eliminated. First invented by Prof. Peter Willems in 1957, rotor–stator homogenizing remained relatively unchanged for nearly 35 years.

Plastic Homogenizing Probes

In 1992, Omni International, Inc. (Marietta, GA) invented the first plastic rotor– stator homogenizing probes, known as the Omni Tips™ (Figure 1). This marked the first significant improvement to this venerable technology and completely revolutionized rotor–stator homogenization (patent numbers 6,398,402 B1 and 6,863,431). Stainless steel homogenizing probes, still used in many laboratories, usually require a tedious cleaning step between samples. This may involve complete probe disassembly, especially if autoclaving is necessary. After cleaning, the probes must be carefully dried and reassembled. Even when meticulously cleaned, sample-to-sample contamination cannot be totally eliminated, and the complex geometry of stainless steel probes leaves many places for contaminants to hide. Plastic Omni Tip probes, on the other hand, can be disposed of after use, thereby eliminating the possibility of cross- contamination. Further, the use of modern engineering plastics enables the probes to be cleaned and reused. When chemically cleaned, Omni Tips can be reused dozens of times, and can be autoclaved up to seven times. The probes require no bearings, and simple two-piece construction allows for easy disassembly and cleaning. This simple construction also reduces the potential for cross-contamination by a factor of four when compared to stainless steel probes.

With a trend toward smaller and more valuable samples, sample loss can be a real source of concern for some laboratories. The design of Omni Tip plastic probes offers the additional benefit of a clear outer tube, thereby keeping the entire sample visible during the homogenization process and ensuring that sample is not lost within the probe. A second trend, particularly in laboratories with scarce financial resources, is equipment sharing. With stainless steel probes researchers are never quite sure what was most recently processed, or how clean the probe is. The low acquisition cost of Omni Tip plastic probes means that laboratory homogenizer motors can now be easily shared, while each researcher can use his or her own Omni Tip probes. Further, stainless steel probes can easily cost more than a thousand dollars, and can be damaged if not effectively maintained and cleaned. A mere six-inch drop can permanently damage a stainless steel probe. The Omni Tips can readily survive a drop in excess of six feet and remain fully functional. Many laboratories also value consistent sample-to-sample processing. Stainless steel probes wear with use, particularly in the lower bearing area. The PTFE lower bearing is responsible for centering the probe knife during processing.

As the soft PTFE material wears, the spacing between the rotating knife and the stator begins to change, leading to variable processing results. Omni Tips are molded to rigid standards with virtually no dimensional probe-to-probe variability. As a result, they tend to yield highly repeatable processing results.

The plastic probes are available in a soft tissue version for sensitive cell disruption and in a hard tissue version, tough enough to process frozen tissue as well as most other applications that previously required stainless steel probes. Omni Tips spin at 35,000 rpm, which is the same speed as their stainless steel counterparts. For aerosol containment, a broad range of sealed tube solutions is available to work with the probes.

Omni Tips are very economical to own and use. Their acquisition cost is about 1% of stainless steel probes, which can be significantly reduced when cleaned and reused. This low cost makes batch processing a highly efficient method for homogenization. Samples can be quickly homogenized, and the probes subsequently disposed of, or batch cleaned for reuse. Elimination of the cleaning step between samples also creates an ideal platform for automation, as with the Omni Prep™ homogenizing workstation (Omni International), which is capable of processing six samples simultaneously, with a capacity of up to 250 samples per hour.

Multi-sample Homogenizer

The Omni Prep programmable homogenizing system (Figure 3) is designed to eliminate homogenizing processing bottlenecks, while also addressing the shortcomings of existing approaches to rotor–stator homogenizing methods. The system is small enough to easily fit into a fumehood, and also preserves precious laboratory bench space. It is designed around a rack system that allows six samples to be processed simultaneously. By utilizing a second processing rack, six more samples can be prepared while the first rack is processing, permitting a single operator to process up to 250 samples per hour. Operator fatigue and repetitive motion injuries that can result from homogenizing a large number of samples per day by hand are also eliminated, while a single technician can now perform the work of six. The racks are designed to accommodate a broad range of tube sizes from 1.5-mL microcentrifuge tubes up to 50-mL conical bottom tubes, and are available in fixed or movable configurations. The fixed rack is well suited for processing samples that require sealed tubes, while the movable version is intended for processing larger sample volumes that require probe mobility within the sample. The recommended processing volume for the Omni Prep is .25 mL up to 30 mL. A cooling tray is also offered to keep sensitive or frozen samples cold during the homogenization step. A clear plastic door protects the operator from accidental splashing, and a fan-driven positive airflow pattern move air away from the front of the instrument for exhaustion into a fumehood or through a HEPA filter.

Reference

1. Mace, B.E.; Sullivan, P.M. The Use of Steel Homogenizer Probe Results in Sample Carryover Contamination. Duke University
Medical Center, Durham, NC, 1997.

by Brian E Mace and Patrick M Sullivan
Duke University Medical Center, Durham, NC

Introduction

In any experiment involving different treatment groups or varying starting material it is important to insure no crossover or contamination of sample material occurs. We routinely work with mouse models of varying genotype and then study the effects of different treatments in these mice. More specifically, we measure the levels of apolipoprotein E protein and mRNA in brain tissue using a very sensitive and quantitative assay system. Therefore, we tested two tips probes (Omni International) for potential contamination between tissue samples during the homogenization process. The probes we tested were the plastic tip disposable generator probe (Omni cat # 34725) and the steel tip probe (Omni cat #G7-95st)

Procedure

After removal from the skull the mouse brain is cut in half and then each hemisphere is cut into 5-8 mm pieces before being placed into a cryotube and then immediately frozen in liquid nitrogen. All samples were treated the same with the probe type being the only variable. One half of the brain was homogenized with the plastic probe and the other half was homogenized with the steel probe. The tissue is placed into 1 ml of guanidine buffer (5.0 M guanidine-HCL, 50 mM Tris pH 8.0) and allowed to thaw for 30 seconds before homogenizing on ice using an GLH homogenizer (Omni). Homogenization was done on low speed (setting 1) for 45 seconds, medium speed (setting 3) for 45 seconds, and then on high-speed (setting 6) for thirty seconds in a 2 ml cryogenic vial (cat # 430488 Corning Incorporated, Corning, NY). The probes were then cleaned in water on high speed for two minutes and wiped down with a kimwipe (Kimberly- Clark, Roswell, GA). The probe is then put in to a cryogenic vial containing only 1 ml of guanidine buffer and then turned on for 1 min to “clean” the probe. This is repeated one more time. The primary sample is rocked for 4 hours at room temperature to insure complete solubilization. Homogenates are diluted 1:10 in ice-cold casein buffer (0.25% casein/phosphate buffered saline pH 7.4, 0.05% sodium azide, 1X protease inhibitor cocktail, 0.5 M EDTA, pH 8.0,) and kept on ice. Diluted homogenates are spun in an Eppendorf microfuge at 14,000 rpm for 20 minutes at 4 C and the supernatant assayed for protein content.

The Micro BCA Protein Assay (Pierce) was used according to the manufacture’s protocol. In brief, 150 µl of sample is pipeted into duplicate wells of a 96 well plate (Costar cat # 3596, Corning Incorporated, Corning, NY). 150 µl of working solution was added to each well and the plate was mixed for 30 seconds on a shaker. The plate was covered and incubated at 37 C for 2 hours and the absorbance was measured at 562 nm on a Theromomax microplate reader (Molecular Devices, Sunnyvale CA). The samples were averaged and background subtracted. Protein concentration was determined by using a known concentration of BSA as the standard. To correct for different weights of tissue the following formula was used:

Average protein concentration of wash/average protein concentration of 1/2 brain *100%

These numbers were then used to compare the amount of protein carryover between the plastic and steel probe.

Results

Both the Omni tip disposable generator probe # 34725 and the steel Omni tip probe #G7-95ST homogenized the brain tissue very efficiently. For comparison sake we corrected for different starting amounts of protein by looking at the percent of wash homogenate protein compared to the total protein detected in the 1/2 brain homogenate. The plastic Omni tip disposable generator probe # 34725 had a carry-over of 0.6% (0.12 µ?ug/ml) of total protein from brain homogenate. The steel Omni tip probe #G7-95ST had a carry-over of 2.4% (0.34 µ?ug/ml) of total protein from brain homogenate.

It is worth noting that these numbers were obtained after processing of a single sample. Since the steel probe would be used for all subsequent samples we do not know if this carryover of protein contamination would accumulate over time. Extensive cleaning of the steel probe between each sample may minimize this risk, however the time required for autoclaving, cleaning and technician time does not warrant use of the steel probe in our opinion. Also, SDS-solubilized tissue may show even more carryover, since a much larger fraction of the tissue is not soluble in SDS. Obviously this is not an issue when using the disposable probes. For measuring small differences in mRNA between samples using a RT-PCR method, the plastic disposable probes would clearly be preferred.

Many diverse laboratory homogenization technologies exist, including ultrasonic disruption, mortar and pestle, bead mills, blade systems (blenders), and rotor-stator. Each of these technologies has its own advantages and disadvantages.

Ultrasonic processing works best for very small and very hard-shelled samples such as spores and yeast cells. Ultrasonic processing, however, imparts a great deal of energy into the sample in a very concentrated area. This may result in sample temperature rise and sometimes leading to sample denaturing. Mortar and pestle processing can be quite effective, but is time consuming and can lead to repetitive motion injury. This technology can also yield inconsistent processing results.

Bead mills work well with small and tough to process samples, although the initial sample size must be relatively small. Sample heating is often a concern and samples must be separated from the beads after processing, leading to potential sample loss. Blade systems tend to work well for stringy samples, but generally are not effective for processing small quantities of sample. They are less efficient than rotor-stator homogenizing, and are limited in particle size reduction of about 15 microns.

Rotor-stator technology is a highly efficient method utilized by many laboratories to homogenize, disrupt, emulsify and blend a broad range of samples, including tissue samples. As a result, rotor-stator homogenizers are used whenever possible for most laboratory homogenizing tasks. Sample processing times are generally very short, homogenization is fast and efficient, and sample temperature rise is minimized or eliminated. First invented by Professor Peter Willems in 1957, rotor-stator homogenizing remained relatively unchanged for nearly 35 years.

Omni Tip™ Plastic Homogenizing Probes
In 1992, Omni International, Inc. invented the first plastic rotor-stator homogenizing probes, known as the Omni Tips™ (Figure 1). This marked the first significant improvement to this venerable technology and completely revolutionized rotor-stator homogenization (Patent Numbers: 6,398,402 B1 and 6,863,431).

Stainless steel homogenizing probes, still used in many laboratories, usually require a tedious cleaning step between samples. This may involve complete probe disassembly, especially if autoclaving is necessary. After cleaning, the probes must be carefully dried and reassembled. Even when meticulously cleaned, sample-to-sample contamination cannot be totally eliminated, and the complex geometry of stainless steel probes leaves many places for contaminants to hide.

Plastic Omni Tip™ probes, on the other hand, can be disposed of after use, thereby eliminating the possibility of cross contamination. Further, the use of modern engineering plastics enables Omni Tips™ to be cleaned and reused. When chemically cleaned, Omni Tips™ can be reused dozens of times, and can be autoclaved up to seven times. Omni Tips™ require no bearings, and simple two-piece construction allows for easy disassembly and cleaning. This simple construction also reduces the potential for cross contamination by a factor of four when compared to stainless steel probes (Figure 2).*

With a trend toward smaller and more valuable samples, sample loss can be a real source of concern for some laboratories. The unique design of Omni Tip™ plastic probes offers the additional benefit of a clear outer tube, thereby keeping the entire sample visible during the homogenization process and assuring that sample is not lost within the probe. A second trend, particularly in labs with scarce financial resources, is equipment sharing. With stainless steel probes researchers are never quite sure what was most recently processed, or how clean the probe is. The low acquisition cost of Omni Tip™ plastic probes means that homogenizer motors can now be easily shared, while each researcher can use his or her own Omni Tip™ probes. Further, stainless steel probes can easily cost more than one thousand dollars, and can be damaged if not effectively maintained and cleaned. A mere six inch drop can permanently damage a stainless steel probe. The Omni Tips™ can readily survive a drop in excess of six feet and remain fully functional.

Many laboratories also value consistent sample-to-sample processing. Stainless steel probes wear with use, particularly in the lower bearing area. The Teflon lower bearing is responsible for centering the probe knife during processing. As the soft Teflon material wears, the spacing between the rotating knife and the stator begins to change, leading to variable processing results. Omni Tips™ are molded to rigid standards with virtually no dimensional probe-to-probe variability. As a result, Omni Tips™ tend to yield highly repeatable processing results.

Plastic Omni Tip™ probes are available in a soft tissue version for sensitive cell disruption and in a hard tissue version, tough enough to process frozen tissue as well as most other applications that previously required stainless steel probes. Omni Tips™ spin at 35,000 rpm, which is the same speed as their stainless steel counterparts. For aerosol containment, a broad range of sealed tube solutions is available to work with Omni Tips™.

Omni Tips™ are very economical to own and use. Their acquisition cost is about 1% of stainless steel probes, which can be significantly reduced when cleaned and reused. This low cost makes batch processing a highly efficient method for homogenization. Samples can be quickly homogenized, and the probes subsequently disposed of, or batch cleaned for reuse. Elimination of the cleaning step between samples also creates an ideal platform for automation, as with Omni International’s new Omni Prep™ homogenizing work station, capable of processing six samples simultaneously, with a capacity of up to 250 samples per hour.

Omni Prep™ Multi-Sample Homogenizer
The Omni Prep™ Programmable Homogenizing System (Figure 3) is designed to eliminate homogenizing processing bottlenecks, while also addressing the shortcomings of existing approaches to rotor-stator homogenizing methods. The Omni Prep™ is small enough to easily fit into a fume hood, and also preserves precious laboratory bench space. The Omni Prep™ is designed around a unique rack system, which allows six samples to be processed simultaneously. By utilizing a second processing rack, six more samples can be prepared while the first rack is processing allowing a single operator to process up to 250 samples per hour. Operator fatigue and repetitive motion injuries that can result from homogenizing a large number of samples per day by hand are also eliminated, while a single technician can now perform the work of six. The racks are designed to accommodate a broad range of tube sizes from 1.5ml microcentrifuge tubes up to 50ml conical bottom tubes and are available in fixed or movable configurations. The fixed rack is ideal for processing samples that require sealed tubes, while the movable version is ideal for processing larger sample volumes that require probe mobility within the sample. The recommended processing volume for the Omni Prep™ is .25ml up to 30ml. A cooling tray is also available to keep sensitive or frozen samples cold during the homogenization step. A clear plastic door protects the operator from accidental splashing and a fan driven positive airflow pattern move air away from the front of the instrument for exhaustion into a fume hood or through a hepa filter.

The trend in many of today’s laboratories is toward high throughput and low-volume samples. Competition is driving the need to enhance productivity and efficiency. Many protocols also require the elimination of sample-to-sample cross-contamination, as well as operator protection from pathogens. To satisfy this trend, more and more laboratories are looking toward automation to help manage their sample load.

Rotor stator technology is a highly efficient method utilized by many laboratories to homogenize, disrupt, emulsify, and blend a broad range of samples, including tissue samples. When rotor stator homogenizers are used, sample processing times are generally very short, often requiring only 30–90 sec. However, stainless steel homogenizing probes can require an additional and tedious cleaning step of several minutes per sample, and even then, sample-to-sample contamination cannot be totally eliminated. This additional cleaning step can limit the number of samples processed by an individual technician to 10–15 samples per hour. When plastic probes such as patented Omni Tips (Figure 1) (Omni International, Marietta, GA) are used, batch cleaning or disposal of the probes is possible, thereby increasing the number of samples processed by a single operator in an hour to 30 or more.

These plastic probes offer the additional benefit of clarity, thereby assuring that sample is not lost within the probe. Simple two-piece construction facilitates easy cleaning and reduced cross-contamination. Their low cost makes it practical to own large quantities of probes, which can be collected after processing and autoclaved or chemically cleaned in large batches, reducing cost to pennies per use. For critical samples, the probes can even be discarded after use. They are available in soft tissue versions for sensitive cell disruption, or in hard tissue versions, tough enough to process frozen tissue and for most applications that previously required stainless steel probes. Additionally, a broad range of sealing cap solutions are available to work with the probes.

Robotic systems are available that can increase the number of samples homogenized to 95 samples per hour. These systems are efficient, but can cost in excess of $30,000 and require a large amount of bench space. They utilize stainless steel probes, which require a between sample probe cleaning step, and therefore cannot process samples in sealed tubes, and also may not fit under most laboratory fume hoods.

The Omni Prep Programmable Homogenizing System patent pending (Omni International) is designed to eliminate this processing bottleneck, while also addressing the shortcomings of existing approaches to rotor stator homogenizing methods. The system is 400 mm × 200 mm × 300 mm, which makes it small enough to easily fit into a fume hood, and also preserves precious laboratory bench space. The system is designed around a rack system that allows six samples to be processed simultaneously.

By utilizing a second rack, six more samples can be prepared while the first rack is processing. By continuously feeding, unloading, and loading racks, a single operator can process up to 250 samples per hour. Operator fatigue and repetitive motion injuries that can result from manually homogenizing a large number of samples per day are also eliminated, while a single technician can perform the work of eight.

The racks are designed to accommodate a broad range of sample sizes and tubes, ranging from 2.5 mL to 30 mL, and are supplied in fixed or movable configurations. The fixed rack is well suited for processing samples that require sealed tubes, while the moveable version is suitable for processing samples that require probe mobility within the sample. A cooling tray is also available to keep sensitive or frozen samples cold during the homogenization step. A clear plastic door protects the operator, and a fan-driven positive airflow pattern moves air away from the front of the instrument for exhaustion into a fume hood or through a HEPA
filter.