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Scientific literature reveals an interesting pattern in the way researchers have historically homogenized samples. Foremost, scientists in their traditionally independent manner rely on no specific tool in any one field to disrupt samples. The methods and procedures that are employed support the notion of innovative and independent thought upon which science is built. However, there is a pattern that occurs frequently which is very effective, that being the use of two or more of the methods discussed above. Most sample processing is done with two steps using different homogenizers.
Using two steps to homogenize samples is done out of practicality. The first step in the process is used to reduce the size of the sample to coarse particles while the second step further reduces or obliterates those particles. The second step of the process is less or unsuccessful without the initial processing step. In order to compare single-step and two-step homogenization methods, equal sized samples of mouse muscle were homogenized with the methods sited above and with two-step combinations of those methods. The lysates generated from those processes were then assayed for lactate dehydrogenase (P) which provided data on the relative efficiencies of the method (Fig. 20).
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On microorganisms and cell suspensions, sonication is very effective at lysing cells as compared to bead beating. Perhaps the most apparent tool that does not work on solid samples is ultrasonication, but is very effective when applied to samples first reduced in size. With solid mouse muscle used as a comparative substrate, sonication essentially cooked the muscle. On the solid, no disruption occurred, and in this particular case, no active enzyme was liberated. However, if sonication is used as a second step then the effectiveness was highly increased. Ultrasonication following cryogrinding (Fig. 16) was far superior to either of those methods alone, more than doubling their combined LDH activity (Fig. 20). Indeed the combination of CryoGrinder™ and sonication liberated greatest amount of enzyme and became the standard by which the other methods were measured. |
Figure 16. Cryogenic grinding followed by sonication proved to be the most efficient combination for homogenization with a relative efficiency of 100%. The density of small size of particles exceeded all other methods (see Fig. 20). |
Other combinations also proved to be effective. CryoGrinder™ in conjunction with the Dounce (Fig. 17), Potter-Elvehjem (Fig. 18), and conical glass homogenizers all generated homogenates better than those methods alone. The rotor-stator and sonicator also were an effective combination (Fig 19).
Figure 17. Cryogrinding mouse muscle followed by Dounce homogenizer was 44.6% as efficient as compared to other methods (see Fig. 20). |
Figure 18. Combining the CryoGrinder and Potter-Elvehjem homogenizer on mouse muscle yielded an improved efficiency of 52.1% (see Fig. 20). |
Figure 19. Mouse muscle processed first with a rotor-stator and then sonicator has a relative efficiency of 54%. This is greater than the sum of the individual efficiencies (see Fig. 20). |
The trade off with the two-step homogenization processes is that it slows throughput. Certainly the liberation of analytes is greater, but processing time per sample is increased. As compared to the high throughput homogenizers, two-step homogenization process is more thorough, but it is off-set by lower productivity by the researcher. Consequently, it is necessary to establish a goal of the homogenization process and weigh it against productivity demands. If the objective is to process a vast number of samples, then high throughput methods are warranted. If pure analytical data is required, then two-step processing of samples should be pursued.
Generally the efficiency of sample disruption is inversely proportional to its throughput, the exception being with high throughput bead beating (Fig. 20). High throughput homogenizers (mixer mills) are very effective at disrupting samples, yielding the highest LDH activities for a single step homogenization processes. Manual glass homogenizers can process large samples to generate suspensions that have good LDH activity, extremely small particle sizes, and are readily liquid handled. However, throughput was laboriously slow and residual fibrous tissues often remained adhered to the homogenizer. Mechanical homogenization yielded very fluid samples though resulting particle sizes (observed microscopically) were relatively large. However, samples with removed debris cleared by centrifugation retained considerable enzyme activity.
Most single step processes are less than 50% effective as the most effective two-step combination. The force of grinding balls with the high throughput homogenizers provides the best single-step results, and with that, the large vials and grinding balls suggest that greater force produces better disruption. Sonication, interestingly, borders both sides of the effectiveness scale showing its ineffectiveness on solid samples but is highly efficient at finishing a two-step process. Although many one-step processes are only partially effective as compared to alternatives, it is important to realize that if chemical lysing methods are used subsequently, the resulting homogenization may be completely adequate. This is certainly the case for cryogenic grinding where tissues are effectively dissolved with detergents and chaotropes.
Figure 20. This relative efficiency chart is very revealing as regards the effectiveness of any single method on sample homogenization. The blue bars represent a single processing step while red bars are two-step processes.