Version 1.1 Complete PDF
David W. Burden, Ph.D.
Part I: Abstract, Table of Contents & Introduction
When developing a disruption scheme, it is prudent to define the characteristics of the desired homogenate and then select the methods, reagents, and tools that will help to meet the objectives. There is a vast selection of chemistries and tools that have been endlessly combined to disrupt samples. Detergents, chaotropes, and lytic enzymes are often effective for lysing cells and tissues, many times alone, but also when combined with mechanical methods. Mechanically disrupting samples using homogenizers, which can be grouped into those that grind, shear, beat, and shock, is commonplace when chemical methods alone are insufficient. The examination of methods used to homogenize samples has shown that effectiveness is directly related to the nature of the sample. Samples that start with small particles, such as bacterial cultures, are most effectively disrupted by ultrasonication, but that same method is the poorest for solid muscle. In such cases, samples must first be disaggregated into smaller particles prior to processing. Methods which rely on a single processing step, such as with the high throughput homogenizers, can yield very good sample disruption, but they do not match two-step processes that breakdown samples in a series of steps. The need to process large numbers of samples may require a trade-off with the effectiveness of homogenization.
Disruption is an early and fundamental step in any research which involves analyzing, separating, or isolating some component from an intact sample. This includes the isolation/harvesting of cellular components or quantification of RNA, DNA, proteins, and analytes. Both chemical and mechanical/physical methods are available for disruption, with chemical methods being preferred for many sample types (e.g., E. coli and cultured cells). However, many microorganisms, intact tissues, solid specimens (e.g., seeds), and heavily encased samples are not effectively disrupted chemically. With chemically resistant samples, mechanical and physical methods that rely on grinding, shearing, beating and shocking can be used. Mechanical homogenizers, manual homogenizers, mortar and pestles, sonicators, mixer mills, and vortexers are several of the more common tools used for mechanical and physical disruption.
Sample disruption, or homogenization, is often scantly detailed in protocols even though it does have significant impact on the end results of a process. Many research articles will simply state that a sample was “homogenized” in a defined buffer, without specifying what type of homogenizer was employed. When specific homogenizers are mentioned, such as the Dounce or bead beater, little additional information is provided to detail its use. Consequently, methods used for sample disruption are also not necessarily well understood. Indeed like many well established lab processes, homogenization methods are passed on from researcher to researcher like inheritable family treasures, with little effort expended to decipher the process itself. This leads to significant variation in methodology between laboratories. Being fair, the impact of homogenization on an experiment may be minimal, but at times the choice of tools, chemistries, and their method of use may have a significant impact on the outcome.
Where possible, chemical disruption may be the preferred method, such as lysing E. coli with SDS for plasmid isolation, but it may also introduce unwanted molecules into the lysate. Though useful for nucleic acid isolation, detergents and chaotropes may certainly denature proteins making their application to protein purification impractical. The same is true for the addition of lytic enzymes, which in the case of protein purification, must be subsequently removed. If chemical disruption is impractical or simply does not work, then mechanical and physical disruption of samples is the alternative.
Mechanical/physical methods for disrupting samples include grinding, shearing, beating, and shocking. Grinding, which is done with such tools as a mortar and pestle, involves applying force downward on a sample in conjunction with a separate tangential (i.e., rotating) force. Shearing is like that of a blender where a force is tangentially applied to a sample. Directly impacting a sample with a ball or hammer is beating. Shock is similar to beating, but there is no physical implement contacting the sample, just shockwaves. At times it is difficult to discern between the different forces that relate to each method. For instance, grinding is a combination of shearing and beating, but for the sake of simplicity we will segregate the different tools into these categories.
In practice, scientists mix and match disruption methods to meet their needs. Though an ideal disruption method would require only a single step, it is quite common to see two or more methods being used in tandem to obtain the desired result. For instance, the isolation of subcellular fractions could first involve cutting a tissue with scissors, followed by course shearing with a handheld homogenizer, and then a final dissociation with a glass Dounce homogenizer. If any one of these steps is omitted, then the degree of homogenization would be reduced.
There are several methods we employ to evaluate homogenization methods which will be highlighted in this guide. First, homogenization releases cytosolic enzymes and several of these can be assayed and used as a relatively measurement of sample disruption. Lactate dehydrogenase is routinely used in our laboratories for this assessment. Similarly, measuring soluble protein can be used as an indicator of homogenization efficiency. Microscopic observation of samples also provides useful information on the extent by which samples are homogenized.