The E. coli pathogen is responsible for approximately 96,000 foodborne illnesses and $405 million in healthcare costs every year. (1) Yet in spite of its negative image, this bacterial species plays a critical role in laboratories around the world. Specifically, its cells can be ruptured to expose the intracellular contents for numerous applications. Although multiple cell lysis techniques exist, liquid homogenization is the most popular for cultured cells like E. coli. Here is a complete guide to E. coli cell rupture via homogenization, including the equipment you will need, methods and processes, and accurate analysis.
Prior to homogenization, E. coli cells may need to be mixed with a diluting buffer. The optimal buffer depends on the product’s chemical makeup and stability. Once mixed, a cell suspension is ready for homogenization; this process requires appropriate equipment, and there are a variety to select from. For example, ultrasonic, mechanical, and high-pressure homogenization are a few of the more common techniques. In particular, high pressure homogenization allows for reduced sample sizes and uniform consistency. Scientists should have a clear understanding of the specific application they require equipment for before selecting a homogenization method. (2)
Methods & Processes
Although specific methods may change based on the homogenizer and application, most samples will require some form of pre-homogenization treatment and post-homogenization cooling. Many homogenizers, such as Pion's BEE brand laboratory homogenizer, can be adjusted to optimize results, particularly across cell types and processes. For example, cavitation, shear, and impact are forces that are critical to successful cell rupture. BEE brand homogenizers are structured so that the researcher can adjust these forces to be more gentle or harsh to produce the desired effect. In this way, the optimum results for high yield cell rupture are achieved for the widest variety of cells.
Analysis of Disruption & Particle Size
Analysis of the disruption must take place as close to conclusion of homogenization as possible for an accurate reading. The sample can be viewed with a bright-field or phase-contrast microscope. In additional to its observational strengths, phase-contrast allows the user to gather information on particle size and its impact on downstream processes. The homogenate can then be sedimented to determine protein concentration; these results are to be compared with protein concentrations of prior passes to determine if additional passes are needed. Although a qualitative assessment of particle size can be obtained through a phase-contrast microscope, a quantitative process may be required, specifically in the context of product optimization. Several processes exist to accomplish quantitative analysis, cumulative sedimentation analysis (CSA) is more reliable and requires materials typically found in a laboratory.
Pion's BEE brand Homogenizers for Effective Cell Disruption
As your laboratory prepares to run and analyze cell disruptions, you will begin looking at homogenizers that best suit the required needs. Pion is trusted by pharmaceutical researchers and lab managers around the world. We deliver an array of key benefits, such as production of cell lysates, nano/micro emulsions and dispersions and lipids and suspensions. Our homogenizer processes can be controlled to better suit your product; for example, pressure can be adjusted to be gentler or harsher and the results can be scaled to manufacturing. Finally, our equipment is easy to use, produces higher yield in less time, and results are reproducible and scalable.
Learn more about how BEEI can assist you in cell disruption by visiting us here.