Homogenization, which is one of the golden standards in the laboratory, is the key to obtaining biological material inside the cells. There are several methods used for this purpose, but each of them leads to the loss or weakening of the integrity of the wall or membrane cell, causing disintegration and release of individual elements of the cell.
Isolating structures such as cell organelles, DNA or proteins from biological materials is a challenge, and the first stages of this procedure are crucial for successful further experiments.
Plant and yeast cells, which have additional protection in the form of a cell wall, pose a particular difficulty. The release of the desired components from the inside of the cell is important in the case of diagnostic methods, e.g. when it is necessary to obtain as much as possible of the genetic material of a pathogen isolated from tissues taken from humans. Obtaining lysates cell is also one of the main elements routinely used in scientific experiments based on molecular biology. The choice of cell disintegration method has a significant impact on the further use of material isolated from the cell. In the case of proteins, sometimes a critical property that determines the usefulness of material isolated from the cell is its preservation of enzymatic activity.
The effectiveness and efficiency of the process depends on the precision, the device used, the selection of appropriate operating parameters, accessories, as well as strict control of the reaction environment. Homogenization of biological material is carried out using special homogenizers or other laboratory methods that do not require the use of this type of equipment.
- Ultrasonic homogenizer, also called sonicator, is used for the process of disintegration of particles or cells contained in a suspension using ultrasonic waves (high power and low frequency 20-100 kHz). Loss of cell integrity occurs due to the cavitation effect. This phenomenon is based on the processes of dynamic growth and disappearance of vapor-gas bubbles in the liquid and is caused by changes in pressure at a constant temperature. Mechanical forces – created at the place of collision of cavitation bubbles and the shock wave – generated after their implosion are the main factors responsible for cell damage. Irreversible or reversible damage to the cell membrane (sonoporation effect) is amplified by radical-like substances formed as a result of thermal decomposition of the solvent and particles contained in colliding bubbles. The correct course of sonication depends on the parameters of ultrasound, the shape of the sonication cell, the properties and the amount of medium to be sonicated, including the presence of air bubbles and suspension particles.
- The ball homogenizer is a stationary device and is increasingly appearing in the automated version. Disintegration of cells using it involves the use of the disintegrating material in the form of beads, which are placed in a homogenised suspension (the exception is the so-called “dry procedures”). The structure and properties of grinding balls enable their use in the homogenization of diverse biological material, such as: hard animal tissues (e.g. bone tissue, teeth), hard plant material (e.g. root, seeds), soft animal tissues (liver, heart, brain) , smooth muscles), soft plant material, microorganisms, bacteria and their spores.
- The stator-rotor homogenizer produces homogenate by direct damage to the cell membrane or wall by means of knives located on the rotor head. Due to the way the device is operated, a manual and stationary homogenizer with higher power is distinguished. Stationary homogenizers have a non-automated or automated mode of operation. In laboratories, stator-rotor homogenizers (depending on the construction of the rotor and head) are used in working with soft or hard animal tissues. Homogenization of this type of biological material often requires the addition of cellular protease inhibitors and lowering the temperature of the reaction mixture (0-4 deg. C).
- The pressure homogenizer allows the cells to disintegrate by subjecting their suspension to high pressure – while it is moving through a tight gap – and to rapidly release to atmospheric pressure. This method allows high-performance homogenization of cell suspensions with a volume of approx. 10-30 ml, however, it becomes technically difficult for smaller volumes and too time-consuming for larger ones. This type of homogenizer is mainly used for disintegration of bacterial and yeast cells.
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