The primary goal of homogenization in molecular biology is to disrupt cells to isolate intracellular molecules such as nucleic acids and proteins along with organelles of cell. Many biological samples present a challenge for isolation of these molecules in an active form. The method to disrupt cells may vary depending on the type of cell and its cell wall composition. The method chosen for disruption should be efficient and at the same time it should not be too harsh to destroy the molecules isolated. There are different methods available for cell disruption. They are broadly classified into Mechanical and Non-mechanical homogenization.
Mechanical Homogenization Non-Mechanical Homogenization
Bead Milling Decompression
Fluidization Osmotic shock
French Press Freeze-thaw
Colloid Mill Enzymatic Lysis
For small sample quantities, up to a gram of sample weight, agitation by beads is the most economical way of homogenization. The agitation, either through hand-held vortexing or through mechanical means cause the beads to strike against each other and with the cells. This, in conjunction with the shearing force created by moving liquid phase, disrupts cell structure. This is effective against microorganisms and other free moving cell suspensions.
Bead Beating – Considerations
Excessive force will rupture cells and at the same time will result in DNA shearing and thus will reduce the quality of isolated DNA. Though DNA shearing may not be a factor in PCR amplifications, it will affect the shelf life and be a limitation in certain down stream applications that require relatively larger DNA fragments.
High temperature and excessive shearing will denature proteins and increase the wear of grinding materials.
Hints for successful temperature control include
Hints for successful temperature control include pre-chilling of samples and containers, runs of short duration with rest time to allow for re-chilling samples on ice and fewer beads and/or extra buffer to act as heat sink, and reduced degree of shaking vigor.
Chemical contamination of grinding media is usually rare. To mitigate this problem, chose beads that are inert such as zirconium oxide stabilized with yttria. Glass beads are basically silica and lower in density. Due to wear, bead surfaces could become reactive in the presence of salt solutions that are commonly used in homogenization. Garnet beads are generally inert but have a poor wear resistance. The beads could break up generating dust and contaminating samples.
Bacteria requires bead of 0.1 to 0.5 mm diameter; yeast, algae and hyphae use beads of 0.5 to 1.2 mm. For homogenization of plant and mammalian tissues, chop the tissues and use beads
of 1 to 5.00 mm in diameter.
Our Beads...... Type Density (g/cc)
High density beads for efficient homogenization Glass 2.5
Inert surface chemistry Silica Carbide 3.2
Glass and Zirconium derivatives with inert surface chemistry Zirconia/Silica 3.7
Ideal for nucleic acid and protein isolation Ceramic 3.86
Compatible with mechanical homogenizers Zirconium silicate 4.3
Several different types and size ranges Endure Beads 6.2
Effective against bacteria, spores, yeast, plant and mammalian tissues Steel 7.9