In Part 1, I introduced the types of cell walls out there and what they are made of – now it’s time to learn how to get through them.
1. Mechanical methods of cell disruption
Mechanical cell disruption is really just that: forcing open the cell wall and spilling the contents. The advantage to mechanical disruption is that no chemicals are introduced that might interfere with the substance you want to extract. The drawback however is that the method has to be carefully adjusted as to not destroy that molecule of interest.
Mortar and Pestle
Just give the cells a good old grinding. This does not have to be in suspension and is often done with plant samples frozen in liquid nitrogen. When the material has been disrupted, metabolites can be extracted by adding solvents.
Glass or ceramic beads are used to crack open cells – it might not sound like it, but this kind of mechanical shear is gentle enough to keep organelles intact. It can be used with all kinds of cells, just add beads to an equal amount of cell suspension and vortex!
Ultrasonic homogenizers work by inducing vibration in a titanium probe that is immersed in the cell solution. A process called caviation occurs, in which tiny bubbles are formed and explode, producing a local shockwave and disrupting cell walls by pressure change. This method is very popular for plant and fungal cells but comes at a disadvantage: It’s very loud and has to be performed in an extra room, otherwise you will be very unpopular.
Homogenizers use shearing forces on the cell similar to the bead method. Homogenization can be performed by squeezing cells through a tube that is slightly smaller than them, thereby shearing away the outer layer (French Press) or by using a rotating blade like in a blender (Rotor-Stator Processors).
Freeze-thaw cycles work by formation of ice crystals and cell expansion upon thawing, ultimately leading to rupture. Used for algae and soft plant material. The drawback is that it is very time-consuming.
High temperatures (Microwave, Autoclave)
High temperatures (and pressure) disrupt bonds within cell walls, but also denature proteins. Although it is quick, you better find another method if your application is affected by the damage heat does to the rest of the cell.
2. Non-mechanical methods
Non-mechanical methods involve the addition of enzymes or chemicals that specifically break down cell wall components. They are often used in combination with mechanical force to ensure complete disruption of the cell. The disadvantage to their use is that they often have to be removed from the sample afterwards.
Naturally occurring enzymes can be used to remove the cell wall specifically, for example when isolating the protoplast (cell without the wall). Depending on what organism you work with, that can be cellulases, chitinase, bacteriolytic enzymes like lysozyme (destroys peptidoglycans), mannase, glycanase (etc. ).
Organic solvents like alcohols, ether or chloroform can disrupt the cell wall by permeabilizing cell walls and membranes. They are especially handy if you want to extract hydrophobic molecules (like plant pigments) because they will be collected in the solvent. Often used on plants in combination with shearing forces.
EDTA can be used specifically to disrupt the cell walls of gram negative bacteria, whose cell walls contain lipopolysaccharides that are stabilized by cations like Mg2+ and Ca2+. EDTA will chelate the cations leaving holes in the cell walls.
Now you are armed with ideas and ready to attack your cells! If you want to go back to finding out what your cell wall is likely to consist of before you chose a method, read it here!