Biology Reference
In-Depth Information
Homogenization Methods
1. Use of Enzymes
Cells that are encased in thick, tough cell walls (e.g. plant, fungi, and some bacteria)
present a particularly challenging set of problems. The cell wall must be removed while
doing as little damage as possible to the more delicate underlying plasma membrane.
Once the cell wall is removed, the remaining cell wall-free protoplast can be homogenized.
A variety of commercially available enzymes are used to digest the cell wall
[7,12,13]
. These
enzymes include cellulase, pectinase, lysozyme, lysostaphin, zymolase, glycanase, mannase,
mutanolysin, and many more. Unfortunately, it is almost impossible to remove a cell wall
without doing considerable damage to the delicate molecules residing below, which are
attached to the outer surface of the plasma membrane. Also, the enzymatic methodologies
for cell wall degradation are not applicable to large-scale preparations, and the enzymes
must be inactivated or removed before the protoplasts are to be homogenized.
2. Shear Force (Mortar and Pestle)
The most commonly employed cell homogenization technique involves use of a mortar
and pestle. The basic concept involves forcing cells through a gap between two very close
surfaces. The resulting shear force tears the cell apart
[14]
. The shear force will increase as
the gap is narrowed or as the movement of the pestle relative to the fixed mortar is increased.
Very accurate control of the shear force applied to the cells is essential. If the shear force is
insufficient, cells will not be disrupted. If the force is too great, the entire cell, including its
organelles, will be broken into many different microsomes. At extreme shear force, biochem-
icals may even be destroyed.
Several commercial mortar and pestle homogenizers are at the heart of most cell
membrane studies. The most commonly used homogenizers are the Ten Broeck and Dounce
homogenizers (
Figure 12.1
), both of which are glass mortar and pestles that are purchased
with preset, controlled gap sizes. Cells are forced through the gap by manual manipulation
of the pestle. The process is normally performed in ice to prevent heat-induced destruction of
cell components. The addition of a motor driven Teflon pestle is part of the Potter-Elvehjem
homogenizer. Although Van Rensselaer Potter (
Figure 12.2
) and Conrad Elvehjem are now
best known for their homogenizers, both made major unrelated contributions to the life
sciences. Potter pioneered and named the field of Bioethics while Elvehjem discovered
Niacin.
3. Blenders
Another very commonly employed type of homogenizer is generally referred to as
a 'blender'. Blenders can vary from simple, inexpensive household food blenders or juice
extractors to sophisticated, expensive instruments that employ high-speed blades and
specialized sample chambers.
The first simple food blender was introduced to the public in 1936 by big band leader and
popular radio and television personality, Fred Waring, and was an immediate hit. However,
Waring did not actually invent the blender. This was accomplished in 1922 by Stephen
Poplawski and was later improved by Fred Osius in 1935. The Osius blender became the orig-
inal Waring blender. In fact Fred Waring was only the shill that popularized the appliance.
Originally Waring called his blender the Miracle Mixer but later changed it to the Waring
