Biology Reference
In-Depth Information
Isotonic solutions contain equal concentrations of impermeable solutes on either side of the
membrane. The swelling or shrinking rate of an artificial lipid bilayer vesicle (called a 'lipo-
some', discussed below) is an excellent osmometer
[15,16]
.
In most cases, intracellular organelles are more easily extracted if the cell is slightly
swollen, enhancing its breakage by other methods
[17]
. Cells are more susceptible to
breakage when placed in hypo-osmotic buffers and are more stable in hyper-osmotic buffers.
While there are countless homogenization buffers, historically the major osmoticum has been
either ~250 mM sucrose or ~120 mM KCl, lightly buffered to pH 7.4.
5. Sonication
Ultrasonication employs high frequency sound waves, typically 20
50 kHz, to disrupt
cells
[18,19]
. Ultrasonication can be used by itself for cell homogenization or more commonly
in conjunction with other techniques. Light application of ultrasonication can even be used to
separate cells from one another or to remove cells from a tissue culture substrate (glass or
plastic).
There are three basic types of ultrasonication: tip, cup horn, and bath (
Figure 12.4
). In
a tip sonicator, the high frequency is generated electronically and the energy transmitted
directly to the sample via a metal titanium tip that oscillates at high frequency. The rapid
tip movement results in alternating very low pressure areas (cavitation) and high pressure
areas (impaction) that can tear the cell apart. The basic concept had its origins in the
1950's and technical advances have reduced required minimum sample volumes down
to less than 200
e
l. Although tip sonication remains the most popular for cell disruption,
it does have some serious problems. As the titanium tip oscillates, it slowly disintegrates.
As a result the tip must be replaced regularly and the generated titanium particles must
be removed from the cell sample. In addition the rapid tip oscillation generates a lot of
heat that must be dissipated quickly to prevent destruction of the delicate biological
material.
To avoid problems associated with inserting the titanium tip probe directly into the
sample, another method has been developed where the tip is placed in a water bath adja-
cent to a chamber that holds the sample. A commercial device that accomplishes this is
referred to as a 'cup horn' (
Figure 12.4
). Since the sonic energy must pass through a water
m
(a)
(b)
FIGURE 12.4
Common types of sonication homogenizers: (a) Tip Sonicater,
Courtesy of Giltron
and; (b) Cup
Horn Sonicater,
Courtesy of Pontina Elettronica
.
