O
perating procedures for the Parr Cell Disruption Vessel
can be adjusted to suit individual requirements. The
procedures described here will be suitable for most
applications, but they are not intended to be either complete or
restrictive. Each operator will develop refinements which produce
the best results with his particular materials, while still observing
the basic vessel handling instructions and safety precautions
described below.
Preparing The Cell Suspension
Individual cells such as lymphocytes, leukocytes, tissue culture
cells or very fragile bacterial cells will not require pretreatment.
Tissues must usually be preminced to ensure that they will
pass through the dip tube and discharge valve. Hand presses,
mechanical homogenizers, pressing through screens or sieves,
slow speed blenders and manual dissection have all been used
successfully for this purpose. Tougher tissues will require finer
premincing than will softer tissues. Best results will be obtained
if any connective tissue which might block or plug the discharge
passages are removed or finely divided at this time.
The intended use of a homogenate generally determines the
composition of the suspending medium. Isotonic solutions are
commonly used. Solutions with higher concentrations will tend to
stabilize the nucleus and organelles. Conversely, very dilute solu-
tions will prestretch the cells by osmotic pressure and will render
them more susceptible to disruption by the disruption method.
Several investigators have reported that very small quantities
of calcium chloride, magnesium acetate, or magnesium chloride
added to the suspending medium will stabilize the nuclei when
differential rupture is desired. Ratios of approximately 10 mL of
suspending medium to one gram of wet cells are commonly
used to prepare the cell suspension. Higher or lower ratios can
also be used.
Charging The Vessel
Instead of pouring the cell suspension directly into the vessel
cylinder, most users will find it convenient to hold the sample
in a beaker or test tube which can be placed inside the larger
vessels. A supplementary container is particularly useful when
working with small samples. As a general rule, the capacity of
the inner container should be approximately twice the volume
of the suspension to be treated. Larger samples may, of course,
be poured directly into the cylinder. If an inner container is used,
it must be positioned so that the dip tube leading to the release
valve reaches to the very bottom of the container. Plastic test
tubes, centrifuge tubes and beakers work well as auxiliary sample
holders. They are not only unbreakable but they also can be
floated on ice water within the vessel to keep the suspension cool
during equilibration and to bring the bottom of the sample holder
up to the tip of the dip tube to ensure complete sample recovery.
Sealing and Pressurizing The Vessel
After placing the cell suspension in the vessel, set the head on
the cylinder with the dip tube extending into the sample holder
and attach the split ring clamp. No wrenches, holders or special
tools are needed in this closing operation. The two ring sections
slide together from either side of the vessel and latch into place.
To pressurize the vessel, attach the filling connection to a
commercial nitrogen cylinder and connect the flexible Nylon
pressure hose to the vessel inlet valve. A quick disconnect fitting
on the hose makes it easy to complete this connection and to
remove the vessel from the filling system after pressure has been
applied. A gage on the filling connection shows the pressure in
the supply cylinder while a gage on the vessel head shows the
pressure applied to the sample.
It should be noted that the vessel does not have to remain at
a fixed location during the loading, closing and filling operations.
These steps can be performed anywhere in the laboratory and
the vessel can then be carried to a different location for pressure
equilibration and sample recovery if the user wishes to do so.
The amount of pressure used will vary with different samples
and with different disruption or homogenization procedures.
Large samples will often absorb a significant amount of nitrogen,
making it necessary to re-pressurize the vessel one or more times
in order to maintain the desired pressure level. Also, a significant
pressure drop may occur as the homogenate is released from the
vessel, making it necessary to add additional nitrogen in order to
treat all of the sample at the same pressure level. Such additions
can be made easily using the control valves provided on
the vessel head and on the filling connection.
Operating Procedures
1831 Nitrogen Filling Connection
B u l l e t i n 4 6 3 5
5
1 - 8 0 0 - 8 7 2 - 7 7 2 0
C e l l D i s r u p t i o n V e s s e l s
