homogenate. Electron microscopy of the microsomal pellets
showed the particles to be smaller and more uniform in size for
the decompression method. In summary, these authors stated
that the nitrogen decompression method was more efficient
and probably less variable than the Teflon pestle and glass tube
methods.
Comparison with pestle and tube methods.
In an applica-
tion at the Veterans Administration Research Hospital in Chicago,
a homogenate that had required eight hours to produce with
the pestle and tube was prepared in fifteen minutes with a cell
disruption vessel. In another laboratory, up to 12 kilograms
of brain per day are being homogenized with a cell disruption
vessel.
Wallach and his associates
5
have used the nitrogen decom-
pression method to obtain complete cell fractionation with
minimum nuclear damage. Working with Ehrlich Ascites
Carcinoma Cells using a 0.0002M magnesium acetate buffer, they
have studied the cellular distribution of phospholipides. Wallach
has published many other papers in which the decompression
technique has been used to prepare cell membranes.
Vaccine Preparation
A number of commercial laboratories have found that the
nitrogen decompression technique is extremely effective for
releasing virus from fertilized eggs. This method can be scaled up
for commercial production using larger disruption vessels which
are offered for this purpose by Parr.
Bacterial Cells
While some notable successes have been achieved using
this technique with bacterial cells, the disappointments certainly
outweigh the successes. Remember the technique is a “gentle
method” which depends upon disolving a sufficient quantity
of nitrogen into the cell to cause the rupture as the pressure is
released. Bacterial cells have small volumes of liquid and tough
cell walls; not a combination readily receptive to the nitrogen
decompression technique.
Fraser
7
in 1951
published some of the earliest studies on
nitrogen decompression and its effect on E Coli. Fraser’s work
was limited because his vessel was restricted to 900 psi operating
pressure. Nevertheless, he was able to obtain 75% rupture in
one pass and over 90% rupture in two successive passes using
E Coli harvested during the log growth phase. Results with other
bacteria and organisms with tough cell walls have been mixed.
There are several ways in which bacterial cells with tough
walls can be treated to facilitate disruption by the nitrogen
decompression method. These include: (1) harvesting the cells
during an early growth phase before the full wall is developed;
(2) growing the cells in the presence of an agent which will inhibit
the formation of the cell wall; (3) using a lysozyme to weaken the
wall prior to processing; or (4) using a mechanical pretreatment
to weaken the cell walls before applying the nitrogen decom-
pression method. Although these techniques have been applied
successfully to many bacteria with heavy cell walls, they are not
equally effective for yeast, fungus, spores and similar cells
with very heavy or hard walls. Vigorous mechanical methods
are generally required to break down the cell structure in these
hard-walled materials since they generally do not respond well
to treatment by the nitrogen decompression method.
Plant Cells
Loewus and Loewus
10
have published a number of papers in
which they describe the application of nitrogen disruption proce-
dures to plant cells and to tissue cultured plant cells. They also
report considerable success in breaking diatoms by this method.
References
(1) Hunter, M. J. and Commerford, S. L., 1961. “Pressure
homogenization of mammalian tissues.” Biochim, Biophys.
Acta,
47
:580-6.
(2) Dowben, R. M., Gaffey, T. A. and Lynch, P. A., 1968.
“Isolation of liver muscle polyribosomes in high yield after
cell disruption by nitrogen cavitation.” FEBS Letters, Vol. 2,
No. 1, pages 1-3.
(3) Dowben, R. M., Lynch, P. M., Nadler, H. C. and Hsia, D. Y.,
1969. “Polyribosomes from L. Cells.” Exp. Cell Research,
58
:167-9.
(4) Short, C. R., Maines, M. D. and Davis, L. E., 1972.
“Preparation of hepatic microsomal fraction for drug metab-
olism studies by rapid decompression homogenization.”
Proc. Soc. Exper. Biol. Med.,
140
:58-65.
(5) Wallach, D. F. H., Soderberg, J. and Bricker, L., 1960. “The
phospholipides of Ehrlich and ascites carcinoma cells
composition and intracellular distribution.” Cancer Research,
20
:397-402.
(6) Manson, L. A., Foshi, G. V. and Palm, J., 1963. “An asso-
ciation of transplantation antigens with microsomal pipopro-
teins of normal and malignant mouse tissues.” J. Cell and
Comp. Physiol.,
61
:109-18.
(7) Fraser, D., 1951. “Bursting bacteria by release of gas
pressure.” Nature,
167
:33-4.
(8) Avis, P. J. G., 1967. “In subcellular components, preparation
and fractionation.” (Ed. Birnie, G. D. and Fox, S. M.) Chapt. 1,
Pressure homogenization of mammalian cells. Published by
Plenum Press, New York.
(9) Manson, L. A., 1972 “Extraction of membranous transplan-
tation antigens by pressure homogenization.” (Ed. Kahan,
B. D. and Reisfeld, R. A.) Chapt. 9, Transplantation Antigens.
Published by Academic Press, New York.
(10) Loewus, M. W. and Loewus, F., 1971. “The isolation and
characterization of d-glucose 6-phosphate cycloaldolase
(NDA-dependent) from acer pseudoplatanus L. cell cultures.
Plant. Physiol. (1971)
48
:255-260.
B u l l e t i n 4 6 3 5
7
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
