138
FREEZE—DRYING
—
This has been described indetail in Rebhun 1961 and 1965. Basically, the tisSue is frozen iii «‘a
‘
cylinder covered on the lower face with a porous cloth. This is suspended in the quenchig
uid contained in a beaker suspended in liquid nitrogen in such a way as to maintain the
temperature of the quenching uid at approximately — 155 °C (in the case of Freon 22);
The cells are frozen (asdescribed below) in the uid contained in the cylindei‘. The latter is
then lifted into the cold nitrogen vapor (the whole assembly being in a large Dewar),
allowing moSt of the Freon to drain. Five ml. serum bottles are precooled, lled with liquid
nitrogen and suspended in the DeWar partlysubmerged in liquid nitrogen. The frozen cells
are transferred by means of a precooled forceps from the freezing cylindèr, through the
gaseous, cold nitrogen to the serum bottles. The serum bottle caps are provided with appro—
priate hypoder‘mic needles to e"ect transfer of low temperature uids. The bottles containing
thefrozen material are rapidly transferred into the freezer, the remaining nitrogen, poured
off, the bottles capped, and lled with appropriate substitutingfuid at lowtemperatüïe:
The technique allowstransfer of tissue with no chance of warming to temperatures higher
:
than that of
_
thesubstitüting uid.
-
'
‘»
D. Embedüng
f_*
’
“_
Most of the results were obtained With Araldite 502 or Epon 812 embedded tissue.
E. Results
-
…
…
-
The Whole point of developing these
Was to attempt to eliminate the ice-crystal
'
mdrpholOgy. The result of all the above Work can be sumDaarizedin a simple statement : '
there was essentially no change in frequency of
of the two different types of
morphology obtained. With respect to the ice—crystal morphology, the size of the spaces
was considerably smaller on the average than before,
iCe Crystals and these
preparations looked better on the’light microscope level. This Was to be. expected. However,
the fact that reduction of the volume of frozen tissue up»to
smaller than the
s‘amplesrst used, and the general fact that
all aspects of the technique,
result in any general increase in oŒur'ence ,, of non—ice «crystal artefact pïeparations,
was mostdijsturbing. It suggested to us that the…variabilitywas probably induced not…so
much by our techniques
butbÿ =some:accident, uneontrôlled in our process. It
occurred to us, in lookihgovèr our techniques in detail and especially in going over the
protocols of ‘sucçessful’,experiments, that…sone variable we did not control, was accidental
waterremoyalprior to freezing. Thatis, àitpc”cfurred to usthat ourte0hniqœsdidnot prevent
evaporation of
to controlthis
_,
_
__
,
,
FT1ssueHanlmgTæhmm‘e
._
,
—rsopg;
terme ti'8sue, were constructedmthe
manner
inch lengths Of 75
Wire “Were bent“ into 1“oô'ps” by
around 3
glass tubing. The free Lend“ of the'loop “hadlé”Was 1nsertedmalä=moh
length of #29hypOde‘rmic tubing (stainlesS
crana'pèd
Sûchâ
with“ fsmallsahemo
were coatedw1thf
made
from 05%
ethylene
iliàiitb‘f}jfeü water surfacefrom
colors
-pu1apîé:a î: fi: ? >î?ï :'
FREEZE—DRYING
—
This has been described indetail in Rebhun 1961 and 1965. Basically, the tisSue is frozen iii «‘a
‘
cylinder covered on the lower face with a porous cloth. This is suspended in the quenchig
uid contained in a beaker suspended in liquid nitrogen in such a way as to maintain the
temperature of the quenching uid at approximately — 155 °C (in the case of Freon 22);
The cells are frozen (asdescribed below) in the uid contained in the cylindei‘. The latter is
then lifted into the cold nitrogen vapor (the whole assembly being in a large Dewar),
allowing moSt of the Freon to drain. Five ml. serum bottles are precooled, lled with liquid
nitrogen and suspended in the DeWar partlysubmerged in liquid nitrogen. The frozen cells
are transferred by means of a precooled forceps from the freezing cylindèr, through the
gaseous, cold nitrogen to the serum bottles. The serum bottle caps are provided with appro—
priate hypoder‘mic needles to e"ect transfer of low temperature uids. The bottles containing
thefrozen material are rapidly transferred into the freezer, the remaining nitrogen, poured
off, the bottles capped, and lled with appropriate substitutingfuid at lowtemperatüïe:
The technique allowstransfer of tissue with no chance of warming to temperatures higher
:
than that of
_
thesubstitüting uid.
-
'
‘»
D. Embedüng
f_*
’
“_
Most of the results were obtained With Araldite 502 or Epon 812 embedded tissue.
E. Results
-
…
…
-
The Whole point of developing these
Was to attempt to eliminate the ice-crystal
'
mdrpholOgy. The result of all the above Work can be sumDaarizedin a simple statement : '
there was essentially no change in frequency of
of the two different types of
morphology obtained. With respect to the ice—crystal morphology, the size of the spaces
was considerably smaller on the average than before,
iCe Crystals and these
preparations looked better on the’light microscope level. This Was to be. expected. However,
the fact that reduction of the volume of frozen tissue up»to
smaller than the
s‘amplesrst used, and the general fact that
all aspects of the technique,
result in any general increase in oŒur'ence ,, of non—ice «crystal artefact pïeparations,
was mostdijsturbing. It suggested to us that the…variabilitywas probably induced not…so
much by our techniques
butbÿ =some:accident, uneontrôlled in our process. It
occurred to us, in lookihgovèr our techniques in detail and especially in going over the
protocols of ‘sucçessful’,experiments, that…sone variable we did not control, was accidental
waterremoyalprior to freezing. Thatis, àitpc”cfurred to usthat ourte0hniqœsdidnot prevent
evaporation of
to controlthis
_,
_
__
,
,
FT1ssueHanlmgTæhmm‘e
._
,
—rsopg;
terme ti'8sue, were constructedmthe
manner
inch lengths Of 75
Wire “Were bent“ into 1“oô'ps” by
around 3
glass tubing. The free Lend“ of the'loop “hadlé”Was 1nsertedmalä=moh
length of #29hypOde‘rmic tubing (stainlesS
crana'pèd
Sûchâ
with“ fsmallsahemo
were coatedw1thf
made
from 05%
ethylene
iliàiitb‘f}jfeü water surfacefrom
colors
-pu1apîé:a î: fi: ? >î?ï :'