’
'
INTERPRETATÏON OF
141
destruction of the ribosomes. This might suggest some special condition
the water in
these regions. We will return to this point later.
_
.
.
_
The origin of the bundles of microtubules we feel to be the dissociàted mitotic apparatus.
That is, the treatment with hypertonic sea water appears to break down the mitotic apparatus
leaving the microtubules intact. Although no direct proof of thiswas obtained, microtubules
when seen in these eggs after freezing in non—mitotic stages are always randomly arranged
and single, rather than organized into bundles.
'
K. Morphology of Pancreas Dehydrated in Full Strength Glycerol
_
The morphology of pancreatic cells dehydrated as described in Section I and subsequently
freeze-substiæted is illustrated in Figures «7 and 8, although there is cosiderable variation in
morphology in dierent blocks, most still possessing some areas of ice-crystals. Thus the
degree of dehydration is highly variable by this method. In many areas we see the nuclei
with morphology as that in gure 7. Note the chr'omatin blocks, nucleolus and nuclear pore
regions. Note also the wide space surrounding the nucleus, the cytopleismic ribosomes and
the general lack of obvious membrane associated With the ribosomes. The mitochondria
(and ER) in other parts of the tissue look more like those in‘osmotiCally dehydratedeggs,
Figure 8. Some cells appear extremely dense and'shrimkèh (easily seen in the light as well as
electron microscope level) and these do n0t show bœomes—althoùgh the membranes of
the ER are often visible. .We feel that certain of the reports of the absence of ribosomes after
freezing are reports of Severaly dehydrated cells such as theSe, undoùbtedly accideny
dehydrated by evaporation prior to
’
‘
'
_
’
Despite the variability of these results, we feel that theyt'in with the general conclusion
reached with marine eggs; namely that morphology similar (althoüghhot identical) to that
seen after chemical xation can be attained only after a“ “notinc0nsiderable amount of
dehydration, probably, in the light of theresults of Luÿet! and Keane, 1953, of the order of
removal of 40 % of the water.
’
'
'
L. Morphology of Marine Eggs Impregnäted with
'
'
The above work on dehydration was not entered
with the idea that a general technique
applicable for microscopy would result,
ofdeciding:whetherour
results
could possibly have resulted from accidental dehydrati0n. Taken with thepreviously men—
»
tioned result that prevention of evaporation eliminated‘ all but ,theice crystal morphology,
the implication seems relatively well supported that ésomev‘Cell, water,—must be-temoved or in
some way prevented from crystalli‘zingif cells are,
Witha reasonable morpho—
logy. Our results with impregnation of pancreas:and liver
agents are pres—
ented below. Here we mention some preliminary results on marine eggS.
'
_
,
'
,
Eggs (unfertilized) were equilibrated in sea water at room temperature, in 20 % ethylene
glycol or dimethyl sulfoxide, and frozen.ln
% of the eggs, ice'crystals are
present. In some cases the crystals
very small-or are absent, and ribosomes, ER,
annulate lamellae, etc., may be seen, although thiemorphol‘0gy isby
no 'means‘f‘g00d”.
‘
However, microtubules may be Seen
at this early
stage of observation
random, Figure 9:This
:
I‘
-
… !:ï
-*
ï*
_
’
'
INTERPRETATÏON OF
141
destruction of the ribosomes. This might suggest some special condition
the water in
these regions. We will return to this point later.
_
.
.
_
The origin of the bundles of microtubules we feel to be the dissociàted mitotic apparatus.
That is, the treatment with hypertonic sea water appears to break down the mitotic apparatus
leaving the microtubules intact. Although no direct proof of thiswas obtained, microtubules
when seen in these eggs after freezing in non—mitotic stages are always randomly arranged
and single, rather than organized into bundles.
'
K. Morphology of Pancreas Dehydrated in Full Strength Glycerol
_
The morphology of pancreatic cells dehydrated as described in Section I and subsequently
freeze-substiæted is illustrated in Figures «7 and 8, although there is cosiderable variation in
morphology in dierent blocks, most still possessing some areas of ice-crystals. Thus the
degree of dehydration is highly variable by this method. In many areas we see the nuclei
with morphology as that in gure 7. Note the chr'omatin blocks, nucleolus and nuclear pore
regions. Note also the wide space surrounding the nucleus, the cytopleismic ribosomes and
the general lack of obvious membrane associated With the ribosomes. The mitochondria
(and ER) in other parts of the tissue look more like those in‘osmotiCally dehydratedeggs,
Figure 8. Some cells appear extremely dense and'shrimkèh (easily seen in the light as well as
electron microscope level) and these do n0t show bœomes—althoùgh the membranes of
the ER are often visible. .We feel that certain of the reports of the absence of ribosomes after
freezing are reports of Severaly dehydrated cells such as theSe, undoùbtedly accideny
dehydrated by evaporation prior to
’
‘
'
_
’
Despite the variability of these results, we feel that theyt'in with the general conclusion
reached with marine eggs; namely that morphology similar (althoüghhot identical) to that
seen after chemical xation can be attained only after a“ “notinc0nsiderable amount of
dehydration, probably, in the light of theresults of Luÿet! and Keane, 1953, of the order of
removal of 40 % of the water.
’
'
'
L. Morphology of Marine Eggs Impregnäted with
'
'
The above work on dehydration was not entered
with the idea that a general technique
applicable for microscopy would result,
ofdeciding:whetherour
results
could possibly have resulted from accidental dehydrati0n. Taken with thepreviously men—
»
tioned result that prevention of evaporation eliminated‘ all but ,theice crystal morphology,
the implication seems relatively well supported that ésomev‘Cell, water,—must be-temoved or in
some way prevented from crystalli‘zingif cells are,
Witha reasonable morpho—
logy. Our results with impregnation of pancreas:and liver
agents are pres—
ented below. Here we mention some preliminary results on marine eggS.
'
_
,
'
,
Eggs (unfertilized) were equilibrated in sea water at room temperature, in 20 % ethylene
glycol or dimethyl sulfoxide, and frozen.ln
% of the eggs, ice'crystals are
present. In some cases the crystals
very small-or are absent, and ribosomes, ER,
annulate lamellae, etc., may be seen, although thiemorphol‘0gy isby
no 'means‘f‘g00d”.
‘
However, microtubules may be Seen
at this early
stage of observation
random, Figure 9:This
:
I‘
-
… !:ï
-*
ï*
_
’