289
289
A
(Kultschitzky) Cells
Cells in
in
A Study
Study of
of the
the Argentaffin
Argentaffin (Kultschitzky)
frozen-dried
by Phase-Contrast
Phase-Contrast Microscopy
Microscopy
frozen-dried Tissue
Tissue by
and
Ultra-Violet Light
Light
and Ultra-Violet
By
A. C.
C. CHRISTIE
CHRISTIE
By A.
(From
London. Present
Present address,
address, Royal
Royal Hospital
Hospital for
for Women,
Women,
(From the
the Royal
Royal Cancer
Cancer Hospital,
Hospital, London.
Paddington,
Sydney, Australia)
Australia)
Paddington, Sydney,
With
one plate
plate (fig.
(fig. 1)
1)
With one
SUMMARY
SUMMARY
1.
intestine have
have been
been studied
studied by
by phase-contrast
phase-contrast
1. The
The argentaffin
argentaffin cells
cells in
in guinea-pig
guinea-pig intestine
microscopy
Frozen-dried tissue
tissue has
has been
been used.
used.
microscopy and
and in
in ultra-violet
ultra-violet light.
light. Frozen-dried
2.
One
such
cell
in
a
section
5-7^
thick
was
selected
and
studied
throughout.
In an
an
2. One such cell in a section 5-7^ thick was selected and studied throughout. In
unfixed
the cytoplasm
cytoplasm is
is packed
packed with
with fine
fine granules
granules which
which
unfixed section
section mounted
mounted in
in nonane,
nonane, the
emit
in ultra-violet
ultra-violet light
light of
of wavelength
wavelength 2,750
2,750 A.
A. There
There
emit aa greenish-yellow
greenish-yellow fluorescence
fluorescence in
is
absorption of
of light
light of
of this
this wavelength.
wavelength.
is also
also photographic
photographic evidence
evidence of
of absorption
3.
the fluorescence
fluorescence changes
changes to
to orange-yellow
orange-yellow and
and bebe3. After
After formaldehyde
formaldehyde fixation
fixation the
comes
of light
light by
by the
the granular
granular cytoplasmic
cytoplasmic contents
contents is
is
comes much
much weaker.
weaker. The
The absorption
absorption of
also
also greatly
greatly reduced.
reduced.
4.
no photographic
photographic evidence
evidence of
of absorption
absorption of
of light
light of
of
4. There
There is
is no
no fluorescence
fluorescence and
and no
wavelength
cytoplasmic contents,
contents, either
either before
before or
or after
after
wavelength 2570
2570 A
A by
by the
the granular
granular cytoplasmic
formaldehyde
fixation.
formaldehyde fixation.
5.
in these
these cells
cells is
is unaltered
unaltered by
by formaldehyde
formaldehyde
5. The
The nuclear
nuclear chromatin
chromatin pattern
pattern in
fixation,
in photographs
photographs taken
taken in
in ultra-violet
ultra-violet light
light of
of both
both
fixation, and
and is
is well
well demonstrated
demonstrated in
the
the wavelengths
wavelengths mentioned
mentioned above.
above.
INTRODUCTION
INTRODUCTION
I
T
that the
the granular
granular material
material in
in the
the argentaffin
argentaffin
T has
has recently
recently been
been reported
reported that
(Kultschitzky)
artifact (anon.,
(anon., 1954).
1954). Also,
Also, Eros
Eros (1932)
(1932)
(Kultschitzky) cells
cells is
is aa formalin
formalin artifact
reported
formaldehyde-fixed tissue
tissue fluoresce
fluoresce in
in ultraultrareported that
that the
the granules
granules in
in formaldehyde-fixed
violet
was re-examined
re-examined by
by Jacobson
Jacobson (1939)
(1939) in
in the
the
violet light.
light. This
This phenomenon
phenomenon was
case
human argentaffin
argentaffin cell
cell (so-called
(so-called 'carcinoid')
'carcinoid')
case of
of formaldehyde-fixed
formaldehyde-fixed human
tumour,
of absorption
absorption being
being in
in the
the region
region of
of 2,700
2,700 A.
A.
tumour, the
the maximum
maximum amount
amount of
Lison
fluorescence is
is only
only obtained
obtained after
after formalformalLison (1953)
(1953) stated
stated that
that the
the yellow
yellow fluorescence
dehyde
dehyde fixation.
fixation.
Erspamer
claimed that
that the
the substance
substance 'enteramine'
'enteramine' within
within
Erspamer and
and Asero
Asero (1952)
(1952) claimed
the
They, employing
employing the
the picrate
picrate
the argentaffin
argentaffin cells
cells is
is 5-hydroxytryptamine.
5-hydroxytryptamine. They,
derivative,
and Page
Page (1948),
(1948), and
and Rapport
Rapport (1949),
(1949), working
working
derivative, and
and Rapport,
Rapport, Green,
Green, and
with
complex, studied
studied the
the ultra-violet
ultra-violet ababwith the
the tryptamine-creatinine-sulphate
tryptamine-creatinine-sulphate complex,
sorption
that each
each compound
compound shows
shows maximal
maximal absorption
absorption
sorption spectrum
spectrum and
and found
found that
at
formaldehyde at
at any
any stage.
stage. It
It is
is therefore
therefore
at 2,750
2,750 A.
A. They
They did
did not
not employ
employ formaldehyde
important
in fact,
fact, the
the granular
granular argentaffin
argentaffin substance
substance
important to
to ascertain
ascertain whether,
whether, in
does
light and
and absorb
absorb such
such aa wavelength,
wavelength,
does or
or does
does not
not fluoresce
fluoresce in
in ultra-violet
ultra-violet light
without
formalin.
without previous
previous treatment
treatment with
with formalin.
[Quarterly
Science, Vol.
Vol. 96,
96, part
part 3,
3, pp.
pp. 289-293,
289-293, 1955.]
1955.]
[Quarterly Journal
Journal of
of Microscopical
Microscopical Science,
290
Christie—A Study of the Argentaffin (Kultschitzky) cells
Although the granular material within these cells in human and guineapig material is fixed by osmium tetroxide (Christie, 1955) and by potassium
dichromate (unpublished observation), it was considered advantageous to
avoid chemical fixatives if possible. Frozen-dried tissue was the obvious choice,
provided the granular material is retained in the unfixed cell in sections thus
prepared. This was found to be the case, and a study of the argentaffin cells
both by phase-contrast microscopy and in ultra-violet light will be the subject
of the present communication. First, cells were studied in unfixed tissue; then
the same cells were re-examined after formaldehyde fixation, and finally
stained by Gomori's (1948) hexamine silver nitrate technique to ensure correct identification of the cells under examination.
MATERIAL AND METHODS
After a guinea-pig had been killed instantly, a piece of duodenum (or upper
jejenum) was transferred to liquid propane at —1850 C. within 45 seconds.
Frozen-dried material was obtained by drying the tissue at —400 C. for 3 days
and then embedding in paraffin wax at 580 C. over a period of 2 minutes.
Sections 5-7/x thick were cut and mounted on quartz slides. After removal
of the paraffin by flooding the section with nonane, a quartz coverslip was
applied and sealed round the edges with molten paraffin wax in order to
prevent the nonane from evaporating. The following procedures were then
carried out:
(1) Examination under ordinary light, first by ordinary and then by phasecontrast microscopy, and finally by ultra-violet light of wavelengths 2,750 A
and 2,570 A.
(2) After removing the coverslip carefully and allowing the nonane to
evaporate, the section was exposed to formaldehyde vapour (from a solution
of commercial formalin of 40% strength in a Coplin jar) for 4 hours and then
immersed in 10% formaldehyde with 1% calcium chloride for 16 hours.
After washing in distilled water for half an hour the section was remounted
in nonane and examined as in (1) above.
(3) The coverslip was again removed and the section stained by Gomori's
(1948) hexamine silver nitrate technique for approximately 18 hours, when
the granules in the argentaffin cells appeared a light brown colour against a
practically unstained background.
RESULTS
In frozen-dried unfixed paraffin sections of guinea-pig small intestine, the
argentaffin cells are clearly visible under ordinary light microscopy, but are
even more clearly discernible when phase-contrast is used. In fig. 1, A a plump,
roughly spherical cell can be seen filled with fine granules. On formaldehyde
fixation and subsequent staining it is seen to contain the silver-reducing granules typical of argentaffin (Kultschitzky) cells, a confirmation of its
identity.
Christie—A Study of the Argentaffin (Kultschitzky) cells
291
Under ultra-violet light of wavelength 2,750 A this cell emits a clearly
visible greenish-yellow fluorescence, and contains cytoplasm which strongly
absorbs light of this wavelength (fig. 1, B).
After formaldehyde fixation this cell shows some shrinkage (fig. 1, D), but
no more than is apparent in nearby cells. However, when viewed under ultraviolet light of wavelength 2,750 A there is a considerable difference in the
cell's appearance. The bright greenish-yellow fluorescence has now changed
to orange-yellow and its intensity is considerably diminished. Fig. 1, E shows
that the intensity of absorption of light of this wavelength by the granular
cytoplasmic contents is now not detectable photographically.
The reduction of the intensity of both the fluorescence and the absorption
after formaldehyde fixation could possibly be explained on the basis of alterations in the intensity of the ultra-violet light source overnight (for a lapse of
this time is required for formaldehyde fixation to be accomplished). To overcome this possible source of error, sections from the same paraffin block of
frozen-dried unfixed material were cut and examined in conjunction with the
formaldehyde fixed ones at the same time. Over a period of about 15 minutes
the sections, fixed and unfixed, were repeatedly interchanged and examined
by myself and two colleagues, without knowledge as to which one was fixed
and which not so treated, in order to eliminate the possibility of personal
factors prejudicing decisions as to colour and intensity changes, as well as
eliminating errors due to changes in the intensity of the light source. A change
in both the colour and the intensity of the flourescence after formaldehyde
fixation was clearly apparent.
The unfixed cell was also examined in light of wavelength 2,570 A, both
before and after formaldehyde fixation. Fig. 1, F depicts the same cell after, and
fig. 1, c the cell before formaldehyde fixation, when photographed in light
of this wavelength. In both figures strong absorption by the nuclear chromatin
is shown, but not by the granular cytoplasmic contents; nor do they fluoresce.
There is also absorption of light of wavelength 2,750 A by the nuclear
chromatin, and comparison of fig. 1, B and 1, c with 1, E and 1, F show that
formaldehyde fixation has not materially altered the typical nuclear pattern;
or rather, since the pattern was first depicted in formaldehyde-fixed tissue as,
for example, in Ciaccio's (1906) illustration, it is more correct to say that the
characteristic pattern is not produced by such fixation.
The granules have the same appearance and are of about the same size in
unfixed and fixed tissue, when studied by phase-contrast microscopy, as can
be seen from fig. 1, A and 1, D.
DISCUSSION
Gomori (1948) maintained that formalin either alone or in mixtures is
essential for the fixation of the argentaffin cell with its content of cytoplasmic
granular material. On the other hand, Cordier (1926) reported that he could
see and stain the granules with neutral red, in teased fresh tissue suspended
in 'serum artificieF.
292
Christie—A Study of the Argentaffin (Kultschitzky) cells
It has now been shown that, in frozen-dried guinea-pig tissue, the granules
are present in a morphologically similar form to those seen in formaldehydefixed tissue. In such unfixed tissue sections prepared by the Altmann-Gersh
technique they also emit a greenish-yellow flourescence when viewed in
ultra-violet light of wavelength 2,750 A. After formaldehyde fixation the colour
changes to orange-yellow and the intensity is diminished. The intensity of
light absorption as assessed by photographic means is also greatly diminished
by treatment with formalin. In the cell depicted in fig. 1, E it was not sufficient
to affect the photographic plate, though in other argentaffin cells examined
in the same and in other sections there was often slight darkening of the cytoplasm. Thicker sections, such as can be employed when one is examining
carcinoid tumours, would almost certainly show considerable absorption
amongst groups of cells rich in granular material; but throughout this work
only single cells in sections 5-7 /x. thick have been examined.
These observations are opposed to the hypothesis that the granular
material is a formalin artifact, if by this is meant that in tissue fixed in fixatives not containing formalin the granules are not demonstrable. However,
they confirm that formalin almost certainly has some chemical action on the
granules—a deduction previously made from the observation that previous
fixation by formaldehyde prevents the subsequent darkening of them by
osmium tetroxide (Christie, 1955).
It appears that the above findings favour the hypothesis of Cordier (1926),
who also worked with guinea-pig material, that the argentaffin substance is
present in granular form in fresh tissues examined immediately after removal
from the animal.
I should like to thank Mr. R. King for taking the photographs, and Miss
Shirley Charter for preparing the frozen-dried sections.
FIG. 1 (plate), A, a frozen-dried, unfixed, and unstained paraffin section of guinea-pig
duodenum, showing a crypt of Lieberkiihn with a relatively large, spheroidal argentaffin cell
containing cytoplasm packed with fine granules. (Phase-contrast.)
B, the same crypt of Lieberkiihn as shown in fig. 1, A, viewed by ultra-violet light of
wavelength 2,750 A. The cytoplasmic contents of the argentaffin cell show strong absorption
of this light. Although it is better seen in the next figure, the typical chromatin pattern within
the nucleus can be discerned.
C, the same crypt as in fig. 1, A, B, photographed in ultra-violet light of wavelength 2,570 A.
Although there is still strong absorption by the nuclear chromatin, the cytoplasmic contents
of the argentaffin cell do not absorb light of this wavelength.
D, the same crypt viewed by phase-contrast microscopy after formaldehyde fixation. Apart
from considerable shrinkage, the cytoplasmic contents of the argentaffin cell have been
otherwise unaltered by fixation.
E, the same crypt viewed by ultra-violet light of wavelength 2,750 A, after formaldehyde
fixation. Although the nuclear chromatin still shows strong absorption and reveals a pattern
unchanged by fixation, the cytoplasmic contents now show no photographically detectable
absorption.
F, the same crypt, now photographed in ultra-violet light of wavelength 2,570 A, after
formaldehyde fixation. Nuclear chromatin still shows strong absorption, but, as in fig. 1, c,
there is no cytoplasmic absorption by the argentaffin cell.
Christie—A Study of the Argentaffin (Kultschitzky) cells
REFERENCES
ANON., 1954. Lancet, Annotation, 2, 372.
CHRISTIE, A. C , 1955. (In the press.)
CIACCIO, M. C , 1906. C.R. Soc. biol. Paris, 60, 76.
CORDIER, R., 1926. Arch. Biol. Paris, 36, 427.
EROS, G., 1932. Allg. Path. Anat., 54, 385.
ERSPAMER, V., and ASERO, B., 1952. Nature Lond., 169, 800.
GOMORI, G., 1948. Arch. Path., 45, 48.
JACOBSON, W., 1939. J. Path. Bact., 49, 1.
LlSON, L-, 1953. Histochimie et cytochimie animales. Paris (Gauthier-Villars).
RAPPORT, M. M., GREEN, A. A., and PAGE, I. H., 1948. J. biol. Chem., 176, 1243.
RAPPORT, M. M., 1949. Ibid., 180, 961.
293