Further Observations on Amoeba diseoides.
By
Catherine Hayes, S.N.D., B.Sc, Ph.D.
From the Zoology Department, University of Glasgow, and the
Laboratory of Notre Dame, Dowanhill, Glasgow.
With 3 Text-figures.
INTEODUCTION.
WHEN in 1938 I wrote 'An Account of A m o e b a d i s e o i d e s , its Culture and Life History', I was aware of some
lacunae in the work. These were due to lack of sufficient
material at that date. Because the large, free-living amoebae
grow so slowly it takes considerable time and constant care to
accumulate strong, healthy, laboratory cultures from a very
limited original source. During the intervening years, however,
I have had excellent success in the laboratory cultivation of
this amoeba and in consequence abundant material at every
stage of the life-cycle to enable me to make deeper and more
detailed studies of each stage. The observations recorded in
the following pages are the results of these studies which complete the life-history of Amoeba d i s e o i d e s .
1. CYTOPLASMIC DIVISION.
When the amoeba is about to divide it withdraws all the long
pseudopodia, fastens itself to the sub-stratum and becomes
spherical, the sphere being covered all over with very short,
blunt pseudopodia (Text-fig. 1 A).
Compared with the original size of the amoeba in its creeping
or floating condition this sphere is small, which fact suggests
that the cytoplasm has become condensed. The first sign of
the actual division is the appearance of a clear area in the
cytoplasm at about the middle of the sphere and at right
angles to the equator (Text-fig. 1 B). Since sufficient depth of
water must be allowed for division to take place normally and
uninterruptedly the observations have to be carried out under
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a no. 3 objective. With this magnification it is impossible to
tell whether the clear area just referred to is a real break or
only a clearing in the cytoplasm. Whatever its nature it spreads
gradually northwards and southwards to the poles (Text-fig.
1 c); then as a general rule it closes up again and the amoeba
sphere looks as if no division had taken place. After a minute
TEXT-FIG. 1.
Diagrammatic representation of fission in A m o e b a d i s c o i d e s .
A, Spherical form assumed before division—numerous short,
blunt pseudopodia. B, First appearance of the clear area in the
cytoplasm, c, The clear area has spread to the poles, D, Actual
division accomplished.
or two large pseudopodia are pushed out, the cytoplasm begins
toflowrapidly and the two daughter amoebae move slowly away
from each other, the actual dividing line being the originally
observed clearing or break (Text-fig. 1 D). Almost at once the
daughter amoebae look and are in actual linear measurement
as large as the mother amoeba, suggesting that the cytoplasm
is now very thinly spread out and has probably absorbed water,
perhaps in the short time which elapses between the completion
of the division and the moving apart of the individuals.
One division-sphere was seen to divide into three individuals.
Ordinary division into two took place first; then, immediately
after, one of the daughters again divided into two. This probably often occurs in Amoeba discoides since in fixed and
stained individuals two or three large nuclei are quite common.
I have also seen multiple mitosis in stained preparations.
2. MITOSIS.
When division is about to take place the nucleus loses its
staining capacity, the nuclear membrane becomes exceedingly
OBSERVATIONS ON AMOEBA DISCOIDES
197
thin, and the karyosome disappears. The chromatin masses are
now evenly distributed (Text-fig. 2 A) ; but they are small, and
because of their inability to stain it is exceedingly difficult to
discover the nucleus in the stained amoeba. This phase may be
looked upon as early prophase. The next stage (Text-fig. 2 B),
TEXT-FIG. 2.
Mitosis in A m o e b a d i s c o i d e s . nm, nuclear membrane; cb,
chromatin blocks, A, Early prophase: membrane thin, chromatin
blocks evenly distributed, karyosome has disappeared, B, Late
prophase: spindle fibres condensing out. C, Metaphase: all the
very small numerous chromosomes round the equator of the
nucleus, D, Late anaphase: polar caps dome-shaped. E, Telophase: chromosomes fused into a band. (Drawing more or less
diagrammatic.)
although still more difficult to detect, shows indications of a
late prophase in which the achromatic spindle threads begin to
appear. In metaphase (Text-fig. 2 c) the very small chromosomes are arranged on the equator of a more or less barrelshaped nucleus. The nuclear membrane persists and for the
first time the chromosomes are easily visible though they are
too small and too numerous to be counted. There are no visible
centrosomes and no asters. In anaphase the two daughter
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sets of chromosomes travel to the poles (Text-fig. 2 D). The
spindle poles are dome-shaped, and in this respect Amoeba
d i s c o i d e s differs from A m o e b a l e s c h e r a e (Taylor and
Hayes, 1944) and from Amoeba p r o t e u s , where they are
conical (Dawson et al., 1937).
In telophase (Text-fig. 2 E) the chromosomes sometimes
coalesce and appear as a deep-staining band around the circumference of the base of each dome.
The initiation of division of the cytoplasm causes the two
daughter telophasic-nuclei to be carried to opposite ends of the
' fission-amoeba' where the daughter nuclei are reconstructed
into the resting stages.
3. THE EMISSION OF CHEOMIDIA, PREPARATORY TO
CYST FORMATION.
On p. 474 of my paper (1938) I stated that I had not seen
ehromatin blocks actually escaping from the nucleus of
Amoeba d i s c o i d e s . Since then I have often seen chromidia, i.e. ehromatin blocks, just outside the nucleus and
scattered through the cytoplasm of many fixed and stained
adult Amoeba d i s c o i d e s . These preparations had always
been made by the usual long and laborious method of fixation,
dehydration, clearing, &c.
My thanks are due to Dr. Pontecorvo for calling my attention
to the acetic-alcohol plus aceto-carmine technique; for, by its
use, large numbers of adult amoebae considered due to undergo
cyst formation can rapidly be examined.
After 24 hours in acetic-alcohol the amoebae can be examined
at once in aceto-carmine when the escaping chromidia can be
detected quite easily in those individuals where the phenomenon is taking place (Taylor and Hayes, 1944).
4. EXCYSTATION AND EARLY DEVELOPMENT OF
AMOEBA DISCOIDES.
M e t h o d of O b t a i n i n g C y s t s .
In December 1942, while examining a Petri-dish culture
which had been in a flourishing condition for several months,
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199
I noticed that most of the individual amoebae were packed with
very large nutritive spheres and I suspected that these individuals were ready to form cysts (Taylor and Hayes, 1944).
Consequently several slides were prepared by placing on each
five or six of these large amoebae. The preparations were then
placed in a damp chamber and left for some weeks, care being
taken to add a little water when necessary. When the preparations were examined it was found that most of the amoebae
had disintegrated, giving rise to cysts, excysting amoebulae,
and early stages in the development of the excysted amoebulae.
The amoebae not removed from the original culture gradually
disappeared and an examination of the bottom of the Petridish revealed cysts and developmental stages similar to those
found on the slides.
D e s c r i p t i o n of C y s t s a n d of Y o u n g E n c y s t e d
Amoebae.
The living cysts of Amoeba d i s c o i d e s are spherical with
a diameter varying from 9 n to 12 /x. They are provided with
a very thin outer wall and a much thicker inner one (Text-fig.
3 A). The inner thickened wall appears to be the outer surface
of the encysting amoeba, for when excystation is completed
the outer thin wall alone remains behind. The interior of the
cyst consists of highly concentrated, viscid, non-granular
cytoplasm in which the newly differentiated nucleus is the first
definite structure to be seen. Soon after this a small contractile
vacuole begins to pulsate at regular intervals (Text-fig. 3 B).
The inner cyst wall now becomes permeable, that is, it is
gradually becoming an integral part of the central mass of
cytoplasm. The fact that the contractile vacuole is able to
function indicates that there must be some communication
between the interior of the cyst and the surrounding water,
although no visible break in the outer wall can be detected
(Taylor and Hayes, 1944). Later the cytoplasm becomes
capable of amoeboid movement, contracting slightly from the
cyst wall (Text-fig. 3 c). It is less viscid and is now ready to
emerge from the cyst. The newly emerged amoebula, though of
a fluid-like consistency and of extreme transparency, moves
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but little at first and these slight movements are of the nature
of little convulsions. It does, however, engulf a few very
small food particles during this period (Text-fig. 3 D). It next
tends to float, numerous pseudopodia are pushed out and a few
TEXT-FIG. 3.
A, Cyst o f A m o e b a d i s c o i d . e s . B, Differentiation begins in cyst.
Nucleus distinguishable, c, Preparation for hatching—inner wall
of cyst 'dissolved'. Contractile vaeuole functioning, D, Newly
hatched amoebula, ingestion of food, E, Amoebula begins to move
actively by means of long pseudopodia. F, Later stage, a,
Amoebula assumes the 'Iimax' stage . . . ectoplasm and endoplasm clearly differentiated, H, Older amoeba, metabolic products
as well as food-vaeuoles. cv, contractile vaeuole; ec, ectoplasm;
en, endoplasm; fv, food vaeuole; iw, inner wall; mp, metabolic
products; n, nucleus; ow, outer wall. All drawings made from
living material.
more particles are engulfed. The cytoplasm though becoming
gradually coarser is so thin that it is exceedingly difficult to
distinguish ectoplasm from endoplasm (Text-fig. 3 B to P).
The next stage in development may be called the 'Iimax'
stage as all the pseudopodia are withdrawn and the amoebula
begins to creep about in Iimax fashion. The cytoplasm, colourless and very fluid, moves easily and rapidly. The ectoplasm
OBSERVATIONS ON AMOBBA DISCOIDES
201
hag become ftilly differentiated and is voluminous and very
transparent; the endoplasm is by now quite granular. Numerous
food particles are always present. The nucleus is conspicuous
and large in comparison -with the siae of the amoeba. By contrast -with that of Amoeba l e s c h e r a e the karyosome is
large.
From now onwards growth manifests itself by increase in
bulk rather than increase in length. The endoplasm shows
metabolic products in addition to food vacaoles (Text-fig. 8 H) S
the ectoplasm is less voluminous. Periodically, as in the adult,
the actively moving amoebae assume a resting stage during
which they are more or less spherical with numerous blunt
pseudopodia, but from this condition they easily pass again
into the 'limax' state.
Further stages in development have already been described
in my former paper (1988) on Amoeba d i s e o i d e s . I had
not at that time seen the excystation and early stages described
above.
REFERENCES.
Dawson, J. A., Kesaler, W. R., and Silbeistein, J. K., 1935.—Bid. Bull.
69, 447.
1937.—Ibid., 72,125.
Hayes, Catherine, 1938.—Quart. Joum. Mior. Soi., 8©, 459.
Taylor, Monica, and Hayes, 0., 1944.—Ibid., 84, 295.