Amoeba proteus: Some new Observations on
its Nucleus, Life-history, and Culture.
By
Monica Taylor, S.N.D., D.Sc.
With 11 Text-figures.
INTRODUCTION.
WHEN reading over the manuscript of ' Nuclear Divisions in
A m o e b a p r o t e u s ' (14), a kind office for which, on this
casion, I am once more indebted to him, Professor Graham
Kerr expressed a wish that I should undertake a more detailed
study of the behaviour, during division, of the chromatin
blocks described in that paper. At the same time I became
interested in a series of publications by Drs. Atkins and Lebour
in connexion with hydrogen-ion concentration, two of which
(1 and 2), particularly, seemed to open up a useful line for me.
Having a large number of pedigree Amoeba cultures, with
field-notes of their history extending back to 1916-17, as well
as cultures of other micro-organisms, there Avas plenty of scope
for testing the value of this physico-chemical factor in the
cultivation of micro-organisms under laboratory conditions.
I should like to take this opportunity of thanking the authors
for their courteous response to my many inquiries, Dr. Atkins
having given me the benefit of his expert advice in matters of
a physico-chemical character.
An intensive study of the hydrogen-ion concentration 1 of my
numerous Amoeba cultures, coupled with a more intimate
examination of the nuclear divisions, has led to the elucidation
of the encystment phenomena in A. p r o t e u s (' Y ', Carter
(5), Schaeffer (10)). For many years I have known that after
a period of depression countless minute Amoebae appeared in
the cultures. These grew up to maturity and underwent many
1
See Part II of this paper for details.
120
MONICA TAYLOR
fission divisions, thus forming a luxuriant culture which, after
a varying period of time, once more underwent a period of
depression.
It was evident that these periods of depression Avere associated
with an encystment phenomenon of some description. It was
natural to suppose that this encystment method would be
similar to that described by Sister Bernardine (Dr. L. A. Carter)
for A. p r o t e u s ' X ' (A. d u b i a , Schaeffer). In spite,
however, of the fact that I have been observing cultures of
A. p r o t e u s ' Y ' for so many years, I have never found cysts
of such a type, although the presence of minute Amoebae has
been constantly recorded in the field-book. Nor have I any
records of mitotic figures such as those described by Carter (3)
and Doflein (6), although countless specimens have been ' fixed '
and examined.
In my 'Nuclear Divisions in A. p r o t e u s ' (14) I put
forward as a hypothesis (p. 42) that ' the published figures of
mitosis in A. p r o t e u s belong to the sporulation cycle of
the life-history '. To anticipate what is to be described later
on, this hypothesis receives no support from the facts to be
narrated.
I have not yet discovered any cases of mitosis, which failure
appears to me to add additional weight to the reasons put
forward by Asa Schaeffer (10) for separating A. p r o t e u s
' X ' and ' Y ' into distinct species—A. d u b i a for ' X ',
A. p r o t e u s for ' Y '.
In the ' Technique of Culturing A . p r o t e u s ' (11) it was
pointed out that it is possible to have two types of laboratory
culture :
(A) A culture in which the onset of the period of depression
is uniform for all the individuals in the culture—a culture
therefore which contains no adult Amoebae during practically
six months of the year.
(B) A culture which having been inoculated with material
from various sources contains adult Amoebae at any time during
the year—though, as explained before, adults are not so
numerous during the spring months (12 and 13).
AMOEBA PROTEUS
121
A culture of type A is obviously more suitable for a study
of the phenomena connected with the period of depression,
i. e. tho encystment period. It was in such a culture that the
discovery of the encystment apparatus was actually made. It
has, of course, been subsequently examined in other cultures.
In the field-book readings of culture 24 the appearance of the
Amoebae was noted, October 6, 1923. On November 27,
1923, they are recorded as being adult, fine, sturdy creatures,
and feeding voraciously on large Paramecia almost exclusively,
which fact rendered the task of examining the microscopical
details much easier. (Quantities of this material were fixed
in strong Flemming solution.) There was no risk of confusing
the encystment phases with small ingested organisms partly
digested, &c. On December 10 most of the individuals had
commenced undergoing the encystment phenomena to be
described.
The field-book records the appearance of these individuals
as being extremely white in reflected light, and black-looking
in transmitted light. In the endoplasm were innumerable
bluish spheres as large as the larger so-called excretion spheres
(10, pp. 215, 221).1 Nucleus-like bodies could be seen inside
these spheres. The pH of culture 24 was 7-4. In other
Amoebae, where these nucleated spheres could not be seen so
easily, or were absent, the cytoplasm seemed to be undergoing
a sort of cytolysis, rounded masses of protoplasm, highly
vacuolated and without a cyst-wall, could be seen in the cytoplasm. Quantities of Amoebae from this culture were fixed in
Bouin's fluid (Duboscq Brasil 1905 modification).
On account of the superficial likeness between the nucleated
spheres and the large nutritive spheres, and the different
reactions of these latter to fixatives and stains, Bister Carmela
has undertaken an exhaustive study of the action of various
fixatives and stains on A m o e b a p r o t e n s generally, the
results of which are included in an appendix to this paper.
1
In view of the results to be recorded later on in this account it is proposed, henceforth, to call these bluish spheres nutritive spheres instead of
' excretion ' spheres.
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MONICA TAYLOR
PART I.
NUCLEAR DIVISIONS.
Technique.—Material from cultures of type B when the
Amoebae were so numerous that they could be skimmed off
with practically no extraneous material, was fixed in strong
Flemrning solution. For this purpose the Amoebae, freed
from algae and moulds as completely as possible, are put into
a solid watch-glass, allowed to settle down, and to expand in
a warm place (65° F.). As much water as can possibly be
removed, without causing the Amoebae to contract, is then
pipetted off and the fixative applied quickly. Since there was
an almost unlimited supply of material the Amoebae were
transferred to centrifuge tubes, where they were stained and
dehydrated. (With care, it is possible to obtain very beautiful
preparations of whole Amoebae by this method.) In the
meantime Mr. P. Jamieson had prepared a thick creamy
solution of celloidin in clove oil. The Amoebae were cleared
in clove oil and gently centrifuged, the supernatant clove oil
being then removed and replaced by the solution of celloidin
in clove oil. They fell to the bottom on being gently centrifuged, when the excess of celloidin in clove oil was removed.
Some melted paraffin was poured into a solid watch-glass and,
when nearly hard, a small depression was made with the rounded
end of a glass rod. The Amoebae in their celloidin-clove-oil
fluid were then pipetted into this depression, which was at
once flooded with chloroform. In a few seconds the little
' block' of celloidin was washed out of the depression and
transferred to another watch-glass containing chloroform.
After two hours the celloidin ' block ' was embedded in paraffin
in the usual way, and serial sections cut. These were stained
on the slide, the stains employed being Heidenhain's or Delafield's haematoxylin, light green being applied as a counterstain
in some cases.
A renewed study of the nucleus, with the aid of the technique
described above, has yielded some additional details of interest,
which it is the main object of this paper to describe. It will
be remembered that the nucleus is a large discoid body, which
is rolled about passively by the streaming endoplasm. Its
AMOEBA PROTEUS
123
membrane is strongly marked. Immersed in the nuclear sap
is (1) a conspicuous, centrally placed, plate-like ' karyosorne '
which lies in a highly vacuolated achromatinic substance, and
(2) the chromatin (Text-figs. 1A and 1B : cf. PI. 2 (14)). The
chromatin is confined to the blocks, situated normally just under
the nuclear membrane. The ' karyosome', being plate-shaped,
may appear circular or band-shaped according to the point
of view. It consists of two substances : (1) a ground substance
which does not stain so deeply as (2) a substance in the form
of small blocks or rods which stain like chromatm (Text-figs. 1A
and 1B ; 8). (Cf. PI. % figs. 1, 2, 3 (14).)
One outcome of this renewed study is to establish the likenessx
between the ' karyosome ' with its vacuolated network and the
ordinary cytoplasm—in sections the material for which has
been fixed in Plemming and stained in Heidenhain's haematoxylin. It is simply an achromatinic framework or nuclear
reticulum, which is denser in the plate-like portion where the
knots in the reticulum lie close together, and the granules are
more numerous. The ' karyosome ' is then simply a more
elaborate form of the achromatinic framework or nuclear
reticulum as defined by Minchin (8, p. 77). When viewed in
optical-section in a stained preparation of a whole Amoeba,
the chromatin blocks heighten the mottled appearance of
the so-called karyosome. In newly hatched Amoebae the
so-called karyosome is very conspicuous, and much more
homogeneous, i.e. less granular and less reticulate (Textfig. 10). In smear preparations of these young Amoebae,
where the Amoebae have been allowed to dry completely before
fixation, this ' karyosome ' appears to be quite similar to the
ordinary cytoplasm—just a special nucleoplasm enclosed within
the nuclear membrane.2 Minchin (8, p. 76) is of opinion that
the nuclear membrane is formed from the achromatinic framework—and that it is always a structure very readily absorbed
and reformed, and it appears to present no difficulty to the
passage of substances from the nucleus into the cytoplasm
and vice versa. That the nuclear membrane of A. p r o t e u s
1
i. e. in adult Amoebae.
More detailed investigations of the role of the karyosome, especially
in the growing (i. e. non-adult) Amoebae, are at present being conducted.
2
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MONICA TAYLOR
forms no exception to this rule is fully supported by observation (cf. Text-figs. 1A and 1B, and 3).
The viscosity of the nuclear sap seems to vary with the age
of the nucleus. It is greatest when ' chromidia ' formation is
in progress.
T13XT-FIG. l A .
Two stained preparations of whole Amoebae drawn by Miss
Margaret Curran, M.A., B.Sc. (from culture 3). A sub-culture
(culture 10) having been secured, most of the material of culture 3
was fixed in warm corrosive acetic and used for the preparation
of twenty-seven slides. On each slide there are several Amoebae
almost all of which are in the condition illustrated in the figure,
i. e. in process of shedding ' generative chromidia' from the
The chromatin blocks (Text-fig. 1B, C) lying in the nuclear
sap just under the nuclear membrane consist of two
morphologically distinct substances, one with a great affinity
for chromatin stains—the chromatin proper—and the other
a ground substance not easily stained by Delafield or
other haematoxylins, but coloured by counterstains, e.g.
light green. This substance would seem to be the piastre
described by Minchin (8, p. 77). The structure of these
AMOEBA PROTEUS
125
blocks recalls the very beautiful analysis of the chromosome
made by Martens in the ' Cycle du Chromosome somatique '
(7), where this duality of substance is seen to be likewise
a characteristic of plant chromosomes. The beaded character
of the chromosomes in many metazoa is also recalled by a
TEXT-PIO. 1B.
• j ™ v » % t *
. • • • • . - •
•
-
- , - • • • ' • •
•
•
* " i
nucleus. The stain used waa a modified carmine stain (14, p. 41)
for the formula of which I am indebted to Dr. J. S. Dunkerly.
a, nuclear membrane ; 6, reticulum of nucleoplasm, so-called
' Karyosome ' ; c, chromatin blocks ; d,fissiondivision of nucleus
commencing, two rows of chromatin blocks (cf. PI. 2, 14) ;
e, nuclear membrane absorbed in part; ' generative chromidia',
i. e. chromatin blocks, escaping.
study of the chromatin blocks in A. p r o t e u s. It is significant
to note that certain cytoplasmic inclusions in Amoeba, which
are unstained by haematoxylins after Mernming fixation,
and which are certainly metabolic products, have a very
similar colour to the plastin in sections. This suggests that
plastin may be material of a nutritive character, especially
in view of the fact that it disappears as the chromatin increases
126
MONICA TAYLOR
in bulk. The likeness of cytoplasmic inclusions to the plastin
may perhaps account for the observation made by Goldschmidt
(8), in ITastigella, of the extrusion of plastin from the
nucleus into the cytoplasm, to serve as a matrix for the
chromatm, which passed out from the nucleus subsequently.
iff
6.
5.
Diagram to illustrate the ' resting' and ' dividing ' stages of the
chromatin blocks. 1, ' resting ' block, chromatin and plastin
indistinguishable ; 2, chrornatin becoming more distinct ;
3, fully condensed chromosome ; 4, division of chromosome into
two ; 5, formation of four ' daughter ' chromosomes ; 6, four
' daughter ' blocks ; 7, ' daughter ' blocks going into ' resting '
stage, a, reticulum of nucleoplasm ; b, plastin ; e, chromatin ;
d, nuclear sap.
In Amoeba proteus plastin and chromatin do not separate,
as will be explained later.
The chromatin blocks (Text-fig. 2) pass through the phases
familiar to the cytologist as ' resting ' and ' dividing '. In the
resting condition the ' block ' has a mottled appearance and
does not stain so precisely. It looks as though the chromatin
AMOEBA PROTEUS
127
had been converted into an extremely fine reticulum almost
indistinguishable from its ground substance of plastin. The
block is always surrounded by a clear area, the achromatinic
network (Text-fig. 2, b) being displaced to make way for i t ;
it thus lies freely in the nuclear sap. As the time for division
approaches the chromatin condenses into a small chromosome
which stains very definitely. The plastin is completely hidden
at this stage. It is either covered entirely by the chromatin,
or it has been absorbed by it. The purple chromosome stands
out conspicuously in its clear sphere of nuclear sap. A block,
about to divide, can thus quite readily be picked out from the
blocks in the resting condition. Division in the blocks is not
synchronous, but, as already described (14, p. 43), begins in
a little patch, the process gradually extending. There is a real
division of each block into two and then into four daughter
chromosomes. The daughter masses of chromatin separate and
the achromatinic network stretches between them. Gradually
the chromatin in each daughter block assumes the appearance
of the resting condition, the plastin becoming again conspicuous.
It is thus easy to understand that Goldschmidt's observation
on M a s t i g e l l a may have a different interpretation from the
one he gives. What he considered to be chromatin only is,
in A. p r o t e u s at any rate, chromatin overlying and completely masking its groundwork of plastin, just as in the fully
condensed chromosome the ' element achromatique' of
Martens (7) is masked during metaphase and early anaphase.
When the chromatin block goes into the ' resting ' condition
the plastin reappears. It will be shown, later on, that the
chromatin blocks become the ' generative chromidia '.
The Amoeba nucleus grows as the cytoplasm increases in
bulk, by the increase in the number of chromatin blocks. The
rate of growth is in all cases dependent upon the food supplies
of the Amoebae and varies enormously. When the Amoeba
has attained its adult size, and is about to undergo fission
divisions, the achromatinic network divides into two, each
daughter element along with its complement of chromatin
blocks forming a daughter nucleus (14, p. 43).
Ill
MONICA TAYLOR
As will be shown later in Part II, the condition of the nucleus
may be greatly modified by alteration in the environment of
the Amoeba.
Encystment.
It has been repeatedly pointed out that, unless precautions
be taken, cultures of A. prot eu s undergo depression periods.
TEXT-FIG. 3.
Portion of section (4M) through an Amoeba from culture 2, in
region of nucleus. Fixation Flemming, stain Delafield's haematoxylin. a, nuclear membrane ; b, portion of nuclear membrane
absorbed ; c, e(l) earliest stage of nuclei of young Amoebae (in
c (1) note groundwork of plastin and peripheral chromatin); d, e,f,
very young nuclei of young Amoebae in different stages of development ; g, chromatin block in nucleus of ' Mother' Amoeba ; h,
nuclear sap in ' Mother' Amoeba ; k, nucleoplasm in ' Mother '
Amoeba. Compare with Text-fig. 1B, e, Text-fig. 6, (1-3).
(Some experiments which seem to throw a little light on the
causes of this onset will be described in Part II.) The fission
divisions cease, and preparations for encystment commence.
The onset of this phase is heralded by the fact that the nuclear
AMOEBA PROTEUS
129
membrane is absorbed in one or more areas on the nucleus
(Text-figs. 1 and 3). The chromatin blocks in the neighbourhood of this absorbed nuclear membrane begin to show signs
of approaching ' division ' (of. Text-fig. 2). Out of the mass
of plastin and chromatin which constitutes a block (Textfig. 2, (1)) in the ' resting ' stage there emerges a much stouter,
more deeply staining, and more conspicuous chromosome than
that which would normally have arisen if the block had been
' dividing ' inside the nuclear membrane, merely to increase
the size of the Amoeba nucleus, or as a preliminary to a fission
division of that nucleus. This chromosome takes on an appearance as though it divided into two, but very quickly the plastin
reappears, the staining capacity of the chromatin is lessened,
and the one-time block is converted into a spherical mass with
the plastin in the centre and the chromatin round the periphery
(cf. Text-fig. 3, c (1)). This structure escapes into the cytoplasm. Similarly the other blocks near the absorbed membrane
(Text-figs. 1 and 3) behave in the same way, each at first
lying freely in the cytoplasm near the nucleus, but eventually
being carried away gradually by the streaming movements of
the endoplasm to considerable distances from it. It is thus seen
that these ' generative chromidia ' are really chromatin blocks
that have escaped from the nucleus into the cytoplasm.
The condition of the culture is wholly responsible for the
degree of rapidity in this process of ' extrusion of chromatin
blocks '. Sometimes large numbers are found to be escaping
in rapid succession. In other cases the escape is difficult to
detect so few is the number of blocks shed.
Arrived in the cytoplasm each block forms the rudiment of
the nucleus of a young Amoeba. This rudiment grows in size
(Text-fig. 3) by repeated division of the initial block. I
have counted as many as eight chromosomes in the rudiment, but on the whole I am inclined to think that the number
varies slightly (cf. Text-fig. 6, (1-5)). It will be seen later on
that the young Amoebae vary slightly in size. The reserve
products of the ' Mother ' Amoeba are called upon to supply
food for the development of the numerous nucleus rudiments.
NO. 273
K
130
MONICA TAYLOR
There is a steady decrease in the number of' nutritive ' spheres
as the encystment processes proceed (Text-figs. 4 and 5).
The next stages in the formation of the young Amoebae are
difficult to follow, and take place quickly. Each nucleus
rudiment, by successive divisions (Text-fig. 6, (3 and 4)), having
TEXT-FIG. 4. (Cf. Text-figs. 5, 6.)
Portion of a lobopod of an A. p r o t e u s . From preparation of
a whole Amoeba fixed in Bouin, stained in Delafield's haematoxylin (culture 10). The nutritive spheres stained black purple
by this technique can be distinguished from young individuals
with cysts not yet completely differentiated.
become provided with its complement of ' blocks ' (this,
varying in number, as already explained) becomes vacuolated
in the centre and in such a way that the chromatin material is
brought into more intimate communication with the cytoplasm
of the ' Mother' Amoeba in which it evidently initiates
activities. The first of these seems to be the differentiation
of a layer of new cytoplasm for the young Amoeba (Text-
AMOEBA PROTEUS
131
fig. 6, (5) b). Almost immediately the structure (i.e. the
early stage of the definitive young Amoeba) becomes very
vacuolated (Text fig. 6, (6)). The nucleus is in consequence
no longer clearly distinguishable in the sections of this stage.
This vacuolization is due to rapid absorption of nutritive
TEXT-FIG. 5. (Cf. Text-figs. 9 and 10.)
Section 4fi through A. p r o t e u s (from culture 24). Material
fixed in Bouin stained in Delafield's haematoxylin. The nutritive
spheres, stained black purple by this technique, are diminished
greatly in number in correlation with the cysts being so numerous.
[Consult Text-fig. 6 legend for explanation of various stages in
development of cyst shown on a smaller scale in the above
figure.]
material, i.e. of nutritive spheres. Next, a cyst wall is differentiated apparently from the cytoplasm of the ' Mother ' Amoeba
(Text-fig. 6, (7)). Nutritive material is enclosed within the cyst
wall(Text-fig.6,(7)<2). If a living ' Mother' Amoeba be examined
when the cysts are at the stages represented by Text-fig. 6,
(7) and (8), these latter are seen to contain structures which are
clearly not the differentiating Amoebae. They (Text-fig. 6,
.(8) d) vary in size, and are absent from the fully differentiated
cyst. Bach is a mass of nutrient material appropriated from
the endoplasm of the ' Mother ' Amoeba, and is apparently
K
2
TEXT-FIG. 6.
Successive stages in the differentiation of the young A. p r o t e u s .
(1), (2), (3), (5), (6), (7), (8), (11), from sections fixed in Bouin,
stained in Delafield's haematoxylin and light green ; (4) from
section fixed in Flemming ; (9), (10), from smear preparations of
whole Amoebae (culture 24). N.B.—When Amoebae containing
nearly ripe, and fully ripe, cysts are placed on a slide and allowed
to expand in a warm place (about 65° to 70° F.), they tend to grip
the slide as the water evaporates. If fixative be carefully run
over them many of the Amoebae are cemented to the slide when
AMOEBA PROTEUS
133
wholly used up during the ensuing later stages of development.
In the fully formed cyst the young Amoeba has attained the
characteristic appearance of an A. p r o t e u s (Text-fig, (i,
(10), although the ' karyosome' of the nucleus is not yet
clearly differentiated from the nuclear sap. After this follows
a typical cyst-stage (Text-fig. 6, (11)), i.e. the whole structure
becomes gradually smaller, the cyst wall resisting the entrance
of fixatives and refusing to stain : indeed, the encysted young
Amoeba bears a superficial likeness to a gregarine sporocyst.
The reserve products of the ' Mother ' Amoeba have been
completely used up by this time (Text-figs. 7 and 8). The
resources of the nucleus, however, do not seem to be exhausted
(Text-rig. 8). The achromatinic network remains and often
there appear to be chromatin blocks in the nucleus when the
' Mother ' Amoeba is packed with cysts. It must be remembered, however, that the achromatinic network of the Amoeba
nucleus is very voluminous, stains readily, and is a conspicuous
object even when the chromatin has been removed from it.
The remains of the cytoplasm of the ' Mother ' Amoeba form
a shroud round the mass of cysts for a time, but this thin covering quickly disintegrates and the encysted Amoebae are dispersed
throughout the aquarium where they can only with difficulty
be distinguished from the innumerable encysted organisms of
other kinds, Protozoa, plant-spores, &c.
In favourable circumstances the young Amoebae may hatch
out of their cysts at once ; on the other hand, they may remain
quiescent for a varying period of time. The rupture of the
the preparation can be treated as a ' smear ' preparation (cf.
Text-fig. 7). (1), (2), (3), increase in size of plastin-chromatin in
developing nucleus of young Amoeba ; (4), nucleus with vacuole
and blocks (e) in periphery ; (5), blocks {a and b) in ' dividing '
condition (of. nucleus in / , Text-fig. 10), commencement of
differentiation of cytoplasm of young Amoeba ; (6), group of
differentiating Amoebae ' vacuolated' stage (c, nutrient sphere ;
d, vacuole of nutritive material) ; (7), cyst wall just formed,
nutrient material in form of a globule (d), Amoeba nucleus not
clearly distinguishable from cytoplasm ; (8) nutrient material (d)
no longer globular ; (9) cyst wall (/) fully formed, no trace of
nutrient material ; (10), young Amoeba in cyst, nucleus (7i) and
cytoplasm (</) segregated ; (11), cyst fully formed, unstainable.
184
MONICA
TAYLOR
cyst wall seems to be brought about by the action of a ' hatching
ferment ' (15, p. 81). This collects in a vacuole which impinges
on that area of the cyst Avail which is eventually ruptured. If
TEXT-FIG. 7.
i
(Cf. Text-fig. 6.)
i i I I
'Mother' A. p r o t e u s containing fully differentiated cysts.
a, ectoplasm ; 6, marks position of nucleus ; c, cysts.
some of the material from, an aquarium where encystment
phenomena are known to be in progress be carefully pipetted
on to a slide, microscopical examination will reveal large
numbers of empty cysts in the neighbourhood of unhatched
Amoeba. The young Amoeba floats about in the water, and
AMOEBA PBOTEUS
135
in keeping with this habit its pseudopodia become long, stiff,
and radiate. In fact it often bears a superficial resemblance
to an Actinophrys. It can be made to grip the substratum by
reducing the quantity of water on the slide when it creeps
about in typical A. p r o t e u s fashion (Text-fig. 9).
I have no evidence of gametic formation such as is described
TEXT-FIG.
8.
•O2> rrv.m
Section (4/i) through a n A. p r o t e u s (culture 24) in which the
nucleus of t h e ' M o t h e r ' Amoeba is still present.
Fixation
Bouin, stain Delafield's haematoxylin a n d light green, a, nuclear
m e m b r a n e ; b, young Amoebae a t stage represented in Textfig. 6, (6) ; c, nuclear reticulum (' karyosome ' in elevation) ;
d, chromatin blocks ; e, ripe cysts (at stage represented in Textfig. 6, (11)) ; / , stage corresponding to t h a t represented in Textfig. 6, (5) ; g, nutritive spheres.
for P e 1 o m y x a . I have never seen the Helizoon-like Amoebae
unite in pairs to form a zygote, as is said to happen in the case
of P e l o m y x a (8, p. 228).
Ths presence of good supplies of bacteria and other minute
food-organisms is an important factor in the rearing of these
young Amoebae. (That these young creatures sometimes,
however, ingest relatively large food-organisms can be appre-
IB6
MONICA TAYLOR
ciated by an inspection of Text-fig. 10, g, where the clear area
represents the remains of an ingested flagellate within which
the nutritive spheres are making their appearance. This figure
also illustrates the conspicuousness of the nutritive spheres
when stained in Delafield's haematoxylin.) This early period
of development is a critical time; large numbers perish before
TEXT-PIG. 9.
%
ir
From culture 68, sub-culture of twenty-four made by treatment
with tartaric acid (see Part II). a, b, c, d, newly hatched A. p r ot e u s (a and d floating form, 6 and c creeping form); e, twomonths-old A. p r o t e u s ; f, nucleus of same after staining;
g, outline of A. p r o t e u s when three months old, drawn after
specimen had been allowed to expand on a slide, and to creep.
attaining maturity. The newly hatched Amoeba is only visible
under the high power. Its contractile vacuole is very characteristic, its nucleus can be detected in the living animal, its
ectoplasm is voluminous and extremely hyaline. These young
Amoebae have the same habit as their adults have of becoming
AMOEBA PROTEUS
187
temporarily perfectly spherical when the ectoplasm has the
appearance of a thin cyst wall.
The chroma-tin blocks as seen in a stained preparation of
young Amoebae when in the ' resting ' condition (cf. Textfig. 2, (7)) are by no means conspicuous (Text-fig. 10, a and d).
Probably tbis fact accounts for the relatively inconspicuous
TEXT-FIG. 10.
Stained preparations of recently hatched and young Amoebae
(A. p r o t e u s ) to show nuclei in which the blocks are in the
' resting ' and ' dividing ' stages, a, b, fixed in absolute alcohol,
stained in Ehrlich's haematoxylin, from culture 19, blocks in' resting ' condition, so-called karyosome is conspicuous ; c, d, e, / , g,
fixed in Bourn, stained in Delafield's haematoxylin, cleared in
clove oil, from culture 52 ; blocks in c, d, g in ' resting ' condition ; blocks in e, f in ' dividing ' condition ; so-called ' karyosome ' clearly distinguishable from chromatin material in peripheral blocks ; n.s., nutritive spheres in process of formation.
character of the nucleus as a whole. It requires careful
differentiation, i. e. overstaining and then destaining, to make
good permanent preparations of young Amoeba nuclei. When
the blocks proceed to divide they are seen to be larger in
proportion to the size of the Amoeba (Text-fig. 10,/) than they
138
MONICA TAYLOR
are in the adult. (Compare also the relatively conspicuous
size of the chromosomes in the differentiating nucleus, Textfig. 6, (5).) As the growth of the Amoeba proceeds, the blocks
become smaller in proportion to the increase in their number
until the Amoeba becomes adult, when there is a sort of rough
proportion between the age of the nucleus and the size of the
block, i.e. the older the Amoeba, the larger the block.
PART II.
AMOEBA CULTURES AND HYDROGEN-ION
CONCENTRATION.
Thirty pedigree cultures in all have been used for this
investigation. Most of these were contained in cylindrical glass
vessels (diameter 8 in., height 4 in.), the volume of water present
being from one and a half to two litres. Some few were in
vessels of smaller dimensions.
The hydrogen-ion concentration is recorded as is usual in
terms of pH—the symbol pH denoting the logarithm of the
number of grams of hydrogen ion per litre. The colorhnetric
method was used for the determinations, the range of readings
obtained being sufficiently great to make an accuracy of 0-2
quite adequate.
Adult Amoebae for the most part are to be found on the
bottom of the aquarium or on the surface of the debris which
collects there (young Amoebae float just above the debris,
as do likewise those adults that have become temporarilyspherical for the purpose of fission). There is, therefore,
a comparatively large bulk of water above the Amoebae.
The pH of each culture recorded has been obtained by gently
but thoroughly stirring the water of the aquarium and then
allowing the debris to subside, a sample of the water (10 c.c.)
being then taken off in a test-tube and treated with the
indicator.
Since all the cultures were stored in the laboratory the temperature is fairly uniform for the greater part of the year,
i.e. 58° to 60° P. The aquaria were shaded from direct sunlight.
Numberless readings taken from flourishing Amoeba cul-
AMOEBA PBOTEUS
139
tures show that in Glasgow the pH of the water in the aquarium
(after it has been stirred up) when most of the Amoebae are
adult and undergoing fission is 6-6.
This then may be regarded as the optimum pH. A diurnal
A^ariation due to photosynthesis can be obviated by keeping
the cultures in the shade. The most practical method of maintaining this pH 6'6 is by sub-culturing once at least in three
months. When a culture is in such a condition that a pipette
full of material (5 to 7 c.c.) from the bottom of the aquarium
put into a solid watch-glass and viewed under the low power
of a Greenough binocular shows 50 to 100 adult Amoebae, then
a sub-culture should be made from it. For this purpose an
infusion of boiled wheat grains (5-7 to 100 c.c. of water) should
be put into an incubator (or near the radiator, or in any warm
place—65° to 70° P.) for one or two days, when the infusion
should be inoculated with from 5 to 10 c.c. of the inoculation
material, more water being added every few days to compensate for evaporation and bring the bulk of water gradually
to about a litre.
A sub-culture, successfully made, is at its prime in about
three months, its pH is 6-6 (in Glasgow), and it is then ready
to be used in its turn for further sub-cultures. The original
stock cultures, if fed regularly with additional wheat grains,
will undergo periods of depression and luxuriance, and will
form useful stock that can be called upon in case of accident.
Amoebae can live in water whose pH is higher than 6-6,
but the struggle for existence seems to be greater ; the higher
pH of the water favouring the growth of a variety of rotifers,
ciliates, &c, not useful for Amoeba food. Adult Amoebae
can of course devour quite large Paramecia and the smaller
rotifers, but large rotifers, Prontonia, &c, are inimical to
young Amoebae. In such cultures the Amoebae have, so to
speak, to take turns in the cycle of dominating organisms, and
fit in their cycle of changes and complete their life-history
in intervals when the enemy organisms are less active (encysting, or producing eggs).
Amoebae can live in water whose pH is as low as 4. The
140
MONICA TAYLOR
field-book records of culture 10 show that it has been in a
flourishing condition at a pH of 4 ; the pH of this particular
aquarium water never rises beyond 5, and falls as low as 3-2
when the Amoebae are encysting.
The pH of unsuccessfully inoculated cultures has been very
usually 4 in my experience.
I have not yet accumulated sufficient evidence of good
results to recommend the raising of pH by means of the addition
of chemicals.
Experiments being conducted on the pH of the water in
which the various moulds and algae that crop up in Amoeba
cultures are being grown, seem to show that acid-producing
plants are largely responsible for low pH and for fluctuations
in the pH readings. If these gain the upper hand the Amoebae
succumb, or they encyst until the other organisms have reacted
and so brought about a less acid condition. A voluminous
fungoid matting of a dirty greyish colour that often accumulates
round freshly added wheat seems to be inculpated and should
be removed. Similarly a mould of the nature of a whitish
incrustation that accumulates on the surface of the water
and which can be removed by placing pieces of paper on to it
and skimming it off, by removing the paper from contact with
the water, is a herald of a low pH. Other mould spores, on the
contrary, are greedily devoured by Amoebae. The evidence
at present available shows that sunlight favours the growth
of certain of these acid-producing plants.
The individuals of a culture that has been obtained by
a successive series of sub-cultures from one initial culture
are often found to be supercharged with storage products—
metabolic substances. The nucleus, too, is often irregular,
very lobed. These Amoebae tend to bud off lumps of cytoplasm when they are being transferred to a slide. These
characteristics are due to the artificial frustration of the encystment phenomena. The most beautiful and typical of individuals
are those that are just adult, i.e. about six months old.
The pH of a culture can be lowered without damage to the
Amoebae by means of tartaric acid. The results obtained from
AMOEBA PROTEUS
141
the use of this acid, which I chose on account of its employment
in cooking operations, confirm the subsequent discovery made
by Pan tin on the marine Amoebae (9).
All the adult Amoebae in a culture whose pH is 6-6 may be
killed by lowering the pH to 3 by the addition of tartaric acid.
The encysted Amoebae are unharmed by this treatment and
begin to hatch out in due course, when their growth can be
studied. Another method of studying the emergence of the
young Amoebae from their cysts is to put several large old
Amoebae into a solid watch-glass with water from the aquarium
out of which they were taken, the watch-glass being covered up
to prevent evaporation. The adult Amoebae, after a varying
period of time, begin to undergo the encystment phenomena,
and when this is completed they can no longer be recognized
under the low power of a Greenough binocular—by reflected
light. Under the high power in transmitted light the bottom
of the watch-glass is seen to be covered with cysts, and as a rule
there is a great growth of green flagellates. After three weeks
or a month from the time of starting the experiment, an
examination under the high power of an ordinary microscope
of the material from the bottom of the watch-glass will reveal
the presence of cysts, cysts ready to open, and newly hatched
Amoebae.
The onset of a period of depression is often heralded by a rise
of pH to 7'3 and upwards to 7-8. If now a sub-culture be made,
the change of temperature, lack of food-supply, &c, may
accelerate the encystment phenomena, instead of causing the
Amoebae to go on with their fission divisions. In such a case
the success of the inoculation cannot be judged without
recourse to the high power of a microscope. This sub-culture
will not contain adult Amoebae for six months at least.
The starving of Amoebae after they have fed voraciously will
accelerate the formation of cysts. This starvation is sometimes brought about under more or less natural conditions by
the wheat decomposition products being absorbed by algal
growths.
A micro-organism culture which is known to contain encysted
142
MONICA TAYLOR
Amoebae but which, in addition, is inoculated with a variety
of organisms such as F r o n t o n i a l e u c a s , Paramecia, large
Brachionus, and other large rotifers, which are detrimental
to prolific development of Amoebae, may be converted into
a good Amoeba culture by a lowering of the pH to 4 by means
of tartaric acid. All these organisms are killed off by the
treatment, and their decomposition products, together with
that of the wheat, constitute a good pabulum for the Amoebae,
which then have a chance of thriving.
In concluding, I Avish to record my indebtedness to Miss
Isabella McGuire, B.Sc, for much assistance in the routine
work of taking pH readings.
SUMMARY.
1. Additional detail of the minute structure of the nucleus
of A . p r o t e u s has been given.
2. It has been shown that growth in the size of the nucleus
and fission division of the nucleus are consequent upon a
previous division of chromatin material situated in the blocks.
3. This division of the chromatin blocks has been described.
4. The history of the formation and development of the
young Amoebae, encystment, hatching, rate of growth has been
traced out.
5. Some recent modifications in the methods of making
laboratory cultures of A. p r o t e u s have been recorded.
6. Amoeba culture in relation to hydrogen-ion concentration has been discussed.
BIBLIOGRAPHY.
1. Atkins, W. R. G., and Lebour, M. V. (1923).—"The Habitats of
Limnaea truncatula and L. pereger in relation to Hydrogen-ion
Concentration ", ' Soient. Proc. R. D. S.', vol. xvii, N.S., no. 41.
2.
(1923).—" The Hydrogen-ion Concentration of the Soil and
of Natural Waters in relation to the Distribution of Snails ", ibid.,
no. 28.
AMOEBA PROTEUS
148
3. Carter, Lucy A. (1913).—" Notes on a Case of Mitotic Division in
Amoeba proteus, Pall.", ' Proc. Roy. Phys. Soc. Edin.', vol. xix,
no. 4.
4.
(1915).—" The Cyst of Amoeba proteus ", ibid., no. 8.
5.
(1919).—"Some Observations on Amoeba proteus", ibid.,
vol. xx, part 4.
6. Doflein, IF. (1818).—"Die vegetative Fortpflanzung von A. proteus,
Pall.", ' Zool. Anzeiger', Bd. xlix, no. 10.
7. Martens, P. (1922).—" Le cycle du chromosome soinatique dans le
Paris quadrifolia " , ' Acad. R. de Belgique. Bulletins de la Classe
des Sciences ', no. 3, pp. 124-30 ; also ' La Cellule ', torn, xxxii,
2 e fascicule.
8. Minchin, E. A. (1912).—' An Introduction to the Study of Protozoa.'
9. Pan tin, C. F. A. (1923).—" Amoeboid Movement ", ' Journ. of the
M. B. Association of the United Kingdom ', vol. xiii, no. 1, December
1923.
10. Schaeffer, A. A. (1916).—" Notes on the Specific and other Characters
of Amoeba proteus, Pall. (Leidy), A. discoides, spec, nov., and
A. dubia, spec, nov.", ' Archiv fur Protistenkunde ', Bd. xxxvii,
1916.
11. Taylor, Monica, and Hayes, C. (1921).—" The Technique of Culturing
Amoeba proteus ", ' Journ. Roy. Micr. Soc.', pp. 241-4.
12. Taylor, Monica (1920).—"Aquarium Cultures for Biological Teaching",
' Nature ', 105, p. 232.
13.
(1919).—" Note on the Collection and Culture of Amoeba proteus
for Class Purposes " , ' Proc. Roy. Phys. Soc. Edin.', vol. xx, part 4.
14.
(1923).—" Nuclear Divisions in Amoeba proteus ", ' Quart.
Journ. Micr. Sci.', vol. 67, part i, April.
15. Wintrebert, P. (1922).—" Titres et Travaux scientifiques."
144
MONICA TAYLOR
APPENDIX.
Nutritive Spheres in Amoeba.
By
Sister Carmela Hayes, S.N.D., B.Sc.
INTRODUCTION.
SCHABPFBR (2) states that A . p r o t e u s is 'characterized
by the occurrence of a large number of ^clear bluish spheres
which in occasional individuals reach a size of 10 microns in
diameter. They occur in greater number and more constantly
in this species under varying conditions than in any others that
I have examined.'
Later on, in speaking of A. d i s c o i d e s , he goes on to say
that ' constantly occurring inclusions are spheres of a pale blue
colour, the so-called excretion spheres, which are connected
somehow with digestive processes, as earlier observers have
indicated. The number of these spheres varies according to
the amount of food eaten and digested and the rate of division,
as has been noted for other species.'
Because of the superficial resemblance in the living Amoeba
of the large so-called excretion spheres to encysting young
Amoebae, and their varying behaviour to the fixatives and
stains ordinarily employed in cytological investigation (and
also because of an artifact occurring in certain stained preparations of whole Amoebae), Sister Monica asked me to undertake
an exhaustive examination of the effects of these various
fixatives and stains on A. p r o t e u s and to determine if
possible the nature of these cytoplasmic inclusions observed
by Schaeffer.
As explained in the foregoing paper these so-called excretion
spheres have been there alluded to as nutritive spheres. This
term will similarly be employed to designate them in the
following record.
AMOEBA. PROTEUS
145
With some thirty pedigree cultures at my disposal I have been
able to make a large number of preparations—temporary and
permanent—and to cut sections of Amoebae under various
conditions, the field-book records of these cultures supplying
me with their complete history.
I have been able to confirm Schaeffer's observations that
these spheres vary in number according to the amount of food
eaten and digested.
Sister Monica has shown that the nutritive spheres play an
important part in the phenomena of encystment, and that in
certain preparations of whole Amoebae—notably those stained
in Delafield's haematoxylin after fixatives other than Flernming's—they can easily be distinguished from the various
stages in the differentiation of the encysting young Amoebae.
In other cases of whole preparations, however, it is not quite
so easy to clearly distinguish the ripe ' spores ' from the large
nutritive spheres. No such difficulty exists in the interpretation of sections.
THE NUTRITIVE SPHERES.
(a) T e m p o r a r y P r e p a r a t i o n s . — I f a solution of
iodine in potassium iodide be run under the coverslip of a slide
on which there is an Amoeba which has not yet begun to undergo
fission divisions, i. e. about six months old, an abundance of
minute starch granules become visible in the cytoplasm.
If an Amoeba, old and containing many nutritive spheres,
be crushed under a coverslip and then treated with iodine
the smaller spheres are stained dark brown. The larger spheres
—less deeply stained—seem to form centres around which the
starch granules collect. Each large pale-brown sphere with the
particles adhering to it forms a striking object.1
In aceto-carmine preparations the spheres are unstained.
(b) P e r m a n e n t Preparations.—If an Amoeba be
put on a slide, and a smear preparation be made of it, the
1
A somewhat similar phenomenon is evident in permanent preparations
treated with iodine solution after Ehrlich's haematoxylin.
NO. 273
L
146
MONICA TAYLOR
larger spheres lose their spherical form and become irregular
patches which stain deeply in thionin and Delafield's haematoxylin.
Old Amoebae overloaded with large nutritive spheres fixed
in modified Bouin,1 corrosive alcohol, or corrosive acetic solutions and stained in borax carmine behave as do the Amoebae
that have been crushed and treated with a solution of iodine
in potassium iodide. The starch granules are either attracted
to the large spheres or the adhesive nature of the substance
in these spheres retains the starch grains in their immediate
neighbourhood. Consequently when viewed in permanent
preparations stained in carmine stains the nearly colourless
groundwork of the sphere covered with the stained granules
(purplish blue in good daylight, blackish in artificial light)
give a superficial resemblance to a ripe Amoeba cyst. The
artifact is very conspicuous, forming a striking contrast to
the general red of the cytoplasm. That these coloured particles
adhering to the nutritive spheres are not symbiotic bacteria such
as have been described for P e l o m y x a (4), can easily be seen
from careful examination of the preparations, accompanied by
a constant reference to temporary preparations and to the
living animal.
After fixation with any of the more ordinary fixatives—
absolute alcohol, absolute alcohol plus corrosive sublimate,
absolute alcohol plus corrosive plus acetic acid, aqueous
corrosive acetic, modified Bouin, or formalin 10 per cent., and
staining with Delafield's haematoxylin (as supplied by British
Drug Houses, Limited), the spheres show up in a very striking
manner—they appear as black blobs measuring from 1 to about
S/tt in diameter (Schaeffer records spheres of 10/a diameter, but I
have seen none quite this size), and somewhat resembling the
yolk-globules seen in sections of young embryos stained in
iron haematoxylin. In preparations of whole Amoebae where
the nutritive spheres are very numerous these dark purple
masses mask the other structures.
Treatment of Amoebae containing numerous large spheres
1
Formula of Duboscq Brasil. 1905.
AMOEBA PROTEUS
147
with osmic acid solution and with Sudan III proved that fat
was not a constituent of these bodies. Moreover, all the
ordinary methods of fixation and up-grading in alcohols and
xylol or clove oil left these spheres intact and undissolved,
which is another proof of their not being fat (1).
Amoebae from culture 20—crowded with large blue spheres—
fixed in aqueous corrosive acetic and then soaked for two and
a half days in water (which was changed three or four times
per day) showed no trace of the spheres when stained in
Delafield's haematoxylin; presumably they bad dissolved
out in the water. This solubility of the spheres in water would
suggest that they are of the nature of glycogen. The mere
fixation in aqueous corrosive acetic is not sufficient to dissolve
the spheres.
Numbers of Amoebae, at many stages in their life-history
from cultures 19, 29, and 37, fixed in absolute alcohol, were
stained in Ehrlich's haematoxylin and then treated with iodine
solution according to the method described by Gatenby (1).
.Others from the same cultures and having the same fixation
were treated with Best's carmine after Ehrlich's haematoxylin
(method given in (3) and, though both these methods were
described for staining sections on slides, the results obtained
in the bulk staining were quite satisfactory, the spheres giving
somewhat of the glycogen reaction in both cases, i.e. yellow to
reddish brown in iodine solution, and red in Best's carmine. The
Amoebae stained in Ehrlich's haematoxylin plus iodine solution
make especially pretty pictures, as the yellow to reddish-brown
spheres form a pleasing contrast to the beautiful blue of the
Ehrlich in nucleus and cytoplasm. Similarly pretty pictures
are obtained by overstaining in Delafield's haematoxylin
differentiating in slightly acidified alcohol and overstaining in
light green when the purple of the nutritive spheres is in strong
contrast to the green of the general cytoplasm.
That the colour of the spheres varies so much in different
individuals—from bright yellow through brown to reddish
brown—shows that the glycogen-like substance which they
contain must vary in composition; probably, in course of
148
MONICA TAYLOR
being formed in some, at an optimum in others, and being used
up in yet a third set.
Stained in Ehrlich's haematoxylin alone the spheres are
reddish. After fixation in Flemming's solution the spheres are
not stained by the haematoxylin dyes used (Ehrlich, Delafield,
Heidenhain). No matter what the fixative, they are not
stained red by borax carmine nor by picro-magnesium carmine.
It may be noted here that the carmine stains, especially Best's,
give an opaqueness to the preparations which is absent from
those stained in haematoxylin.
EFFECTS OF FIXATIVES.
Since A. p r o t e u s is so largely used by the elementary
student as well as by the scientific investigator the following
results may be of value.
1. Absolute Alcohol preserves the natural form very
well, also the sphere-like inclusions, but it rapidly coagulates
the ectoplasm of adults as well as young individuals and so
forms a cyst-like skin round the Amoeba. The achromatinic
framework or nucleoplasm is not fixed so effectively as by
stronger fixatives.
2. Corrosive Absolute and Corrosive Absolute
plus a little (less than 3 per cent.) Acetic Acid are both
good fixatives for the reticulum, both cytoplasmic and nuclear
also for chromatin and the sphere-like inclusions.
3. Aqueous Corrosive Acetic may be used with
success.
4. Modified Bouin (Duboscq Brasil, 1905) is fairly
good for nucleoplasm and good for chromatin, but it tends to
make the cytoplasm unnaturally transparent. It is useful for
penetrating the cyst wall of encysting Amoebae.
5. Formalin 10 per cent, as usual did not prove a good
nuclear fixative. The nutritive spheres do not take on such
a dark purple stain in Delafield's haematoxylin after this
fixative.
6. Hot Water can with care be safely employed for those
AMOEBA PROTEUS
149
physiological purposes where alcoholic and acid fixatives would
interfere with the investigation being pursued.
The blue spheres stain readily in Delafield's haematoxylin
after any of the above-mentioned fixatives ; but no matter
what the fixation may be they are not stained by ordinary
borax carmine or by the picro-magnesium carmine used, useful
though these latter stains are for nucleus and reticulum.
7. P l e m m i n g ' s S o l u t i o n is a useful fixative for whole
preparations and sections, but the proverbial difficulty of
staining after Flemming holds good here, and it is remarkable
that the blue spheres which after other fixatives are so deeply
stained by Delafield's haematoxylin are unstained by it after
this fixative. Although unstained, however, their presence is
easily recognized in sections.
Prom what has been said above it would appear then that
the pale blue spheres of A. p r o t e u s contain a glycogen-like
substance, and the number of spheres present and their exact
chemical composition depend on the stage of its life's cycle
at which the individual is and also on its physiological condition.
LIST OF EEFERENCES IN APPENDIX.
1. Gatenby, J. Bronte.—" The Identification of Intracellular Structures ",
' Journ. Roy. Micr. Soc.', pp. 93-118, 1919.
2. Sehaeffer, A. A.—" Notes on the Specific and other Characters of
Amoeba proteus, Pallas (Leidy), A. discoides, spec, nov., and
A. dubia, spec, nov.", ' Archiv fur Protistenkunde ', 1916.
3. Bolles Lee.—' The Microtomist's Vade-Meoum', edition by Gatenby,
1921.
4. Minchin, E. A.—' An Introduction to the Study of the Protozoa ', 1912.