Observations on Protozoa:
I. The impermanence of the contractile vacuole
in Amoeba Vespertilio.
II. Structure and mode of food ingestion of
Peranema.
By
Libbie H. Hyman.
Laboratory of Experimental Biology,
American Museum of Natural History, New York.
With 7 Text-figures.
I.
IMPERMANENCE OF THE CONTRACTILE VACUOLE IN
AMOEBA VESPERTILIO.
THE origin, permanence, and function of the contractile
vacuoles of Protozoa remain questions of perennial interest and
debate. Whereas in some Protozoa, such as many ciliates, the
vacuoles are undoubtedly fixed and permanent organelles, this
is not so in others, especially Ehizopoda. Even in this group
the vacuoles may occupy a fixed position, and reappear in the
same spot after each systole, as in Heliozoa and the shelled
Lobosa. In amoeba, the origin of the vacuole d e n o v o after
each contraction from a group of minute droplets, or of granules
which dissolve to form droplets, has been reported by Metcalf
(1910), Howland (1924), Botsford (1926), Day (1927), and
Herwerden (1927). Metcalf states that the vacuole reappears
after each contraction in a definite group of' excretory granules';
but others have been unable to find any special granules
associated with the vacuole, and indeed most observers record
that the vacuole does not necessarily reappear at the same spot
where it disappeared. On the other hand, Gelei (1933) finds
that in A m o e b a l i m a x the vacuole reforms after systole
in the same place where it disappeared, from a minute remnant
of the preceding vacuole. I also, in a small amoeba closely
resembling Penard's (1902) description and figures of A.
g u t t u l a , have noted the reformation of the vacuole after
44
LIBBIE H. HYMAN
each systole from the fusion of two or three small vacuoles
which come into view in the same spot where the preceding
vacuole discharged.
Eecently an amoeha has come to my attention which has
a type of vacuole behaviour differing from anything in the literature, except the recent observations of Gelei (1938) which will
be referred to later. This amoeba appeared in fair abundance
in a culture started in the fall of 1984 for D a p h n i a , and
consisting of boiled lettuce leaves to which were added from
time to time additional lettuce leaves and small bits of raw liver
or dead tadpoles. At times also the water (spring water) was
changed without disturbing the bottom debris or the algal
coating on the walls. This culture reached a state of equilibrium
in which the wall became covered with a dark green film composed of diatoms, green algae, and other low plant forms, and
supporting for many months a moderate number of small
amoebae of several kinds, A r c e l l a , P e r a n e m a , V o r t i c e 11 a, rotifers, and a few ciliates.
My attention was attracted to the largest of the small
amoebae by the presence in its endoplasm of a number of water
vacuoles. This amoeba has been identified with certainty as
A. v e s p e r t i l i o Penard, 1902. It corresponds in all details
with Penard's description and figures, further with the description and figures of A. v e s p e r t i l i o in Brown's report (1910)
on the rhizopods of the English Lake District, and in Wailes
and Hopkinson's manual (1919) of the British fresh-water
rhizopods. A. v e s p e r t i l i o was previously recorded for the
vicinity of New York City and for Augusta, Ga., by Wailes
(1912). The source of this species in my culture cannot be stated
with certainty, but presumably the amoebae were introduced
with the D a p h n i a which were purchased in the aquarium
department of a large store, and which in all probability were
collected near New York City.
The structural details and pseudopod type of A. v e s p e r t i l i o have been repeatedly described, and on these matters
I have nothing new to add except that the endoplasm contains
a few scattered highly refringent bodies which appear to be
crystals (Text-fig. 1, C), and which under high magnification are
OBSERVATIONS ON PROTOZOA
45
seen to be double (Text-fig. 2), consisting of two oval bodies
adhering laterally. My specimens were small, fluid, and very
active, having a length of 50-70 /x when extended in locomotion.
The species habitually feeds upon diatoms and unicellular green
algae, and usually contains a large number of such bodies in
TEXT-FIGS. 1 and 2.
Fig. 1.—Amoeba v e s p e r t i l i o in locomotion, free-hand. C, crystals;
N, nucleus with an endosome; V, vacuoles.
Fig. 2.—Crystals enlarged.
process of digestion. It may also, however, as noted by Lapage
(1922) eat small amoebae. A specimen was seen on one occasion
to ingest an A. g u 11 u 1 a of about one-third its own diameter,
and another time an A. v e s p e r t i l i o half-way inclosed an
A. g u t t u l a before abandoning it. Two or three other individuals were noticed each of which contained an A. g u t t u l a
in process of digestion inside a large vacuole. As A. v e s p e r t i l i o apparently never devours its own species, Lapage's term
'cannibalism' seems scarcely appropriate. The indications are
that A. v e s p e r t i l i o feeds only upon organisms which are
motionless or practically so. The small spherical amoebae
(about 25/i diameter), here termed A. g u t t u l a from their
46
LIBBIB H. HYMAN
correspondence with Penard's (1902) description, move with
excessive slowness. Ivanic's (1933) account of the formation
of a 'gullet' in A. v e s p e r t i l i o when ingesting food seems
to me somewhat exaggerated. Naturally a relatively small
amoeba must form a deep food-cup when ingesting a relatively
large elongated type of diatom.
The endoplasm of A. v e s p e r t i l i o always contains a
variable number of water vacuoles, which are more numerous
the larger the specimen, and any one of which, as will be seen
shortly, can function as a contractile vacuole. Penard's figures
show two to three vacuoles, and he states that there appears
to be one principal vacuole and many others which appear
and disappear like contractile vacuoles. Brown also figures
three vacuoles, and remarks that the species has several
vacuoles which tend to fuse to one. Wailes and Hopkinson
record one to three contractile vacuoles and additional small
vacuoles. According to Lapage, A. v e s p e r t i l i o frequently
has several contractile vacuoles, but specimens with only one
contractile vacuole are at least as common. None of these
observers has seen the truth of the matter nor interpreted the
vacuoles correctly. As to Doflein's statement that A. v e s p e r t i l i o has but one contractile vacuole which requires 10 to 20
minutes to discharge, I can only suggest that he must have
%
wrongly identified his specimens.
For accurate observation of the vacuoles, specimens free from
food bodies must be studied under an oil-immersion lens. It
can then readily be seen that vacuoles continually arise de
n o v o in the endoplasm, particularly in the posterior end, as
minute droplets. These increase in size and often two of them,
usually not more than two, will fuse on coming in contact.
Such fusions occur only among the smaller vacuoles. The larger
vacuoles do not unite even when pressed together by endoplasmic currents. The number and size of the vacuoles present
at any one time is highly variable. Often the smaller specimens
have only three or four noticeable vacuoles, but those of maximum size frequently contain fifteen or twenty vacuoles of all
sizes. The vacuoles while growing in volume seem to be mere
droplets of fluid without definite walls, for they are easily dis-
OBSERVATIONS ON PROTOZOA
47
torted into all sorts of shapes when pressed against by more
solid objects in the endoplasm or caught against the gelating
sides of the amoeba. The vacuoles move about freely while
below maximum size, but tend to remain in the rear half of the
animal.
When any one of the vacuoles has attained maximum size its
behaviour alters so that one can forecast that a contraction is
imminent. It seems to develop a stiffened wall, for it now remains
perfectly spherical and can no longer be contorted from the
spherical shape by contact with other objects. It also lags
behind more and more into the rear end of the amoeba. The
wall appears thick and of the same bluish-green hue as the
endosome. Finally, such a vacuole lags so much that it comes
into the gelating margin of the posterior end, where it slowly
discharges, apparently through pressure from the surrounding
ectoplasm undergoing gelation. Vacuoles which are about to
contract are drawn in my figures with thickened walls.
No new vacuole reforms in the spot where a vacuole has
discharged. This spot, which appears very dense and jelly-like,
mingles with the general ectoplasm and is soon lost to view.
Meantime one of the other submaximal vacuoles attains maximum size and goes through the changes just described, and this
behaviour continues indefinitely. Bach vacuole in turn grows,
stiffens into spherical form, lags into the rear, and discharges,
and in the meantime new vacuoles form as minute droplets in
the endoplasm so that a number of vacuoles is always at hand.
Text-figs. 3 to 6 attempt to depict the series of events. In
Text-fig. 3 vacuole 1 is about to discharge, as indicated by its
thickened wall, and the next in size, nos. 2 and 3, are still rather
small. In Text-fig. 4 nos. 2 and 3 are fusing into one, and other
vacuoles, nos. 4 and 5, are increasing in size. In Text-fig. 5
the single vacuole formed by the fusion of 2 and 3 is about to
discharge. In the interval between Text-figs. 5 and 6 nos. 4
and 5 fused, and in Text-fig. 6 this vacuole is about to discharge,
while other vacuoles have grown in size.
It is, of course, impossible to prove that we are not dealing
with multiple vacuoles which disappear and reappear, but I consider this highly improbable. Schaeffer (1918) claims that A.
48
LIBBIB H. HYMAN
b i g e m m a has multiple vacuoles, and that he has seen several
in systole simultaneously. I have made some observations on
an amoeba collected near New York City which corresponded
closely to Schaeffer's description of A. b i g e m m a ; it is my
opinion that this species does not have multiple vacuoles, but
that its vacuoles behave as in A. v e s p e r t i l i o . In the latter
species I have never seen but one vacuole in systole at any one
TEXT-FIGS. 3-6.
Figs. 3-6.—Successive growth and discharge of vacuoles, free-hand.
Tig. 3.—Vacuole 1 discharging, 2 and 3 small.
Fig. 4.—Vacuoles 2 and 3 fusing, 4 and 5 growing.
Fig. 5.—Vacuole formed of the fusion of 2 and 3 discharging.
Fig. 6.—Vacuole formed by the fusion of 4 and 5 discharging. Vacuoles about to discharge indicated by a thickened rim.
time, although the intervals between successive vacuolar contractions vary considerably. This variation seems to me further
evidence that we are not dealing with multiple vacuoles in
A. v e s p e r t i l i o , for if so we should expect the vacuoles to
recur at rather definite intervals.
With this point in mind I have timed the intervals between
successive systoles with a stop-watch in two or three specimens
and have made observations on many others. The actual figures,
in seconds, for one specimen were: 45, 30, 40, 50,10, 33,10, 52,
45, 53, 10, 12, 50, 43, 55, 17, 10, 33, 42, 33, 25, 32, 43, 13, 32,
10, 37, 50, 27, 58, 15, 50, 40, 63, 37, 65, 58; for another: 25,18,
7,15, 95,13,12, 40, 60, 35,110, 65,15, 35, 35,15, 40, 35, 60, 40.
What happens is this. If the amoeba by chance has a number
of large vacuoles at about maximal size, then these contract in
rapid succession at intervals of 5 to 20 seconds as a rule. When,
OBSERVATIONS ON PROTOZOA
49
however, these have all discharged, there are no vacuoles of
sufficient size ready in the endoplasm. Then an interval of 45
to as much as 90 or 100 seconds elapses until some of the smaller
vacuoles have had time to grow to the maximum size. Thus
periods of several discharges in rapid succession are nearly
always followed by longer intervals between discharges.
It is my impression that the vacuoles do not contract from
any intrinsic mechanism, but are forced to discharge by the
gelation pressure of the surrounding ectoplasm. Vacuoles are
seldom seen to discharge until they have manoeuvred themselves into a position against the surface membrane of the rear
end or the sides of the posterior third of the animal, as shown
in Text-figs. 3, 5, and 6. Often the vaeuole bulges out the
membrane at this place (Text-fig. 6), and then slowly collapses
seemingly under pressure from its surroundings. Often the
vaeuole, which has reached the necessary position and discharges,
is slightly smaller than another which was expected to contract
first but which had not yet come close enough to the periphery.
On one occasion an amoeba drew out into two masses connected
by a narrow strand. A vaeuole caught in this strand discharged
as the strand narrowed down to approximately the diameter
of the vaeuole. Small vacuoles do not, however, discharge when
they happen by chance to get into the right situation, but simply
slip out again into the general endoplasm since their lack of
a definite wall permits them to assume a dumb-bell or other
shape and so to slide through narrow places. A certain rigidity
in the wall is essential to the discharge.
In A. v e s p e r t i l i o , then, we are dealing with an amoeba
which continually forms and discharges water vacuoles. The
observations here presented seem almost conclusive proof that
the main function of the contractile vaeuole is to prevent the
accumulation of too much water in the cytoplasm, that this
organelle is in fact a water-regulating mechanism. The state
of affairs noted in A. v e s p e r t i l i o is probably attributable
to the fact that this amoeba is very fluid, with a thin surface
membrane, and consequently is poorly protected against inflow
of water. Its small size is not a factor, since other much smaller
types of amoeba present in the culture were observed to have
NO. 313
E
50
LIBBIB H. HYMAN
but one vacuole, which reappeared at the point of disappearance.
These amoebae were, however, slow-moving forms of stiff consistency and thus less subject to water intake.
I venture to predict that a vacuolar behaviour similar to that
of A. v e s p e r t i l i o will be found in other fluid, rapidly moving
types of amoebae. I have already seen it in A. b i g e m m a , as
noted above, and also in two other kinds of small (25/u) but
active forms. Very probably all the species said to have multiple
vacuoles are really continually forming and discharging new
vacuoles. Gelei (1933) has described a new species, A. p l u r i v e s i c u l a t a , in which the vacuolar behaviour, as far as can
be determined from a brief paragraph, is identical with that
of A. v e s p e r t i l i o , from which it is certainly specifically
distinct. Gelei also suggests a correlation between ectoplasmic
consistency and permanence of the vacuolar system. I should
perhaps mention that my observations on A. v e s p e r t i l i o
were completed and the conclusions stated above were reached
before I had seen Gelei's paper.
In view of the foregoing facts, it is not surprising that Haye
(1930) failed to find any definite wall or pore for the vacuole of
A. v e s p e r t i l i o .
Observations such as those here reported demonstrate the
futility of arguments about the permanence, mode of formation,
&c, of the contractile vacuole. Obviously these matters vary
from species to species, a.nd facts verified for one species cannot
be dogmatically applied to others. The type of vacuolar system
in any protozoan species is correlated with the consistency and
very likely other properties of its ectoplasm.
II.
STRUCTURE AND MODE OF POOD INGESTION OF PERANEMA.
P e r a n e m a t r i c h o p h o r u m is one of the most studied of
the free-living flagellates, but no agreement has yet been reached
concerning its morphology and manner of feeding. This flagellate was common in the A. v e s p e r t i l i o culture, and many
hours were spent observing this interesting animal under an oil
immersion lens. The activity of the animal and its tendency
to exhibit at any moment suddenly euglenoid contractions
render exact observation under high powers difficult. Specimens
OBSERVATIONS ON PROTOZOA
51
with very short flagella are less active and the most suitable
for observation.
The morphology of P e r a n e m a has been described in recent
years by Hall and Powell (1927, 1928), Brown (1930), and Hall
(1933), but Brown and Hall do not agree on the details of the
anterior end. In my opinion neither of these observers is entirely
correct, but Brown is more nearly correct than Hall. The anterior
end presents two principal structures, the gullet and the
pharyngeal-rod apparatus. In agreement with Brown, and in
disagreement with Hall, I find these two structures to be unrelated to each other. My observations (Text-fig. 7), like those
of Brown, have convinced me that the gullet opens at the
anterior tip of the animal slightly asymmetrically, so that one
lip protrudes a little more than the other. Prom the gullet
opening, which I consider (contrary to Brown) to be the mouth,
the neck of the gullet leads inward and expands into a spherical
base, usually termed the reservoir. Theflagellumpasses through
the mouth and neck of the gullet, which it almost fills, and then
curves parallel to the bottom wall of the reservoir to fasten
upon the side of the latter. The attached end of the flagellum
is considerably broader than the diameter of the flagellum at
the mouth, and hence I consider it probable that the flagellar
attachment is double as maintained by Brown and not single
as stated by Hall and Powell.
The pharyngeal-rod apparatus is plainly double, as all recent
observers agree. The two components are shaped like pins
rather than rods; that is, they have a rounded head from which
the stem tapers posteriorly to a pointed end. The two rods must
be fastened together somehow, as figured by Brown, since they
always move as one object. Possibly they are really the thickened edges of a plate-like structure. At the heads of the rods
there is a sickle-shaped filament, the falcate trichite, F., Textfig. 7, which I saw clearly at times, but which I should probably
not have noticed had I not read about it in the literature. It
appears to be fastened to the rod-heads, since it moves with
the rods.
My observations agree with those of Brown, that the rods
and falcate trichite have no relation to the gullet entrance or
52
LIBBIB H. HYMAN
gullet wall and are, in fact, some little distance removed from the
gullet. Hall and Powell (1928) show the rod-heads situated at
the gullet entrance which they place considerably below the
anterior tip. I am certain that Hall and Powell are incorrect
in their conception of the morphology of these parts. The gullet
entrance is not situated at the rod-heads but at the anterior tip
M
TEXT-FIG. 7.
The anterior end of P e r a n e m a t r i c h o p h o r u m . F., falcate trichite;
<?., gullet; M., mouth; R., pharyngeal rods; V., vacuole.
of the animal, and the flagellum positively does not pass into
the interior at the rod-heads but does pass through the gullet
entrance at the anterior tip.
At the rod-heads a clear round area is noticeable, especially
if the magnification is not too high. This area is considered by
Brown to be the mouth, and hence he concludes that P e r a n e m a
has a mouth distinct from the gullet entrance. Hall (1933)
argues against this view of Brown's, but does not seem to realize
that he places the mouth at exactly the same spot where Brown
places it (namely, at the rod-heads) and that it is he himself
who is in error in considering this spot to be also the gullet
entrance and the place of attachment of the flagellum. It is
clearly neither the gullet entrance nor the place where the
flagellum passes inside.
OBSERVATIONS ON PROTOZOA
53
It is my opinion that the clear spot at the rod-heads between
these and the falcate trichite is not an opening and is not the
mouth. This view is based on observations on the feeding of
P e r a n e m a . I have never seen any evidence of passage of
food into the animal at the rod-heads, and I am reasonably
certain that the food is taken in by way of the gullet entrance.
The clear spot at the rod-head seems to result from the close
proximity of the heads to the periplast at this point.
My observations, therefore, in the main agree with those of
Brown, except that I find no mouth other than the gullet entrance. The diagonal striations of the periplast figured by both
Brown and Hall have been regularly seen, particularly in the
anterior part of the animal. No trace could be detected of a
second flagellum said by some authors to be present, and I
strongly doubt its existence. I find the contractile vacuole to
be larger than either Hall or Brown figure it and at diastole
almost to equal the size of the reservoir, which it partially
overlaps (Text-fig. 7). I confess, however, that I was not able
to come to any definite decision regarding the vacuolar system.
It frequently seemed to me that there is a partially contractile
reservoir distinct from the gullet base (which it covers) and that
a small contractile vacuole may discharge into the former, that
is, that there are three vesicles—gullet base, reservoir, and
vacuole—instead of two.
P e r a n e m a was observed to feed on many occasions, but
its extreme contortions during the process and its tendency
to get the food object between itself and the slide, and so present
its rear end to the observer, render exact observations difficult.
Contrary to the statement of Brown, that P e r a n e m a will
grow only in cultures containing other euglenoid flagellates,
P e r a n e m a flourished for months in my cultures, which were
devoid of other flagellates. In agreement with Hall, I found that
P e r a n e m a eats algae, general debris, and zoogloea containing
algal and other minute cells. Specimens were frequently seen
with ingested diatoms. Apparently P e r a n e m a feeds only on
quiescent organisms, plant, or animal.
The feeding behaviour depends on whether the animal attacks
a food object larger or smaller than its own diameter. In the
54
LIBBIE H. HYMAN
former case, if the object is too large to be swallowed whole, the
P e r a n e m a sucks the contents. Tannreuther (1923) states that
in so doing the animal first pierces the prey with its pharyngeal
rods. No one else has been able to verify this observation, and
I believe it to be incorrect. I have never seen the pharyngeal
rods protruded beyond the periplast, and I do not think they
can be so protruded or that in fact they have any power of
independent action. They move only with the adjacent soft
parts. I repeatedly saw P e r a n e m a feeding on what seemed
to be the very slow, scarcely moving A. g u t t u l a , at any rate
upon some rounded object containing gross inclusions, mostly
green algal cells. In attacking such an object the P e r a n e m a
expands the lips of the gullet entrance to an astonishing
diameter, applies the lips to the object, and sucks much after
the manner of a person sucking a fruit. The inclusions can be
seen passing down the gullet and immediately thereafter
shooting into the endoplasm where they accumulate, chiefly
in the posterior part of the body. I am quite certain these
inclusions went down the gullet. The pharyngeal rods could be
seen held to one side and taking no part in the operation. The
flagellum, bent to one side by the stretching of the gullet
entrance, keeps up a wild swirling during the ingestion, and
the sucking movements are brought about by active body
contortions.
When ingesting smaller objects or feeding on a general mat
of algae or zoogloea the animal grasps single objects by the
gullet lips, and works them down the gullet by means of body
contortions; or shovels the general material into the gullet. In
the latter case the pharyngeal rods have been seen to act as
a sort of scraper. Owing to the stretching of the gullet entrance
in the feeding process, the rods take on a diagonal position so
that the rod-heads are brought into the more projecting or
upper lip. This lip, stiffened by the rod-heads, has been seen to
shovel material into the gullet entrance. I therefore agree with
Brown that the pharyngeal rods of P e r a n e m a serve the same
purpose as the trichites of the gymnostomatous ciliates, that is,
they aid in getting a grasp on food and they strengthen and
support the food-ingesting passage. I suggest that they be
OBSERVATIONS ON PROTOZOA
55
called simply trichites. They cannot move of themselves but
can be moved in definite ways by the feeding movements of
the anterior end.
Hall reports the formation of food vaeuoles in the bottom of
the gullet, but I was unable to observe such a process. In at
least many cases food objects, easily followed because of their
green colour, were seen to pass immediately through the gullet
into the endoplasm, where they accumulate in the posterior half
or two-thirds of the body.
III.
SUMMARY.
1. In A. v e s p e r t i l i o Penard (1902) there is a continuous
d e n o v o formation of water vaeuoles, each of which in turn
increases to maximum size, acquires a stiffened wall, lags into
the rear end of the amoeba, and discharges apparently through
pressure from the gelating ectoplasm. No continuity exists
between successive vaeuoles.
2. The observations indicate that the contractile vacuole
system serves to discharge water necessarily taken in in hypotonic medium. The more fluid the ectoplasm and the thinner
the surface membrane, the less permanent is the vacuolar
system of a protozoan.
3. In P . t r i c h o p h o r u m Ehrb. the gullet opens and the
flagellum passes inward at the anterior tip of the animal, not
at the heads of the two pharyngeal rods. No evidence was found
for the existence of a mouth at the rod-heads as maintained
by Brown (1930), and the gullet entrance is believed to be the
mouth.
4. In feeding on large objects P e r a n e m a applies the
greatly expanded gullet lips to the object and sucks in the
contents; smaller objects are swallowed whole. The pharyngeal
rods are not used to pierce the food object, but serve as trichites
to assist the upper or more protruding lip in grasping objects
and to stiffen the food-ingesting parts. Food is swallowed by
peristaltic contortions.
56
LIBB1E H . HYMAN
L I S T OF E B F E R B N C E S .
Botsford, E. F.r(1926).—"Contractile vacuole of Amoeba", 'Journ. Exp.
Zool.', 45.
Brown, James (1910).—"Freshwater Rhizopods from English Lake District", 'Journ. Linn. Soc. Zool.', 30.
Brown, V. E. (1930).—"Cytology and binary fission of Peranema",
'Quart. Journ. Micro. Sci.', 73.
Day, H. C. (1927).—"Formation of contractile vacuoles in Amoeba
proteus", 'Journ. of Morph.', 44.
Doflein, F. (1907).—"Naturg. der Protozoen V. Amobenstudien", 'Arch.
f. Protistk.', Suppl. 1.
Gelei, J. von (1933).—"Wandernde Exkretionsvacuolen bei den Protozoen", 'Arch. f. Protistk.', 81.
Hall, R. P. (1933).—"Ingestion in Peranema", ibid., 81.
Hall, R. P., and Powell, W. N. (1927).—"Morphology and systematic
position of Peranema trichophorum", 'Trans. Amer. Micro. Soc.', 46.
(1928).—"Morphology and binaryfissionof Peranema", 'Biol. Bull.',
54.
Haye, A. (1930).—"Exkretionsapparat bei den Protisten", 'Arch. f.
Protistk.', 70.
Herwerden, M. A. (1927).—"Umkehrbare Gelatinierung durch Temperaturerhohung bei einer Siisswasseramobe", 'Protoplasma', 2.
Howland, R. B. (1924).—"Experiments on contractile vacuole of Amoeba
and Paramecium", 'Journ. Exp. Zool.', 40.
Ivanic, M. (1933).—"Nahrungsaufnahme einiger Susswasseramoben",
'Arch. f. Protistk.', 79.
Lapage, G. (1922).—"Cannibalism in Amoeba vespertilio", 'Quart. Journ.
Micro. Sci.', 66.
Metcalf, M. M. (1910).—"Studies upon Amoeba", 'Journ. Exp. Zool.', 9.
Penard, E. (1902).—'Fauna Rhizopodique du Bassin du Leman.' p. 92.
Schaeffer, A. A. (1918).—"Three new species of Amebas", 'Trans. Amer.
Micro. Soc.', 37.
Tannreuther, G. W. (1923).—"Nutrition and reproduction in Euglena",
'Arch. Entw. mech.', 52.
Wailes, G. H. (1912).—"Freshwater Rhizopoda and Heliozoa from the
States of New York, New Jersey, and Georgia", 'Journ. Linn. Soc. Zool.',
32.
Wailes, G. H., and Hopkinson, J. (1919).—'British Freshwater Rhizopoda
and Heliozoa.' Vol. IV. Supplement to the Rhizopoda, p. 10.