EXPERIMENTS ON THE SURFACE LAYER OF THE AMEBA
In the preceding chapters we have discussed the streaming of the
endoplasm in various representative species of ameba, and its
transformation into ectoplasm at the anterior end. We have observed that
the details of streaming are not quite the same for any two species of
ameba, and that in consequence the character of locomotion also is
specific for every ameba. All the observations prove that movement in
ameba is always associated with streaming, and streaming (in locomotion)
with ectoplasm formation. It follows therefore that the form of movement
observed in amebas depends invariably upon the streaming of the
endoplasm accompanied by the formation of ectoplasm.
There is however another element which, although it appears to be a
consequence of ectoplasm formation, must nevertheless be included in any
account of ameboid movement because of the light it is bound to shed on
the physical processes concerned in streaming. This element is the thin
outer layer which separates the water in which the ameba lives from the
ectoplasm. It is the properties of this layer to which we may now direct
our attention.
That such a layer exists was indicated by observations of Bütschli (’92)
and Blochmann (’94), as already mentioned; but neither of these authors
stated definitely whether they considered a third layer actually to
exist or whether the ectoplasm as such moved forward. Jennings (’04), as
has been pointed out, concluded that no third layer exists and that the
particles clinging to the outsides of amebas, which are carried toward
the anterior end, are carried by the ectoplasm. Gruber (’12) concluded
however that an outer layer exists, composed of gelatinous substance,
which moves ahead at about the same rate as the ectoplasm (p. 373).
According to Gruber’s view the outer layer is a permanently
differentiated layer of material. Schaeffer (’17), on the contrary calls
it a layer of protoplasm, which moves forward faster than the forward
advance of the ameba.
It is a very simple matter to demonstrate the existence of this layer.
Although any insoluble non-toxic substance of low specific gravity such
as carmine or soot, when reduced to very small particles and mixed with
the water in which the amebas to be examined live, will cling to the
outside of the ameba so that the movement of the outer layer can be
observed; in my experience the best as well as the most convenient
substance to use is the dried flocculent colloidal sediment from ameba
cultures, rubbed to powder with the ball of the finger. This powder
swells up in water into flocculent masses which are large for their
weight and do not show such active Brownian movement as particles of
carmine or india ink, and they consequently adhere more easily to the
ameba. Moreover no foreign substances are thereby introduced into the
water.
[Illustration: Figure 13. _Amoeba sphaeronucleosus._ In locomotion. Note
the nucleus, contractile vacuole, ectoplasmic ridges. This ameba is not
known to form pseudopods. Length, 120 microns.]
Of the more common species of amebas, those with the firmer ectoplasms
are the most favorable for studying the movements of the outer layer. We
may therefore first take up several observations on _Amoeba
sphaeronucleosus_ (Figure 13). This ameba resembles the more common _A.
verrucosa_. It is about 120 microns long and is usually of an oval shape
in locomotion. It is more active and less disturbed by jars than
_verrucosa_.
Figure 14 represents a _sphaeronucleosus_ with a small particle attached
to the middle of the upper surface of the ameba. As the ameba moves
forward, shown by successive outlines, the particle likewise moves
forward, but, as will be observed, at a more rapid rate. Measuring the
distance from particle outline 1 to 4, and from ameba outline 1 to 4, it
is seen that the rate of movement of the particle compares with the rate
of movement of the ameba as 2.48 to 1.
[Illustration: Figure 14. Illustrating the movement of a particle on the
upper surface layer of an _Amoeba sphaeronucleosus_. Length of the
ameba, 120 microns.]
[Illustration: Figure 15. An _Amoeba sphaeronucleosus_ with two
particles attached to its upper surface film, one in the middle and one
at the side. _a_ moved 2.6 times as fast as the ameba while _b_, lying
nearer the side, moved only 1.9 times as fast as the ameba. Length, 100
microns.]
Particles lying near the side do not move forward as rapidly as those
lying in the middle. Figure 15 shows two particles, one of which, _a_,
lying near the middle of the ameba, moved 2.6 times as fast as the ameba
advanced in the region of the particle; while particle _b_ moved only
1.9 as fast as the ameba in front of the particle. The speed ratio of
particle _a_ to particle _b_ was as 1.26 to 1.
[Illustration: Figure 16. Illustrating more rapid movement of the
surface film in the middle of _Amoeba sphaeronucleosus_ than near the
edge. The vertical lines connecting the particle with the ameba outlines
were drawn only for convenience of reference. Length of ameba, 120
microns.]
Figure 16 shows a particle lying still more to the side than in the
preceding figure. In the first six stages the particle moved 1.85 times
as fast as the ameba. The particle then came to the edge. From stage 7
to 10 the particle moved more slowly than the ameba. At stage 11 the
particle had come to lie in the posterior half of the ameba, where the
tendency of the surface layer is to travel toward the middle of the
upper surface. In stage 12 the particle had gotten away from the edge of
the ameba and already shows a gain in speed. From stage 13 to 16 the
particle moved again about 1.83 times as fast as the ameba. But at stage
16 the edge was reached with a consequent decrease in speed of the
particle.
The direction of the path described by a particle carried on the back of
an ameba depends upon what part of the ameba is most rapidly forming
ectoplasm. That is, the particle tends to
[Illustration: Figure 17. Illustrating the different speeds with which
particles move when attached to the surface film of an _Amoeba
sphaeronucleosus_, depending upon their location. Particle _a_ moved 3.5
times as fast as the ameba and _b_ 2.7 times as fast. Length of ameba,
110 microns.]
move toward that part of the anterior edge that is advancing most
rapidly. Figures 17 and 18 illustrate this point. Figure 17 shows an
ameba with two particles on its back, and with an unequally advancing
anterior edge. Particle _a_ moved more rapidly than _b_ because: (1) it
was moving away from a more rapidly receding posterior region; (2) the
right anterior edge was advancing more rapidly than the left anterior
edge; (3) the particle was nearer the anterior edge. The rapidly
advancing right edge in stage 4 accounts for the veering of the particle
_a_ to the right. The more rapid advance of _b_ from stage 3 to 5 is due
to the remoteness of the anterior right edge, which, because of its
nearness to particle a pulls on it to a much greater extent than on
particle _b_. That is to say, when a particle lies somewhere _between_
two rapidly growing regions on the anterior edge, leading in different
directions, that particle is attracted to the edge less rapidly than a
particle lying immediately back of either advancing region. As may
readily be observed each change in speed or direction of movement of the
particle _b_ finds its explanation in the amount and location of
ectoplasm formation at the time. Large particles like _a_ do not so
readily reflect changes in the direction of pull of the surface layer.
The rapid rate of movement of particle _a_--3.5 times as fast as the
ameba--finds its explanation in an actively advancing anterior edge that
was unusually wide. Particle _b_ moved at a slower rate, 2.7 to 1. It
started from near the posterior edge where it moved comparatively slowly
for a short distance.
[Illustration: Figure 18. Illustrating the effect on the path of a
particle attached to the surface film of an _Amoeba sphaeronucleosus_
when the ameba changes its direction of movement. From stages 3 to 5 the
ameba veered to the right, also the particle. From stages 6 to 9 the
ameba turned sharply to the left, and this change of direction was
reflected in the movement of the particle. Length of the ameba, about
120 microns.]
Figure 18 shows more pronounced changes in the direction taken by a
particle attached to the back of an ameba. The change in direction at
stage 6 was caused by a wave of ectoplasm thrown out at the left side,
and cessation of movement at the anterior edge. At 7 a small wave was
thrown out at the anterior edge and a large wave on the left. At stages
8 and 9 the direction of the particle was again a response to the waves
of ectoplasm thrown out at the left anterior edge, which thus became the
anterior end.
[Illustration: Figure 19. Illustrating the rapid movement of the upper
surface of an _Amoeba sphaeronucleosus_ under the most favorable
conditions. The particle moved 3.56 times as fast as the ameba. Length
of the ameba, 130 microns.]
The movement of particles on the under side of an _Amoeba
sphaeronucleosus_ depends upon what part of the ameba is attached to the
substratum. Where the ameba is attached there is of course no movement
of the surface layer and the particles remain stationary. In an ameba
attached as shown in figure 20, _a_, there was a very slow movement of
particles forward near the middle of the attached region (x), but
whether this was related to the movement of the outer layer of the upper
surface was not determined. The movement of these particles was
considerably slower than the movement of the ameba. In another ameba
attached at the anterior and posterior ends (Figure 20, _b_) no movement
of particles on the under side could be discerned. The small particles
showing Brownian movement, with the surrounding water, are dragged along
as a mass. This movement is purely mechanical, and is what would be
expected on purely physical grounds, when a more or less cup-shaped
object is moved along in water in close contact with a flat surface.
Such particles as have become attached to the surface layer on the under
side of the ameba, because of their slower movement than that of the
ameba, eventually bring up at the sides near the posterior end, as the
ameba moves along. From here they are carried forward in the manner
already described. Thus there comes about a “rotation” of particles
adhering to an ameba as described by Jennings (’04) and Dellinger (’06),
though the explanation is different from that given by Jennings (l. c.)
as we shall see further on. No case of a similar rotation of larger
particles which had sunk into the ectoplasm, as described by Jennings
(’04, p. 142), has come under my observation.
[Illustration: Figure 20. _Amoeba sphaeronucleosus_. _a_, the under side
of the ameba. The part of the ameba attached to the substratum is
stippled. Particles attached to the surface film at _x_ moved slowly
forward. _b_, the under side of the ameba, showing the attached parts
stippled. The particles suspended in the water at _x_ moved slowly
forward with the ameba. _c_, a cross section of an ameba of shape shown
in _b_, showing the ridges on the surface. Length of the ameba, about
100 microns.]
The movement of the surface layer in _A. verrucosa_ is quite like that
of _sphaeronucleosus_. Figure 21 shows a group of three particles
carried by a _verrucosa_ while changing its direction of locomotion. The
particles changed position with regard to each other and they moved at
different speeds. Particles _a_, _b_, _c_, moved respectively 2.40,
3.26, 2.85 times as fast as the ameba advanced. Other experiments
indicate that the outer layer of _verrucosa_ moves at about the same
speed, compared with the speed of the ameba, as that of
_sphaeronucleosus_.
Amebas with so-called limax-shaped bodies do not possess surface layers
that carry particles forward with the same speed as those amebas with
broad bodies. It is only occasionally that large amebas such as
_proteus_ are found in a limax or clavate shape. One of the most
favorable of the large amebas in this respect is _discoides_. It is
frequently found in clavate shape and it possesses the further advantage
in being nearly cylindrical in cross section. It is also more in the
habit of loping along the surface in the manner described by Dellinger
(’06, p. 57) so that what is observed to take place in _discoides_ in
the clavate shape, holds likewise for free pseudopods extended into the
water out of contact with a solid support (Figure 22).
[Illustration: Figure 21. Illustrating the similarity of the movement of
the surface layer of _Amoeba verrucosa_ with that of _A.
sphaeronucleosus_. A group of three particles, connected by dotted lines
for reference, change their relative positions as the ameba
(_verrucosa_) changes its direction of movement. Length of the ameba,
150 microns.]
[Illustration: Figure 22. Illustrating the movements of an _Amoeba
proteus_, after Dellinger. At _c_ in stage 2 a pseudopod is projected
which fastens itself to the substratum as shown at _c_, 3, while _a_, 2,
is pulled loose. In 4 another pseudopod is projected which fastens
itself at _d_. The ameba is not in contact with the substratum at all
points on its under side.]
In figure 23 is shown a clavate _discoides_ with a small particle
attached to its side. The particle moved forward until it came to lie at
the anterior edge, 10. The speed of the particle from 1 to 10 was 1.36
times as fast as that of the ameba, a much slower rate than was observed
in _sphaeronucleosus_. At 6 a new pseudopod was projected for a short
distance, thus increasing the amount of new ectoplasm forming in
proportion to that of the whole ameba. This change was reflected in the
increased speed of the particle, which moved 1.64 times as fast as the
ameba from 5 to 6. At 10 the anterior end again spread out and again
the particle moved faster--twice as fast as the ameba from 9 to 10.
Stages 11, 12, 13 are added to show that the particles do not tend to go
to the under surface but remain at or very near the tip. The slight
irregularity of the waves of hyaloplasm pushed out at the anterior end
accounts for the changing position of the particle after it has reached
the anterior edge. The particle remained at the edge of the advancing
ameba for several minutes after the stage drawn at 13.
[Illustration: Figure 23. Showing the movement of a particle on the
surface layer of an _Amoeba discoides_. The particle remained on the
anterior end of the ameba for several minutes after stage 13. The ameba
was about 320 microns long.]
In another observation the effect of a narrowing of the advancing tip of
the ameba is shown very well. In figure 24 the ameba was advancing with
a broad anterior end, as shown at 1 and 2. From 2 to 4, the region where
new ectoplasm was made, narrowed down very considerably. These changes
in the width of the anterior end are reflected, as in Figure 17 by a
decrease in the relative speed of the moving particle. Thus the particle
moved 1.75 times as fast as the ameba from 1 to 2 while from 2 to 4 the
particle moved only 1.27 times as fast as the ameba.
[Illustration: Figure 24. Showing the effect of a narrow anterior end on
the rate of movement of the surface. Length of the ameba, about 320
microns.]
The movement of the third layer in _proteus_ is difficult to study owing
to the formation continually of ridges, as explained on page 20. Even in
clavate shaped amebas, waves of protoplasm are pushed out on the sides
and on the tip with consequent formation of ectoplasm, so that the ameba
grows in width slowly at the same time that it grows in length. A
typical shape of a _proteus_ in clavate form is slightly tapering toward
the anterior end. This shape is maintained by gradual extension of the
sides of the anterior half or two-thirds of the ameba as it moves along.
These conditions are just the reverse of what was seen to be the case in
_sphaeronucleosus_ and _verrucosa_, where the anterior edge was wider
than any other part of the body. But _discoides_, although free from the
ridges and grooves characteristic of _proteus_, frequently has an
anterior edge that is narrower than any part of the body, thus
necessitating extension of the sides as the ameba moves forward.
Let us now see what is the effect of ridge formation upon the movement
of the surface layer. Figure 25 shows a _proteus_ and a narrow anterior
end in _proteus_ with two pseudopods and a particle attached to the side
of the ameba at 1. Both pseudopods advanced until stage 4 was reached,
but the particle was not appreciably deflected from an approximately
straight path by the small pseudopod at the other side of the ameba.
Reference to the figure shows that the particle travelled much faster
while the pseudopod on the side was extending than after it began to
retract. The particle moved 1.43 times as fast as the ameba from 1 to 4.
But from 4 to 7 the particle moved only 1.06 times as fast as the ameba.
[Illustration: Figure 25. _Amoeba proteus._ Rate of movement of the
surface layer as compared with the rate of movement of the ameba. The
pseudopod on the right was extended to stage 5; from then on it was
retracted, as indicated by the outlines. Length of the ameba, 400
microns.]
In the earlier stages the outer layer was pulled toward the tip of both
pseudopods, in the later stages only toward one, and in this lies the
explanation for a more rapid movement of the particles in the earlier,
and a slower movement in the later stages. This effect was also observed
in _discoides_, but the fact that the particle in the later stages moved
only very little faster than the ameba is due to a narrow anterior edge
and to the formation of ectoplasm in the ridges over the surface of the
ameba. The effect of ridge formation on the movement of particles
attached to the surface film is well seen when an ameba has two forward
moving regions opposite each other. Under such conditions particles
located equidistant or nearly so between such regions, move only very
slowly or not at all, the pull upon the film being nearly or quite
equal. In a similar manner the ridges which are constantly forming on a
_proteus_ are continually competing with the anterior end in their pull
upon the surface layer, thus preventing rapid forward movement.
[Illustration: Figure 26. Showing the comparative rate of movement of
the surface film over the retracting parts of the ameba. In figures 2 to
8 only a part of the ameba is shown. Length of the ameba, 500 microns.]
Figure 26 shows that the surface layer flows away from the tip of a
retracting pseudopod that is located near the anterior end. The particle
moves slowly until the body of the ameba is reached, when movement
becomes more rapid, 8, 9. This proves that the third layer moves away
from the retracting parts of an ameba, no matter how large the total
area of these parts may be in proportion to the area of new surface that
is being made. But whether the speed of the moving third layer changes
in correspondence with a larger or a smaller ratio between building and
retracting ectoplasm has not been ascertained.
Figure 27 shows that the relative positions of particles attached to the
surface layer may readily change while the ameba deploys its psuedopods.
Three particles marked _a_, _b_, _c_ and connected
[Illustration: Figure 27. A part of an _Amoeba proteus_ illustrating
what is perhaps the most characteristic quality of the surface layer of
amebas, its fluid nature. Three particles, _a_, _b_, _c_, were moving
forward along an actively growing pseudopod. In stage 2, particles _b_
and _c_ had arrived nearly at the tip of the pseudopod. A pseudopod was
then thrown out on the right, which resulted in the movement of _a_ in
the same direction, while _b_ and _c_ remained nearly stationary. Later
on this pseudopod was retracted. _b_ and _c_ were drawn back toward the
main body of the ameba while _c_ remained behind, moving only very
slowly. Thus the relative positions of these particles was completely
changed.]
by a line for convenience of reference, were in the position indicated
at 1 when the forward end of the ameba occupied the position indicated
by outline 1. As the ameba moved forward the particle _c_ gained
slightly on _a_ and _b_ for no ascertainable reason, unless it was on
account of the projection of the large pseudopod on the opposite side.
At stage 2 a new pseudopod was started on the right, which at stage 3
had grown to large size while streaming in the original pseudopod was
arrested. At stage 3 particles _a_ and _b_ retained the same position
they had in stage 2, except for a slight turning to the right. Particle
_c_ however moved across the base of the original pseudopod and on to
the middle of the new pseudopod. At stage 4 _a_ and _b_ had again only
slightly moved to the right of the position they occupied in stages 2
and 3, while _c_ moved rapidly toward the tip of the new pseudopod. The
new pseudopod was then retracted and at stage 5 the particles had begun
to move back toward the main body of the ameba. Particles _a_ and _b_
now gained considerably on _c_ because they were located further away
from the tip of the retracting pseudopod. Particles _a_ and _b_ were
drawn to the middle of the retracting pseudopod because of the
continuous enlargement of the large pseudopod on the right, below,
through which the ameba moved on.
The most important feature of this observation is the change in the
position of the particle _c_ with respect to that of _a_ and _b_. The
latter particles retained their relative positions with very slight, if
any, change, while _c_ swung around _a_ and _b_ nearly 180°, and at the
same time changed the distance very greatly between itself and the other
particles. Moreover, _b_, at stage 5 led the procession of particles,
while at stage 1, _a_ led. No further demonstration is necessary to show
that the surface layer is distinctly fluid and dynamic, and not at all
such a static structure as an elastic permanent skin, as Jennings (’04)
and Rhumbler (’14) maintained.