p^iss}^^

^>^.

THL FUNCTION OF THE NUCLEUS OF THi:. LIVING CELL

Dissertation
Submitted to the Board of University Studies

of

The Johns Hopkins University

In confornity with the resiuirements for the degree

of
Doctor of Philosophy

toy

Vernon Lynch

Baltimore, lol?.

THE FUNCTION OF THE NUCLEU3 OF THE LIVING CELL

Vernon Lynch ,
(From the Physiological Laboratory of the Johns Hopkins University)

Contents

i. Introduction Page 2

II. iiethod 2-1-

ill. Relation of the Nucleus to Moveiaent 26

IV. ourvival of the Non-Nucleated Cell 29

V. The Cultivation of Ameba by Solutions 32

VI. The Nutrition of the Non-Nucleated Cell 42

VII. Respiration in the Non-Nucleated Cell 4fi

VIII. The Effect of Temperature f^l

XI. iv'etabolic Rate in the Non-Nucleated Cell 56

All. oummary and Conclusions 61

aIII. Discussion 62

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I. Introduction.

It is a well recognized fact that the science of phys-
iology, and in fact all biological science, has not attain-
ed to that degree of exactness which is characteristic of
the sciences of physics and chemistry. To the question,
how may physiology be made a more exact science, there are
as many answers as there are methods of investigation: but
there is one line of attack, based upon simple logic and
upon the analogous development of those sciences which are
nearer the goal, which may rightly claim special attention.

Ke who surveys the development of the sciences of phys-
ics and chemistry, and especially the rapid advances of
recent years, must be struck with one phase of this devel-
opment. With the formation of the atomic theory, both sci-
ences were able to make a great advance. They were now in
possession of a unit by means of which they could conceive
of the occurrence of phenomena, explain them, and make
conjectures and even predictions. And with the subsequent
intensive study of this unit of matter, the advances in
physics and chemistry were marvelously accelerated, and
their progress was roughly parallel to the progress in the
study of the atom. Deprived of the conception and knowlege
of the atom, modern chemistry and modern physics could

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scarcely be called sciences. In a quite analogous manner,
he v/ho surveys the development of biological science can
not but be struck by the great advances which followed the
formation of the cell theory. IVhat the atom is for physics
and chemistry the living cell is for biology: and it is
not surprising that the discovery and identification of
the unit of living matter was followed by just as inarvel-
0U8 a development in the science of living matter as succeed-
ed the conception of the unit of non-living matter. But when
we turn to look for the intensive study of living matter,
and the scientific advancement which would just as surely
follow this study, v/e are doomed to some disappointment.
The intensive study of the living cell has for years been
limited tcr one method: namely, the study of the dead cell
with the hope of finding out more about the living cell.
The results which have been obtained by the use of this
method have been very instructive as far as they have gone^
but it is to be expected that this method alone would have
its limitations; and in fact it would seem that this aspect
of cell study has already been pushed as far as may profit-
ably be done. It is a logical conclusion that the biolo-
gist must now turn his attention to the more difficult task
of the direct study of the living cell: but may he not feel
confident that the:^.e studies will be rewarded by a progress
quite ev^ual to the recent progress in physical science?

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All of the phenomena occurring in living matter, the
phenomena which it is the avowed purpose of physiology to
study, are ultimately referable to changes occurring in
the living cells of which it is composed. Many of the phen-
omena occurring in living matter, such as secretion, move-
ment, irritability, and growth, can readily be referred to
the more fundamental changes in the living cells; but the
attempt to interpret rmy single property of a living cell,
such as growth, division, contraction, in terms of physics
and chemistry, or in any other way, has thus far led to no
result satisfactory to the minds of scientific inquirers.
Nevertheless, it seems that this is the task which lies
before the science of physiology on its way toward becoming
an exact science: the task of interoreting and explaining
the fundamental properties of living cells.

One who turns his attention to the direct study of the
living cell may be at a loss how to begin. It is undoubted-
ly ov/ing to the technical difficulties of the subject that
it has not already been further advanced. We are dealing
with tiny objects of an almost inconceivably delicate con-
stitution, morphologically very complex, and chemically of
far greater complexity than any other knovm substance; and
to these mysterious little objects are to be attributed
all of the. various phenomena of living matter; for all of
the activities of living matter may be ultimately referred

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to activities of its constituent units, the living cells.
Nothing will admit of so little manipulation, and all of the
lore of physical science must be drawn upon for the care-
ful disentanglement of tht maze of phenomena exhibited by
a single living cell. A tiny speck of jelly, more than three-
fourths water, it may move, feed, digest, grow, reproduce,
and respond to all manner of changes in its surroundings,
but the slightest change in a single condition of its en-
vironment may snuff out its life.

But since we must begin somewhere, and since it is the
phenomena of the living cell which we are to study, let us
start by dividing these phenomena roughly into two classes:

1. Those phenomena which are characteristic of living cells
in general, and which are exhibited by most or all living
cells. Such are respiration, growth, irritability.

2. Those phenomena which are peculiar to certain types of
living cells, and not exhibited by the great majority of
cells. Such are ameboid and ciliary movement, photosyn-
thesis, secretion, and perhaps contractility.

Now while both of these classes of phenomena must be stud-
led and are of great importance on account of their wide-
spread occurrence and significance, it seems that in the
present state of our knowlege of living cells, ignorant

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as we are of those general phenomena which all living
cells exhibit, we could most profitably turn to the study
of those phenomena common to most or all living cells. The
experiments presented in this paper represent such a study.

In various branches of biological science, progress
has been made by first studying structures md then attempt-
ing to discover the significance of these structures, and
while there is no intrinsic reason why it should, it seems
plausible that cell physiology may advance along the same

b

lines. At any rate, it can not be denied that the prolem
of the function of the -eeti nucleus, the most character-
istic structure of the cell, is one of the most alluring
problems of cell physiology, and as such it has excited
much speculation, and been the subject of some experiment-
al work.

Before reviewing the experiments of previous workers
in this field, it might be well to call attention to a few
matters of common knowlege and observation which bear
upon the problem. The most obvious fact with regard to the
nucleus is that it almost invariably occupies a central
position in the cell, and no biologically trained man,
accustomed as he is to regard the smallest acts of nature
as significant, would fail to appreciate the importance of
this fact. Reflection upon the central position of the
nucleus leads to the conclusion that the nucleus must

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play a central role in the activities of the cell, and
that it must be of great importance in the life and ac-
tivities of the cell as a whole. Moreover, when the nu-
cleus does not occupy a central position, the ex-centric
position which it does occupy nay also be of significance.
In basement membranes and other epithelial cells of higher
organisms the nucleus is found toward the side of the cell
which is closest to the blood supply, and this, together
with the fact that in other cells the nucleus may be seen
in close proximity to, or even engulfing nutritive mater-
ial, leads to the conclusion that the nucleus must play an
important role in the nutrition of the cell. The careful
provision which is made for the equal division of the nu-
cleus, and apparently for even parts of the nucleus, when
the cell divides: the complex but exact changes v/hich the
nucleus undergoes in preparation for reproduction; the care-
ful allotment of certain nuclear material from each parent
for the formation of the new individual, and the subsequent
union of this nuclear material from each side, all go to
show that the nucleus is important in the properties and
characteristics, the individuality, and even the existence
of the cell. Nature has also furnished us, in the case of
the red blood corpuscle, with the interesting experiment
of a cell without a nucleus, and we know that its life is

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limited, its days are n\imbered, just as are those of the
medullited nerve fiber which has been severed from its
nucleus.

ciuch were the facts which the older biologists had at
their disposal in the attempt to solve the proMem of the
function of the cell nucleus: and these facts together
with certain subsequent experimental developments have
led to the formation of two main theories with regard to
the function of the cell nucleus. The first and oldest
theory I have been unable to find clearly stated by any
■earlier writer than Claude Bernard: it asserts that the
nucleus is responsible for the building up processes, the
synthetic or anabolic activities of the cell. "II semble
liue la cellule qui a perdu son noyau soit st^rilise'e au
point de vue de la generation, c'est-a-dire de la synthese
raorphologisiue, et iiu'elle le soit aussi au point de vue
de la synthese chimi^iue, car elle cesse de produire des
principes imme'diats, et ne peut guere qu'oxyder et detru-
ire ceux qui s*y etaient accumules par une elaboration
ante'rieure du noyau. II semble done que le noyau soit le
germe de nutrition de la cellule; il attire autour de lui

et elabore les mati»riaux nutritifs." "Le protoplas-

ma circumnucleaire, d' autre part, renfermerait tous les
produits de 1 • elaboration synthetique du noyau, c'est-a-
dire les principes immediats destine's a se detruire et

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s'oxyder." (Ber?iard, 18P7, p525). The second and more

recent theory is attributed to Jacques Loeb, a well known

It
advocate of the theory* aftd asserts that the nucleus is

the center of oxidations of the cell, the organ where the
oxidative processes are most prominent and rapid. " It
seems to me therefore, that all the facts which are knovm
thus far very naturally support the idea that the nucleus
is the org:cn of oxidation of living matter: and that frag-
ments of cells without a nucleus are not able to regenerate
because their oxidative activity has fallen to too low a
point, ouch pieces die slowly from asphyxia." (Loeb^'^oi;.

While it may be that both of these theories fall wide
of the mark, it is well, in reviewing the experimental data
at hand, to bear in mind their relation to the two views:
that the nucleus is the synthetic organ or the oxidative
organ of the cell.

In seeking an experimental proof of the function of
the cell nucleus, only one method has been extensively
used: that of dividing the living cell into two parts, and
making a comparative study of the part v/ith a nucleus and
the part without a nucleus, the differences being attributed
to the presence or absence of the nucleus. The other exper-

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iment which suggests itself, that of isolating the nucleus
from a cell and studying the isolated organ, has been un-
successful, owing, no doubt, to the very rapid death of

a

the nucleus which occurs nfter its removal from the cell."

But the fruitful and suggestive results of studying the
chemistry of the isolated orn;an are well Imown.

Altho the nucleus has, since its discovery by Robert
Brown in l8oi, been the subject of much morphological in-
vest! ('-ati on, most observatios and experiments of a phys-
iological n:iture upon this subject are of a comparatively
recent date. The earliest experiments appear to be those

A

of K. lirandt (1887) upon the rhizoPod Actinosphaerium
Eichhornii. After division of this organism, he observed
that the pieces which contained nuclei regenerated to com-
plete individuals, while those which lacked a nucleus always
died without regent ration. Similar observations were made upon

It

plant cells by Schmitz (187P). Upon rupture of the membrabes
of cells in the alga Valonia utricularis and Siphonocladus
Wrisbergi, the protoplasm rounded up into little globules,
some of which contained nuclei and some did not. '.'/hile

"The author has frequently observed a nucleus, which

had been isolated fi*om an ameba by destruction of the
cytoplasm, undergo a rapid darkening, becoming distinctly
more opaque in a 'few seconds. While the significance of
thi3 phenomenon in not understood, it may be the result
of an oxidative change terminating in the death of the
organ.

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the nucleated pieces soon forced ripw rrembranes and con-
tinued to live, tic 'on-nucleated pieces died withou-tlref;en-
erating a membrane. These observations were extended and
confirmed by the careful experiments of Nussbaum (1RR5).
With a fine needle he divided the infusoria Oxytricha and
Gastrostyla into a nucleated and non-nucleated piece. The
non-nucleated piece died in the course of a few days,
without any manifestation of regeneration; but the nuclear
part was regenerated into a complete cell, and continued
to grow and reproduce by division. From these experiments
it is evident that for the phenomena of growth and division,
and for the regeneration of lost parts, the nucleus is ab-
solutely essential: and upon these facts all subsequent
investigators are agreed. The observations of Gruber
(1884) upon Spirogyrat those of Balbiani fl8P2) and Verworn
(1888) upon many infusoria; those of Tofer (18^0), Stole
(j-QlO), and the author upon Ameba proteus: and many other
direct and indirect observations lead inevitably to the
conclusion that without the nucleus cell grov/th and cell
division as well as the regeneration of lost parts are
absolutely impossible. Wiatever additional function the
nucleus may have, its relation to the phenomena of growth
is beyond question.

Altho there is no such uniformity of opinion with
regard to the part played by the nucleus in other activ-

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ities of the cell, the disagreement is, in some cases,
readily understood. The phenomena of movein^ent, being the
ones most readily observed, have been studied most extens-
ively. Nussbaum observed that in the enucleated pieces of
infusoria the cilia continued to beat up to the onset of
death. In like manner Balbiani and verworn observed that
the normal activity of the cilia continued after the nu-
cleus had been removed, until death changes set in. '.Vith
regard to ameboid movement, however, there is some differ-
tnce of opinion. Gruber observed that enucleated pieces
of Araeba proteus ceased making normal movements soon after
the operation, and this was confirmed by Hofer (IRPO) and
by Willis (ioiG). 3tolc (iQiO) however, apparently using
a different variety of Ameba proteus, observed normal
movements for days after removal of the nucleus. But the
most striking experiments upon the influence of the nu-
cleus upon normal movement were made by Verworn, using
Liacrymaria olor, in which the movements are very complex.
This flask-shaped ciliate extends its long neck and waves
it about, the cilia upon the head end beating actively,
and then suddenly retracts like an elastic band, to re-
peat the performance later. If stimulated it swims away.
Non-nucleated parts of this organism, regardless of which
part is selected, exhibit the same characteristic -ind

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complex movements and re sponge to stimuli is n'hen they
formed a pirt of the intact org-mism. We are thus led to
conclude that altho removal of the nucleus does in some
cases interfere with norni.nl 'Movement, at any rate perfect-
ly normal movement and irritability are 4uite possible
withou*. the presence of the nucleus.

Closely related to the phenomena of movement are the
phenomena of respiration, and there are many interesting
observations which bear more or less directly upon the re-
lation of the nucleus to eel"! respiration. Aside from the
fact that, in aerobic organisms, normal movement implies
normal respiration, the activity of the contractile vac-
uole indicates that respiration is not interfered with by
remov tl of the nucleus. There is a gene^'a4r «greemen% that
the function of the contractile vacuole is that of a res-
piratory organ, aiding in the removal of carbon dioxide
from the interior of such large cells as protozoa. V/hen
a protozoon is divided into two parts, the part which
lacks a contractile vicuole soon Tovtr^ one, regardless of
whether it contains a nucleus or not, Balbiani , Hofer,
Stole, Penard). The vacuole contained in the non-nuclear
fragment pulsates witl^ about the same frequency as that in
the nuclear fragment, but gradually becomes slower with

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the inevitable changes of death (Balbiani, Hofer, Stole ).■;'?

That the non-nuclear red blood cell respires is a
well known fact, and Tashiro (I9l7) has recently demonstrat-
ed that the non-nuclear nerve fiber produces carbon dioxide
and that this cai-bon dioxide production is increased by
stimulation and decreased by anesthetics, .'."oreover, meas-
urements of the respiration of nerve ganglia showed that
the part of the cell which contained the nucleus produced
no more carbon-dioxide than the non-nuclear p-:'rtions, and
possibly not as much.

Loeb ;'i905), however, basing his opinion in part upon
the supposed presence in the nucleus of nucleo-proteins
which contain iron, suggests "that the nucleus is the organ
of oxidation of living matter; and that fragments of cells
without a nucleus are not able to regenerate because their
oxidative activity has fallen to too low a point'.' But he
adds "I do not beleive that without the nucleus all processes

The regeneration of the contractile vacuole does not,
as might at first sight anpear, belie the fact stated
above: that regeneration of parts is impossible without
the nucleus; for the contractile vacuole is not to be look-
ed upon as a structural element of the cell, such as the
oral groove, but rather Is its appearance to be regarded as
the expression of a certain condition in the protoplasm.
The presence of certain granules in the locality where
the vacuole has repeatedly appeared (Metcilf , 1910) may
be the result rather than the cause of its appearance
at this point, for the granules evidently accumulate at
the edge of the formed vacuole.

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of oxidation cease in the protoplasm." In support of this
view, Osterhout (lPx7) has recently published some inter-
esting observations upon the cells of the leaf of the
Indian Pipe. He finds that these cells become dark when
they are injured, owing to the oxidation of a pigment
which they contain; but that the darkening, and hence the
oxidation, occurs most readily in the nucleus. Prom this
he concludes that oxidation is most rapid in the nucleus of
the uninjured cell. These experiments will be discussed
later.

That it is the protoplasm rather than the nucleus
which is concerned in respiration is indicated by tlie
experiments of Demoor (1895). Under conditions which
are known to depress oxidations, such as cold, and lack
of oxygen, or the presence of anesthetics, lemoor brot
the protoplasm of spirogyra cells into a condition of
inactivity; but the activity of the nucleus continued as
shown by its repeated divisions. In like manner the nu-
cleus of a frog's leucocyte continued its ameboid movements
after the cytoplasm had been rendered inactive by chloroform. "

Child (1015) calls attention to the fact that anes-
thetics affect most ^uickly those regions which have the
most rapid respiration.

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With regard to the ability of the non-nucleated cell
to secrete, there are some interesting observations upon
Ameba proteus. In normal movement as seen upon a glass
slide, this organism is attached to the bottom and can
thus move from place to place. This ability to stick to
the bottom, as well as to stick to food particles, seems
to depend upon a secretion which is present upon the sur-
face of Ameba. Without this secretion the ameba is not able
to move from place to place, but can only stretch out pseud-
opods in different directions. That the production of this
secretion is in some way dependent upon the presence of
the nucleus is shovm by the fact that the secretion is
absent shortly after the removal of the nucleus, as shown
by Hofer.

Further evidence upon the question of the ability of
the enucleated cell to produce secretions is found in ex-
periments upon digestion. Hofer, Gtr^lc, and others have
observed more or less normal digestion of food particles
535^H after removal of the nucleus: but observations made
upon the digestion of particles ingested before enucleation,
and to which the digestive secretions have already been
added, are little more to the point than observations
made upon digestion in a test tube. Some crucial experi-
ments v/ere performed by Hofer, hcvever. Hofer removed the

(16)

nucleus from ameba proteus as soon after the ingestion
of a Paramecium as possible. If the operation vas perform-
ed immediately the Paramecium wis not killed, but escaped,
owing doubtless to the lack of certain secretions. But even
when the operation was not performed until the death of the
Paramecium, the digestion was much more slow and incomplete
than normal. Hofer concludes that "protoplasm can produce
secretions only with the aid of the nucleus."

Other substances than digestive secretions are no
longer formed after the removal of the nucleus. Thus Klebs
(1887) observed that when ::ells of Spirogyra virere treated
with strong sugar solutions, and snail globules of protonlasn
were formed as the result of the plasmolysis, only those
fragments which contained nuclei made new cellulose mem-
branes. Later Verworn (1888) showed that pieces of Poly-
stomella crispa, a protozoon which secretes about it a
snail shell of lime, were able to form a new shell pro-
vided they contained a nucleus, but the pieces which lack-
ed a nucleus, tho showing the normal ameboid movement,
were quite unable to form a new sliell. The relation of the
nucleus to the form.atlon of these substances is further
born out by the observations of Haberlandt (1887): that
whenever there is a localised formation of new cell wall

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in plant cells the nucleus is founi in close juxtaposition
to the point of this formation. Xorschelt (1889) also
observed that in the egg cells of the water beetle, Dyt-
iscus marginalis, where the eggs are supplied with nutri-
tive granules by surrounding cells, the nucleus sends
pseudopod like processes in among these granules. There
are also numerous observations of an exchange of granules
between nucleus and cytoplasm.

In apparent contrast t-. these observations, "^alla
(1890) round that in the root hairs and pollen tubes of
some phanerogamic plants, enucleated bits of protoplasm,
when selected from a growing part, were able to form a new
cell wall: the "cellulose reaction." These observations may
not, however, be as contradictory as they seem, for the
importance of selecting a growing part suggests that we
may be dealig with an after effect of the nucleus: that
the formative substances produced by the nucleus may have
been already present in the protoplasm at the tine of its
separation.

ouch an explanation is not applicable, however, when
we come to the f ormati m of starch. In the experiments of
Klebs quoted above, the interesting discovery was -lade
that non-nuclear protoplasm, nrovided it contained chlor-
ophyl, was ^uitc capable of forming starch in the light-

(IB)

These observation were later confirmed by Gerassimoff
(1890). This is the one outstanding case of an organic
synthesis which occurs quite Independently of the presence
of a nucleus. But it is to be remembered that this syn-
tliesis is performed by certain specific organs , the chlor-
oplasts, which are themselves not unlike nuclei; and in
fact that one step in this syntliesis may be performed by
chlorophyl which has been extracted from the leaves
(Usher and Priestly).

'While the series of experiments which have been report-
ed show clearly that the nucleus is necessary for the prop-
er performance of certain functions by the protoplnsm, and
for the growth and continued life of the nrotoplasT^, the
converse fact must not be overlooked: that the protonlasm
is necessary for the performance of the functions of the
nucleus, and even for its continued existence. Thus in the
formation of new substances by the cell, the activity of
the cytoplasm is quite as essential as that of the nucleus-
This is strikingly shown by the experiments of Demoor cited
above. 'Alien by means of cold, narcotics, and the like, he
inhibited the activity of the cytoplasm, the activity of
the nucleus continued, but the formation of a new cell wall
was prevented. Moreover, Verworn (18^2) showed that nuclei
isolated from ihalassicolla nucleata soon disintegrated to

(19)

a granular mass. The nuclei ilso died if they were inject-
fcd into norm il or enucleated protoplasm, which indicated
that the nucleus is very -lUicHy injured when reiioved from
its protoplasm.

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II. Method.

Of the different methods which have been employed to
solve the problem of the function of the cell nucleus, the
one which has given the most illuminating results is the
method of extirpation: removing the nucleus and comparing
the enucleated cell with a normal nucleated cell. In order
that the nucleus may be removed from a cell, or that a cell
may be cut in half, it is necessary or expedient that a
cell of considerable size be selected; but at the same
time it must be a cell which can be kept alive or cultivat-
ed for several generations if necessary. The cells which
meet these requirements most exactly are certain unicell-
ular organisms, certain protozoa. Among such a large and
diverse group of organisms, it is not difficult to find
one which is peculiarly suited for the oroblem at hand.
For the experiments which follow, Ameba proteus vas select-
ed. Owing to the slow movements and absence of shell in
this organism, it can readily be cut in half: or if desir-
ed the nucleus nay, after some practice, be removed with
as little as one tenth or less of the protoplasm. Moreover,
the lack of differentiation in Ameba is a decided advantage,
for we are cei-tain that in removing oart of the protoplasm

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with the nucleus, we are removing no important organ of the
cell. The one disadvantage in the use of this fascinating
living cell is the difficulty of cultivating it. Like all
rhizopods amebas are difficult to raise, but many invest-
igators have succeeded in keeping healthy cultures in the
laboratory for years.

In view of the difficulties which are so often met in
cultivating amebas, it may not be out of place to adi one
moi'e method to tliose which have been described for rearing
these valuable experimental animals. It is almost liter-
ally true that each investigator has a method of raising
amebas, but that no one can use it except himself. This is
not surprising when it is seen that "water" is employed
in making the cultures, with little regard for its purity.
kVhile amebas nay flourish in the "water" of one locality,
they may tiuickly die in the ^vater of another locality.
This difficulty was avoided by the use of distilled water.
The method which follows is based upon one which has been
employed by Miss Hyman of the University of Chicago-

The prejudice of many biologists against the use of
distilled v/ater, and the prevalent notion that it is toxic
are not supported by experiments upon the toxicity of
distilled water. (Daniel . 19'-')'

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A small amount of hay, including both stalks and leaves,
is cut into pieces ;ibout three inches long; four grafts of
this material is then olaced in a large beaker. To this
is added 0.2 gram of dry bread crumbs and 500 cc of dist-
illed water. The material is boiled for a few minutes,
and then poured into flat dishes to a depth of 1 to 2 cms.
The dishes are covered to keep out dust, and if water
evaporates from them it can be replaced by distilled water.
After the infusion has cooled, several pipettes full of
fluid are added from a culture which contains healthy
araebas, or from a pond or stream where amebas are found.
In doing this, care is taken to av-^id taking up any of
the sediment, for it is the sediment rather than the
super-natant fluid which is apt to contain enemies to
Ameba. This in^oculation may be repeated, and in the course
of two weeks the culture becomes cloudy .vith bacteria,
zoogloea, and small ciliates and flagellates: the food of
Ameba. A small dish of the culture fluid is now seeded
with amebas, being careful to avoid large organisms such
as Crustacea and worms. If living amebas are not present
a week later, more of the fluid containing amebas should
be added. This culture is used to innoculate the rest of
the infusion. The addition of a little hay occasionally
will keep the amebas present in great numbers. The room
should not be allowed to become very hot or very cold.

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Ihe amebas were transferred to different media by-
means of a fine glass c-^pillary pipette, the sharp end
of which had been made smooth by passing thru a flame.
In most of the experiments the animals were kept in drops
of fluid upon slides with a depression at each end- The
slides were kept in a moist chamber in a room whose temp-
erature did not vary greatly from 20 °C. For cutting the
amebas in half, or cutting the nucleus away from the rest
of the cell, a fine glass needle was dravm off at a slight
angle from the end of a glass rod. The cells were divided
under a binocular microscope.

dince the animals would not live long in the tap
water '^f this city, a search was made at the outset for
a solution in which they could be kept in healthy, active
condition. Ten amehas were removed from a culture, freed
from food material and debris, and transferred to the solu-
tion to be tested. Each day they were examined and trans-
ferred to a fresh solution, the experiment being continued
until all of the animals were dead. It appeared at first
that distilled water was the most satisfactory medium
that could be obtained. Thus ten amebas lived (without
food) for an average of 13.2 days in distilled water,
whereas animals kept in tap water, for example, soon assum-
ed an abnormal appearance, and could seldom be kept alive
longer than one v/eek. But it was later discovered that

(24)

the distilled water was greatly improved by the addition
of one tenth the volume of spring water. Not only was the
average life somewhat longer, (14^/p days), but the con-
dition of the animils during this time was greatly improv-
ed. In the distilled water, the animals soon cease to be
attached to the bottom, and are hence unable to m ;ve from
place to place in the normal manner; but in the ''lO"^ spring
water", after it became saturated with air, they remained
attached up to a few days before death. The spring water
used contained a trace of calcium, but not as much as was
contained in the tan witer. In the following experiments
the amebas were, unless otherwise noted, kept in W^ spring
water.

(25)

Relation of the Nucleus to Movement.

The movement of normal amebas in a suitable mediujn,
such as the dilute spring vater, when placed upon a glass
slide, was found to be of the limax type. The ameba is
attached to the slide by means of some sticky secretion,
and flows steadily forward, sending out pseudopods at the
anterior end, often from alternate sides. Frequently
araebas are found which are not attached to the slide, and
such amebas may project pseudopods in any direction, but
are unable to move from place to place. These animals
will in time, however, adhere to the slide, unless they
are abnormal or the medium is unsuitable. IVhen these
active amebas are stimulated roughly, as by pressure with
a glass needle, they retract into a more or less spherical
shape, and small droplets of protoplasm may be seen project-
ing from the surface.

The effect of amputation of the nucleus upon the
movement of the cell may be seen from the following typ-
ical experiment:

5:81 P.M.- An active ameba which was attached to the
slide was divided into two approximately
e^ual parts, the nucleus remaining in the
posterior half. Both parts went into the
typical condition of sli'iulation.

5:25 P.i/:.- ooth pieces have put out pseudopods and are
active, but the non-nuclear part, which was
cut from the anterior end, is much nore active
than the nuclear and posterior oart.

3:27 P.f,:.- Both pieces are moving in the typical limax

(26)

fashion, but the movement of the non-nucleated
piece h:"T.s become perceptibly sloviev.
5:28 P.i-i.- The non-nucleated piece ceases its progressive
moveiTients and slowly retracts into a somewhat
corrugated sphere. Only an occasional very
blunt pseudopod is now produced, and slight
a^^itation shows that the fragment is no longer
attached to the slide. The nucleated piece

differs from a normal ameba only in size.

*

The retraction into the spherical shape is an invar-
iable phenomenon, and is of such a definite character that
the tine of its appearance nay be accurately determined.
Thus in 15 amebas which were so divided that the nucleus
remained in the posterior half, the retraction occurred
at an average of 9 minutes after the onrration. To the
observer it resembles strongly the response to stimulation,
except for the absence of the protoplasmic droplets mention-
ed above. The protoplasm is still capable of movement, as
is shown by transferring it to another medium, such as
distilled water, to which it responds by a change of shape.

This effect of amputation of the nucleus upon move-
ment is not always permanent. If a number of amebas are
cut in half, and the nucleated and non-nucleated pieces
observed each day, on the first or second day after the
operation it will be noticed that many of the non-nucleated
fragments are moving in the typicxl limax fashion, and in
fact there is sometimes no great difference between the

(27)

movements of the nucleated and non-nucleated pieces,
olight agitation, however, shov/s that the non-nuclear
amebas are scarcely attached to the slide; the slightest
distur'iance serves to dislodge the'^. This fact, which
must be due to the failure to produce the sticky secretion,
is probably partly responsible for the difference in movement
between the nuclear and non-nuclear cells.

The fact that a non-nucleated ameba may, under any
conditions exhibit normal movements, justifies us in
concluding that the nucleus is not necessary for movement.!
In ameba, however, movement is affected in some indirect
way by removal of the nucleus.

(28)

The Survival of the Non- Nucleated Cell.

All observations made upon cells which have been
deprived of their nuclei prove conclusively that non-
nucleated protoplasm is destined to ^pow- without growing
or dividing. But in most cases, particularly in ameba, it
has been impossible to get this protoplasm to take in food,
and even that food which is already included in the proto-
plasm is but imperfectly digested. Consequently it is fair
er to compare the survival of an enucleated cell with one
which has been isolated without food, rather than with an
actively feeding cell. When this is done, it is found
that the non-nucleated oell lives almost as long as the
nucleated one, provided there is no difference in size.
If an ameba is cut into unequal parts, the nucleus, owing
to its central position, will usually be found in the
larger part. Since large fragments live longer than small
ones, special car^e must be taken to avoid a difference in
size.

Fifteen amebas were cut in half, and both halves
were kept without food in either distilled water or in
water taken from the culture. The number of days which
each piece lived after the operation is given in the
following table:

(29)

Survival of Survival of
Medium nucleated non-nucleated

part part

Distilled

water

7

days

6 days

II

7

7

II

10

6

ti

9

4

tf

7

4

t«

5

3

ti

9

6

Culture

water

7

5

II

8

6

»

6

6

If

9

7

H

11

8

If

7

8

n

7

7

tf

/■erage

7

days

7

A^

7.

?F

Gdays

It v/ill be noted that in some cases the non-nucleated
fragments lived as long as those which contained nuclei.
In fact, by cutting the ameba in such a way that the piece
containing the nucleus was smaller than the piece which
lacked it, the non-nucleated cell could be made to outlive
the nucleated one. Thus ten amebas were divided in such
a way that the nucleated piece was from one-half to one-
fourth the size of the other. The results are tabulated
below :

(30)

Survival

of

Survival of

micleat(

D'i

non-nucleated

part

part

6

10

7

7

S

13

6

5

5

6

6

7

7

7

6

8

7

7

6

10

Average- 5.7 days 8 days

We may conclude that a cell deprived of its nucleus

may survive as long as a cell deprived of food.

(31)

The Cultivation of Araeba by Substances in Solution.

The apparent resemblance of enucleated amebas to amebas
deprived of food suggested that some of the phenor^ena ex-
hibited by the non-nucleated cell might be the result of
starvation, and have no direct connection with the absence
of the nucleus. Accordingly, an attempt was made to supply
the amebas with adequate food substance.3. Since it v/as not
possible to get the enucleated organisms to take in food
particles, an effort was made to provide a medium in which
amebas could be nourished by the absorption of substances
in solution.

It has been pointed out that no artificial synthetic
medium has ever been provided which was adequate to nourish
an animal cell ( Surrows and Neymann, loi"^), althc the
blood and lymph apparently constitute a natural synthetic
medium for the cells of higher' organisms. Even cells cul-
tivated in vitro depend upon the autolysis of neighboring
cells for their nourishment, and the attempt to cultivate
protosoa in nutritive organic media has met with complete
failure (Biedermann, 191G, pf;78; Doflein, p ro8). In the
light of recent experiments in nutrition, however, it
seems possible that such negative results rna-Y tie due

(52)

to the failure to supply certain substances which are
necessary for proper nutrition. An attempt was therefore
made to provide an adequate medium for the primitive an-
imal cell, Ameba proteus.

Since amebas were found to move m.ore norm.ally and
to live longer in distilled v/ater to which had been added
one- tenth the volume of spring water, this "10''^ spring
water" was used in making up all solutions. 'Various car-
bon compounds, including several sugars, were nov/ tested
with the hope of finding a source of energy.

The method used vas as follows: A number of amebas
were selected from the same culture, and freed from food
and bacteria by transferring repeatedly to fresh solutions
of lOfJ spring water. They were then transferred to slides,
piecing 10 in 10"^ spring water as a control, and 10 in
each of the solutions to be tested. The anim.als were
examined and counted each day, washed in water, and trans-
ferred to fresh solutions to prevent the development of
bacteria. The most promising substances found were the
hexoses, glucose and levulose, t>-e effect of which may
be seen by the following experiments:

(33)

Nvunber of amebas living
Date

Water 1% Glucose ifo Levulose

Feb. 7

20

20

20

8

20

20

20

9

le

20

20

10

15

20

20

11

15

20

20

12

15

20

20

15

15

20

20

14

15

20

20

15

15

20

20

16

15

20

19

17

15

20

Ip

18

15

20

18

19

15

20

18

20

11

18

17

21

11

17

17

22

11

17

15

25

9

17

15

24

7

17

15

25

6

16

14

26

6

16

12

27

4

12

11

28

o

12

8

lar. 1

2

11

8

2

2

10

2

6

0

4

1

4

5

1

S

6

1

0

7

i

8

0

Average
life

12.6 days

21.8 days

19.4 days

Not only did the amebas live longer in the sugar
solutions, but during the early part of the experiment
they were much more active. At the end of a week, hov/ever.

(34)

many of the amebas began to take on an opaque appearance,
and ceased normal movements. The substance which was

u

formerly a food now act^ as a poison. Child (1015) has
shown that susceptibility to poisons increases during
starvation.

Altho these cultures v/ere not sterile, it v/as possible
to keep the bacteria fi-om becoming numerous, and thus pre-
vent their becoming an appreciable source of food for the
amebas.

Having found a carbohydrate food for Ameba, a search
was now made for a source of nitrogen. In such a primitive
cell, the possibility that simple compounds of nitrogen
may be used, at once suggests itself. Of various compounds
of nitrogen which were tried, the best results were ob-
tained with ammonium nitrate and urea; and of the.se two,
urea was much the better, owing to its comparatively lov/
toxicity. The effect of these substances is seen in the
follov;ing experiment. (The comparatively short life of the
animals is to be attributed to the use of an inferior
culture) .

(35)

Number of a^^ebas living

Date 1"^ Glucose 1'^ Glucose 1*^ Glucose -^

■^0.1'^ urea O.Ol'^ NH4NO5

Feb. 24

10

cJay-3

10

(ik^jfes

10 d-ftjfcs

25

9

10

8

26

9

11

8

27

9

12

8

28

6

1 2

8

Mar. 1

5

12

8

2

5

15

8

4

12

8

4

4

12

8

5

4

12

8

6

4

10

8

7

• 4

10

8

8

4

10

8

0

4

8

7

iO

4

6

7

11

4

6

7

12

4

6

4

13

S

5

3

14

2

4

2

15

1

3

2

16

1

5

1

17

1

3

0

21

0

1

22

^

27

0

Average

life

9.

. 1 days

16.

.6 days

12.9 da

Vr

That these compounds are of some use to the amebas
is indicated by the increased length of life when they
are added to the solution; but that they are not an ad-
eiiuate source of nitrogen is indicated by the fact that
the animals eventually die and that no growth can be

(56)

detected. The shrinkage caused by the glucose would
render the detection of grov/th difficult even if present.

Of special interest, however, are the three cases
of cell division seen in the urea solution. Altho hundreds
of amebas have been kept under daily observation until
they were completely disintegrated, no single case of
cell division was ever observed unless food substances
were supplied; and with tv/o possible exceptions, no divis-
ion v/as observed unless a source of nitrogen was added.
On the other hand, divisions have been repeatedly observed
in solutions containing urea or certain amino acids. That
the division is not simply the result of the stimulating
action of the urea upon the nrotoplasm is shown by the ef-
fect of urea solutions to which no glucose is added. In
such solutions, tht amebas not only do not divide, but
they die more quickly than in water alone. Urea in the
absence of glucose is simply a mild poison, but w^hen urea
and glucose are used together, the animals live longer,
have a more normal appearance, and may even reproduce.
The necessity of using the two substances together indicates
that they are built up or combined to form some more com-
plex substance which is of use to the organism.

if this is a true c ise of organic synthesis, it woulci

(57)

be interesting to see whether it occurs in the cell which
has been deprived of its nucleus, in order to thrw light
upon the supposed synthetic function of the nucleus.
This point is taken up later.

Having failed to obtain growth with simple compounds
of nitrogen, the organisms were nov/ supplied with amino-
acids. The use of single amino-acids was not promising,
oome reproduction v.-as obtained with a saturated solution
of tyrosine, but the animals soon died. A mixture of
amino-acids was then prepared in the following way:

Five grams of Hammarsten's casein was heated for
twenty hours with 100 cc of a molecular solution of sulph-
uric acid. The hydrolysis was perfor^.ed on a water bath
in a flask fitted with a reflux condenser. At- the hydrol-
ysis, when the solution no longer gave the biuret reaction,
a saturated solution of barium hydroxide was added until the
reaction v/as slightly alkaline, and the solution boiled to
remove ammonia. Dilute sulphuric acid was added until very
slightly acid, and the solution filtered hot. The barium
sulphate precipitate, which had adsorbed much of the humus
substance, was not washed. The solution was exactly neutral-
ized with s'odium hydroxide, and the resulting solution,
whose volume v/as 700 cc, had the following properties:

(58)

A clear, straw-colored fluid, with a bitter-3weet taste.
The reactions for barium, calcium, iron, reduced sulphur,

and tryptophane were negative; phosphate v/as present, and

react f OK
the xantho-proteic -«^-r4- was positive. When exposed to the

air, bacttria develop^_^with remarkable rapidity. The solu-
tion v/as not toxic to amebas, even when evaporated to one
half the volume, but evaporation to one fifth the volume
resulted in a solution which was slightly toxic.

This solution of ^.mino acids is lacking in glycine,
which is absent in casein, and in tryptophane and cystine,
which have been destroyed.

In cultivating amebas in this solution, it was dif-
ficult to prevent the development of bacteria, but a meth-
od was devised which served to exclude them almost com-
pletely. The amebas were washed five to ten times in
sterile water, and the slides upon which they v/ere kept
were boiled. The culture solutions were kept in small
flasks fitted with capillary tubes. By inverting the
flask and warming with the hand, a few drops could be
placed upon the slide. After using, the flask was boiled

The toxicity of amino acids observed by Burrows and
Neymann (1917) upon embryonic chicken cells was probably
due to the relatively concentrated solutions used.

(39)

and a test tube placed over its neck. The cultures v/ere
examined d-iily, the amebas washed in sterile water, and
transferred to new solutions.

The hydrolysed casein solution proved to be a fairly
good medium in which cases of cell division v/ere occasion-
ally observed, but there was never any growth, and the
changes which preceded death could be seen in about ten
days. The addition of glucose resulted in some improve-
ment, but it did not prevent death.
Number of amebas living.

Date

Hydrolysed

Hydrolysed

casein

casein +
0.2'^ glucose

May 1

5

5

2

5

6

b

6

6

4

6

6

5

6

7

6

6

8

7

6

8

8

Dying

8

9

Discontinued

P

10

8

11

8

12

8

15

8

14

7
Dying

Am.ebas healthy
Somewhat abnormal

Exp. discontinued

Since this death might be due to the absence of some

foodstuff, various substances were added to the "diet".

(40)

The addition of various salts was tried, but no improve-
ment was observed. Cystine and tryptophane, so i^nport-
ant in the nutrition of highei- animals, were added to
the solution in concentrations which were shov/n not to
be toxic. A small amount of milk was added to provide the
unknown "accessory" food substances and certain salts.

Hydrolysed casein

+

1'% glucose

Date

-1-

0.

.1'% tryptophane

-^

0.

.0?'''^ cystine

-+-

0.

.2^ milk

May 2Z

5

24

5

25

6

26

6

27

6

28

6

po

6

50

6

51

6

une 1

2

6

5

5

4

3

5

6

1

7

0

No adequate synthetic medium w.-is found for Ameba
proteus. The failure may be due to some peculiarity
of the cell, such as its enormous size, or a low
permeability.

(41)

The Nutrition of the Non-Nucleated Cell.

Since, as v/aa shovm above, ^■ilucose prolongs the life
of amebas, and apparently acts a3 a food, it would be
interesting to know whether it has a similar effect upon
the cell from which the nucleus has been removed.
To determine this, the nucleus was removed from a number
of amebas, care being taken to remov^e as little protoplasm
as possible. Under favorable circumstances it is possible
to cut the nucleus from the cell vnlthout removing more
than one- tenth of the protoolasm. Some of the amebas were
now transferred to water, while others of equal size were
kept in glucose solutions. The usual precautions were
taken to prevent the development of bacteria.

Number of amebas living
Date

Water O.S's glucose

Dec

9

9

4

9

o

5

9

9

6

9

9

7

9

9

8

9

9

Q

9

9

10

9

9

11

8

8

12

4

7

15

5

6

14

o

4

15

0

rr

16

1

17

0

'1

Average life- 8.9 days 10. P days

(42)

Altho the amebas kept in glucose lived sonewhat longer
than the controls, the effect was not very striking,
oince there are great differences between different amebas,
even when selected from the same culture, a somewhat more
satisfactory experiment was performed as follo'."s: The
nucleus was removed from a numbi r of amebas, and then the
amebas were divided in half. One half was kept in water,
as a control, while the other half was kept in a solution
of glucose.

Number of amebas living
Date Water I't glucose

April 5 IP IS

4 12 12

5 12 12

6 12 12

7 11 11

8 10 11

9 9 11

10 6 10

11 o 6

12 2 4
15 0 5
14 0

Averare life- 6.0 days 7.7 days

The non-nucleated cells live longer in glucose solu-
tions, and prob-ibly use it as a food. This is in agree-
ment with the work of Rous and Turner (1^15), who used
glucose for the preservation of the non-nucleated red

(43)

blood corpuscles.

If the life of the non-nucleated cell is prolonged
by the use of glucose, is it further prolonged by the
use of urea or ammonium nitrate ?

The nucleus was removed from 2" amebas, and after
washing, ten were placed in glucose, ten in glucose + urea,
and nine in glucose + ammonium nitrate.

Number of amebas living

Date

Glucose

Gl

ucose

-+-

Glucose -t-

0.

1':^ urea

O.Ol'^ NH4NO5

Kov. If)

10

10

9

20

10

10

9

21

10

10

9

22

10

9

9

25

10

9

8

24

10

7

8

25

10

5

8

.'•-•6

Q.

2

7

27

6

1

5

28

0

2

29

4

2

Dec. 2

0

1

>j

ays

days

0

'erage li

fe- 8.

.6 d

6.

.5

8.2 days

Q

Thus in the non-nucleated cell, the addition of
urea to the glucose was not beneficial: on the contrary
it was harmful. ITie addition of amnonium nitrate v/as
without effect.

(44)

Evidence was offered above that the normal, nucleated
ameba was able to form sone combination between glucose
and urea ( or some derivative of urea ). If it is true
that the beneficial effect of glucose ->- urea depends upon
a synthesis, we may conclude that the non-nucleated cell
is unable to perform, this synthesis.

(45)

Respiration in the Non-Nucleated Cell.

It haa been suggested (Loeb, 1-^05) that the nucleus
is the organ of oxidation of the living cell; and that
peculiarities of the non-nucleated cell, such as lack of
the pov/er to synthesize or to regenerate lost parts, are
the result of a lowered oxidative activity. The non-
nucleated pieces, it is said, "die slowly from asphyxia."

If this theory is true, we should expect to find a
marked difference between the effect of depriving the
non-nucleated cell of oxygen, md the effect of depriving
the nucleated cell of oxygen. V/e should expect the cell
in which oxidations were occurring most rapidly to be
most affected by its removal, whereas the cell which was
using very little oxygen should not be gre'itly affected.
According to Child (1P15), regions in which respiration
is rapid are more susceptible to lack of oxygen, and die
sooner than regions in which respiration is slower.

The effect of depriving the nucleated and non- nucle-
ated cells of oxygen v/as investigated in the following
manner: Five or more amebas were divided into two pieces
of as nearly equal size as possible. After leaving them
for a longer or shorter period in air, they were placed

(4G)

in hanging drops, side by side, in an Englemann gas-chamher .
Nitrogen, which had been washed in 'vater, was then passed
thru the chamber, and this atnosphere was maintained until
the death of the organisms. In the third exnerinent, the
nitrogen was first bubbled from a capillary tube thru
strongly alkaline pyrogallic acid, to remove po 3Sible
traces of oxyf-en. In the first experiment, the temper-
ature of the water jacket surrounding the chamber was
raised to 2G''C. in order to accelerate the experiment.
The other experiments were performed at 20' C.

Exp. 1

Time

5:00

P.M.

9:50

A.M.

10:00

11:00

12:00

1:00

P.M.

1:20

1:30

2:00

2:25

2:50

2:55

2:45

Number of amebas living
Nucleated Non-nucleated

Average life-

Amebas

divided

Placed

in

nitrogen

7

7

7

7

7

6

7

4

7

5

6

1

4

0

5

2

. 1

0

4.2 hrs.

5.1 hrs

)1j

(47)

Time

Number of amebas living
Nucleated Non-nucleated

Lxp. 2 10:50

A.

M.

Amebas

divided

11:05

Placed

in

nitrogen

12:00

5

5

1:00

P.

M.

5

5

2:00

5

5

5:00

5

5

4:00

5

5

5:00

5

5

6:00

5

5

7:00

■ 5

4

7:50

5

2

8:30

4

2

9:00

4

2

10:00

4

1

11:00

4

1 •

12:00

4

1

8:00

A,

M.

5 •

1

9:00

3

1

9:30

3

0

10:00

3

10 : 15

1

11:00

1

12:00

1

1:00

p

.M.

1

2:00

life-

0

17.5

hrs

Average

11.7 hrs

Exp. 3 9:00

A.

Amebas

divided

10:20

Placed

in

nitrogen

11:00

5

5

12:00

5

5

1:00

P

.M.

5

5

5:00

5

4

4:00

5

2

5:00

5

2

8:00

1

9:0 0

4

1

9:50

4

0

10:00

2

11:00

2

12:00

2

6:00

A
1:

Lfe-

0
15.4

hrs

Avera.ee

7.7 hrs.

If

(48)

Thus there is a distinct diffe^-ence in the suscept-
ibility to lack of oxygen in the nucleated and non-nuc-
leated cell, but this difference is just ooposite to what
v/e should expect if oxidations were proceeding more rapid-
ly in the nucleated half. Not only does the non-nucleated
cell die more quickly when deprived of oxygen, but it is
the first to a^.sume a spherical shape and to cease putting
out pseudopods.

The opposite experiment, that of increasing the supply
of oxygen, is also of interest. Were it true that in remov-
ing the nuc lexis we have removed the organ of oxidation, and
that the cell ia slowly dying of asphyxia, it should be
possible to delay the death of the non-nucleated cell by
supplying it with more oxygen. On the contrary, it was
found that when the nucleated and non-nucleated halves of
amebas were kept in an atmosphere of oxygen, both died in
less than twelve hours, and the non-nucleated pieces were
killed quite as rapidly as those which contained nuclei.

The experiment was performed in the following
manner: Five amebas were cut in half with a fine glass
needle, and each half was transferred to a hanging drop
in a gas chamber. Oxygen (Linde Air Products) which had
been thoroly washed was now passed thru the chamber, and

f4n)

the organisms kept under observation until all had dis-
integrated. The animals were divided at 10 A.M. Oxygen
was passed thru at 10:50 A.M.

Time of death
Nucleated Non-nucleated
cell cell

1:30

2:00

2:00

2:10

4:15

3:23

5:00

5:27

6:50

4:15

5.3 hours

4.5 hours

V

The injurious action of oxygen, which is doubtless
the result of an increase in the oxidations of the cell,
took effect somewhat more rapidly upon the cell which lack-
ed a nucleus. It is difficult to see how a cell could be
killed in a few hours by an atmosphere of oxygen if its
oxidations have become depressed.

(50)

The Effect of Temperature upon the Non-Nucleated Cell.

In the experiments hitherto reported, a fairly con-
stant temperature of 20' C. had been maintained. An attempt
Wiis now made to determine the effect of temperature upon
the nucleated and non-nucleated cells, with the hope of
discovering a possible difference in the rate at which
chemical changes were occurring in the two cells. Two
methods of investigation suggest themselves:

(1) The temperature of the medium is gradually raised
until death occurs.

(2) The organisms are kept at a rather high temperature,
and observed until death occurs.

Both of these methods were used in the order named.

In the first method, amebas were placed upon a

Pfeiffer warming stage, thru which water of any desired

temperature could be passed. After dividing the ameba

into two e-iUal parts, the temperature of the warming stage

was raised at the rate of about one degree Centigrade per

minute. The rate at which the temperature is raised is

of importance, for by raising the temperature rapidly, the

organisms may be killed at a comparatively low temperature.

As the temperature rises, the at^ebas soon withdraw all of

their pseudopods and assume a spherical shape. This does

not, however, indicate the death of the organism., for if

at this point the temperature is slowly lowered to 20 C,

the animals will, in a few hours, send out pseudopods and

resume normal movement. If the temperature is raised

(51)

high enough, the cells will either disintegrate completely
or coagulate, v/ith the result that the cell boundary can no
longer be seen. The ttmoerature at which the spherical
shape was assumed, and the temperature at which death
occurred are recorded below.

Lxp.

Spherical shape assumed

Nucleated

N

on-

-nucleated

1

59= C .

38 C.

2

40-

39-'

5

40"

40"

4

srage- £9'. 5

44'^

40 ; 2

Death

Nucleated Non-nucleated

•^r C.

50

40° •
40". 2

50 C.

a

50-^

52''
48'

50 U

The effect of gradual cooling was not so easily deter-
mined. When the temperature of the fluid was lowered great-
ly and even super-cooled, there was no injury provided crys-
tallization did not take place. '.'men the solution crys-
tallised, however, as the result of adding a small crystal
of ice, the amebas were injured mechanically, and disinte-
grated as soon as the solution nelted.

out the effect of cooling could be shown in a some-
what different manner. The nucleated and non-nucleated
parts of an ameba were slowly warmed tn 44° C, and then
cooled in ten minutes to 22'^ C . Two minutes later, both
fragments disintegrated at the same time: the result of

(52)

the rapid cooling.

Thus no difference could be found in the susceptibil-
ity of the nuclear and non-nuclear fragments to a rise or
fall of temperature. The other method 'vas now employed:
keeping ameb ts at different temperatures until death occurred

Eleven amebas were freed from food particles and debris,
and divided into two eiiual parts. Both the nuclear and non-
nuclear fragments were kept in a moist chamber at an aver-
age room tem.perature of 20" G.

Number of amebas living
Date
Lxp.l. 20° C. Nucleated Non-nucleated

Oct ^

10

11

11

11

11

11

32

11

11

IS

8

10

14

7

6

15, A.M.

7

3

15,P.?.U

5

2

16

4

2

17

4

2

18

4

2

1Q,A.W.

5

1

19, P.M.

1

0

20

0

,ge life-

Pi. 2 days

4.3 days

Thus, as was shown before, the life of the non-nuclear
fragment at ordinary temperature isalmost as long as that
of the nuclear fr^agment.

Ten amebas //ere now divided and each placed in a moist
chamber which was kept in a thermostat at 30° C.

(55)

iLXp . 2 . 30° C .

Number of -imebas living
Date

Nucleated Non-nucleated

Oct. 22 10 10

7
7
4
2
1
0

22

10

23^>''-

10

25V-V-.

9

24

9

25

9

26

6

S€J-

5

27

m

29

3

30

3

51

1

1

0

Nov.

Average life- 5.2 days 2.1 days

Apparently the life of the nuclear fragments was not
shortened by exposure to high temperature, but this was
probably due to the use of healthier amebas in Experiment
2. Further experiments showed that the life of nucleated
amebas was shortened by raising the temperature, but the
life of non-nuclear amebas was shortened somewhat more.

Twelve amebas were now divided and placed in a moist
chamber at iCC The moist chamber was illowed to warm
slowly to room temperature before removing the slide for
examination.

(54)

Number of amebas living
£xp. 5. 10° C. Date

Nucleated Non-nucleated

Oct.

17

12

12

18

12

12

19

12

12

20

12

12

21

12

12

22

12

12

23

11

11

24

11

11

25

11

11

26

11

11

27

11

11

2P,

A.M.

11

9

2Q,

P.iM.

11

8

SO,

A.M.

11

5

50,

P . iVi .

11

4

SI,

A.M.

11

3

SI,

P.M.

10

2

1,

A.M.

0

1,

Xm.

S

2

life-

0 •

ige

12. G days

10. R days

llie life of both fragments was prolonged by cold in
this case, out in two other cases it was distinctly short-
ened. This may have been due to the use of different cultures

if susceptibility to high or low temperature may be
used as an index to the rate of metabolism, there is no
very great difference between the two fragments. There is
some evidence that cells in which metabolic processes are
rapid show a greater susceptibility to high temperature
than do cells with a low metabolism (Child, 1^15, p 66).
If this is true, there is some slight indication of a more
rapid metabolism in the non-nuclear fragment.

(55)

Metabolic Rate in the N on- Nucleated Cell.

llie rate at which metabolic processes are occurring
in different parts of an organism or tissue has been very
successfully studied by determining their susceptibility
to various poisons, especially to potassium cyanide (Child,
1915, 1015 j. Child has brot together a great deal of direct
and indirect evidence to show that, in general, regions of
high metabolism are more susceptible to poisons than are
regions of low metabolism. Accordingly, the susceptibility
of the nucleated and non-nucleated cell was investigated
with a view to determining how the rate of metabolism was
affected by the removal of the nucleus.

The amebas were cut in half, and one hour after the

M
operation they were transferred to a fresh — •- solution

50
of KGN upon a ho llov;- ground glass slide. The drop of solu-
tion was icm.ediately covered with a cover slip to prevent
the escape of HCN. Since this could not be entirely pre-
vented, the solution gradually became weaker, and as a

result, those amebas which were not killed in two or three
minutes usually lived for more than an hour. Jince small
fragments of ameba were found to be more susceptible than

(56)

large ones, an effort was made to divide the amebas into
equal parts. After more than 50 amebas had been treated
with cyanide, it was obvious that some unknown factor was
playing a part in the death of the organisms; for altho
the non-nucleated fragments usually succumbed first, there
were many conspicuous cases in v/hich the nucleated part
died much sooner. It seemed possible that the unknown
factor might be a difference between the anterior and
posterior end. The amebas v/ere divided v/hen they u^ere
moving along the bottom in the "limax" condition, and in
some cases the non-nuclear fragment was taken from, the
flowing anterior end, while in other c^ses it was taken
from the retracting posterior end. In order to determine
whether this made a difference, a number of amebas v/ere
divided in such a way th^t the nucleus remained in the
anterior half, while others were so divided that the
nucleus remained in the posterior half.

The number of minutes which the nuclear and non-
nuclear fragments were able to withstand treatment with
cyanide is tabulated below:

(57)

Duration of life

Nucleus in anterior end Nucleus in posterior end
Nucleated Non-nucleated Nucleated Non-nucleated

96 min

8 m:

5

34

45

15

200

200

190

190

4-5

170

260

240

1

2

290

530

580

62

6

180

r:

140

260

1

260

25

soo

15

2£0

3

2b0

140

;^2o

■ 305

87

2

230

6

185

41

255

4ro

450

250

o

445

4

100

305

1

10

1

120

1

1

0.5

410

410

550

530

320

1

1°0

min

83 min

155

10

145

5

85

1

170

167

5

5

145

115

146

2

105

2

100

1

98

7

5

S

1.

5

1

1.

Q

0.8

76

2

74

2

6

2

80

11

eo

10

75

i

0.5

12

1

Q

3

25

25

•^

Average isg. 4- min

1313 min

74.8

mm

19.3inin

(58)

ViTien the nucleus was in the anterior end, the non-
nuclear fragment died first in 1^ cases, the nuclear
fragment died first in ? cases, and in the remaining 4
cases each part disintegrated at about the same time.
When the nucleus was in the posterior end, however, the
nuclear fragment was never the first to disintegrate: the
death of both fragments occurred at the same time in two
instances, and in the remaining 22 cases the non-nuclear
fragment was the first to disintegrate. The complicating
factor is now understood. The anterior end of Araeba is
more susceptible to cyanide than the posterior end.
Essentially the same conclusion was reached by Miss Hyman
(1917) in an investigation of the metabolic gradient in
Ameba.

V«hen the non-nucleated half is taken from the poster-
ior end, its greater susceptibility to cyanide is not
very definite because of the difference which exists be-
tween the anterior and posterior parts. When this dis-
turbing factor is removed, however, by cutting the non-
nuclear fragment from the anterior end, the non-nuclear
fragment is seen to be much more susceptible to cyanide
than the nuclear fragment.

(^9)

The sane difference in susceptibility between the
nucleated and non-nucleated cells was found when they
were kept for 20 hours after the operation before treat-
ment with cyanide.

Whether we can conclude that the rate of metabolism
is increased by removal of the nucleus is doubtful. It is
possible that removal of the nucleus decreases the resistance
to cyanides for some other reason. But at any rate, the
evidence such as it is opposes the view that respiration
and metabolism are depressed by removal of the nucleus.

(60)

oummary and Conclusions.

1. An arneba from v/hich the nucleus has been removt d

may at times exhibit perfectly normal movement; in gen-
eral, however, movement is somewhat affected by removal
of the nucleus.

2. An ameba deprived of its nucleus lives almost as long
as an ameba deprived of food.

o. i:,vidence is offered that Ameba can use glucose in solu-
tion as a food. There is also evidence that Ameba can
synthesize glucose and urea, or some derivatives of
these substances, to form a product which is of nutri-
tive value.

4. Glucose is also of some benefit to the enucleated ameba, but

the supposed synthesis of glucose and urea can not be
performed.

5. The non-nucleated cell is injured more quickly by either

a lack or an excess of oxygen than is the normal nucleated

cell.
G. The non-nucleated cell is somewhat more susceptible to high

or lov/ temperature than the nucleated cell.
7. The non-nucleated cell is more susceptible to cyanide than

the nucleated cell.

(61)

Discussion.

in the introduction, the two important theories of
nuclear function were stated: the theory of synthesis and
the theory of oxidation. According to the former theory,
the non-nucleated cell is unable to construct, build up,
synthesize nev/ substances; while according to the latter,
it is more or less unable to oxidise the substances already
present. In reviewing the experiments here reported, it
is seen that they not only offer strong evidence for the
validity of the synthesis theory, but they constitute a
practical proof that the oxidation theory is not true.

From the outset there has been no satisfactory evidence
in support of the oxidation theory. It was apparently suggest-
ed by some experiments reported by Jpitzer (18^7). Spitzer
obtained from various cells a preparation of nucleoprotein
which contained a small amount of iron and which had the
property of decomposing hydrogen peroxide. From this it has
been concluded not only that all nuclei contain iron, but
even that all nucleoproteins contain iron. The latter
statement is of course false, and the microchemical evidence
in support of the former statement fMacallum, 1892) is very
unsatisfactory. 'i'he presence of iron in the nucleus was

From a very pure preparation of the sperm heads of the
Great Lakes Whitefish, the author was unable to find a trace
of iron in 0.5 gram of the dry material by the sulphocyanide
test. The ashing was done in the electric muffle and by
Neumann digestion, so that there was no question of "mas]:ed"
iron. Unpublished.

(62)

taken as an indication that the nucleus olnyed an important
role in oxidations, but the presence of this iron is ooen
to doubt. 3pitzer's cata-lase may have been derived from the
cytoplasm.

The chemical evidence for the oxidation theory is very
slight, and the physiological evidence is equally so. The
assumption that the organ which is furth^/est removed from
the supply of oxygen is the organ of oxidation is not logical
in the first place. "It should be born in mind that oxy,r;en,
in order to reach the nucleus, must penetrate a layer of
cytoplasm containing reducing substances." (R. Lillie, 1^11,
p 722). Moreover, as was sho\m in the above experiments, the
non-nucleated cell may exhibit perfectly normal movements,
oince the energy for movement in aerobic organisms is deriv-
ed from the oxidation of organic matter, it is unlikely that
oxidations have become depressed. But if there is a depression
of oxidations we should certainly expect f cf Loeb, I'^'^S)
to find some improvement in the cell when we increase its
supply of oxygen. On the contrary, as has been shown, the
cell becomes spherical and dies in a few hours. Also the
susceptibility to lack of oxygen, the susceptibility to
cyanide, and the susceptibility to high and low temperature
all indicate that respiration and rate of metabolism are
quite a'3 rapid in the enucleated cell as in the normal cell.

Recently, however, experiments have been reported which

'-35)

seem to give strong support to this theory: namely, the
experiments of Osterhout on the leaf of the Indian Pipe.
When the leaf of this plant is cut, the cells '."hich have
been injured soon become dark. Osterhout has shown that
this darkening is an oxidation, and since it occurs first
in the nucleus, argues that the nucleus is the center of

A.

oxidations. These experiments, however, admit of another
interpretation. It is reasonable to bel'elve that at the
center of the cell there is a strong reducing action, and
that very little oxygen is present. After the death of
the cell, however, oxygen, like other substances, may enter
freely, an J. in the dying cell it would be expected that ox-
idations would be very rapid in the neighborhood of the
nucleus, wliere the reducing action is greatest. In Oster-
houts experi^Tents, the cells ?/hich show the oxidative dark-
ening of the nucleus are those which have been injured by
cutting the leaf. Granted that the nucleus is the center
of oxidations in the cell v/hich is injured or dying, that
does not prove that it is the center of oxidations in the
normal living cell.

Moreover, Kite and Chambers (1012) found, in cells
which were apparently uninjured, that reduction was most
rapid in the region of the nucleus. ITie dye janus green
becomes red (or— G-o-l-o-i'4-«-&e) when reduced. Kite and Chambers
observed that this reduction occurred first at the centro-

(64)

some and second in thf- chroiiatin of the nucleus.

The experinents here reported offer strong support to
the theory that the nucleus is the organ of synthesis.
Ahen a cell is depri /cd of food, the processes of synthesis
must, to a great extent, come to a ston, altho synthesis
of intermediary products of metabolism Is still possible,
it is probably not a coincidence that a cell deprived of
its nucleus lives almost as long as a cell deprived of food.

An opportunity to put the synthesis theory to a direct
test was afforded by nutritional experiments. W>->en amebas
are kept v/fthout food, they v/ill die in from one to two
weeks, but if glucose is present in solution their life is
somewhat prolonged. It appears that the glucose is used as
a food. When urea was added the organisms lived even longer,
and in some cases divided. Urea alone, wit'-iout glucose, had
no such effect, a fact v/hich is interpret ted as meaning that
the nitrogen compund is combined by the cell with the carbon
compound. When nitrogen v/as already com.bined with unoxidised
carbon, as in the amino-acids, the addition of glucose was
unnecessary.

When these experiments were repeated upon the e-ucleated
cell, it was found that glucose was some "hat beneficial, and
was apparently used as a food. But the further addition of
urea was not only of no benefit, but actually hastened the
death of the protoplasm. Apnarently non-nuclear protoplasm

(65)

is unable to corr.blnf, carbon and nitrogen: the power of' syn-

ii

thesis is absent.

There is further evidence of the loss of the newer of
synthesis in the failure to produce tht sticky secretion by
means of vvhich Ameba attaches itself to various objects.
Normal amebas are fir'ly attached to the slide, and frequent-
ly stick to the sides of a pipette when drawn up into it.
Non-nucleated amebas, however, sh'-irtly after the repoval
of the nucl&us, are but lightly if at all attached to the
slide, and never stick to the sides of a pipette.

In conclusion, the enucleated cell rnay move, respire,
di^^est, respond to stimuli, and exhibit any activity which
is dependent solely upon catabolic or destructive processes
of protoplasm. The one group of phenomena which it never
shov.'S, ai-r. the phenomen'i of growth, and the related phenom-
ena of regeneration and division. The phenomena of growth
are essentially phenomena of organic synthesis, and the de-
pendence of grov/th upon the nucleus involves the dependence
of organic synthesis upon the nucleus.

The configuration of atoms: G-N-C, so characteristic

of the nucleic acid moleciiie, is also the configuration

v/hich occui-3 in the protein molecule at the points v/here

the amino-acids are joined. This fact may be not wit'nout
significance.

(66)

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Microgr. 4.
Bernard (1679)- Lepons sur les Phenomenes de la Vie.
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Brandt (1895)- Actinospherium Eichornii. Inaug. Diss. Halle.
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..-.:. for embryonic chicken cells. J. Exp. Med. 2^
Child (1913)- (Cyanide method) Jour. Exp. Zool. 14.

(1915)- Senescence and Rejuvenescence.
Doflein ( )- Lehrbuch der Protozoenkunde.
Demoor (1895)- Contribution a 1'6'tude de la physiologie de

la cellule. Arch.de Biol. 15, 1G5.
Gerassimoff (1890)- Einige Bemerkungen Uber die Function des

Zellkernes. Bull, de la soc Imp^r. des
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(1895)- Mikroskopische Vivisection. Ber. Naturf.
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ungen an Amoeba proteus. Arch.f.Prot. "5.
Hofer (1890)- Einfluss des Kerns. Jena. Zeit. Natur. 24, 105
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Science, N.3. 56, G39.
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Korschelt (1907)- Regeneration und Transplantation. Jena.
Lillie, F.(1895)- Smallest Parts of Stentor Capable of Re-
generation. Jour. Morph. 12, 239.
Lillie, R. 3. (1911)- Physiology of Cell Division. J.Moroh. 22.
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Hech. 8, G89.
(1905)- Studies in General Physiology. II. 505.
Macallum, A.B.(l892)- On the demonstration of the presence

of iron in chromatin by michrochemical
methods. Proc . Royal Soc. London, 50.
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Morgan (1901;- Regeneration of Stentor. Biol. Bull. 2, 311.
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Arch. f. Prot. 3, 44.
^Palla (1890)- Beobachtungen uber Zellhautbildung an des

Zellkerns Beraubten Protoplasten. Flora, 73.
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(67)

'Penard (1912)- Amibes du groupe Terricola. Amputation of

Nucleus. Arch. f. Prot. 28, 78.
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der Siphoniocladiaceen. Fest. d. Nat .Ges. Halle
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(68)

Vernon Lynch was born in Baltinore on Fay 17, IR'^'S.
After a preliminary education in the public schools of
Baltimore, he entered the Johns Hopkins University in
1910, and was graduated as a Bachelor of Arts in 1914.
The year 1914-15 he spent as a graduate student in
Physiology at Johns Hopkins, and the following year was
Fellow in Physiological Chemistry at the University of
Chicago. In 1916, he resumed his studies at the Johns
Hopkins University as an assistant in physiology and
candidate for the degree of Doctor of Philosophy.

4^^

^

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^

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