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THE DECENNIAL PUBLICATIONS OF
THE UNIVERSITY OF CHICAGO

THE DECENNIAL PUBLICATIONS

ISSUED IN COMMEMORATION OP THE COMPLETION OP THE FIRST TEN
YEARS OP THE UNIVERSITY'S EXISTENCE

AUTHORIZED BY THE BOARD OP TRUSTEES ON THE RECOMMENDATION
OP THE PRESIDENT AND SENATE

EDITED BY A COMMITTEE APPOINTED BY THE SENATE

EDWAED CAPPS

STABE WILLABD CUTTING BOLLIN D. SALISBUBY

JAMES BOWLAND ANGELL WILLIAM I. THOMAS SHAILEE MATHEWS

CAEL DAELING BUCK FEEDEEIC IVES CAEPENTEE OSKAE BOLZA

JULIUS STIEGLITZ JACQUES LOEB

THESE VOLUMES ARE DEDICATED

TO THE MEN AND WOMEN

OP OUR TIME AND COUNTRY WHO BY WISE AND GENEROUS GIVING

HAVE ENCOURAGED THE SEARCH AFTER TRUTH

IN ALL DEPARTMENTS OF KNOWLEDGE

STUDIES IN GENERAL PHYSIOLOGY

STUDIES IN GENERAL
PHYSIOLOGY

JACQUES LOEB

FORMERLY OF THE DEPARTMENT OF PHYSIOLOGY

NOW PROFESSOR OF PHYSIOLOGY IN THE

UNIVERSITY OF CALIFORNIA

THE DECENNIAL PUBLICATIONS
SECOND SERIES VOLUME XV

PART II

CHICAGO

THE UNIVERSITY OF CHICAGO PRESS
1905

o

Copyright 1905

BY THE UNIVERSITY OF CHICAGO

TABLE OF CONTENTS

PART I

I. The Heliotropism of Animals and its Identity with

the Heliotropism of Plants - 1

II. Further Investigations on the Heliotropism of Ani-
mals and its Identity with the Heliotropism of
Plants - 89

III. On Instinct and Will in Animals 107

IV. Heteromorphosis - 115
V. Geotropism in Animals 176

VI. Organization and Growth - 191

VII. Experiments on Cleavage - 253

VIII. The Artificial Transformation of Positively Helio-
tropic Animals into Negatively Heliotropic and
vice versa - - 265

IX. On the Development of Fish Embryos with Sup-
pressed Circulation 295

X. On a Simple Method of Producing from One Egg
Two or More Embryos Which Are Grown
Together 303

XI. On the Relative Sensitiveness of Fish Embryos in
Various Stages of Development to Lack of Oxygen
and Loss of Water - 309

XII. On the Limits of Divisibility of Living Matter 321

XIII. Remarks on Regeneration 338

XIV. Contributions to the Brain Physiology of Worms 345
XV. The Physiological Effects of Lack of Oxygen - - 370

TABLE OF CONTENTS

PART II

XVI. The Influence of Light on the Development of

Organs in Animals - 425

XVII. Has the Central Nervous System Any Influence

upon the Metamorphosis of Larvae? 436

XVIII. On the Theory of Galvanotropism 440

XIX. The Physiological Effects of Ions. I - 450

XX. On the Physiological Effects of Electrical Waves 482

XXI. The Physiological Problems of Today - 497

XXII. The Physiological Effects of Ions. II - 501

XXIII. Why Is Regeneration of Protoplasmic Fragments

without a Nucleus Difficult or Impossible ? 505

XXIV. On the Similarity between the Absorption of Water

by Muscles and by Soaps - 510

XXV. On Ions Which Are Capable of Calling Forth

Rhythmical Contractions in Skeletal Muscle 518

XXVI. On the Nature of the Process of Fertilization and
the Artificial Production of Normal Larvae
(Plutei) from the Unfertilized Eggs of the Sea-
Urchin 539

XXVII. On lon-Proteid Compounds and Their Role in the
Mechanics of Life-Phenomena.— The Poison-
ous Character of a Pure NaCl Solution - 544

XXVIII. On the Different Effects of Ions .upon Myogenic
and Neurogenic Rhythmical Contractions and
upon Embryonic and Muscular Tissue - - 559

XXIX . On the Artificial Production of Normal Larvae from
the Unfertilized Eggs of the Sea -Urchin
(Arbacia) - 576

XXX. On Artificial Parthenogenesis in Sea-Urchins 624

XXXI. On the Transformation and Regeneration of Organs 627

XXXII. Further Experiments on Artificial Parthenogenesis

and the Nature of the Process of Fertilization - '-638

TABLE OF CONTENTS

XI

XXXIII. Experiments on Artificial Parthenogenesis in
Annelids (Chsetopterus) and the Nature of the
Process of Fertilization - -

XXXIV. On an Apparently New Form of Muscular Irrita-
bility (Contact-Irritability?) Produced by Solu-
tions of Salts (Preferably Sodium Salts) Whose
Anions Are Liable to Form Insoluble Calcium
Compounds

XXXV. The Toxic and the Antitoxic Effects of Ions as a
Function of Their Valency and Possibly Their
Electrical Charge -

646

692

708

XXXVI. Maturation, Natural Death, and the Prolongation
of the Life of Unfertilized Starfish Eggs
(Asterias Forbesii) and Their Significance for
the Theory of Fertilization .728

XXXVII. On the Production and Suppression of Muscular
Twitchings and Hypersensitiveness of the Skin
by Electrolytes 748

XXXVIII. On the Methods and Sources of Error in the

Experiments on Artificial Parthenogenesis - 766

INDEX - - - - - - . ' . . . - 773

PART II

XVI

THE INFLUENCE OF LIGHT ON THE DEVELOPMENT
OF ORGANS IN ANIMALS1

I. EAKLIER EXPERIMENTS

Two GREAT series of experiments which Nature herself
has made are at our disposal for answering the question as
to what effect light has on the development of animals;
namely, the intra-uterine development, and the development
of animals living in caves. The fact that intra-uterine
development goes on in complete darkness proves that the
formation of the embryo and its organs, histological differ-
entiation, and considerable growth can occur and continue
for a long time in the absence of light. As far as animals
living in caves are concerned, some of them differ from the
same forms which live in the light in the development of
single organs, such as eyes, antennse, and pigment. It has
not, however, as yet been proved that this peculiarity of the
cave inhabitants is a direct effect of the lack of light upon
their development; but granting that it is the direct result
of lack of light, it follows from a summation of the facts in
hand that where light has any direct effect whatsoever on
development, it evidently makes itself felt only upon the
development of individual organs and not upon the develop-
ment in general.

It is strange that, notwithstanding the defmiteness of
these facts, experimental work on the influence of light on
the development of organs in animals has been directed
mostly to the question whether light promotes or inhibits
development and growth of animals in general.

iv, Vol. LXJII (1895), p. 273.
425

426 STUDIES IN GENERAL PHYSIOLOGY

It is not to be wondered at that the various investigators
arrived at diametrically opposite results.

Edwards states that frogs' eggs do not develop and soon
die when inclosed in a dark box, while the development
takes place in an open box which is exposed to the light,
According to Edwards, even the young larvae require a longer
time to develop in the dark than in the light. Edwards's
statements are based on very few experiments. Dutrochet
repeated the experiments of Edwards, and found that
where the supply of oxygen was sufficient and the tempera-
ture was the same in the two boxes, the eggs of Batrachians
developed as well and as rapidly in the dark as in the
light. These facts indicate that in Edwards's experiments
the eggs suffered from lack of oxygen and exposure to a low
temperature.

Be"clard published a short communication on the influence
of light on the development of the eggs and the larvae of
flies. He placed the eggs under colored bell-jars, and found
that after four or five days development was most advanced
under the violet and blue jars, and least under the green. I
cannot understand the experiments of Be"clard, as fly larvae
hatch in about two days in summer, and up to this time their
size depends on the size of the eggs, since further growth
takes place only when the larvae find food in which to bury
themselves. The statements of Be"clard regarding the influ-
ence of light on the production of carbon dioxide in animals
are also doubtful.

It is rather strange, though characteristic, that in the
scientific literature of our subject one frequently finds serious
mention of the investigations of General Pleasanton, made
on six pigs. The general put three pigs into a stall with
violet windows, and three into a stall with ordinary windows.
While the three pigs exposed to violet light gained 398
pounds in four months, the others gained during the same

THE INFLUENCE OF LIGHT ON ORGANS 427

time 386 pounds in weight. From this observation Pleasan-
ton concluded that violet light is favorable to the growth of
pigs. General Pleasanton's book is printed in blue type, and
gives an explanation of all natural phenomena, from love
down to the activity of a volcano.

Emil Young made a series of experiments on the influ-
ence of colored light on development. He concluded that
violet light hastens to a certain degree the development of
frogs' eggs, and the growth of embryos, while green light is
fatal to or greatly retards development. Young's results are
incomprehensible. The larvae of frogs develop naturally in
daylight, and the latter contains more green light than light
which has passed through a green screen, and should accord-
ing to Young be fatal. We might, perhaps, assume that
some other light counteracts the effect of the green light
sufficiently to do away with this fatal effect. From Young's
experiments, however, this does not seem to be the case. It
is quite possible that other conditions (such as the develop-
ment of micro-organisms) affected the results of Young's
experiments, which lasted through several weeks.

Driesch used monochromatic light and carried out the
same experiments as Young on freshly fertilized eggs of
Rana, Echinus, and Planorbis, and found, in all cases, that
light "has no influence on the segmentation or the forma-
tion of organs; under otherwise similar conditions these
phenomena occur with the same velocity in darkness, in
white, in green, in violet, or in other lights."

In my experiments on heteromorphosis in Naples, I
observed that the polyps of Eudendrium racemosum are
positively heliotropic, and that the number of polyps which
develop is apparently dependent on the intensity of the light.
It seemed that fewer polyps were developed in weak light
than in strong light. This accidental observation led me to
study the influence of light on the development of organs

428 STUDIES IN GENERAL PHYSIOLOGY

iu Eudendrium racemosum more closely in Woods Hole.
The results of these observations are briefly reported in the
following pages.

II. NEW EXPERIMENTS

1. The species of Eudendrium studied in Woods Hole
has the same name as that in Naples — namely, Eudendrium
racemosum; it is, however, not certain that the two forms
are identical. The following statements hold for the form
in Woods Hole. When fresh stems of Eudendrium are put
into an aquarium, all the polyps soon fall off, probably due
to unavoidable injury in collecting and handling the mate-
rial. In the course of a few days, however, with a good
supply of oxygen and a sufficiently high temperature, new
polyps are developed. It was the dependence of this new
development on light which was studied.

A large quantity of vigorous colonies was collected each
time. Long stems were picked and put in separate vessels,
ten being distributed into each vessel, all of which contained
an equal quantity of sea- water. Each of the stems usually
formed from ten to twenty polyps. The different vessels
were exposed to various kinds of light. In each experiment
I therefore dealt, not with the development of a single
polyp, but with a large number of them. I thought it
necessary, furthermore, to make another set of control ex-
periments by exposing the same stems successively to differ-
ent kinds of light.

Experiment 1. — On August 8 a number of stems of the
same culture of Eudendrium was divided as equally as pos-
sible between two vessels, in the manner described above.
One of the vessels was exposed to diffuse daylight; the other
was placed in a dark box which was ventilated every even-
ing. The supply of oxygen was the same in the light as in
the dark, and the temperature was always the same in the
two vessels.

THE INFLUENCE OF LIGHT ON ORGANS 429

On August 14 over fifty polyps had developed in the
vessel which was exposed to light, while in the vessel kept
in the dark not a single polyp had been formed. The
experiment was continued until September 1; the polyps
thrived and increased in number in the light, while in the
dark not a single polyp had yet been formed. The stems
which up to this time had been in the dark were now
exposed to the light. On September 6 — that is to say, in
five days — several polyps developed on each stem. The
number of polyps increased from day to day. The same
stems, which in three weeks had been unable to form a
single polyp in the dark, developed a great number of them
in five days when exposed to the light. The control ani-
mals in the light had developed polyps from the first.

Experiment 2. — On August 16 the stems of a new colony
were divided equally among three vessels, two of which were
placed in the dark and one in the light. As usual, numerous
polyps were formed in the light in the course of five days;
no polyps were formed at first in the dark. This experi-
ment was also continued until September 1. By that time
no polyps had yet developed on the stems kept in one of the
darkened vessels ; two stems in the other darkened vessel had
developed six polyps. The animals were then exposed to
light; in five days all the stems had produced new polyps.

Experiment 3. — On August 25 one half of a Eudendrium
colony was placed in diffuse light, while the other half was
put in the dark. On September 1 a large number of polyps
had been developed in the light ; but in the dark only roots
and no polyps had been developed. Conditions remained
the same until September 5, when the animals which had
been kept in the dark were exposed to the light. On the
following day they were accidentally killed.

These experiments show that light favors the develop-
ment of polyps in Eudendrium ; that no polyps, or only very

430 STUDIES IN GENERAL PHYSIOLOGY

few, are developed in the dark ; that darkness does not, how-
ever, interfere with the development of roots.

2. It was of interest now to establish which rays of the
visible spectrum favor the development of polyps. The
effect of light on plants is known to be very strikingly a
function of the wave-length. Assimilation and in part the
formation of chlorophyll are pre-eminently functions of
the long wave-lengths. The heliotropic phenomena are
essentially a function of the blue rays. According to Sachs,
the ultra-violet rays are of special importance in the forma-
tion of blossoms in certain plants. I have shown that the
short light-waves are most effective heliotropically in animals
also. From this, however, no conclusions can be drawn as
to which rays influence most especially the formation of
polyps. I therefore studied this subject experimentally.
Unfortunately, these experiments were hampered because
no other means of obtaining monochromatic light were at
my disposal than the use of blue and red glass. I had
special boxes made for these experiments which were painted
black inside and one wall of which was formed by blue or
red glass. The dark-red glasses which I employed yielded a
light which was fairly monochromatic ; the dark-blue glasses
allowed some red to pass through. In the case of the light-
red and light-blue glasses the light was far from monochro-
matic.

Experiment 1. — On August 31 a large number of Euden-
drium were divided between two vessels, one of which was
placed in a box in dark-red light, the other in a box in dark-
blue light. As nearly as I could judge, the red and blue
glasses allowed about equal quantities of light to pass
through. The old polyps perished within three days, but
somewhat sooner in the red than in the blue light. On
September 4 I discovered the first new polyp in the blue
light. The number of new polyps in the blue light steadily

THE INFLUENCE OF LIGHT ON ORGANS 431

increased, but not a single one was formed in the red light.
On September 8, seventy new polyps with stems 3-10 mm.
long had developed in the blue light. Not a single polyp
had developed in the red light, but a few roots had been
formed. Conditions did not change during the following
days. After nine days not a single polyp had developed in
the red light, while over seventy thriving polyps had been
formed in the blue light.

In order to test whether the stems in the red light would
develop polyps if brought into the blue light, I substituted
a blue glass for the red glass on September 9. Two days
later, on September 11, indications of new polyps were
already apparent, and on the following day thirty-two com-
plete polyps had been formed; on the next day the number
had increased to sixty-six. From these experiments it is
seen that in the development of polyps red light acts more
like darkness, while blue light acts like mixed daylight, just
as in heliotropic phenomena.

Experiment 2. — On August 22 a large number of stems
of Eudendrium were equally divided, as in the preceding
experiments, between two vessels, one of which was placed
in blue, the other in red light. After the old polyps had
fallen off, the first new polyps appeared in the blue light on
August 27. At about the same time roots began to develop
in the red light, but no polyps. On August 29 forty vigor-
ously growing polyps had developed in the blue light, while
in the red light no polyps but only roots had developed. On
August 31 the culture in the blue light had formed a forest
of new, well-developed polyps, while the culture in the red
light had only developed several roots. The culture in the
blue light had also developed a few roots. On August 31 I
replaced the red glass by blue. On September 11 the first
new polyps began to form, whose number from now on
steadily increased.

432 STUDIES IN GENERAL PHYSIOLOGY

This time I also made the reverse experiment. The
polyps formed in blue light were exposed to red light (the
blue glass was replaced by a red one on September 6). After
five days all the newly developed polyps had perished. By
September 13 only a few diminutive polyps had developed.

Experiment 3. — On August 25 eight stems of Euden-
drium culture were placed behind light-red glass (which
allowed also some blue light to pass through), and nine
stems of the same culture behind blue glass which was not
very dark. On August 30 a number of polyps had devel-
oped, not only behind the light-blue, but also behind the
light-red glass. The light-red screen was then replaced by
a dark-red one; while the number of polyps constantly
increased in the blue light, development soon ceased behind
the dark-red screen. On September 1 the eight stems in the
red light had only sixteen small polyps, while the nine in the
blue light, which were of the same size and from the same
culture, had eighty polyps. On the following day eighteen
polyps had developed in the red light, while the stems in the
blue light were literally covered with them.

On September 5 the glasses were exchanged ; the animals
which up to this time had been behind the red screen were
now exposed to blue light, while those which had been
exposed to blue light were placed behind the dark-red screen.
The number of polyps on the stems in the blue light (which
had formerly been in the red) increased rapidly ; on Septem-
ber 9 the number had grown to 27, on the 10th to 40, and
so on. The polyps on the animals in the red light (which
had formerly been in the blue) not only did not increase in
number, but they began to die, and on the llth only a few
were left which looked sickly.

A fourth experiment corroborated the result that numerous
and vigorous polyps are formed behind a dark-blue screen,
while in red light only roots are formed.

THE INFLUENCE OF LIGHT ON ORGANS 433

We must therefore conclude that not all the rays of diffuse
daylight influence the formation of polyps equally, but that
only the more refrangible (blue) rays of the visible spectrum
favor the development of polyps, while the less refrangible
(red) rays act as darkness — a fact similar to that established
for heliotropism.

3. We may finally raise the question whether or not light
influences the development of Planula larvae. My studies in
this direction are not yet completed, but I found that in
isolated cases, in spite of an exposure to darkness for several
weeks, during which time no new polyps were formed, the
development of the larvae progressed as under normal condi-
tions; at least they were normal and not retarded in their
development. I was able to make some experiments with
the larvse themselves. These larvae are pear-shaped, and are
able to move forward very slowly by means of cilia. They
are, as I have already shown, energetically positively helio-
tropic. The blue rays are in these experiments more effective
than the red. About forty-eight hours after they begin to
swim, the larvae attach themselves to some solid base, and
within the next twelve hours their pointed ends begin to
grow and to form polyps, while their blunt ends form roots.
When lighted from one side only the whole organism bends
toward the light. The question therefore arose whether the
Planula larva can develop a polyp in the dark. This is the
case when a larva has developed in the presence of light.
When brought into the dark such a larva develops a polyp
within twelve to twenty-four hours. In this, however, we need
not see any contradiction to the other experiments, as in
these the development of the polyps required three to four
days. It would be interesting to determine whether Planula
larvae, if their whole development occurs in the dark, can
develop polyps in the absence of light.

In conclusion I wish to mention that the polyp-bearing

434 STUDIES IN GENERAL PHYSIOLOGY

and growing stems are energetically positively heliotropic.
Only that part of the stem immediately behind the polyp
bends heliotropically. While these curvatures are usually
produced in less than two hours in blue light, no curvature
takes place in dark-red light even after two days; nor do
the heliotropic curvatures appear when the polyps are cut
off. I shall return later to this and some other facts bearing
on the theory of heliotropism. A stem of Eudendrium which
is illuminated from one side only develops more polyps on the
lighted side than on the shaded side — a thing which explains
itself from the foregoing.

III. EXPERIMENTS ON FUNDULUS EMBRYOS

A large number of experiments on Fundulus embryos
show that they develop as completely and as quickly in the
dark as in the light; only the supply of oxygen must be the
same in both cases. In one experiment the eggs were kept
in the dark in a small, tightly closed vessel ; those exposed to
the light were kept in a large vessel; in this case the eggs
developed more quickly in the light than in the dark. Con-
trol experiments showed very clearly that it was not the
light, but the better supply of oxygen to the vessel exposed
to the light, which caused this difference in the development
of the eggs. Only one constant difference exists between
the eggs cultivated in the light and in the dark, and this
concerns their color. As I have stated repeatedly, a large
number of black and red chromatophores are formed in the
membrane of the yolk-sac, which gradually creep upon the
blood-vessels and surround them like a sheath. Since the
number of these chromatophores progressively increases, the
egg, if developed in the light, finally becomes very dark. In
contrast to this, the eggs kept in the dark are very light and
transparent. This difference may possibly be due to a con-
traction of the chromatophores in the dark, but I am not cer-

THE INFLUENCE OF LIGHT ON ORGANS 435

tain of this. The other possibility is that in the dark a
smaller number of pigment cells are formed. In embryos
which develop in the light toward the end of development
the pigment cells form a sheath around the blood-vessels.
When the eggs develop in the dark, only isolated chromato-
phores are found upon the blood-vessels; the vessels are for
the most part free from pigment.

I found no noticeable difference in the development of
pigment in the embryo itself. The pigment cells of the
retina, for example, developed apparently as numerously and
contained the same amount of coloring matter in the dark as
in the light. The yolk-sac alone showed the influence of
the light.

V

XVII

HAS THE CENTRAL NERVOUS SYSTEM ANY INFLU-
ENCE UPON THE METAMORPHOSIS OF LARVAE?1

GUSTAV TOENIER has just published a hypothesis which
is to explain how the acquired characteristics of parents are
inherited by their offspring. This hypothesis is as follows:

In the more highly organized animals every adaptation of a
functioning peripheral end -organ is accompanied by a corresponding
and equal adaptation in the central nervous system ; the central ner-
vous system carries the acquired characteristic to the sexual organ,
which forms with it a functional and nutritive unit, especially to
the sexual cells, in that it compels the latter to undergo similar
transformations. If the sexual cells give rise to new individuals,
the descendants inherit the acquired characteristics of the parents.2

Tornier's paper is very clear, and even though I cannot
agree with his hypothesis, I consider it important that
Tornier through his precise presentation of his subject has
directed the attention of investigators to the question of the
significance of the central nervous system in the processes
of development.

If Tornier's idea is correct, then every alteration in the
central nervous system must be accompanied by a similar
change in the end-organs. Before the appearance of Tornier's
paper I had already made a series of experiments in which
I divided the spinal chord of Amblystoma larvae in order to
determine whether in the change of the Iarva3 to the sexually
mature form the animals with the divided spinal cord would
behave as one or two separate animals; in other words,
whether in an animal with a divided spinal cord the meta-
morphosis of the /nterior and the posterior portions would
occur simultaneously as in the case of the uninjured animal.

i Archiv filr Entwickelungsmechanik der Organismen, Vol. IV (1896), p. 502.
2"tfber Hyperdaktylie," etc., ibid., Vol. Ill, p. 180.

436

THE METAMORPHOSIS OF LARV.E 437

Amblystoma is well adapted to an experiment of this kind.
The larva loses an organ at each end in the process of
metamorphosis — the three large external gills at the head
end of the animal, as well as the so-called tail fin at the
tail end. Both disappear simultaneously in normal animals
in a few days; at the same time an alteration occurs in the
pigmentation and marking of the skin. One could not state
a priori how a division of the spinal cord would influence
the processes of development, for it is well known that
immediately after the division of the spinal cord in dogs
severe changes usually occur in the condition of the skin of
the posterior portions of the body, which may lead to the
formation of abscesses, which later again disappear (Goltz).

Division of the spinal cord (close behind the cervical
region) did not have even the slightest effect in a single
instance upon the processes of development; metamorphosis
occurred just as though the animal were uninjured. This
was the more remarkable as in some cases a metamorphosis
occurred immediately after the division of the spinal cord,
while the wound was still open. I believed at first, on the
basis of these experiments, that division of the spinal cord
might accelerate the metamorphosis of the larvae, or cause it
directly. I found, however, that this was not the case. One
animal, for example, was still in the larval stage six weeks
after the operation, while the uninjured control animals had
already completed metamorphosis.

Since the experiments showed without exception that
division of the spinal cord had no effect upon the meta-
morphic processes, we must state in detail what evidence we
have to show that the cord was indeed severed entirely.
First of all the operation was made in such a way that the
separation of the two ends of the spinal cord could be seen
directly in the bottom of the wound. Secondly, the pos-
terior part of the body was paralyzed, that is to say, it was

438 STUDIES IN GENERAL PHYSIOLOGY

dragged along in the movements of the anterior portion of
the animal as though it was an inanimate mass. A pecu-
liarity exists here, of course, which Friedlander1 and I2 have
observed in worms, in a more pronounced way, namely, that
a sort of indirect co-ordination may occur between the
anterior and posterior portions of the animals in spite of the
division of the cord. Thirdly, that division was perfect was
proved through galvanotropic experiments. The constant
current brings about associated changes in the position of
the anterior and posterior extremities in the normal animal,
while these associated changes in position do not occur in an
animal in which the spinal cord has been severed.3

These observations show without a doubt that, in spite of
the division of the spinal cord, metamorphosis occurs as
though the central nervous system were intact. If Tornier's
idea were strictly correct, one would expect that the division
of the central nervous system would be followed, not only by
a division of the motor and sensory functions of the anterior
and posterior part of the animal, but also by a division of the
morphogenetic functions. Since this is, however, not the
case, this conclusion at least may be drawn, namely, that the
morphogenetic functions in animals with a central nervous
system do not depend so strictly upon the central nervous
system as do the motor and sensory functions.

The objection might be raised that the central nervous
system of Amblystoma is not highly enough developed to
decide the question of the influence of the spinal cord upon
the formative processes. Observations are, however, at hand
which leave no room for doubt that conditions are similar in
the dog. Goltz has found that all the individual milk-glands
in a pregnant bitch develop equally after division of the

i Biologtsches Centralblatt, Vol. VIII.

iPflilgers Archiv, Vol. LVI.

3 LOEB UND GARRET, II, ibid., Vol. LXIV.

THE METAMORPHOSIS or LARV.E 439

spinal cord or even after the removal of a piece.1 In this
case also morphogenetic processes are independent of the
central nervous system.

This objection may also be raised, however, that Tornier
has assumed in the development of the larvse a formative
influence of the central nervous system only upon the sexual
cells, and not upon the body cells. This objection must be
granted, but it must also be remembered, on the other side,
that it is risky to assume such a formative effect of the
central nervous system where we have no means to ascertain
the facts, while we can show that such an influence does not
exist where the facts can be studied experimentally. That a
set of muscles cannot functionate without the corresponding
segments of the central nervous system is certain, but it is
equally certain from the experiments of Goltz and Ewald
that the blood-vessels behave differently. It is, at present,
to say the least, just as probable that the central nervous
system does not influence the sexual cells as that the opposite
assumption of Tornier is true.

If it be assumed, on the other hand, that the formation of
organs, or that morphogenetic processes in general, are
determined through chemical substances which are formed
in the metabolism of the animal and circulate through the
animal, it is clear that, in spite of the division of the central
nervous system, metamorphosis must occur simultaneously in
both portions of the animal anterior and posterior to the cut.
This assumption also does away with the necessity of formu-
lating independent laws for the development of organisms
with and without the central nervous system. In various
papers I have pointed out that the facts of morphogenesis can
be explained on a chemical basis, as Sachs has first suggested.

I will not here discuss the question of the inheritance of
acquired characteristics.

i GOLTZ UND EWALD, P fingers Archiv, Vol.LXIII.

XVIII
ON THE THEORY OF GALVANOTROPISM

ON THE POLAR STIMULATION BY THE CONSTANT CURRENT OF
THE GLANDS OF THE SKIN OF AMBLYSTOMA1

1. IN experiments on the galvanotropism of adult Am-
blystomse I noticed a polar stimulation of the glands of
the skin which soon interested me more than the galvano-
tropic reactions themselves ; for I found that the glands were
always stimulated on the anodal side of the animal, and that
there exists here a similar exception to Pfltiger's law as that
which Ktihne discovered in Actinosphserium. The phenomena
were, however, of great interest in another direction. The
activity of the glands of the skin was determined in part
through a polar stimulation of the central nervous system by
the galvanic current. It could easily be shown that in this
case the central nervous system behaves as a homogeneous
whole. Both facts, the stimulation at the anode as also the
behavior of the central nervous system just mentioned, are,
of course, of significance for the theory of galvanotropic
phenomena.

The skin of the fully developed Amblystoma contains a
large number of glands which give out a mucoid secretion
when stimulated in certain ways. The secretion forms a
white layer upon the black skin. If a descending constant
current of about 3 milliamperes, having a density of about
3S, is sent through the animal, a secretion is formed upon
the anterior half oi; the head (Fig. 126, ab). Small white
dots appear, which become more distinct the longer the cur-

iPflilgers Archiv, Vol. LXV (1896), p. 308. This paper was the third in a series
of communications on galvanotropism which, however, are not reprinted in these
volumes. [1903]

440

ON THE THEORY OF GALVANOTEOPISM 441

rent is kept up ; finally a tiny mucous plug is noticed in each
of the glands.

If the current is sent through the animal in an ascending
direction, a profuse secretion occurs upon the tail (Fig.
127, cd). The amount of the secretion formed is, with the

FIG. 126

same density of current, much greater than that formed
upon the head, and within a short time a thick white layer
is formed which drops off readily when the animal moves.
The secretion may be profuse enough to render the water in
the trough turbid in a short time.

FIG. 127

If the current is passed in a transverse direction through
the animal, secretion occurs only upon one- half of the body
— upon the half directed toward the anode.

We see, therefore, that in all three cases the glands of the
skin upon the anodal side or the anodal end of the animal
are always the ones to begin to secrete. The results are the
same whether metallic electrodes or unpolarizable electrodes
are used.

442 STUDIES IN GENERAL PHYSIOLOGY

That we are, indeed, dealing with the effects of a con-
stant current is shown by the fact, first of all, that the longer
the circuit is closed, the longer does secretion last, the
greater is the amount of the secretion, and the greater
the region in which the secretion occurs; secondly, that the
secretory phenomena are brought about when the intensity
of the current is raised very slowly. The breaking of the
current, on the other hand, has no effect.

2. The next question that presented itself was whether
we dealt in this case with the effects of the current upon the
skin or the glands themselves, or whether we had to do with
a stimulation of the central nervous system. We next in-
vestigated the action of the current upon animals in which
the spinal cord had been cut. When a current was passed
longitudinally through such animals, they behaved as though
they consisted of two fully independent pieces. If we call
that portion of the animal lying anterior to the cut the
anterior animal, and that lying behind it the posterior
animal, then the end of both the anterior animal and the
posterior animal which was directed toward the anode secreted
during the passage of a current longitudinally through the
animal. In Figs. 128 and 129 S marks the position at which
the spinal cord was severed. In a descending current not
only the piece ab (Fig. 128) at the head end of the animal
secretes mucus, as in the case of the normal animal, but
also the anterior region cd of the posterior part of the
animal, which under otherwise similar conditions never
secretes in the uninjured animal. When the current passes
in an ascending direction, secretion occurs not only at the
tail (cd, Fig. 129), as in the uninjured animal, but secretion
occurs also at the posterior end of the anterior part of the
animal (a&, Fig. 129). The secretion in the latter case often
limits itself to the hindmost edge of the immediate vicinity
of the wound, and does not extend forward until the current
has passed through the animal for some time.

ON THE THEORY OF GALVANOTROPISM 448

This reduplication of the secreting regions after the
sectioning of the spinal cord is noticed not only immediately
after severance of the cord, but at any time, even eight
weeks after the operation — I have not aa yet studied these
animals for a longer time than this — after the wound has

FIG. 128

healed so perfectly that it is impossible to recognize the
position of the incision. These experiments render it very
probable that the polar secretion of the glands of the skin,
when a current is passed longitudinally through the animal,
is determined by a stimulation of the central nervous system.

FIG. 129

It also seems as though the entire central nervous system
behaves like a homogeneous whole, and as though the stimu-
lation with a constant current occurs at the anode.

3. In order further to test this possibility, I cut a number
of Amblystomse into small pieces. It was, of course, impos-
sible to keep these pieces alive, so they had to be experi-
mented upon immediately. In this way it was found that
every piece of the animal which still possesses its correspond-

444 STUDIES IN GENERAL PHYSIOLOGY

ing portion of the central nervous system shows the polar
stimulation of the glands of the skin at the anode end when a
current is passed through the piece in a longitudinal direction.
Furthermore, an interesting difference was found between
the head and the tail end of the animal in regard to the size of
the secreting region. When an ascend-
ing (homodromic) current was sent
through an isolated head, the secretion
was limited to the outer edge at the
anodal side (cd, Fig. 130) — the cut end.

FIG. 130 T „ . , *, °, ' , , .

If, on the other hand, a descending
current was sent through the head, almost the entire head
secreted (a&, Fig. 131). When an isolated tail was traversed
by a descending current, the glands of only a small region in
the neighborhood of the wound secreted (a&, Fig. 132). If an
ascending (homodromic) current was
sent through the tail, the glands in the
whole region (cd, Fig. 133) secreted. It """
might be thought that the wound has an
inhibiting effect upon the secretion of
the glands lying in., its neighborhood,
and that for this reason the isolated head secretes most freely
under the influence of a descending current, while an isolated
tail secretes most freely under the influence of an ascending
current. It can easily be shown, however, that the wound

does not play this
role, but that in
general those por-
FiG.132 a b tions of the animal

lying near the head secrete more freely and over a larger
region when a descending current is used than when an
ascending current is used, while the posterior pieces secrete
most strongly under the influence of an ascending current.
For when the head of an animal is amputated, and the pos-

ON THE THEORY or GALVANOTEOPISM

445

terior portion is cut off at a short distance behind the
anterior legs, the remaining piece (Fig. 134) has a wounded
surface at each end. The fragment, however, belongs to the
anterior end of the animal, and therefore behaves like the
head ; that is to say, under the influence of a descending cur-
rent the secretion
— covers the extensive

region ab ^Fig< 131^

FIG. 133 while under the influ-

ence of an ascending current secretion is limited to the nar-
row zone cd (Fig. 135). When the corresponding experi-
ment is made upon a piece cut from
the posterior half of the animal, a
more extensive secretion is obtained
when an ascending current is used •—
than when a descending current is
employed.

4. What becomes of the galvanic FIG. 134

secretion when the spinal cord is destroyed ? Pieces cut from
the trunk in which I had destroyed the spinal cord showed
no polar excitation of the glands of the skin when the cur-
rent was passed in a longitudinal
direction. The effect on the central
nervous system is shown in a still
more striking way when the latter is
destroyed only in part. We experi-
mented upon a piece, as shown in
Fig. 134. We convinced ourselves
FIG. 135 l first of all of the fact that secretion

occurred anteriorly in about the region ab when a descend-
ing current was sent through the animal. We then de-
stroyed the anterior part of the spinal cord (Fig. 136) from
the point S to >SX by means of a needle. When a current
was again sent through the piece in a descending direction,

446

STUDIES IN GENEKAL PHYSIOLOGY

FIG. 136

no secretion occurred at the anterior end (in the region
$$); a secretion occurred instead close behind S in the
region a&, that is to say, in the regions directed toward the
anode of the animal, which still possessed a spinal cord.
The destruction of the anterior portion of the spinal cord
SS had no effect upon the secre-
tion when the current was sent in a
homodromic direction. In both cases
secretion occurred in the region cd
(Fig.* 137). I have often repeated
this remarkable experiment, always
with the same result. It might be
thought that the destruction of the
spinal cord had an inhibiting effect upon the secretion of
those glands of the skin which are connected with the de-
stroyed portion of the spinal cord, and I was myself inclined
to believe this. When I subjected pieces of Amblystomse, in
which the spinal cord had been destroyed either entirely or
in part, to a transverse current, I found to my surprise that
secretion still occurred at the anode, it mattered not whether
the spinal cord was destroyed or not. In view of this fact
only one other assumption remains, namely,
that two sources exist for the stimulation
of the glands of the skin through the gal-
vanic current. One of these is stimulation
of the central nervous system; the second,
the direct stimulation of peripheral organs,
either the peripheral nerve fibers which go
to the glands of the skin, or the nerve end-
ings in the skin, or perhaps the glands themselves. In
dealing with a current running longitudinally we have to do
with only the first source, and polar stimulation of the
central nervous system occurs at the anode. In the case of
a transverse current we have to deal, in addition to this, or

FIG. 137

ON THE THEORY OF GALVANOTROPISM 447

entirely, with a stimulation of peripheral elements. That
these elements are not excited by a current passing longi-
tudinally may depend upon a fact which Maxwell and I
discussed more fully in our paper, "Zur Theorie des Gal-
vanotropismus,"1 namely, the orientation of the elements.

5. In the experiments just described, especially in those
in which the central nervous system is traversed by a longi-
tudinal current, the central nervous system behaves like a
homogeneous whole, one side of which is entirely in anelec-
trotonus, the other in catelectrotonus. This result differs
from those obtained by Maxwell and myself in Crustaceans.
In these it seemed as if the phenomena observed could be
explained in a satisfactory way under the assumption that
the central nervous system does not go into electrotonus as a
homogeneous whole, but that the individual elements (seg-
ments?) are each composed of a catelectrotonic and an
anelectrotonic portion, and that the effect of the current
as a whole is made up of the effects of the current upon
the individual elements (segments?). Must we now do away
with this assumption, which is most probable in the case of
the galvanotropism of Crustaceans, because in another class
of phenomena, namely, the excitation of the glands in a differ-
ent class of animals (salamanders), we find a different be-
havior of the central nervous system? I think not. I
have often convinced myself of the fact that even closely
related animals, even different varieties of the same species,
may behave absolutely differently heliotropically, geotropi-
cally, and stereotropically ; not to speak at all of our experi-
ence concerning the artificial reversal of tropisms. I also
consider it entirely possible or probable that in one class of
animals the central nervous system may behave as a homo-
geneous whole toward the current, while in another class it
may behave as though composed of a series of individual

iPflilgers Archiv, Vol. LXIII (1896), p. 121.

448 STUDIES IN GENERAL PHYSIOLOGY

elements each of which may be considered as in its own
state of electrotonus. As a support for the correctness of
this idea I quote the interesting observation of Koux, to
which this investigator has recently again called attention:

Every cell in an egg which is divided into a smaller or larger
number of cells (morula or blastula) reacts individually, giving rise
to altered electrical fields (special polarization) as long as the egg
is vigorous. If, however, the vitality of the egg is reduced by
cooling or poisoning (not sufficiently to cause death), the com-
plexus of cells reacts as a whole, in other words, like an egg which
is not yet divided into cells.1

If these two opposite conditions can occur in one and
the same animal (under only different conditions), it is per-
haps not extravagant to assume that the central nervous
system of Amblystoma may behave like a homogeneous
whole when subjected to the constant current, while the cen-
tral nervous system of Crustaceans may behave as a series of
separately irritable elements in its galvanotropic reactions.
Whether the central nervous system of Amblystoma behaves
in its galvanotropic phenomena as in its secretory processes
is a question still to be investigated.

It seems to me that our experiments on the polar excita-
tion of the glands of the skin of salamanders may lead to a
different explanation of the liquefaction of protoplasm ob-
served by Kuhne at the anodal side of ActinosphsBrium
from that which he has given.2 Kuhne compares this process
with the tetanus of contractile elements. Might we not
rather in this case be dealing with processes similar in nature
to phenomena of secretion? One can readily understand
how violent phenomena of secretion brought about through a
strong electrical current might lead to a disintegration of
the substance of a tender Protozoon, since the much tougher
epidermis of Amblystoma goes to pieces at the anodal side

1 Pfliigers Archiv, Vol. LXIII, p. 542.

2 Untersuchungen iiber das Protoplasma (Leipzig, 1864).

ON THE THEORY OF GALVANOTROPISM 449

(where the secretion occurs) in such experiments. The- fact
that the disintegration of Actinosphserium and the secretion
in Amblystoma both occur at the anode must also be thrown
into the balance.1

It was an entirely unexpected discovery that ia these ex-
periments stimulation always occurs at th© anodal side of
the central nervous system. This is the. only exception to
Pfliiger's law which has, to my knowledge, been found in
vertebrates thus far.

i Birukoff has called attention to the fact that cataphoretic effects of the
current form the basis of the anodic disintegration of Actinosphserium as well as
of the secretion of the glands of Amblystoma. His idea is entirely 'within the
limits of possibility. [1903]

XIX
THE PHYSIOLOGICAL EFFECTS OF IONS. I1

I. INTKODUCTION

THE works of Van 't Hoff, Arrhenius, and Ostwald on
osmotic pressure and on the dissociation of electrolytes
mark the beginning of a new epoch in science. The effects
of their work have scarcely made themselves felt as yet in
animal physiology. Several years ago I tried to utilize the
theory of osmotic pressure in explaining the hypertrophy of
muscle through activity. The increase in the volume of the
muscle cells during growth demands energy — a fact which
has not as yet been considered in animal physiology.
Physiologists were satisfied with the statement that the
working muscle received more blood, and in consequence
assimilated more strongly, than the non-working muscle,
although it is well known that the best-nourished muscle
does not hypertrophy without work. I have pointed out
the fact that the processes of hydrolysis which can be shown
to take place in the active muscle must lead to an increase
in its osmotic pressure.2 Since the muscle substance acts as
a semi-permeable wall, a source of energy is demonstrated
in this way for furnishing the energy for the work of growth.
The increase in the osmotic pressure in active muscle, or
rather the increase in the amount of water absorbed by the
active muscle, has been proved directly not only by Ranke,
but also by Miss Cooke. This theory has received no notice ;
even the thought that a source of energy is necessary for

1 Pflilgers Archiv, Vol. LXIX (1897), p. 1.

2 It is possible that this is not the only source of energy for the increase of volume
in the muscle. Surface energy or other forms of energy may also play a r6le here.
[1903]

450

THE PHYSIOLOGICAL EFFECTS OF IONS 451

the work done in growth has never once been considered.
We need not go far to find the reason for this. Even in the
newer text-books of physiology the attraction of salts for
water is still spoken of as was the case fifty years ago; the
idea of osmotic pressure and the work of Van 't Hoff have
not yet worked their way into this territory.1

One of the most fertile results of the theory of dissocia-
tion is the idea which has been brought forward, chiefly by
Ostwald, that those reactions of acids in aqueous solutions
which are common to all acids, and only to these, are
dependent upon the activity of the positively charged hydro-
gen ion, and that in a similar way the universally specific
effects of bases are determined by the negatively charged
OH ion. The relative strength of acids and. bases is there-
fore dependent upon the number of H and OH ions con-
tained in the unit volume of the solution, and this number
is determined by the degree of dissociation of the electrolyte
under consideration. A further important result of the
theory of dissociation is the fact that in completely disso-
ciated salt solutions the properties of the solution are the
sum of the properties of the ions contained in it. The ion
itself, however, represents a new species of molecules, namely,
atoms or groups of atoms which are charged with a definite
amount of electricity. It is the object of this and further
papers to determine the physiological effects of individual
ions.

Kahlenberg and True were the first to make such experi-
ments in physiology. 2 These authors investigated the toxic
properties of acids and salts in dilute aqueous solutions upon
growing plants. As a measure of the toxic effects these
authors used that concentration of the given electrolyte in
water which just allowed germinating beans to grow sixteen

1 Since these lines were written matters have changed. [1903]

2 Botanical Gazette, Vol. XXII (1896), p. 81.

452 STUDIES IN GENERAL PHYSIOLOGY

to twenty-four hours. They found that aqueous solutions
of HBr, HC1, HNO3, H2SO4, and KHSO4 which contained
the same number of hydrogen ions in the unit volume were
equally toxic; that, in other words, the toxic exect of these
acids was determined solely by the hydrogen ions and not
by the anions (nor by the molecules which, at the dilution
employed, were all dissociated electrolytically). They could
show in a similar way that the toxic effects of salt solutions
employed in their observations were determined by their ions.
I do not doubt the correctness of the principal conclusions
of Kahlenberg and True. But I do not believe that these
authors determined the limit of the poisonous action sharply
enough. They found,Jx>r example, that the acids mentioned
above were no longer toxic when 1 gram-molecule (in univ-
aleiit) or ^ gram-molecule (in bivalent acids) was dissolved
in 6,400 liters, but ihat when this amount was dissolved
in 3,200 liters they were poisonous; that is to say, the
germinating lupines cease to grow in them after from sixteen
to twenty-four hours. More accurate determinations than
this they did not make. If someone should say that the
acids studied in these experiments acted, not according to
the number of hydrogen ions, but according to the percentage
of acid, such a criticism could not be overcome by the figures
of Kahlenberg and True. These concentrations for HC1,
|H2SO4, and HNO3 behaved, for example, as 36.5:49:63;
they lie therefore between the values 1 : 2. Kahlenberg and
True have chosen for these purposes a very unsatisfactory
physiological reaction. The exact time at which growth
ceases cannot be determined accurately ; and when, in addi-
tion, experiments must be continued through the night, the
determination of the exact time becomes so uncertain that,
according to my idea, the method is a questionable one for
the quantitative determination of the toxicity of different
solutions.

258.*-

THE PHYSIOLOGICAL EFFECTS OF IONS 453

I undertook the determination of the physiological effects
of the ions of a series of electrolytes on frog's muscle. It
seemed essential to my mind, however, to choose such reac-
tions as are capable of exact quantitative determination. As
such reactions I chose first of all the amount of water ab-
sorbed by a muscle under the influence of certain electrolytes ;
for I had found that the gastrocnemius muscle of the frog,
which has, as is well known, usually about the osmotic pres-
sure of a 0.7 per cent. NaCl solution, increases considerably
in weight upon the addition of a trace of an acid or an alkali
to this sodium-chloride solution. This increase in weight is
chiefly due, no doubt, to the absorption of water. The second
reaction which I chose was the influence of the electrolyte
on the threshold of stimulation. As a source of stimulation
I used induction shocks, and as the measure of the threshold
of stimulation, the greatest distance at, which the secondary
coil could be moved away from the primary and just appre-
ciable contractions still take place. I attach less value to the
latter method, because it seems to me that the irritability of
the individual muscle fibers does not vanish at the same
moment. When the electrodes are placed at one point, no
effect may be appreciable; when they are moved, however,
a slight twitching of individual fibers may yet be brought
about. This method can, therefore, not be considered as
accurate as the first method.

II. EXPEKIMENTS WITH ACIDS

1. The normal solutions used in these experiments were
made with the greatest care by Dr. Bernhard, assistant in
the chemical laboratory. Solutions of the acids and alkalies
were made which were one-tenth normal with reference to
the H and OH contained in them. The HC1 solution, for
example, contained 1 gram-molecule (1 mol.) HC1 in 10 liters
of water; the H2SO4 solution, only ^ mol. H3SO4 in 10

454 STUDIES IN GENERAL PHYSIOLOGY

liters of water. Of these solutions 5, 10, 15, or 20 c.c. were
added to 100 c.c. of a 0.7 per cent. NaCl solution. The gas-
trocnemius muscle of a frog was laid bare without injuring
the muscle, its surface rapidly dried between sheets of filter
paper, and the tendon cut. The muscle was placed between
two watch crystals, weighed, and then introduced into one of
the solutions described above. The muscle remained in the
solution for exactly one hour, when it was removed, again
carefully dried between filter papers, and again weighed.
The method is accurate to within about 5 mg. The follow-
ing circumstances are, however, to be considered in using
the method. As the exchange between the substances con-
tained in the muscle and in the solution occurs chiefly at the
surface of the muscle, it is necessary to use for the same
series of experiments only muscles of approximately the same
weight, which have, in consequence, about the same surface.
A light muscle (with its relatively larger surface) will show
a relatively greater difference in weight at the same osmotic
difference between the muscle and solution, after remaining
one hour in the latter than a heavier muscle (with its rela-
tively smaller surface). Secondly, the fact must be remem-
bered in utilizing this method that the osmotic pressure of
the gastrocnemius of the frog is subject to not inconsiderable
variations, depending upon whether the animal was previously
quiet or active. If the effect of acids is to be compared,
care must therefore be taken that frogs are used which have
been kept for twenty-four hours under as nearly as possible
the same conditions (light, water, temperature, oxygen sup-
ply). Even then individual differences continue to exist.
This difficulty can, be overcome only by making a large
number of experiments.

2. I will first give five series of experiments which show
that the acid effects of HNO3, HC1, and H2SO4 are the same
when the same number of hydrogen atoms are contained in

THE PHYSIOLOGICAL EFFECTS OF IONS 455

the same volume of the solvent. In these experiments the
solvent was not a 0.7 per cent. NaCl solution, but a 0.585 per
cent. NaOl solution. To each 100 c.c. of this solution were
added 5 c.c. of a 0.365 per cent. HC1, or a 0.49 per cent.
H2SO4, or a 0.63 per cent. HNO3 solution — that is, 5 c.c.
of a one-tenth normal solution of these acids. The Roman
numerals indicate the series of experiments ; the Arabic fig-
ures indicate here, as in the following tables, the increase in
weight which each muscle shows in percentage of its original
weight after remaining for one hour in the solution. The
equation F=210 means in this case, as in the following
tables, that 1 or ^ mol. of the electrolyte is dissolved in 210
liters of water.

TABLE I

( F= 210)

I

II

III

IV

V

Average

HNO3.
HC1

8.6$
8.2

7.6£
7.8

7 ^£/
/ .0%

7.6

7.6#
7.6

8.0#
7.6

7.8£
7.7

^ H2SO4 . .

SA

6.7

7.9

7.9

9.6

8.1

It can easily be seen that the results in each series of
experiments harmonize to such an extent that one could
easily forget that we are dealing with effects on living
tissues. The variations between the values of the individual
series of experiments can be explained by the differences in
the state of the experimental material. In general, all the
results of the same series of experiments show relatively
high values (I and V), or relatively low values (for example,
II). The temperature has much to do with these variations,
but is not the only circumstance of importance.

From these experiments it can be said that solutions of
these three acids, which contain the same number of hydrogen
atoms in the same volume, have quantitatively the same
effects on the increase in the weight of (the amount of water
absorbed by) the muscle.

456 STUDIES IN GENERAL PHYSIOLOGY

3. Can we now say that in these experiments we are deal-
ing only with the effects of the hydrogen ions? In order
to decide this question, we must see which fraction of the
molecules of acid is dissociated at the dilution employed.
If we designate that fraction of the molecules which are

dissociated by a, then, as is well known, a = — - , where

/*„ represents the molecular conductivity of the electrolyte at
the dilution v, p^ the molecular conductivity at infinite
dilution.

' According to Ostwald,1 the speed of migration of the H
ions at 25° = 325, that of the 01 ions = 70.2. According to
Kohlrausch's law /^ is for HC1 therefore = 395. In the
same way we find for HNO3, p^ =390. According to
Kohlrausch, the speed of migration of H ions at 18° = 290.
That of SO4 can be determined indirectly as about 132. 2
/-too is therefore at this temperature 712 for H2SO4.

When F=200, /*„ for H01 = 377,-for HNO3 =377. At
18°, and when F = 333, pv for H2SO4, according to Kohl-
rausch, = 600.2. In our experiments F=400. For V=
400, pv would lie between 600 and 610.

In our solutions, therefore

a = HI = 0.95 for HC1 (25°);

a = f ft = 0.96 for HNO3 (25°);

a = m = 0.8 (inaccurate) for H2SO4 (18°).

The effect which the presence of NaCl has on the dis-
sociation of the acids is neglected in these values.

If we study the figures somewhat more closely, we see
that they are not far removed from unity; that, in other
words, about 95 per cent, of the hydrochloric and the nitric
acid molecules have dissociated into ions, and something less

1 OSTWALD, Lehrbuch, 2d ed., Vol. II, p. 675.
2KOHLBAUSCH, Wiedcmann's Annalen, Vol. L.

THE PHYSIOLOGICAL EFFECTS OF IONS 457

than 5 per cent, of the acids exist in the molecular form. It
is therefore only logical to attribute the action of the acids
on the absorption of water by the muscle to the dissociated
molecules. Furthermore, since HC1 and HNO3 show the
same degree of dissociation, and the acid effects upon the
muscle are the same, we may further conclude that at the
dilution employed only the hydrogen ions are active, while
the anions Cl and NO3 have no physiological action.

The same probably holds also for sulphuric acid, even
though the degree of dissociation is somewhat less here. We
therefore come to the conclusion that the effect of the acid
upon the absorption of water by the muscle is determined
by the H ions and is independent of the anions SO4, NO3,
Cl, and, further, of the undissociated molecules H2SO4,
HNO3, HC1.

The rest of the experiments in this paper are all made
with a 0.7 per cent. NaCl solution, instead of the 0.585 per
cent. NaCl solution used in the foregoing experiments. An
experiment with 0.7 per cent. NaCl solution when F=210
gave the following results:

For HNO3, 5.7 per cent.
For HC1, 6.1 per cent.
For iH2SO4, 6.5 per cent.

I should like to emphasize again how nearly the values
are identical. The difference between these values and those
obtained with the 0.585 per cent. NaCl solution is determined
by the difference in the osmotic pressures, as we shall see
later.

4. The next problem was to determine how the acid
effects increase with an increase in the concentration of the
acids. In the two experiments of Table II, 10 c.c. of the
one-tenth normal solution of the acid was added to each 100
c.c. of the 0.7 per cent. NaCl solution. V therefore =110.

458

STUDIES IN GENERAL PHYSIOLOGY

TABLE II
( F=110)

I

II

Average

HNO3 .
HC1

99<f
. £%

9.5

8.8$
8.6

9.0#
9 0

^HoSO, .

8.0

9.3

8.6

When r=UO, ^ is about 375 for HC1 instead of 377.
The degree of dissociation is therefore only slightly different
from that when F— 210. Nevertheless, the effect of the
acid is not doubled when the concentration is doubled, but
only increased about 50 per cent. Pronounced toxic effects
already show themselves when F=100. I need not again
point out how well the values agree with each other.

I wish finally to give a series of experiments with
KHSO4 andNaHSO4. One-tenth normal solutions of these
acids were used of which 10 c.c. were added to 100 c.c. of
a 0.7 per cent. NaCl solution. The amount of water
absorbed by the muscle is given in Table III.

TABLE III

( V = 110)

J_

II

III

IV

Average

KHSO4

7 Q%

8 2$

7 9^

NaHSO4

8 1

9.8

7 8$

7 2$

8 2

The values are quantitatively almost equal to those of the
other acids, especially sulphuric acid. In these concentrations
the lesser degree of dissociation of the sulphuric acid when
compared with nitric or hydrochloric acid is clearly shown.

If we sum up the results of all these experiments, we can
say that the acid effects (when V = 100 or 200} of HCl,
HNO3, JH2SO^ KHSO^, NaHSO±, so far as these effects
are expressed in the absorption of water, or, more accurately,
in the increase in the weight of the muscle, are chiefly ion

THE PHYSIOLOGICAL EFFECTS OF IONS 459

effects, and that these are the effects of the hydrogen ions.
AH these acids have quantitatively the same physiological
effect when the same number of hydrogen ions are contained
in the unit volume of solvent.

5. My experiments on the effects of organic acids have
only just been begun. Thus far I have used only four acids,
namely, acetic acid, lactic acid, oxalic acid, and malic acid.
Ten c.c. of a one-tenth normal solution of one of these acids
were added to 100 c.c. of a 0.7 per cent. NaCl solution.
The increase in weight expressed in per cent, of the original
weight of the muscle is given in Table IV :

TABLE IV
(F=110)

I

II

III

IV

Average

Acetic

4.7$

3.4$

3.3#

4.7$

3 9#

Lactic

6 7

6 8

8 0

8 0

7 4

i^ oxalic

6 4

6 3

6 0

8 8

6 9

i^ malic.

4 0

4.4

5.4

6 8

5 1

In order to decide whether we are dealing here with the
effects of H ions, and. in how far, we must first determine the
degree of dissociation of these solutions. We use for this
purpose Ostwald's determinations.1

For acetic acid, /* 100 = 15 (about), /* oo = 364 ;
For lactic acid, /* 100 = 38 (about), /* oo = 358 ;
For oxalic acid, /*200 = 338 (about), /* oo = 365 ;
For malic acid, /*200 = 85 (about), /* oo = 356.

If we calculate from these figures the fraction of mole-
cules that are dissociated, the following values are found for
a for the concentrations employed:

For acetic acid, a = -3Yr = 0.04 ;
For lactic acid, a = -$& = 0.10 ;
For oxalic acid, a = Iff = 0.92 ;
For malic acid, a — -^j- = 0.23.

i Abhandlungen der Sachsischen Gesellschaft der Wissenschaften, Vol. XV (1889).

460 STUDIES IN GENERAL PHYSIOLOGY

The relative number of dissociated molecules varies con-
siderably. In the case of oxalic acid it reaches a value
which approximates almost that for hydrochloric acid. We
find, also, that the acid effect of oxalic acid approximates
that of inorganic acids. For |H2SO4, KHSO4, andNaHSO4,
when F — 210, we obtain increases in weight which average
•8.6, 7.9, and 8.2 per cent, (see Tables II and III). For
oxalic acid we find an average of 6.9 per cent. The action
is somewhat weaker than that of the inorganic acids. We
may in this, perhaps, already see the effects of the anion.
In the main, however, we may yet attribute the absorption
of water under the influence of oxalic acid to the hydrogen
ions.

In the case of lactic acid, however, this is out of the ques-
tion. Only one -tenth of all the molecules were dissociated,
yet the absorption of water was at least as great as in the
case of oxalic acid. It is impossible that we are dealing here
with the mere effects of the H ions of the lactic acid. The
lactic-acid molecules bring about secondary effects in this
case which add themselves to the effects of the H ions.

Acetic acid acts more than half as strongly as oxalic acid,
and yet only 4 per cent, of all the molecules present are
dissociated, while 92 per cent, of the oxalic-acid molecules
are dissociated. The molecules of the acetic acid must there-
fore have secondary effects, and these in even greater degree
than in the case of lactic acid, which add themselves to the
effects of the few H ions which are already present.1

Malic acid, finally, is six times as strongly dissociated as
acetic acid ; its activity is, however, only a little greater than
that of the la.tter.

I am not inclined to admit that these apparent irregulari-

1 It is also possible that acetic acid migrates comparatively more rapidly into
the muscle fiber than oxalic acid. But it is also possible that these acids undergo
or cause chemical changes in the muscle substance* which increase their efficiency.
[1903]

THE PHYSIOLOGICAL EFFECTS OF IONS 461

ties must compel us to question whether we are indeed
dealing with the effects of ions in the case of the inorganic
acids and oxalic acid. The idea, which I have already
suggested, that we are dealing with secondary effects in these
cases, seems to me preferable.

Among these secondary effects the relations to the fer-
mentative processes which are going on at all times in living
tissues are the first to be considered. We know from the
work of Emil Fischer how much fermentability is a function
of the geometric configuration of the molecule. It is possible
that the apparent difficulties which these organic acids at
present offer to the assumption of the physiological effects of
ions may yet become a fruitful support for the theory of
ionization.

III. EXPERIMENTS WITH BASES

LiOH, NaOH, and KOH were especially considered. A
few series of experiments were also made with ^Ba(OH)2
and ^Sr(OH)2. The experiments again showed that these
bases have the same effect upon the absorption of water
by the muscle when they are used in such concentrations
that the same number of hydroxyl ions are contained in
equal volumes of the solutions.

The method was the same as that employed in the case of
acid. Solutions of all these bases were prepared which
were one-tenth normal with reference to OH. Of these
solutions 5, 10, 15, or 20 c.c. were added to 100 c.c. of a
0.7 per cent. Nad solution. The increase in weight which
the muscle suffered in these solutions in one hour was de-
termined and is expressed in the following, tables in per-
centage of the original weight of the muscle. We will
give first of all the experiments with LiOH, NaOH, and
KOH. V indicates again the concentration of the base;
and F=210 means that 1 mol. of the alkali is dissolved in
210 liters of water.

462

STUDIES IN GENERAL PHYSIOLOGY

TABLE v

(F=210)

II

III

Average

LiOH 7.0* 9.0* 8.8* 8.2%

NaOH 11.3 6.4 7.8 8.5

KOH 7.6 3.7 7.1 6.5

TABLE VI

(F = 110)

I II III Average

LiOH.. 17.6* 14.8* 14.2* 15.5*

NaOH 14.2 17.3 16.0 15.8

KOH 10.0 20.8 16.0 15.6

TABLE VII
(F=76.6)

LiOH.. 22.0*

NaOH 19.1

KOH.. 20.6

TABLE VIII

(F=60)

LiOH ....
NaOH....
KOH..

26.4*

26.7

20.7

n

27.5*

27.4

28.2

III

21.3*

27.3

22.4

IV

26.7*

26.7

23.3

25.7*

27.5

22.8

Average

25.5*

27.1

23.5

As can also be seen, the effect increases somewhat more
slowly with an increase in concentration than does the con-
centration itself. In Table VIII the concentration is over
three times as great as that in Table V; the effect of the
alkali, however, only three times as great.

We must now try to answer the question which is of

THE PHYSIOLOGICAL EFFECTS OF IONS 463

greatest importance to us: Do these phenomena rest upon
the effect of the OH ions or the molecules of the bases?
The degree of dissociation of the bases at the concentrations
employed gives us the information we seek regarding this
point.

Without detaining the reader with the mathematical
proof, we can show that the dissociation of these alkalies at
the concentrations employed is nearly complete. From this
it follows that we are dealing with the effects of ions, and
that the effect of the alkalies observed by us on the absorp-
tion of water by the muscle is determined solely by the
negatively charged hydroxyl ions; that the molecules KOH
NaOH, and LiOH, and also the cations K, Na, and Li, play
no part in this effect. For this reason we must obtain
quantitatively equal physiological effects as soon as the
given bases are used in such concentrations that the same
number of hydroxyl ions is present in the same volume of
the solution.

These results are well corroborated by the following ex-
periments with £Ba(OH)2 and ^Sr(OH)2.

TABLE IX
(F=110)

I

II

III

Average

^Ba(OH)3

16.8*
17.5

18.5*
14.5

19.0*
14.3

18 1*
15.4

2 a

TABLE X

(F=60)

I

II

III

Average

UBa(OH).,

27.8*
30.6

29.5*
25.6

24.3*
21.2

27.2*
25.8

^Sr(OH), .

464 STUDIES IN GENERAL PHYSIOLOGY

These are about the same values that we obtained for
NaOH, LiOH, and KOH when F=110 and F=60.

The dissociation of |Ba(OH)2 and £Sr(OH)g in the
concentrations employed here is also so complete that there
can be no doubt that we are dealing solely with the effects
of ions and exclusively with the effects of the hydroxyl ions.
When the effects of acids and alkalies are compared, it is
seen that hydroxyl ions have a greater influence on the ab-
sorption of water than an equal number of H ions in the
same volume of the solvent.

IV. ON THE EFFECTS OF H AND OH IONS ON THE ABSORP-
TION OF WATER BY MUSCLE IN LONG-CONTINUED EX-
PERIMENTS

I had next to convince myself whether the experiments
above detailed take place in living and irritable muscle or in
dead and unirritable muscle. Stimulation experiments
showed that the muscle which had remained for one hour
in one of the alkali solutions even when F= 60 was still
irritable. Irritability was diminished, but it had not dis-
appeared. So far as the acids were concerned, it seemed
that the solution of an inorganic acid when V= 110 is just
as poisonous as that of a base when V= 60, so far as irrita-
bility was concerned. We may therefore perhaps say that
the H ion is much more poisonous for the muscle than
the hydroxyl ion. Of the organic acids which I employed
oxalic acid was the most poisonous, while the remaining acids
were much less poisonous. Oxalic acid approached the
inorganic acids in toxicity. In oxalic acid we deal with the
effects of the hydrogen ions. As I intend to return to this
subject in another -paper, these remarks may suffice at this
time.

It was further necessary to investigate how the absorption
of water by muscle is influenced when the muscle is allowed
to remain much longer in the acid and alkali solutions than

THE PHYSIOLOGICAL EFFECTS OF IONS 465

given here ; so long, indeed, that the muscle may be regarded
as dead. It was found that the increase in weight continues,
even though less rapidly, with an increase in the time.

The following table shows the amount of water absorbed
in twenty-four hours when V = 210 and V = 110:

TABLE XI

F = 210(24hrs.)

F=110(24hrs.)

F=60(18hrs.)

LiOH

47 0#

760$

868$

NaOH

413

722

763

KOH

47.5

77.0

794

One sees again that the quantitative effects of the solu-
tions of the three bases is nearly the same as soon as the
same number of OH ions are contained in the unit volume.
In Table XII is given the amount of water absorbed by
muscles after a residence of eighteen hours in solutions of
acids when V= 110 (or 210):

TABLE XII

F = 110

HC1 . .

47.5$

AHoSO, .

37.7

HNO s 4.

401

Acotic acid

14.2

Lactic acid . . .

27.2

\ oxalic acid
\ malic acid

28.8
20.0

When the muscle is kept for a long time in acids, the muscle
substance becomes liquefied. This is especially noticeable
in the inorganic acids, and in oxalic and lactic acid. The
absorption of water has in itself nothing to do with this pro-
cess, as nothing of the sort is observed in the alkali solutions,
even though the absorption of water is much greater in these.1

II suppose that this liquefaction is due to a hydrolysis which in the presence of
acid is accelerated by the pepsin in the muscle. [1903]

466 STUDIES IN GENERAL PHYSIOLOGY

A comparison of Tables XII and XI shows again how
much stronger the specific effect of the hydroxyl ions is than
that of the hydrogen ions for the absorption of ivater by
muscle.

The increase in the weight of the muscle under the influ-
ence of dilute acids and alkalies therefore continues even
when the muscle has already lost its irritability. As only
small amounts of the acids and alkalies are used, one is
inclined to imagine the effect of the H and OH ions to be of
a fermentative character.

V. IN HOW FAR ARE THE CHANGES IN THE WEIGHT OF THE
MUSCLE A FUNCTION OF DIFFERENCES IN OSMOTIC PRES-
SURE BETWEEN THE MUSCLE AND THE SURROUNDING
LIQUID ?

1. I have availed myself of more than a hundred experi-
ments to show that in a 0.7 per cent. NaCl solution the
gastrocnemius muscle of the frog generally suffers no impor-
tant change in weight during the first hour. Often it loses
a few milligrams in weight ; in rare cases it increases a trifle ;
in the remaining cases its weight remains absolutely con-
stant. The 0.7 per cent. NaCl solution can therefore be
looked upon as approximately of the same osmotic pressure
as the total osmotic pressure of the molecules and ions dis-
solved in the muscle cells. The fact that the presence of an
acid or base in the salt solution compels the muscle to increase
in weight shows that we have to deal in living matter with a
system which alters its osmotic pressure easily, or in which
the permeability of the protoplasmic wall is easily modified;1
One might perhaps think at first that the great increase in
the weight of the muscle upon the addition of acids or alka-
lies is simply determined by the fact that the muscle itself

1 It is possible that the absorption of water caused by alkalies and acids belongs
to those phenomena of imbibition which have recently been studied by van Bemmelen.
[1903]

THE PHYSIOLOGICAL EFFECTS OF IONS 467

takes up these substances. There is no doubt that these
substances enter the muscle, but the increase in the weight
of the muscle amounts to much more than the weight of all
the acid in the solution. I added, for example, 5 c.c. of a one-
tenth normal solution of LiOH to 100 c.c. of a 0.7 per cent.
NaCl solution ; the muscle increased 60 mg. in weight in one
hour and 338 mg. in twenty-four hours. The total amount
of LiOH in the solution was, however, only 12 mg. ! One
might further think that the addition of a dilute acid or
alkali decreased the osmotic pressure of the "physiological
salt solution," and that in consequence its water had to enter
the muscle. I have tested this possibility experimentally,
and have found that the addition of 20 c.c. distilled water to
100 c.c. of a 0.7 per cent. NaCl solution brings about an
increase in weight of from 1 to 3 per cent, of the original
weight of the muscle. The addition of 20 c.c. of a dilute
alkali solution, however, brings about an increase in weight
of about 26 per cent, in one hour!

2. The next task which presents itself here is, first, to
gain an insight into the order of magnitude of the osmotic
forces which come into play in these experiments; and, sec-
ondly, to see how far equal differences in osmotic pressure
bring about equal effects in the muscle.

The concentration of the NaCl solution in which the
muscle of the frog neither increased nor decreased in weight
in an hour varies between 0.62 and 0.72 per cent. Through
activity this concentration may reach 1 per cent, and even
more.

To quarrel over the question as to whether a 0.65 per
cent. NaCl solution or a 0.7 per cent. NaCl solution is to be
designated as a "physiological solution" is absurd, because
the osmotic pressure of the muscle varies so greatly with rest
and activity, temperature, etc., that at one moment a 0.65
per cent. NaCl solution may be isotonic, and one hour later

468 STUDIES IN GENERAL PHYSIOLOGY

a 0.73 per cent. NaCl solution may be isotonic for the same
muscle.

If we assume that a 0.7 per cent, solution is isotonic with
the muscle, we can easily calculate the osmotic pressure in
the muscle, One molecule NaCl in one liter has an osmotic
pressure of 22.3 atmospheres, if no dissociation occurred.
A 0.7 per cent. NaCl solution would therefore have a pres-
sure of 2.67 atmospheres. A certain fraction a of the NaCl
molecules is, however split into ions, and the latter act
osmotically like molecules. If the original number of mole-
cules be N, then, since each dissociated NaCl molecule
yields 2 ions, the proportion of the molecules + ions present
to the number of the original molecules = [.$"(1 — a) + 2 iVa] :
N, or = l + a.

Because of the dissociation, the osmotic pressure therefore
rises from 2.67 atmospheres to 2. 67(1 -f- a) atmospheres.

a can readily be calculated in this case. A 0.7 per cent
NaCl solution corresponds to a concentration of V=8.3.
For V= 10 and T= 18 °, ^ according to Kohlrausch = 86.5.
fj,x at 18° = 103. a therefore = 86.5 — 0.84. The osmotic
pressure of a muscle which is isotonic with a 0.7 per cent.
NaCl solution therefore amounts to about 4. 91 atmospheres.
So far as I know, not a single text-book gives this figure,
which is of much importance for physiology.1

For a muscle which is isotonic with a 0.6 per cent. NaCl
solution the osmotic pressure would amount to 4.2 atmos-
pheres.

One may therefore say that the osmotic pressure of the
gastrocnemius muscle of a frog varies between 4.2 and 4.9
atmospheres.

If the muscle is introduced into a solution the osmotic
pressure of which is higher or lower than 4 or 5 atmospheres,

1 Since this was written, this figure has entered the text-books, but the fact that
it was given here for the first time seems to have been overlooked. [1903]

THE PHYSIOLOGICAL EFFECTS OF IONS 469

the muscle must lose or take up water, and this in proportion
to the difference in osmotic pressure.

3. Three years ago I induced Miss Cooke to make such
experiments. As, however, she weighed the salt without
previously drying it, I considered it well to repeat the experi-
ments with more accurately prepared solutions. The solu-
tions which I used were distilled water (0 per cent.), 0.35,
0.7, 1.05, 1.4, 1.75, 2.1, 2.45, and 2.8 per cent. NaCl
solutions.

The experiments were made at high temperature, 24° to
30° C. Table XIII gives the results. An increase in
weight is indicated by -f , a decrease by — , and in per cent,
of the original weight of the muscles.

TABLE XIII

Concentration

I

II

III

IV

V

o

+28.2$

+30.7$

+29.6$

+30.5$

+29.7$

0 35$

+ 90

+10 0

+ 95

0 70

- 2 0

— 0 7

— 0 5

— 0 5

- o'e

1 05

— 4 7

— 3 7

— 3.1

— 3 9

— 3 8

1 40

— 7.0

— 0.6

- 4.2

- 6.6

— 5 9

1 . 75

- 9.0

- 7.8

- 7.6

- 7.2

- 7.9

2 10

— 7 1

— 8 5

- 7 8

2 45

— 9 5

— 9 5

2 80 ....

— 7.8

- 7.8

We can at once draw an important conclusion from these
results, namely, that the changes in the weight of a muscle in
a sodium-chloride solution do not follow proportionally the
differences between the osmotic pressure of the muscle and
of the solution (when we assume that the osmotic pressure
in the muscle is equal to 4 or 5 atmospheres) ; but that in
hypisotonic solutions the increase in weight occurs more
rapidly with a decrease in the concentration of the outer
solution than the decrease in the concentration of the solu-
tion itself; and that in hyperisotonic solutions the decrease

470 STUDIES IN GENERAL PHYSIOLOGY

in weight occurs more slowly than the increase in the con-
centration of the external solution.

Both results point to the fact that an increase works in
the same sense as a decrease in the amount of water con-
tained in the cells, for both lead, apparently, to an increase in
the osmotic pressure within the cells.

It is generally stated that muscles always twitch in con-
centrated salt solutions in consequence of the loss of water
which they suffer, and Miss Cooke was inclined to see in
these twitchings the cause of the increase in the osmotic
pressure in the muscles immersed in hyperisotonic salt solu-
tions. I noticed, however, that these twitchings always
occurred in 1.05 and 1.4 per cent, solutions; that they occur-
red only rarely in 1.7 per cent, solutions, and that they
never occurred in 2-2.8 per cent, solutions. It is possible
that they occurred in the latter case only during the first few
moments after immersion and then ceased. They might in
this way have escaped my notice. One cannot, therefore,
simply say any longer that the withdrawal of water brings
about contractions in the muscle, but one must add that this
occurs only when the difference in osmotic pressure between
the outer solution and the muscle is not too great, or, perhaps
more accurately, when the concentration of the surrounding
solution is not too high.

The fact that the loss of water by the muscle increases
more slowly than the difference in the osmotic pressures
between the outer solution and the muscle is supplemented
by the behavior of the muscle when left for a long time in
0.7 per cent, and hyperisotonic sodium-chloride solutions.
The decrease in weight soon comes to a standstill, and the
muscle begins to take up water so that in eighteen hours its
weight is greater than its original weight. This occurs even
when the muscle is kept in a 2.8 per cent. NaCl solution!

This fact that the "injured" muscle is able to take up

THE PHYSIOLOGICAL EFFECTS OF IONS 471

water even from so concentrated a solution as a 2.8 per cent.
NaCl solution is of importance in pathology, namely, in the
explanation of oedema. These conditions have thus far
been attributed in the main to changes in blood-pressure
and changes in the vessel walls. I believe, however, that
we are dealing in this case with changes in the osmotic
forces in the tissue cells or changes in their permeability ;
these changes are brought about as in our experiments
through "injuries" (poisons) and lead, according to their
nature, to a swelling of the cells or to a secretion of fluid
into the body cavities.

VI. PROOFS OF THE VALIDITY OF VAN 9T HOFF's THEORY OF
OSMOTIC PRESSURE FOR THESE PHENOMENA

If solutions of LiCl or KC1 are prepared of the same
concentration as a 0.7 per cent. NaCl solution, the muscle
behaves in them, so far as its change in weight is con-
cerned, as in a 0.7 per cent. NaCl solution. The changes in
the weight of a muscle are in this case, therefore, a function
of the osmotic pressure. The degree of dissociation in all
three salts is about the same.

I tested further whether the effect of an addition of 20
c.c. of a one-tenth normal NaOH solution was the same for
"physiological" LiCl, NaCl, and KC1 solutions. The increase
in the amount of water absorbed by the muscle amounted
respectively to 26.4, 26.7, and 20.7 per cent, of the original
weight of the muscle in one hour. In a second experiment
5 c.c. of a one-tenth normal KOH solution was added to 100
c.c. of a 0.7 per cent. NaCl solution and to LiCl and KC1
solutions isotonic with the NaCl solution. The absorption
of water amounted to 8.2 per cent, in LiCl, 8 per cent, in
NaCl, and 7.8 per cent, in KC1. One can scarcely wish that
the results should be more nearly identical.

Should anyone wish to interpret the experiments of the

472

STUDIES IN GENEBAL PHYSIOLOGY

preceding chapter in such a way as though Van 't Hoff's
theory of osmotic pressure did not hold for physiology, these
experiments might perhaps teach him better.

This is especially the case with reference to the experi-
ments with MgCl3, CaCl2, SrCl2, and BaCl2. One mol.
of these compounds was dissolved in one liter of distilled
water and then diluted to the same degree as a 0.7 per cent.
NaCl solution. A muscle was introduced into 100 c.c. of
each of these solutions. Table XIV gives the weight lost in
these solutions in one hour.

Each of the salts mentioned here dissociates into three
ions — two electro-negative chlorine ions and one bivalent
electro-positive ion. According to this, the relation of the num-
ber of molecules actually present to those originally present
is as [N (1 — a) + 3Na~\ : N = 1 -f- 2a. a in this case =
0.6-0.7.

TABLE XIV

I

II

Average

MgCl,

—3 3$

—3 1%

—3 5#

CaCl3

—3 4

—3 5

—3 4

SrCl3

—3 3

—4 7

—4 0

BaCl8

—5.1

—6.7

—5 9

Without dissociation the osmotic pressure of each of these
salt solutions would have amounted to 2.67 atmospheres. In
consequence of the dissociation the osmotic pressure rises to
2.67 (1 '+ 1.3) == 6.14 atmospheres.

The loss in weight which the muscles suffered in these
solutions is nearly equal to the loss in weight which the
muscles suffered in a 1.05 per cent. NaCl solution. In the
latter solution this amounted on the average to 3.8 per cent.
The osmotic pressure of such a solution is about 7.3 per cent.,
that is, therefore, a little higher than that of the MgCl2
solution. When we consider, however, that the condition of

THE PHYSIOLOGICAL EFFECTS OF IONS 473

the protoplasm, and consequently its osmotic pressure, is
affected differently by the different ions — of which we will
say more immediately — the agreement between the values is
satisfactory enough for proving the validity of Van 't Hoff's
theory of osmotic pressure for the life phenomena of cells.
Barium chloride, however, seems to be an exception.

VII. ON THE EFFECT OF POTASSIUM CARBONATE AND SODIUM
CARBONATE SOLUTIONS UPON THE MUSCLE

If solutions of Na2CO3 and K2CO3 of the same concen-
tration as a 0.7 per cent. NaCl solution are prepared, muscles
immersed in them increase markedly in weight. Table XV
gives the increase in weight of the muscles in these solutions
after one hour.

TABLE XV

I

II

Na8CO3.

7.1#

7.1$

K2CO3

5 1

6 8

The muscles showed the glassy appearance of a muscle
which has been in a weak solution of an alkali. The order of
magnitude of the absorption of water also corresponds with
the effects of an alkali. According to the theory of dissocia-
tion, this effect is brought about in the following way: The
potassium carbonate is dissociated in part into potassium and
CO 3 ions; as carbonic acid is a very weak acid — that is to
say is only slightly dissociated — each CO3 ion combines
with two H ions to form carbonic acid. Two negatively
charged OH ions must, in consequence, go into solution,1 and
these bring about both the alkaline reaction of the solution
and the absorption of water by the muscle. We are dealing
in this case, therefore, not with potassium or carbonate
effects, but with the effects of hydroxyl ions.

1 More correctly form 2KOH, which, however, at this degree of dilution is dis-
sociated to a high degree. [1903]

474 STUDIES IN GENERAL PHYSIOLOGY

It is well known that weak solutions of Na2CO3 increase
the irritability of the muscle. NaOH in dilute solution acts
in the same way. Strange to say, these effects are generally
attributed to the Na. We are dealing in both cases, how-
ever, with effects which are brought about solely through
the hydroxyl ions (see sec. iii) and which have nothing to
do with the Na ions or the Na compounds. So far as I
know, this error goes through the whole literature of physi-
ology and pharmacology. The chapter of chemical stimula-
tion as well as the chapter on pharmacology stand in need of
a thorough revision on the basis of .the theory of dissociation.

VIII. THE VELOCITY OF MIGRATION AND THE RELATIVE
TOXICITY OF DIFFERENT IONS FOR THE MUSCLE

It seems to me to be of the greatest interest to compare
the relative toxicities of ions in order to discover if any rela-
tions exist between these toxicities and the other properties
of the ions.

If I make such a comparison of the toxicity of the ions
dealt with in this paper, I do it with two reservations: first,
that although the method of determining the toxicity of ions
for muscle by determining the threshold of stimulation is
superior to the method of Kahlenberg, it is, nevertheless, not as
accurate as that of determining the velocity of migration of
the ions ; secondly, that the observations made thus far refer
to only a very limited number of ions. I hope to overcome
the latter shortcoming by further experiments. But I do
not yet see how the former difficulty is to be overcome.

The most poisonous ion (of the ions which have been dis-
cussed in this paper) for muscle is undoubtedly the hydrogen
ion, which has the* lowest atomic weight, but the greatest
velocity of migration. For the strength of current and the
induction apparatus employed by us, 10-15 c.c. of a one-tenth
normal HC1, HNO3 or |H2SO4 solution (in 0.7 per cent.
NaCl solution) sufficed to do away with the irritability of the

THE PHYSIOLOGICAL EFFECTS OF IONS 475

muscle in one hour. The hydroxyl ions act much more
weakly. When 20 c.c. of a one-tenth normal LiOH, NaOH,
or KOH solution were added to the NaCl solution, the
irritability was greater or just as great as when 10 c.c. of the
acid solution were added. The speed of migration of the
hydrogen ions is, according to Ostwald, 325 at 25°, that of
the hydroxyl ions 170.

If we study the monovalent cations Li, Na, K, lib, and
Cs, it is to be remembered that at the concentration which I
employed of 1 mol. in about 8 liters of water the dissocia-
tion of the chlorides of these elements is so complete (84 per
cent.) that we are dealing chiefly with the effects of ions.
Of these ions Li and Na were not poisonous, so far as the
irritability of the muscle was concerned, for the irritability
remained unchanged. On the other hand, the ions K, Rb,
and Cs, with their greater atomic weight and greater velocity
were decidedly poisonous. The irritability of the muscle in
the KC1 solution (of the same concentration as a 0.7 per
cent. NaCl solution) was almost destroyed after one hour, or
at any rate greatly decreased. The same is true of RbCl
and CsCl solutions of the same concentration as a 0.7 per
cent. NaCl solution.

It cannot be concluded from my observations that the
toxicity of the Rb and Cs ions is greater than that of the K
ions. I am rather inclined to believe that the toxicity of the
three last-named ions for the gastrocnemius of the frog is
about the same. I thought at first that the toxicity of the
ions was a function of their ionic weight. Such a view is,
however, untenable when the atomic weights are considered.
The atomic weight of Li is 7, that of Na 23, that of K 39,
that of Eb 85.2, that of Cs 132.7; and yet there is a great
jump from the toxicity of the sodium ions to that of the
potassium ions. The latter are, however, about as poisonous
as Rb and Cs ions, in spite of the great differences in their

476 STUDIES IN GENERAL PHYSIOLOGY

atomic weights. On the other hand, we notice a beautiful
agreement between the relative toxicity of the individual
members of this group and the velocity of the migration of
the ions. According to Kohlrausch, the speed of migration
of the Li ion at 18° is 33, that of the Na ion 41, that of the
K ion 60. The velocities of the ions of Rb and Cs are equal
to the velocity of K, according to Ostwald. A great jump,
therefore, occurs from Na to K in the velocity of the migra-
tion of the ions, while no such jump occurs from K to Eb
and to Cs, just as is the case in their toxicity. It may
therefore be said that the relative toxicity for muscle of the
ions of this group is rather a function of their velocity of
migration than of their atomic weight. The heavier ele-
ments are, therefore, more poisonous only because their ions
have a greater speed of migration than the Na and Li ions.

On the other hand, the toxicity of K ions is much less
than that of the hydroxyl ions. In our experiments with
alkalies the addition of 10 c.c. of a one-tenth normal alkali
solution to 100 c.c. of a physiological NaCl solution was
sufficient to bring about a decided decrease in irritability.
It did not matter, however, whether the alkali used was
KOH, NaOH, or LiOH. At this concentration only the OH
ions were poisonous, while the toxicity of the K ions did not
appear.

The investigation of the relative toxicity of the bivalent
ions Be, Mg, Ca, Sr, and Ba is rendered difficult by the fact
that a solution of the chlorides of these elements of the
same concentration as a 0.7 per cent. NaCl solution has a
greater osmotic pressure than the sodium-chloride solution.
The muscle, therefore, loses water (as we have seen) in such
a solution, and the loss in water must also decrease the irri-
tability. We are, however, in a position to estimate the
influence of the loss of water on the irritability. The loss
of water in these solutions was about equal to that in a 1.05

THE PHYSIOLOGICAL EFFECTS OF IONS 477

per cent. NaCl solution. While the irritability of the
muscle was decreased but little after remaining one hour in
such a sodium-chloride solution — it fell in one hour from
420 to 380 — it had decreased in a BeCl2 solution, for ex-
ample, to 220 in .one hour. In the remaining solutions
MgCl2, CaCl2, SrCl2, and BaCl2 the decrease in irritability
was still greater. In one experiment, for example, the
muscle just responded, after remaining for one hour in a
MgCl2 solution, when the secondary coil was pushed out
110 mm., while the muscle in the CaCl2 solution could just
be stimulated when the coil was at 0. The muscles which
had been in SrCl2 and BaCl2 did not respond at all to
stimuli after an hour. In another experiment a contraction
could be obtained in a muscle which had been for one hour
in the MgCl2 solution when the secondary coil was at 100;
for the muscle which had been in CaCl2 when the secondary
coil was at 150; for the SrCl2 solution when the secondary
coil was at 50 to 60; while the muscle which had been for
one hour in BaCl2 was absolutely unirritable. These
figures show at the same time that this method of determin-
ing the irritability leaves much to be desired. The follow-
ing conclusion can, however, certainly be drawn from these
experiments: The Be ions are decidedly more poisonous
than the Li ions, even though their atomic weights are
almost the same; in the same way Mg ions are decidedly
more poisonous than Na ions, although their atomic weights
are nearly the same. This fact must also be considered,
that the dissociation of the chlorides of these bivalent ele-
ments is less than that of the chlorides of the Li group, and
that, in consequence, the number of the cations in the Be
group is smaller than in the Li group. Furthermore, since
the SrCl2 and BaCl2 solutions are at least just as poisonous
as the equally concentrated KbCl and CsCl solutions, it is
perhaps right to ask whether the toxicity does not in these

478 STUDIES IN GENERAL PHYSIOLOGY

cases increase simultaneously with the valency. To say that
the toxicity increases in proportion to the increase in atomic
weight would be entirely wrong. The atomic weights are
9.08 for Be, 24.3 for Mg, 39.9 for Ca, 87.3 for Sr, and 136.9
for Ba. If the toxicity of the ions were proportional to
their atomic weights, this would show itself clearly in our
experiments. The velocities of migration correspond much
more perfectly with the relative toxicities of these ions.
According to Kohlrausch, the velocities of the ions at 18°
are about as follows: |Mg = 42, |Ca = 49, |Ba = 53. Sr
is not given, but the velocity of its ion might stand close to
that of Ba. One sees that this series of velocities does not
show the jump which occurred in the former series from Na
to K, and which corresponded to a similar jump in the rela-
tive toxicities. The difference in the velocity of migration
of Mg and Ca is only 10 per cent., while that between the
corresponding members of the Li series, namely, Na and K,
is 50 per cent. We see also that no such jump exists from
the toxicity of Mg to that of Ca. The conclusion seems
justified, therefore, that the relative toxicities of the series
of ions Be, Mg, Ca, Sr, and Ba correspond more nearly to
the velocities of these ions than to their atomic weights.

We have yet to discuss the toxicity of the anions. OH,
with its enormously high velocity of migration, is, as we have
seen, a relatively strong poison for muscle. On the other
hand, the Cl ion, with a speed of migration which is equal
to that of the K ion, is as harmless as the Li and Na ions,
as is shown by the behavior of the 0.7 per cent. Nad solu-
tion, which contains just as many Cl ions as Na ions. The
speed of migration of the NO3 ion is, according to Kohl-
rausch, 60 at 18°; that of £SO4, 66; that of Cl, 62. The
fact that |H2SO4, HNO3, and HC1 show themselves to be
equally poisonous at the same concentration indicates that
in the concentrations which we employed the effect of the

THE PHYSIOLOGICAL EFFECTS OF IONS 479

NO 3 and the SO4 ion does not come into consideration. I
shall return to this point as soon as I have made further
experiments.

It is self-evident, of course, that so close a relation as
proportionality between the velocity of migration and the
relative toxicity of the ions is to be expected only in those
ions which belong to the same narrow group; a group in
which the remaining characteristics of the ions are almost
identical and in which important differences exist only with
reference to atomic weight and velocity of migration. "What
has been said thus far holds only for the members of the Li
series or the Be series among themselves ; it would be wrong
to judge the relative toxicity of K and Ca on the basis of
the velocities of migration of their ions, because in doing
this new differences come into consideration; for example,
the difference in their valencies. It would be entirely
absurd to judge the toxicity of the alkaloids by the velocity
of the ions.

This further fact is to be considered in judging toxicity,
that all our evidence is of a negative character — the cessa-
tion of life or of individual functions, especially irritability.
The most widely differing circumstances may, however,
bring about this result. One poison may act by rendering
impossible processes of oxidation (for example, HCN,
according to Geppert) ; another may bring about molecular
changes which influence the transformations of energy in
the cell, etc. It is to be expected that different groups of
poisons bring about their toxic effects in different ways.
This is a further warning against a too sweeping generaliza-
tion of the relation between velocity of migration and
toxicity. We must finally not overlook the apparent ine-
quality in the permeability of the protoplasm for different
ions, a subject concerning which special experiments are yet
to be made.

480 STUDIES IN GENERAL PHYSIOLOGY

IX. SUMMARY OF RESULTS

1. The addition of small amounts of a very dilute acid
or alkali brings about a great increase in weight (absorption
of water?) in a muscle contained in a physiological sodium
chloride solution.

2. For the inorganic acids, HNO3, HC1, H2SO4,
KaHSO4, NaHSO4 (in a high degree of dilution) this increase
in weight is solely a function of the number of hydrogen ions
contained in the unit of volume of the physiological salt
solution. Solutions of these different acids which contain
the same number of H ions in the unit of volume bring
about a quantitatively equal increase in weight.

3. This simple relation does not hold for organic acids
(acetic acid, lactic acid, malic acid). In these the effect of
the anion or of the undissociated molecules makes itself felt.

4. For the bases LiOH, NaOH, KOH, Sr(OH) 2 , Ba(OH) 2 ,
this increase in weight is solely a function of the number of
hydroxyl ions contained in the unit of volume of the
physiological salt solution. Dilute solutions of these differ-
ent bases which contain an equal number of hydroxyl ions
in the unit of volume bring about an equal increase in
weight.

5. If the muscle is introduced into various solutions of
NaCl the osmotic pressure of which is higher or lower than
that of the muscle, it is found that the change in the weight
of the muscle is not proportional to the difference between
the osmotic pressure in the muscle and that of the surround-
ing solution; in hypisotonic solutions the muscle increases
in weight more rapidly; in hyperisotonic solutions it
decreases in weight more slowly than corresponds to the
differences in osmotic pressure.

6. The validity of Van 't Hoff's theory of osmotic pres-
sure for these processes is proved by the fact that -solutions
of LiCl, KC1, KbCl, CsCl, MgCl2, CaCl2, SrClg, and BaCl2

THE PHYSIOLOGICAL EFFECTS OF IONS 481

bring about approximately the same change in weight as a
NaCl solution of the same osmotic pressure.1

7. Sodium and potassium carbonate bring about an
absorption of water by the muscle in consequence of the
hydroxyl ions contained in the solution of these salts.

8. The relative toxicity of the group of ions Li, Na, K,
Rb, Cs for muscle runs parallel with the velocities of the
migration of these ions and not with their atomic weights.
A similar parallelism exists between the velocity of the ion
and its relative toxicity in the group Be, Mg, Ca, Sr, and
Ba. Such a relation is, of course, to be expected only
between ions which belong to the same narrow group in the
periodic system.

1 When the duration of the experiment is short. [1903]

XX

ON THE PHYSIOLOGICAL EFFECTS OF ELECTRICAL

WAVES1

1. IN the June number of Pflugers Archiv I published
some experiments which were undertaken in the hope of ob-
taining physiological effects through Hertzian waves.2 I
have continued my experiments since then, and will in this
paper publish some new facts and some further confirmations
of the ideas set forth at that time. I will at the same time
enter a little more deeply into the theoretical discussion of
these phenomena.

In the July number of the Archives de physiologie
Danilewsky published a paper on the "Excitation of Nerves
through Electrical Rays." The experiments of this author
coincide partly, so far as the mere facts are concerned, with
the experiments which I have published previously. So far
as his explanation of the experiments goes, it is entirely
wrong.

If we allow a spark to pass between the spheres of the
discharger of an electric machine or a Ruhmkorff coil, we
obtain waves all of which are propagated with the velocity of
light. The wave-lengths, however, depend upon the number
of oscillations per second. If, for example, we separate the
two spheres of the spark discharger of a Ruhmkorff until a
spark no longer passes between them, the number of oscilla-
tions obtained in a second corresponds to the number of
oscillations of the interrupter. In this case we deal with
waves thousands of kilometers in length, and it is of course

1 Pflilgers Archiv, Vol. LXIX (1897), p. 99.

2 "On the Theory of Galvanotropism, V," ibid., Vol. LXVII (1897), p. 483.

482

EFFECTS OF ELECTKICAL WAVES 483

only a faQon de parler if we continue to speak of waves
in this case, just as we might still designate a circle as an
ellipse. If sparks pass across, the length of the waves varies
with the capacity and the self-induction of the discharger.
With small spheres and a short spark discharge very short
waves (a few centimeters in length) can be obtained.
The next shortest waves with which we are acquainted are
the "heat-rays;" after these come the " light-rays" which
act upon the eyes; and finally the ultra-violet rays. The
wave-length is of importance in the effect produced by each
of these waves, in so far as every wave-motion is most effect-
ive when it strikes a body which constitutes a resonator for
the given wave-motion. Under such conditions the energy
of the waves may suffice to cause the body "to vibrate
sympathetically." Definite relations exist between the
wave-lengths and the dimensions of a resonator, according
to which the resonators for light-waves must have almost
molecular dimensions, while the longer waves such as arise
in the discharge of a condensator correspond to larger
resonators.

This fact becomes especially important for the theory of
life-phenomena. The basis of all life-phenomena are chemi-
cal and molecular processes. It is therefore probable a priori
that only such wave-motions will be able to call forth
immediate life-phenomena, the resonators of which have
molecular dimensions. Thus we see, indeed, that light-
waves produce a series of physiological effects. But we
also see that certain functions such as the chlorophyll
functions or the heliotropic effects, etc., depend on definite
wave-lengths. I conclude from this that the heliotropic
functions and the chlorophyll functions are dependent
upon molecules of different dimensions. The electro-
magnetic theory of light will perhaps enable us to deter-
mine the approximate, and later perhaps the absolute,

484 STUDIES IN GENERAL PHYSIOLOGY

dimensions of the molecules in the living cell which are
sensitive to light. With these possibilities in view I have
undertaken at this time more accurate experiments on the
heliotropic activity of the different rays of the spectrum.
My previous experiments in this direction were made in the
i main with colored screens, which

O {-) sufficed for the immediate pur-

i _ pose before me at that time,

namely, to show that the phe-

FIG> 138 nomena of animal heliotropism

are identical with those of plant heliotropism. The latter
had also in the main been analyzed only by the aid of colored
screens.

2. The experiments which I described in the paper
mentioned in the introduction showed, however, clear physi-
ological effects. In these experiments I dealt with oscil-
latory discharges, and the nerve-muscle preparation was
struck by waves the length of which varied in the different
experiments between several centimeters and meters. Yet
I maintained that the oscillatory nature of the discharges
had nothing to do with these physiological effects, but that
the contractions of the muscle were dependent upon the
mere disappearance of the electrostatic charge from the
two spheres of the discharger. It may perhaps be best to
review briefly the chief experiments. We used a Toepler-
Holtz machine. As the living tissue or indicator in our ex-
periments we used two frog's legs with exposed nerves.
Both legs were laid as nearly as possible in the same
straight line, so that the two free ends of the nerves
touched each other. (Fig. 138). In this way a preparation
is obtained in which the capacities are distributed symmetri-
cally upon both sides. Through this fact my preparation
has an advantage over Danilewsky's, who used only one
nerve, connected on the one side with the leg, on the other

EFFECTS OF ELECTRICAL WAVES 485

with the spinal cord. If the nerves are laid parallel to the
spark discharge maximal effects are obtained when the
muscle preparations lie in a symmetrical position with rela-
tion to the two spheres of the discharger (Fig. 138), while
every displacement of the preparation toward one side (Fig.
139) diminishes the effects or causes them to cease entirely.

-f _

- 0 G - -

FIG. 139

If, on the other hand, the nerves of the muscle preparations
are laid at a right angle to the spark discharge, minimal
effects are obtained when the preparation lies in a position
symmetrical with the two spheres of the discharger (Fig.
140), while maximal effects are obtained when the muscle prep-
arations are moved laterally a sufficient distance (Fig. 141).

- -t-

- - , - 0 o -

FIG. 140

The explanation which I gave of these phenomena was
as follows:

a) Let it be assumed that the two muscle preparations
lie parallel to the spark discharge and in a position sym-
metrical with reference to the discharger (Fig. 138). If at
a certain time the one sphere of the discharger is charged
positively, the other negatively, a corresponding distri-
bution of the electricities is induced in the nerve-muscle

486 STUDIES IN GENERAL PHYSIOLOGY

preparations. When a spark passes there is nothing to
keep the electricities in the muscle preparation apart, and a
current consequently passes longitudinally through the
nerves and causes the muscles to contract.

6) The muscle preparations lie at a right angle to the path
of the spark, but in a position symmetrical with reference to
the spheres of the discharger (Fig. 140). The electricities
will now be distributed in the preparation in such a way
that during the passage of the spark a current must pass

•4-

FIG. 141

transversely through the nerve. Since the nerve cannot be
stimulated by a current passing transversely through it, the
effect is in this case minimal.

c) The nerve-muscle preparations again lie parallel with
the spark discharge, but not in a position symmetrical with
reference to it, but toward one side of it (Fig. 139). The
electricities will now be so distributed that during the dis-
charge a current must pass transversely through the nerve.
We must therefore again expect a minimal effect, etc.

The experiment with a mirror which I described in a
previous paper, and which at first seemed to contradict our
explanation, can also be explained in this way. The nerve-
muscle preparations, were placed at right angles with the
spark discharge, but not in a position symmetrical with the
discharger, but slightly to one side of it (Fig. 142). Under
such circumstances only a weak current passes longitudi-
nally through the nerves during the passage of the spark.

EFFECTS OF ELECTRICAL WAVES 487

If now the preparation is so placed with reference to the
spark discharge that a contraction is just rendered impossible
during the passage of the spark, a contraction can again be
brought about when a mirror SS (Fig. 142) is so placed
that one end of it is near that sphere of the discharger
which is farthest away
from the preparation, while
the other end of the mirror
is near that end of the prep-
aration which is directed
away from the spark dis-
charge. The explanation
of the effect of this mirror
is very simple. A distribu- ? 2"~" v

tion of the electricities is FIG. 142

also induced in the mirror, and this induction must increase the
distribution of the electricities in the nerve-muscle preparation.
But it might be imagined that we are in this case dealing
with the effects of waves which are reflected by the mirror.
That this is not the case, but that we are dealing with the
effects of a double induction, is proved by the following
experiment. Let everything be left just as in the previous
experiment, only the spark discharge be turned through an
angle of 90°, so that it is now vertical instead of horizontal
as before. The electric waves will now be reflected by the
mirror just as before, but the strengthening effect of the
induction through the mirror can now no longer exist.
Under these circumstances no contractions occur, whereby
it is proved that the electric waves are not the cause of the
mirror effect. Since my first publication of this experiment
I have supplemented it in two directions which make it more
interesting as a demonstration experiment, and which bring
additional proof of the correctness of our explanation. First
of all I have found that it is not necessary to use a metallic

488 STUDIES IN GENERAL PHYSIOLOGY

mirror, but that a moistened glass plate is just as good a
mirror as a metallic plate. If we were dealing with the
effect of waves, this would not have been the case, as electric
waves penetrate a thin layer of water more easily than a
metal plate. Secondly, the body of the experimenter can be

used in this experi-

O Q ment instead of

a mirror. The ex-
perimenter needs

.* - only to bring one

+ ^— * ~ 1— ^ - hand into the

* neighborhood of

FIG. 143 , ,_.

the sphere a (Fig.

142), the other in the neighborhood of the end b of the
muscle preparations. In this way the muscle can be made
to contract, and the result might convince any believer in
telepathy that his superstition has a scientific foundation.
In the latter case the effects of induction are too apparent,
the effects of a reflection of waves too improbable, to allow
one to think of a wave -effect.

The last series of experiments which I published con-
sisted in the inhibition of the electrical effects through a
screen. The preparation lies parallel to the spark discharge
and in a position symmetrical with reference to the spheres
of the discharger (Fig. 138). The preparation contracted
energetically each time the spark passed between the spheres
of the discharger. If now a metal screen SSt (Fig. 143)
is placed behind the preparation and parallel to it, these
effects disappear. This can be explained as the result of
induction. A distribution of the electricities will be induced
in the mirror by the two spheres in the same sense as in the
muscle preparations. Since the same kinds of electricity lie
opposite each other in the mirror and in the nerve-muscle
preparations, the effect upon the muscle preparations must

EFFECTS OF ELECTRICAL WAVES 489

be weakened by the mirror, and in consequence contractions
do not occur. The explanation through the assumption of
electric waves would be that standing waves are formed, and
that a node exists at the mirror. It was a simple matter to
show that this explanation is wrong. The mirror needs only
to be moved away steadily in order to show that at every
other distance contractions again begin. If we were indeed
dealing with standing waves, periodic inhibitions of the con-
tractions should have occurred when the mirror was steadily
removed from the nerve-muscle preparations.

In this experiment it is also possible to replace the
metallic mirror by a moist glass plate or by the experi-
menter himself. When he stands behind the muscle and
brings one hand near each one of the two free ends of the
preparation, the contractions can easily be inhibited. I
need scarcely mention that this form of demonstration is
especially ' ' impressive. ' '

3. The objection might now be raised against this expla-
nation that we ought to give a more modern representation
of the changes whiclf occur in the preparations in these
experiments. I am glad to fill in this gap in my first pub-
lication, since this gives me an opportunity to enter more
deeply into the chemical theory of electro-physiological
effects, which I have begun to discuss in two previous
papers. The question which is of importance to physiolo-
gists in this case is the following: What is changed in a
nerve-muscle preparation, or any other living substance,
when we say that it has a negative or a positive charge of
electricity, as has happened repeatedly in this paper? It fol-
lows, first of all, from Faraday's law governing conduction
in liquids, that electricity can be conducted in living matter
only through a migration of ions, since only the liquid
portions of a cell are conductors. Ostwald has drawn the
further conclusion from Faraday's law that static electricity

490 STUDIES IN GENERAL PHYSIOLOGY

in a liquid conductor can also be conceived of only as an excess
of negative or positive ions on the surface of the solution.
It seems to me that Ostwald's assumption is a necessary one,
and, corresponding with this, I believe that when we assume
a distribution of electricities in a nerve-muscle preparation,
as shown in Fig. 138, a definite number of positive ions are
distributed over the surface of the right half of the prepa-
ration, and an equal number of negative ions over the sur-
face of the left half of the preparation. The lines of force
which go out from the spheres of the spark discharge to the
preparation can be imagined as the connecting lines between
the centers of polarized elements. These connecting lines
would therefore end at the surface of muscle preparations in
the ions. As soon as the charge disappears from the
spheres of the discharger, the excess of positive ions on the
right side of the preparation and the excess of negative ions
on the left side of the muscle preparation can no longer
remain separated, and a migration of the ions — a current —
must occur in the nerves. In this case (in consequence of
the semi-permeability of certain elements in the preparation ?)
a collection of ions must occur at certain points in the
preparation. The ions are converted into atoms and so
bring about chemical effects either directly or indirectly;
these chemical effects bring about the contractions which
we notice during the passage of the spark.1 One can readily
understand in this way also why the oscillatory nature of
the discharge is of no importance in the physiological effects
produced, as the latter are dependent solely upon the migra-
tion of ions. If our theory is correct, it is therefore to bo
expected that the experiments which have been described
can also be made successfully, when it is possible to do away
with the oscillatory character of the discharge entirely.

1 1 am now inclined to believe that no transformation of ions into atoms occurs
in this case, and that the mere change in the concentration of ions at the surface of
the semipermeable membranes suffices for the result. This harmonizes with a view
expressed since by Nernst. [1903]

EFFECTS OF ELECTRICAL WAVES 491

II

My experiments were published in the June number of
Pfliigers Archiv. In the July number of the Archives de
physiologic B. Danilewsky published two articles under the
title, "Excitation des nerfs par les rayons electriques."
Danilewsky overlooked the circumstance upon which every-
thing depends in this case — the significance of the orienta-
tion of the nerves toward the spark discharge. It will be
seen that Danilewsky's experiments are a further proof for
my assumption that the effects which he and I observed are

FIG. 144

not determined by the oscillatory nature of the discharge.
For his experiments are made in a way which does away
with the oscillatory nature of the discharge almost entirely.
The experiments of Danilewsky apparently fall into three
groups. In reality, however, we are dealing with one and
the same experiment. We will discuss first of all the ex-
periment which he designates as the "interference experi-
ment" (p. 524). Each of the poles of the Ruhmkorff coil is
connected with a smooth metal plate. These metal plates
are set up parallel to each other some 50 to 100 cm. apart.
" If the nerve is placed in a position symmetrical with the
two metal plates, one obtains no effect The symme-
try needs to be altered only slightly, that is to say, the prep-
aration needs to approximate one of the metal plates only
slightly, in order to bring about induced contractions." If
we study the drawing (Fig. 144, according to Danilewsky)

492

STUDIES IN GENERAL PHYSIOLOGY

J

somewhat closely, we find that in this experiment the nerve
accidentally lay parallel to the metal plates and somewhat
outside the area inclosed by the metal plates. This is no
other experiment than that which I have described under 5),
and which is shown in Fig. 140. In order to convince

oneself of this fact one
needs only to turn the
nerve through an angle
of 90°, as I have done,
and lay it parallel to
the line connecting the
electrodes (Fig. 145);
one then obtains maxi-
mal effects when the nerve lies symmetrically with refer-
ence to the metal plates. We do not deal, therefore, with
interference in Danilewsky's experiment, but (as I had
already shown in my first publication) with the well-known
fact that the nerve is insensitive, or only slightly sensitive,
to a current which passes through it transversely. I con-
vinced myself of the correctness of this interpretation in yet
another way. One can also obtain minimal effects when the
nerves lie at right angles to the electrodes and in a
metrical position, if the
nerves are brought
entirely within the area
between the two metal
plates as shown in Fig.
146. In this way cur-
rents are obtained which
pass transversely through the nerves.

The "interference experiments" of Danilewsky are not
only misinterpreted as far as the purely physiological facts
are concerned, but their physical analysis leads also to im-
possibilities. In Danilewsky's experiment a spark does not

v

FIG. 146

EFFECTS OF ELECTRICAL WAVES 493

pass between the electrodes, as it does in my experiments,
but we have to do with a silent discharge. In this way,
however, the oscillatory character of the discharge practi-
cally disappears. As is well known, a tuning-fork emits a
sound only when the prongs, after they have been moved
from their state of rest, spring back quickly. When they
return to a state of rest very slowly, they do not give rise to
sound. It is the same in the case of a discharge. In the
case of a spark discharge we deal with an abrupt release of
the tension which must occur in an oscillatory way. In the
case of a silent discharge we do not deal with oscillations at
all. If Danilewsky speaks of an oscillatory discharge
under such circumstances, it is to be remembered that
in reality the oscillatory character of the discharge dis-
appears so completely in his experiments, that they cor-
roborate beautifully my view that these effects are not
determined through oscillations, but through a single elec-
trostatic discharge.

Of course, it might yet be maintained that electric waves
are present in this case in so far as the interruptions by the
hammer of the Ruhmkorff are periodic. If we estimate these
interruptions in the Ruhmkorff coil as 60 per second (which
is certainly high), then, when the velocity of light is in
round figures 300,000 km., the wave-length is over 5,000
km.! Does Danilewsky expect us to believe that these
waves can interfere between his electrodes which are sepa-
rated from 50 to 100 cm. ? It is also entirely wrong to apply
to such waves the term "rays." We expect rays to move in
a straight line, and for this reason we do not speak of sound-
rays, since these can go around a corner. The "rays" of
Danilewsky would not only go around a corner, but around
the Alps, or around the moon. In a similar way there is no
sense in saying, as Danilewsky does, that in these experi-
ments a "neutralisation des polarite"s electriques" occurs.

494 STUDIES IN GENERAL PHYSIOLOGY

We are dealing simply and solely with the lesser irritability
of nerves to transverse currents.

The second experiment of Danilewsky is intended to show
that we are not dealing with electrostatic effects. He
obtained electrical effects when the secondary coil of the
Ruhmkorff coil was closed by means of the secondary coil of
another induction apparatus. I have repeated this experi-
ment, and have found that it is in reality nothing else than
the experiment discussed before, and that we can therefore
speak as little of the effects of an oscillatory discharge, or of
electric waves, in this case as in the previous experiment.
To convince oneself of this fact one needs only to close the
circuit of the secondary coil of the Ruhmkorff through a
rheostat instead of with the spiral of an induction apparatus.
If a great resistance is introduced into the rheostat, about
20,000 ohms, all the experiments which I have described
under a) and 6) can be repeated between the terminals of
the Ruhmkorff coil. When a resistance of only one ohm is
introduced, no effects are obtained ! In this case, therefore,
we also deal with the production of a high potential at each
side of the resistance through the introduction of a high
resistance. These charges bring about an electrostatic
separation of both kinds of electricities in the muscle prepa-
rations. As soon as the discharge takes place, a current
passes through the nerve which brings about maximal effects
when it passes longitudinally through the nerve; minimal
effects when it passes in a transverse direction. The experi-
ments succeed most beautifully when the secondary spiral of
the Ruhmkorff is closed through a glass tube filled with
distilled water into each end of which dips a copper wire.
In this case the glass tube behaves physiologically exactly
as the path of the spark in my first-described experiments,
and one can here repeat all the experiments which I have
discussed in my first publication. But this is possible only

EFFECTS OF ELECTRICAL WAVES 495

as long as the distance between the copper wires in the glass
tube is great ; that is to say, as long as the resistance is great.
If the ends of the copper wire are brought close together, so
that the resistance between them becomes small, the effects
cease. In this case we also deal with the production of a
difference in potential which brings about a separation of
the ions in the muscle preparations, which leads to the
passage of a current through the nerve when the difference
in potential dissappears. But the oscillatory nature of the
disappearance of the difference in potential has nothing to
do with the physiological effect, since the discharge of the
Ruhmkorff coil is not oscillatory when it is closed through
the tube of water.

The third series of experiments of Danilewsky deal with
unipolar stimulation. One pole of the Kuhmkorff coil is
connected with the earth, the other with a metallic plate.
This experiment differs from the two other methods given
above only in this, that the one pole has a potential of zero,
while the other has a potential twice as high as that in the
other experiments. In regard to the absence of actual
oscillations this experiment is similar to the other two. But
I would especially emphasize that my previous experiments
are in themselves sufficient to show that the oscillatory
nature of the discharge of a Ruhmkorff coil (where this
property is actually present) does not bring about the physi-
ological effect upon the nerve; but that this effect is deter-
mined only through the (single) disappearance of a charge.
These things have, therefore, nothing to do with electrical
waves.

It is perhaps necessary to touch upon the possible prac-
tical application of these experiments in medicine. Dani-
lewsky makes suspicious suggestions in this direction. I
scarcely believe that we can expect much of a practical
application of these experiments. For one can easily con-

496 STUDIES IN GENERAL PHYSIOLOGY

vince oneself of the fact that a moist glass plate will serve as
a complete screen against all electrostatic effects of a charged
body upon the preparation. The entire surface of our bodies
is covered with such a screen in the form of the superficial
layers of the epidermis. In case we are not dealing with
enormous discharges, every thought of utilizing these effects
in medicine is shut out. Since in all these cases we are in
reality dealing only with the effects of currents (even when
we are using an unusually powerful machine), I consider it
more rational to use the galvanic current upon the skin of
the patient directly instead of utilizing the cumbersome
roundabout method of discharging a highly charged body
near a patient.

Finally I must call attention to a fundamental error of
Danilewsky in his idea of the nature of electrical effects upon
protoplasm. Danilewsky believes with Chauveau and d' Ar-
son val that electricity acts only as a "mechanical stimulus."

Nous posse"dons, sur ce point, des indications dans les tres-
interessantes recherches de M. d' Arson val. Dans sa communication
& la Socie"te" de Biologie de Paris, du 4 juillet 1891, M. d Arson val
relate que ses propres recherches sur 1'irritation electrique et me*ca-
nique des nerfs confirment entierement les vues de M. Chauveau
qui, des 1859, de"clarait que l'6lectricit£ agit uniquement comme
excitant me"canique, surtout a son point de sortie et en raison de la
densit^ a ce point.

In contradiction of this idea I should like to point out
that Faraday's idea of electrolysis has become one of the
pillars of modern physics and chemistry. In living matter
it is only the electrolytes which conduct the current.1

1 In a book which Danilewsky has since published he has accepted my view as
far as the ionic conception <?f electrical stimulation is concerned. [1903]

XXI

THE PHYSIOLOGICAL PROBLEMS OF TODAY1

IF it be true that the fundamental problem of physics is
the constitution of matter, it is equally true that the funda-
mental problem of physiology is the constitution of living
matter. I think the time has come for physiology to return
to its fundamental problem.

"Living matter" is a collective term for the qualities
common to all living organisms. Comparative physiology
alone enables us to discriminate between the general proper-
ties of living matter and the functions of specific organs, such
as the blood, the nerves, the sense organs, chlorophyll, etc.
Nothing has retarded the progress of physiology and pathol-
ogy more than the neglect of comparative physiology. Com-
parative physiology shows that secretion is a general function
of all living organisms and occurs even where there is no
circulation. Hence it was a priori false and a waste of time
to attempt to explain secretion from experiments on blood-
pressure. Oxidations occur regardless of circulation, and it
was a priori a waste of time to consider the blood as the seat
of oxidation. Comparative physiology has shown that the
reactions of animals to light are identical with the heliotropic
phenomena in plants. Hence it is a mistake to ascribe such
reactions as the flying of the moth into the flame to specific
functions of the brain and the eyes. Sleep is a phenomenon
which occurs in insects and plants, and it would be a waste
of time to attempt an explanation of sleep on the basis of
phenomena of circulation. The best interests of physiology

i Address delivered at the meeting of the American Society of Naturalists, Ithaca,
1897. This paper was one of seven upon "The Biological Problems of Today," each
speaker being limited to ten minutes.

497

498 STUDIES IN GENERAL PHYSIOLOGY

and pathology demand that the systematic development of
comparative physiology be one of the physiological problems
of today.

I may be pardoned for calling attention to one special field
of comparative physiology which I believe to be especially
fertile. I refer to the field of physiological morphology.
I applied this name to the investigation of the connection
between the chemical changes and the process of organiza-
tion in living matter. Two series of facts allow us to connect
these two groups of phenomena: (1) the fact that phenomena
of fermentation lead to an increase in the number of mole-
cules, and thus bring about an increase of osmotic pressure in
the cells, this increase of osmotic pressure being the source
of energy for the work of growth ; (2) the facts of hetero-
morphosis, i. e., the possibility of transforming in certain
animals one organ into another or substituting one organ for
another through external influences, such as gravitation, con-
tact, light, etc.

The exact and definite determination of life-phenomena
which are common to plants and animals is only one side of
the physiological problem of today. The other side is the
construction of a mental picture of the constitution of living
matter from these general qualities. In this portion of our
work we need the aid of physical chemistry and especially of
three of its theories : stereochemistry, Van 't Hoff's theory
of osmotic pressure, and the theory of the dissociation of
electrolytes. We know that the peculiar phenomena of oxi-
dation in living matter are determined by fermentative pro-
cesses, and we venture to say that fermentations form the
basis of all life-phenomena. It has been demonstrated that
fermentability is a function of the geometrical configuration
of the molecule. Saccharomyces cerevisice is a ferment for
such sugars only as have three or a multiple of three atoms
of carbon in the molecule. Among the hexaldoses only

THE PHYSIOLOGICAL PROBLEMS OF TODAY 499

d-glucose, (i-mannose, and d-galactose are fermentable, while
their stereoisomeres are not fermentable. But the influence
of the geometrical configuration goes farther. Voit has
suggested and Cremer has demonstrated that there is a far-
reaching parallelism between the fermentability and assimila-
tion of carbohydrates. Higher animals as well as yeast cells
are able to form glycogen from such carbohydrates as are
fermentable by yeast. The further development of these
stereochemical relations and their extension to proteids and
nucleins is another of the problems of physiology which will
contribute to the main problem, the analysis of the constitution
of living matter. I believe that the influence of stereochem-
istry will be more or less directly felt in many branches of
physiology, in questions of heredity, as well as in the theory
of space-sensations, as E. Mach has already intimated.

In living organisms chemical energy is frequently trans-
formed into osmotic energy. Van 't HofFs theory of osmotic
pressure permits an application of the law of conservation of
energy to a class of phenomena to which this law was hith-
erto inapplicable, namely, the phenomena of growth, func-
tional adaptation, secretion, absorption, and even pathological
processes such as oedema. The physiologists who thought
that the blood-pressure determined secretion could not under-
stand why secretion took place under a higher pressure tha^n
the blood-pressure. Comparative physiology shows that
secretion does not depend upon circulation, and the theory
of osmotic pressure indicates that the osmotic pressure in the
cells is more than twenty times as high as the blood-pressure.
The work of secretion is done by osmotic pressure and not
by blood-pressure. A prominent physiological chemist has
become a vitalist because he could not explain why the secre-
tions differ from the blood from which he thinks they are
formed. He overlooks among others the fact that the pro-
toplasm possesses the quality of semi-permeability, which

500 STUDIES IN GENERAL PHYSIOLOGY

means that it allows certain substances to pass through and
others not. In my opinion, the working out of a theory of
semi-permeability is one of the main physiological problems
of the day.

The theory of the dissociation of electrolytes is of funda-
mental importance in the analysis of the constitution of living
matter. Pharmacology will feel its influence most directly.
Everything seems to indicate that the specific physiological
effects of inorganic acids are due to the number of positively
charged hydrogen ions in the unit of solution, and the spe-
cific physiological effects of alkalies to the negatively charged
hydroxyl ions. But the universal bearing of the theory of
dissociation upon physiology will perhaps be best seen in the
field of animal electricity. An active element of living mat-
ter is negatively electric to its surrounding resting parts.
We may assume that an acid is formed in the active part, and
that the passive parts are neutral. The positive hydrogen
ions of the acid have a much greater velocity of migration
than the anions. Hence the former will diffuse more rapidly
into the passive tissue than the anions, and the active tissue
will remain negatively charged.1

At no time since the period immediately following the
discovery of the law of conservation of energy has the out-
look for the progress of physiology appeared brighter than
at present. But in order to reap the full benefit of our
opportunities, we must bear in mind that the fundamental
problem of physiology is the determination of the constitu-
tion of living matter, and that in order to accomplish our
task we must make adequate use of comparative physiology
as well as physical chemistry. Pathology, in particular, will
be benefited by such a departure.

1 As far as I am aware, this was the first attempt at applying the principle of
batteries of concentration to the explanation of the current of action. [1903]

XXII

THE PHYSIOLOGICAL EFFECTS OF IONS. II1
THE experiments described in this paper are a continua-
tion of the experiments in my first paper on this subject in
three directions.2 It will be shown by a longer series of
experiments that the organic acids behave differently from
the inorganic. While the physiological effects of the latter
are determined by the concentration of hydrogen ions, this is
apparently not the case with the fatty acids, inasmuch as the
degree of dissociation and the physiological effects of the
fatty acids do not run parallel. We shall not attempt to
explain this apparent exception until we have further experi-
mental data at our disposal. We may, however, think of
the possibility that the fatty acids undergo a partial oxidation
in the muscle.

1 have shown in my first paper that a muscle does not
change much in weight in a 0.7 per cent. NaCl solution
during the first hour, but that it increases greatly in weight
when only a trace of an acid is added. This increase in
weight can be explained by assuming that the hydrogen ions
of the acid act in the muscle hydrolytically like enzymes and
in this way increase the osmotic pressure in the muscle.
While the effect of inorganic acids in sufficiently dilute solu-
tions was quantitatively exactly a function of the number of
hydrogen ions contained in the unit of volume of the solu-
tion, no such relation seemed to exist for the organic acids.
I pointed out that this exceptional behavior from the theory
of dissociation could be explained through secondary chem-
ical processes. I have since made a series of experiments with

iPfliigers Archiv, Vol. LXXI (1898), p. 457.

2 " On the Physiological Effects of Ions, I," Part II, p. 450.

501

502

STUDIES IN GENERAL PHYSIOLOGY

a larger number of organic acids, especially of the fatty series,
which I wish to report here. It is clear that these apparent
exceptions to the theory of electrolytic dissociation in the
case of the organic acids must yield material for understand-
ing the chemical changes which go on in living matter.

The experiments were made as the previous ones. Five
or 10 c.c. of a one-tenth normal solution of the acids were
added to 100 c.c. of a NaCl solution. The gastrocnemius of
a frog was introduced into such a solution and its increase
in weight determined at certain intervals.

The solutions which contained 10 c.c. of a one-tenth nor-
mal acid in 100 c.c. of a 0.7 per cent. NaCl solution — in
which therefore the concentration V equaled 110 — gave more
constant results than those solutions in which V equaled 210.
The following table gives the results of several series of
experiments with a series of acids when V equaled 110. The
Arabic figures give the increase in weight which each muscle
showed, expressed in per cent, of its original weight after
remaining for one hour in the solution.

(F=110)

I

II

III

IV

V

VI

Av'ge

Formic acid .

5 6$

4 6#

5 6#

5 0%

4 2£

5 0#

5 0$

Acetic acid

4 7

3 4

3 3

4 7

3 9

Trichloracetic acid. . . .

7 3

6 8

7 1

Lactic acid

6 7

6 8

8 0

8 0

6 9

7 1

7 2

Valerianic acid

4 4

5 7

4 9

50

Mandelic acid.

6 5

7 5

7 8

7 2

J<2 Oxalic acid

6 4

6 3

6 0

8 8

6 9

2^ Succinic acid

6.1

4 7

6.0

5 6

\& Mialic acid

4 0

4 4

5 4

6 8

5 1

% Dextro-tartaric acid .
jj Racemic acid

5.8
6.3

6.4
5.4

6.6
6.8

7.1

5.9

6.3
6.2

We will now compare with this series the degree of dis-
sociation of these acids when V— HO.1

i Calculated according to OSTWALD, " Ueber die AffinitatsgrOssen organischer
Sfturen," Abhandlungen der Sachsischen Gesellschaft der Wissenschaften, Vol. XV,
p. 89.

THE PHYSIOLOGICAL EFFECTS OF IONS 503

Formic acid

- 0.14

Oxalic acid

- 0.87

Acetic acid -

0.04

Succinic acid

0.08

Tricloracetic acid -

0.94

Malic acid

0.18

Lactic acid -

0.11

Dextrotartaric acid

0.27

Valeria nic acid

0.04

Racenic acid -

0.27

Mandelic acid

0.19

If we compare the effect of the acids on the absorption
of water by the muscle with the degree of dissociation, we
notice that the effects of the acids vary from each other much
less than their degrees of dissociation. Lactic acid, for
example, with only 11 per cent, of its molecules in the ionic
state brings about just as great an absorption of water by
the muscle (7.2 per cent.) as trichloracetic acid, or oxalic
acid in which nearly all the molecules are dissociated.
Mandelic acid acts just as strongly, even though only 19
per cent, of its molecules are dissociated at the concentration
which we employed. The fact that the differences in the
effects of the various acids are so much less than the differ-
ences in their degrees of dissociation might point to the fact
that those acids which are only little dissociated are changed
in the muscle into substances which undergo a greater
degree of dissociation. I will, however, leave the discus-
sion of this possibility until I have made further experi-
ments, especially on the aromatic acids. At that time I will
also give the figures for a greater number of experiments
which I have already made with acids for different lengths
of time and different concentrations. That in these experi-
ments the acids are really absorbed by the muscle was
determined by titration by Mr. Slimmer.1

In HNO3, when F=110, the increase in the weight of a
gastrocnemius weighing 1,731 g. amounted in one hour to
154 mg., that is, to 8.9 per cent, of the original weight of
the muscle. This agrees exactly with our previous values
for this acid, which had given on the average an increase in

i Mr. Slimmer also prepared the solutions mentioned in this paper.

504 STUDIES IN GENERAL PHYSIOLOGY

weight of 9 per cent, at this concentration. In this experi-
ment the solution contained originally 63.3 mg. of HNO3.
After the muscle was removed, the solution had lost 5.1 mg.
of the acid. For trichloracetic acid we found a loss of 14
mg. in two hours. These experiments will be continued.
Besides the fact that the hydrogen ions enter the muscle,
these experiments also show that the increase in weight
which the muscle suffers in the acid solution is much greater
than the weight of the acid absorbed. There can scarcely
be any doubt that the increase in the weight of the muscle
in the acid solution is dependent almost solely, if not entirely,
upon an absorption of water. But how the hydrogen ions
can lead to an absorption of water by the muscle can at
present not be decided definitely.

XXIII

WHY IS REGENERATION OF PROTOPLASMIC FRAG-
MENTS WITHOUT A NUCLEUS DIFFICULT OR
IMPOSSIBLE?1

IT has been shown with certainty by a series of experi-
ments that oxygen is necessary for the development of eggs
as well as for the processes of regeneration. The reason
for this might be sought, among other things, in the fact
that, as I have suggested in two previous papers, synthetical
changes are necessary for these processes, and that the syn-
theses depend upon the supply of oxygen.2 It is well known
that when a cell is cut into several pieces only the pieces
containing a nucleus are capable of regeneration, while the
pieces without a nucleus soon disintegrate. This has been
interpreted by assuming that the nucleus contains specific
formative substances, which it gives off to the protoplasm.
This conclusion is, however, not binding. It might well be
possible that the nucleus is necessary only for the accomplish-
ment of processes of oxidation. The removal of the nucleus
would in this case be associated with an inhibition or a
decrease in the processes of oxidation. This would suffice
to prevent the regeneration of enucleated pieces of the cell.
I wish now to test in how far the facts at hand agree with
such a hypothesis.

To bring about oxidations in living tissues the presence
of catalytic substances is necessary which either "activate"
the atmospheric oxygen, or which render the compounds in
the cell capable of taking up atmospheric oxygen more

lArchivfiir Entwickelungsmechanik der Organismen, Vol. VIII (1899), p. 689
2 " Assimilation and Heredity," Monist, 1898.

505

506 STUDIES IN GENERAL PHYSIOLOGY

energetically. The majority of investigators believe that
oxygen is activated in the tissues, and that in this process
certain substances act catalytically which, after Traube, we
will call "oxygen carriers." It has been possible to extract
these oxygen carriers from the cells and to show that in this
condition the oxygen carriers are still able to bring about
those oxidations which are characteristic of living matter.

The most valuable discovery in this direction was undoubt-
edly made by Spitzer.1 Spitzer has shown that those sub-
stances in extracts of tissues, which favor the transfer of
oxygen (oxidation ferments) belong to the group of nucleo-
proteids. The nucleoproteids are typical substances of the
nucleus. All these nucleoproteids contain iron. Now, we
know that iron salts are especially adapted to accelerate
oxidation. There is no reason to doubt that what is true
for the aqueous extracts of the cells holds also for the
nucleoproteids of living cells. Macallum has proved the
existence of iron in the chromatin substance of the cell
nuclei. The work of Spitzer, therefore, renders it probable
that the nucleus is the organ of oxidation in living matter.

We will now see whether the behavior of cell fragments
without the nucleus corresponds to this assumption. Verworn
describes the changes which occur in the pseudopodia in
these cases as follows : In the pseudopodia of an enucleated
piece of Orbitolites droplets are formed which, in part, flow
together into larger drops. The connection between the
individual drops disappears. "Finally, after about five to
seven hours the central portion forms a round lump without
pseudopodia, around which are scattered a number of larger
and smaller globules, drops, and spindles of protoplasm."
Verworn in adopting and misunderstanding the view of
Berthold has developed a theory of amoeboid movements

1 Pflugers Archiv, Vol. LXVII (1897), p. 615.

2 VEBWOEN, Pflugers Archiv, Vol. LI (1892).

REGENERATION OF PROTOPLASMIC FRAGMENTS 507

according to which the throwing out and the drawing in of
the pseudopodia are attributed to changes in surface tension.
This theory is based on the assumption that the pseudopodia
are liquid.1 This assumption is a physical impossibility.

The pseudopodia of Orbitolites are, according to the
drawings of Verworn, cylindrical; their length perhaps a
hundred times greater than their circumference. Such a
liquid cylinder cannot exist. There is a good reason why
rain falls in drops and not in jets. If r is the radius, h the
height of a liquid cylinder, it can exist only as long as
h ^ 2r7r. For this reason alone the entire contraction theory
of Verworn is wrong. I will not, however, discuss this theory
more closely here.2 From these facts in surface tension it
follows that the pseudopodia of the Rhizopods cannot be
liquid, but must possess a solid framework or a solid mem-
brane. As soon as this solid framework or the solid membrane
becomes liquefied the pseudopods must obey the laws of
surface tension and break up into drops. The latter is,
according to my observations, the process which occurs in
fragments of Orbitolites which contain no nucleus.

Is this liquefaction of solid substances a process which is
indicative of lack of oxygen? This is undoubtedly the case.
Four years ago I showed that the cell- walls of the cleavage
cells of Ctenolabrus become liquid when deprived of oxygen.
When oxygen is readmitted, the formation of cell-walls
begins anew.3 Budgett showed in my laboratory that the
removal of oxygen leads to the solution of the cell- walls in
Infusoria also, and that certain poisons had the same effect.*
Kuhne has observed the same effect of lack of oxygen.5 The

Bedeutung der lebendigen Substanz (Jena, 1892), p. 38.
21 do not here mention the chemotropic ideas of Verworn because they rest
upon no actual foundation whatsoever.

3 " Investigations on the Physiological Effects of Lack of Oxygen," Part I,
p. 370.

4 American Journal of Physiology, Vol. I (1898).
tZeitschrift fiir Biologic, Vol. XXXVI (1898), p. 472.

508 STUDIES IN GENERAL PHYSIOLOGY

liquefaction of the pseudopodia upon the removal of the
nucleus, and the inability of Infusorian fragments with-
out a nucleus to form a new cuticle, correspond entirely
with the assumption that the cell fragments without a
nucleus are in a condition of decreased oxidative activity.

I have expressed this view for several years in my lec-
tures, and I have had it in mind to perform experiments
on pieces of Infusoria without the nucleus under increased
oxygen pressure. If my theory be correct, it should be
possible to prolong the life of fragments of protoplasm
freed from the nucleus by furnishing them a better supply
of oxygen. But it seems to me that this experiment has
already been made. For while Nussbaum, Gruber, and
Verworn found that pieces of Infusoria without the nucleus
go to pieces after two days, and only exceptionally live
several days, all botanists who have made the same ex-
periment on chlorophyll-bearing-cells (for example, Algae)
have found that cell fragments without the nucleus remain
alive five to six weeks.1 Assimilation took place in such
pieces. It seems to me that the comparatively long dura-
tion of life of fragments of AlgaB without a nucleus is of
great importance in judging of the function of the cell
nucleus. As is well known, oxygen is liberated in the
assimilation of carbon dioxide. Pieces of Algae without a
nucleus are therefore, in the light, under better conditions
of oxygen supply than fragments of Infusoria without a
nucleus, for the Infusorian contains no chlorophyll.

It seems to me, therefore, that all the facts which are known
thus far very naturally support the idea that the nucleus
is the organ 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. Such pieces die slowly from asphyxia.

i KLEBS, Biologisches Centralblatt, Vol. VII (1888).

REGENERATION OF PROTOPLASMIC FRAGMENTS 509

The question as to the significance of the nucleus for
development and heredity enters into a new light if these
ideas are correct. Nor can it longer be held that the living
organism is merely a combination of individual cells. "By
cellular structure we understand the fact that there must be
a definite maximal distance, variable, however, for different
forms and tissues, between the elements of the protoplasm
and the nearest nucleus."1 We can now understand the
reason for this. If the distance becomes too great, the
particular protoplasmic element goes to pieces from as-
phyxia. That besides this an interchange of other sub-
stances occurs between the protoplasm and the nucleus I
will, of course, not deny ; it is possible or probable that the
significance of the cell nucleus is not exhausted by its oxida-
tive activity. It is also scarcely necessary to point out par-
ticularly that I do not believe that without the nucleus all
processes of oxidation cease in the protoplasm; it rather
seems from all the facts at hand that it is only markedly
decreased.

i LOEB, Pflilgers Archiv, Vol. LXIX (1897).

XXIV

ON THE SIMILARITY BETWEEN THE ABSORPTION OF
WATER BY MUSCLES AND BY SOAPS l

I

THE difficulty which confronts us in the phenomena of
the absorption of liquids by tissues consists in this, that we
have thus far not been able to find any analogies for these
phenomena in the realm of physics and chemistry. I be-
lieve to have come upon a series of facts which show that a
similarity exists between the absorption of water by muscles
and by soaps. This analogy is, among other things, of im-
portance because it may help us decide which forces come
into play in the. absorption of liquids.

Potassium soaps absorb so much water from the air that
they finally become liquid. The sodium soaps also absorb
water, but much less than the potassium soaps. The cal-
cium soaps are useless for washing purposes because of their
insolubility, or only slight solubility, in water.

If the sodium of a sodium soap is replaced by calcium,
water is usually given off ; on the other hand, if the sodium
is replaced by potassium, water is taken up.

Muscle behaves in exactly the same way. I have shown
in my earlier publications that a muscle neither takes up nor
gives off water during the first hour in solutions of neutral
salts, which are isosmotic with a 0.7 per cent. NaCl solution.
I found slight variations from this rule in a few salts, espe-
cially potassium and calcium salts. These variations were,
though, very slight, and yet too great to attribute them to
errors in experiment. If, however, the muscles remained
in the solutions for more than one hour, these differences

l Pfliigers Archiv, Vol. LXXV (1899), p. 303.

510

WATEK ABSORPTION BY MUSCLES AND SOAPS 511

became very marked; after eighteen hours, for example, it
was found that the muscle kept in an isotonic KC1 solution
had gained from 40 to 50 per cent, in weight; that a
muscle in a CaCl3 solution lost about 20 per cent, in weight;
that the weight of a muscle in LiCl remained practically
unchanged ; while that in NaCl solution had increased a few
per cent, in weight. The following gives a summary of
these results:

TABLE I

Changes in weight in the gastrocnemii of frogs kept in solutions equimolecular
with a 0.7 per cent. NaCl solution. Length of the experiment, 18 hours. An
increase in weight (expressed in per cent, of the original weight of the muscle)
is designated by a + ; a loss in weight by a -.

LiCl ..... -\% NaCl . . ..+ 6* KC1 . . . .+45# CaCl2 . . -20#
LiBr ..... -1 NaBr....+ 7 KBr....+41
Lil ...... +3 Nal ..... +10 KI ...... +45

The figures given in this table were corroborated by a
large number of experiments. Variations due to individual
differences in the muscles and variations in the temperature
are unavoidable, but the order of magnitude of the results
obtained was always about the same as those given above.

Since the difference in the degree of dissociation of the
potassium and sodium salts is very slight, it is of course ex-
cluded that differences in osmotic pressure, due to differ-
ences in the degree of dissociation, should be responsible
for the results obtained.

The potassium and sodium salts of other acids, such as
the sulphates and oxalates, also show these differences,
though not so markedly.

Wallace and Cushny believe that only the anions and
not the cations of salts play a role in the absorption of
water from the intestines.1 If this is true, the absorption
of liquids by the intestine must be governed by entirely
different laws from those which govern the absorption of

1 WALLACE AND CUSHNY, American Journal of Physiology, Vol. I, p. 411.

512 STUDIES IN GENERAL PHYSIOLOGY

water by muscle. I must, however, call attention to the
fact that Wallace and Cushny did not continue their ex-
periments long enough to allow them to draw such a con-
clusion. In the course of one hour a muscle increases about
2 per cent, in weight in a KC1 solution; in a CaCl3 solution
it decreases about 3-5 per cent. ; in a LiCl or a NaCl solu-
tion its weight remains unchanged. The experiments of
Wallace and Cushny lasted less than one hour, and to dis-
cover such slight variations as those given in my experi-
ments is impossible in those of Wallace and Cushny.

In what way, now, can we imagine that potassium salts
bring about a positive, calcium salts a negative, absorption
of water in muscle? When we place a muscle into any of
the solutions mentioned above, Li, Na, K, and Ca ions soon
begin to diffuse into the muscle. Just as in case of soaps
this would lead to the formation of Na, K, and Ca soaps, it
might lead in muscle to the formation of Na, K, and Ca com-
pounds, which behave like the corresponding soaps in at least
one particular — in their power of dissolving water.1

It is of importance, for what has been said, that SrCl2,
BaCl3, CoCl2, and MnCl3 behave like CaCl2. MgCl3,
however, is much weaker in its action than these. Table II
shows the weight lost by muscles when immersed in equi-
molecular solutions of these five salts.

TABLE II

The loss in weight of muscle (in per cent, of its original weight) in solutions equi-
molecular with a 0.7 per cent. NaCl solution. Length of the experiment, eight-
een hours.

MgCl2 .- 4.7^ CoCl2 -35#

BaCl2 -12.0 MnCl2 -39

SrCl2 , -18.0

Perhaps the solutions of all the heavy metals will fall
into this group; that is to say, they will all bring about a
loss of water in muscle.

1 Or of absorbing water.

WATER ABSORPTION BY MUSCLES AND SOAPS 513

The analogy between the behavior of soaps and of muscles
is of importance for the mechanics of the absorption of
liquids. The majority of authors — for example, Hofmeister
—assume that in the absorption of liquids by tissue we
deal with imbibition; that is to say, with capillary phe-
nomena. In the absorption of water by soaps, however, we
deal with phenomena of solution.1 The forces concerned in
the latter are osmotic pressures, and not surface tensions, as
in capillary phenomena.

The antagonism which has been shown by these experi-
ments to exist between potassium and calcium compounds is
of interest in another direction. Ringer has called attention
to the fact that an antagonism exists between calcium and
potassium salts in their effect on the heart. The latter are
supposed to favor the diastole, the former the systole, of the
heart. Howell has adopted the views of Ringer. Possibly
the above-described characteristics of the two substances may
help to explain the phenomena of contractility. I cannot
assent to the view that calcium is the "stimulus" for the
cardiac activity. Years ago this might have been errone-
ously said about oxygen. The cause of cardiac activity (as
that of every automatic activity) is neither calcium nor oxy-
gen, but heat, or, more correctly, its intensity factor, tem-
perature. Calcium and potassium might be of importance
for the changes in the amount of water in various elements,
or for their state of matter, and so be of importance for the
contraction of the heart.

II

I had shown in my earlier publications that when a trace
of acid is added to a 0.7 per cent. NaCl. solution, a muscle
immersed in it absorbs a large amount of water.2 I was

1 This statement will probably have to be modified. Sinee water can be removed
from soaps by pressure, it is at least partly held there by imbibition. [1903]

2 " On the Physiological Effects of Ions," Part II, pp. 450 and 501.

514 STUDIES IN GENERAL PHYSIOLOGY

interested in testing how isotonic calcium and potassium
solutions act upon muscle when acids are added to these
solutions. I usually added 10 c.c. of a one-tenth normal
acid to 100 c.c. of the salt solution, so that the acid was y^-
normal. HNO3 or HC1 were the acids generally employed.
I showed in the article cited above that at this concentration
we are dealing only with the effects of ions, and that the
effect is quantitatively the same whether 10 c.c. one-tenth
normal HC1 or 10 c.c. one-tenth normal HNO3 are added.

The experiments yielded the result that acids markedly
increase the amount of water absorbed by muscles in CaCl2
solutions, but that they have the reverse effect in solutions
of potassium salts, where they diminish the amount of water
absorbed. In one experiment the muscle increased 35 per
cent, in weight in an isotonic KI solution, but only 6.2 per
cent, in an isotonic KI solution to which had been added
the amount of acid indicated above ! The addition of 10 c.c.
of one-tenth normal HNO3 to 100 c.c. KI solution decreased
the amount of water absorbed almost 29 per cent, expressed
in terms of the original weight of the muscle ! In another
experiment the muscle increased 54 per cent, over its origi-
nal weight in an isotonic KC1 solution, but only 39 per cent,
after the addition of the acid, or 15 per cent. less. In an
isotonic solution of K3SO4 the muscle lost 4 per cent, of its
original weight in eighteen hours. By the addition of 10 c.c.
of a one-tenth normal acid to 100 c.c. of the isotonic K2SO4
solution the muscle lost 22 per cent, of its original weight
in the same time !

Exactly the opposite is observed when acid is added to
CaCl2 solutions. While a muscle in an isotonic CaCl2 solu-
tion loses about 20 per cent, of its weight in eighteen hours,
it increases about 30 per cent, when 10 c.c, of a one-tenth
normal HNO3 solution are added to 100 c.c. of the CaClg
solution.

WATER ABSORPTION BY MUSCLES AND SOAPS 515

In regard to the effect of acids, 0.7 per cent. NaCl solu-
tions are more like CaCl2 solutions. As I have shown in my
earlier papers, a muscle absorbs 6 to 8 per cent, of its weight
of water when immersed for eighteen hours in a 0.7 per cent.
NaCl solution. If 10 c.c. of one-tenth normal HNO ~ solution

o

are added to 100 c.c. of the NaCl solution, the muscle
increases about 40 per cent, in weight in the same time.

Ill

When a muscle is immersed in a salt solution of a greater
concentration than 0.7 per cent., it loses water in such a
solution during the first hour or hours. If, however, it
remains in such hypertonic solutions for some time, it steadily
increases in weight, and this the more the greater the con-
centration (within certain limits) of the solution in which it
is immersed. Table III illustrates this paradoxical behavior.
The row of figures on the left gives the concentration ; that
on the right, the increase in weight of the muscle expressed
in per cent, of the original weight of the muscle. The
experiment lasted twenty-four hours.

TABLE III

Increase in weight of muscles

Concentration of in twenty-four hours ex-

the solution pressed in per cent, of their

original weight

1.40 - 6.7

1.75 - 13.0

2.10 17.7

2.45 - 19.0

2.80 23.8

This paradoxical behavior of the muscles is dependent upon
secondary changes which take place in the muscles when
allowed to remain for a long time in the hypertonic sodium-
chloride solutions. I have discussed these changes at length
in my study of oedema.1 I will only point out in passing

iPfliigers Archiv, Vol. LXXI (1898).

516

STUDIES IN GENEEAL PHYSIOLOGY

that one of the changes which occur in the muscle is the
formation of acid. That such a formation of acid, if it
occurs, must increase the absorption of water by the muscle
has been clearly shown in my previous publications. I do
not, of course, believe that the formation of an acid is the
only factor which comes into consideration here. The
observations on the effect of K and Ca on absorption point
to the possibility that the Na ions which enter the muscle
alter its substance chemically.

That this assumption is correct is shown by the following
facts: If some acid is added to a 0.7 per cent. NaCl solu-
tion, it compels a muscle immersed in it to take up much
more water than when the acid is not added. If this same
amount of acid is added to a 2.5-5 per cent. NaCl solution,
it has exactly the opposite effect — the muscle finally loses
more weight in the hypertonic sodium-chloride solution
which contains acid than in the hypertonic solution without
acid. I have not yet determined the exact turning-point.
It lies below 2.4 per cent. NaCl and above 1.3 per cent.
NaCl. These facts show most strikingly that when a
sufficient number of NaCl molecules or Na ions — I suspect
that we are dealing only with the ions — enter a muscle, the
absorptive power of the muscle for water is altered in a
similar way as through the introduction of K ions into the
muscle. Table IV shows the difference in the amount of
water absorbed by muscles in neutral and in T^ normal acid
sodium-chloride solutions in eighteen hours.

TABLE IV

NEUTKAL NaCl SOLUTION

jis NOKMAL ACID NaCl SOLUTION

Concentration

Increase in
Weight in Ejght-
een Hours

Concentration

Increase in
Weight in Eight-
een Hours

4.90*
1.22
0.70

+6#
-2

+7

4.90*
1.22
0.70

-36.0*

+22.2
+40.0

WATER ABSORPTION BY MUSCLES AND SOAPS 517

While acids at one time increase, at another time decrease
the amount of water absorbed by a muscle, depending upon
the nature and the concentration of the salt solution in which
the muscles are immersed, alkalies increase absorption under
all circumstances. Quantitative differences, however, exist
between the different alkalies which I am not yet entirely
able to explain, even though I have made more experiments
with alkalies than with any other group of substances. These
experiments have only served to strengthen my previous
assertion that we deal in these cases only with the action of
the hydroxyl ions.

XXV

ON IONS WHICH ARE CAPABLE OF CALLING FORTH
RHYTHMICAL CONTRACTIONS IN SKELETAL
MUSCLE1

1. IN 1881 Biedermann published a remarkable observa-
tion "On Rhythmical Contractions Brought about in Striated
Muscle through Chemical Stimulation."2 The observation
consists, according to his description, in the following:

The sartorius of a frog, previously poisoned with curare, is
carefully removed from the body, if possible at a low temperature
(0 to 10° C.). If now the muscle preparation fastened vertically
into a muscle clamp, and weighted by its femoral stump, is dipped
into a 0.6 per cent, sodium-chloride solution .... containing
some ordinary alkaline, crystallized sodium phosphate, besides a
small amount of sodium carbonate (in the liter of distilled water
were contained 5 g. NaCl, 2 g. Na2HPO4, and 0.4 to 0.5 g. Na2CO3),
which must be kept at a low temperature (3 to 10° C.), one observes
as a rule, after a longer or shorter period of rest, that the immersed
muscle begins to beat rhythmically.

The twitchings vary in intensity. At times we have to do
with a mere tremor; at times, however, powerful beats and
contortions of the muscle occur; sometimes only individual
fibers are active; sometimes the whole muscle is involved.
Finally, the process may be limited to certain portions of
the muscle ; at other times the whole muscle may be involved.
At low temperatures these phenomena may continue for
days.

Biedermann recognizes the importance of this observa-
tion for the decision of the question whether the heart muscle
is of itself capable of rhythmical activity without the inter-
vention of the ganglion cells.

1 Festschrift fur Professor Fick (Braunschweig, 1899), p. 101.
*Sitzungsberichte der Wiener Akademie, Vol. LXXXII, Part III (1880).

518

RHYTHMICAL CONTRACTIONS IN MUSCLE 519

In his well-known and thorough work on electro-physiology
the same author adds the following:

Strong solutions of Na2SO4, as also very dilute solutions of
NaOH (in 5 per cent. NaCl solution), act in the same way as
Na2CO3,but not so strongly. In view of the similarity in the
effects of those substances upon the heart muscle one is justified in
speaking of a specific action of sodium salts, mentioned above, in
consequence of which the contractile substance of striated muscle
is so altered through the presence of even small amounts of these
substances that it is stimulated to contraction more easily and by
weaker stimuli than is ordinarily the case.1

We also know that Ringer has called attention to the
importance of calcium and potassium salts for the activity of
the heart, so that one might be inclined to believe that cer-
tain salts bring about the activity of the heart directly. In
this connection Ho well considers particularly the calcium
salts.2

2. It therefore seemed of interest to me to determine
whether the power to bring about rhythmical contractions in
skeletal muscle was not the property of certain ions. My
experiments consisted in observing the behavior of the gas-
trocnemms muscles of frogs in a series of solutions. The
muscle was entirely unweighted and unstretched and freed
from all bone. This fact is to be observed in repeating the
experiments. Carefully prepared equimolecular or isosmotic
solutions were used. The chemicals used were chemically
pure, and the water was twice distilled in glass. As we
often deal with only very weak contractions, or, more cor-
rectly, only with a tremor of individual muscle fibers, the
contractions could not be registered graphically, but could
only be observed.

Biedermann has already pointed out that the rhythmical
contractions of muscles in the solution used by him are not
entirely similar to the activity of the heart.

1 W. BIEDERMANN, Electrophysiologie (Jena, 1895), p. 92.

2 W. H. HOWELL, American Journal of Physiology, Vol. II (1898).

520 STUDIES IN GENERAL PHYSIOLOGY

The contractions of a heart preparation fed by an alkaline salt
solution are much more regular and even than the contractions of
a sartorius preparation; it is to be particularly emphasized that in
the first case all the muscle fibers contract evenly and simultane-
ously, while, as has been pointed out above, in a sartorius immersed
in a salt solution it is the rule that the primitive fibers never con-
tract simultaneously and equally strongly; much oftener the con-
tractions observed are local, and the rhythm of the various local
contractions may vary most decidedly.1

This difference between the periodic contractions of the
muscle and those of the heart holds also in the experiments
which I give below. I do not believe, however, that we can
conclude from these differences that there are differences in
the manner in which the rhythmical activities in the two
cases originate. We cannot, for example, conclude from this
that the difference is determined through the influence of
the ganglion cells in the heart. I believe that we deal,
partly at least, with a difference in conductivity. In the heart
muscle the stimulus can pass from element to element. If,
therefore, only one element is stimulated and caused to con-
tract, the whole heart must become active. In skeletal
muscle the sarcolemma prevents such a passage of the stimulus
from element to element, and we obtain in consequence irregu-
lar isolated contractions of the individual fibers. The case is
similar to that observed in Medusae, in which we find a syn-
chronous activity of all the elements when the continuity of
the elements is complete, and a loss in synchrony when the
conductivity between the elements is diminished.2

3. We will now return to our main problem, and endeavor
to answer the question whether only certain ions are able to
bring about rhythmical contractions in muscle.

I tested first of all Biedermann's assertion that these

1 BlEDEKMANN, IOC. tit.

2Engelmann and Romanes have pointed out this fact. A discussion of this sub-
ject is found in my Comparative Physiology of the Brain (New York: Putnam's
Sons, 1900).

RHYTHMICAL CONTKACTIONS IN MUSCLE 521

contractions are due to a specific effect of sodium salts.
Without entering into many details, I will only emphasize
the fact that lithium, caesium, and rubidium also bring about
such periodic contractions. So far as the anions are con-
cerned, rhythmical contractions occur not only in the chlo-
rides of the metals mentioned above, but also in the Br, I,
and F salts. In the last-named varieties of salts contrac-
tions begin even earlier than in the corresponding chlorides.
In a 0.7 per cent. NaCl solution it usually takes more than an
hour before the regular rhythmical contractions begin.
These show themselves at first in a slight tremor at the
femoral end of the gastrocnemius, become stronger, and
finally affect the whole muscle so that the tendon of Achilles
executes pendulum-like movements. These periodic move-
ments can still be observed on the following day even at
room temperature. In a NaF solution, equimolecular with
a 0.7 per cent. NaCl solution, the most vigorous contractions
set in immediately, but last only about half an hour and then
stop. In LiBr or NaBr the contractions are stronger, and
occur earlier than in NaCl. But whether this can be
attributed to the fact that Br ions are more effective than Cl
ions is questionable. As the muscle is always surrounded
by a NaCl solution and contains Cl ions, it is clear that F
or Br or I ions must at first pass in greater number from
the solution into the muscle than the Cl ions. I cannot
say definitely that I ions are more effective than Br ions.
The Rb and Cs ions have a poisonous effect like the F ions;
that is to say, the rhythmical contractions which are pro-
duced at first soon cease. In NaBr, LiBr, Nal, Lil, and
LiCl solutions isosmotic with a 0.7 per cent. NaCl solution
the contractions may continue at room temperature for one
or two days, in which, of course, periods of rest are often
noticed.

4. If the number of ions entering the muscle determines

522 STUDIES IN GENERAL PHYSIOLOGY

the contractions, one would expect that an increase in the
osmotic pressure of the active ions should also be able to
cause rhythmical contractions to set in more quickly. We,
indeed, find that a solution having a greater concentration
than that of a 0.7 per cent. NaCl solution brings about con-
tractions sooner. In a 1.4 per cent. NaCl solution contrac-
tions begin immediately and continue uninterruptedly; in a
1.05 per cent. NaCl solution contractions often do not begin
until several minutes after the immersion of the muscle in
it; in a 0.7 per cent. NaCl solution rhythmical contractions
only begin after one or one and a half hours. In the highly
concentrated solutions the contractions begin immediately,
but they also cease very soon, What has been said here for
NaCl solutions holds good also for the solutions of other
ions capable of calling forth contractions ; for example, NaBr
or LiBr solutions. I had solutions of these two salts which were
isosmotic with the following NaCl solutions: 3.5, 2.8, 2.1, 1.4,
1.05, and 0.7 per cent. In the latter concentration it often took
some time before rhythmical contractions began, while in the
solutions of a greater concentration contractions developed
immediately. In solutions isosmotic with a 1.4 per cent.
NaCl solution maximum contractions set in at once; while
in the next weaker solution, 1.05 per cent, maximum con-
tractions occurred only after some time. In these strongly
concentrated solutions the rhythmical contractions did not,
however, continue for so long a time as in the 0.7 per cent.
NaCl solution, or in solutions of NaBr or LiBr isosmotic
with this.

5. The fact that in salt solutions of high concentration
the contractions soon cease points without doubt to an injury
of the muscular substance through the salt solution used. I
expected that as soon as the contractions ceased in a salt
solution the periodic irritability of the muscle had also
fallen below a certain point. But this was, however, by no

KHYTHMICAL CONTRACTIONS IN MUSCLE 523

means always the case. In a NaF solution equimolecular
with a 0.7 per cent. NaCl solution contractions ceased after
twenty-five minutes. After forty-five minutes I found that
the muscle could still be stimulated at a distance of the
secondary coil of 370 mm. — a degree of irritability which
was not far below the normal. At the same time, however,
I found that a muscle which had been in a 0.7 per cent.
NaCl solution for twenty-four hours still contracted, even
though its threshold of stimulation, as determined by the
same induction coil, had fallen to 300 mm. We have, there-
fore, a different threshold of stimulation for different ions.
One might think that the periodic contractions in the differ-
ent solutions are brought about in that the ions which
penetrate the muscle substance enter into definite combina-
tions with it. That such must exist I have proved in another
paper by showing that Na, K, and Ca ions entering the
muscle produce specific alterations in its osmotic behavior.1
Under this assumption it might be thought that when a
certain number of F ions have entered the muscle, the further
entrance of Na and F ions no longer brings about contrac-
tions, while at the same time an induction current is still
able to bring about marked contractions. I need scarcely
emphasize the fact that a muscle loses its electrical irrita-
bility more rapidly in a NaF solution than in a NaCl
solution isosmotic with it.

6. If in these experiments the rhythmical contractions are
brought about through ions, it was to be expected that no
periodic contractions should occur in non-electrolytes. I
never saw rhythmical contractions begin in chemically pure
distilled water.

I also prepared solutions of glycerin, dextrose, cane
sugar, and milk sugar isosmotic with a 0.7 per cent. NaCl

,1 "On the Similarity between the Absorption of Water by Muscles and by Soaps,"
Part II, pp. 510.

524 STUDIES IN GENERAL PHYSIOLOGY

solution; that is, of an osmotic pressure of 4.91 atmospheres.
In none of these solutions did rhythmical contractions
occur. I cannot as yet, however, say that this is true for all
non-electrolytes. The circumstance that contractions occur
immediately in a NaCl solution of a high concentration
suggests the possibility that loss of water by the muscle
favors the contractions. I therefore introduced the muscle
into glycerin and sugar solutions having two, three, four,
and five times as high an osmotic pressure as a 0.7 per cent.
NaCl solution. In none of them did rhythmical contractions
occur, even though the muscle lost water markedly. What
has been said applies, however, only to rhythmical contrac-
tions. Single twitches — that is, single separate contractions
—were occasionally observed during the first minutes in
glycerin solutions having an osmotic pressure of 4.91 atmos-
pheres or more. Such contractions can also be observed,
however, at times when fresh muscles are laid upon a glass
plate. The glycerin solution is therefore probably only
of a secondary importance.

7. I have above quoted the remark of Biedermann accord-
ing to which the stimulating effect of NaOH, as well as that of
Na2 CO3, is attributed to the "sodium salts." I have, how-
ever, shown that in the specific effects of very dilute alkalies
we are really dealing with the effects of hydroxyl ions.1 I
have since then corroborated this statement in that I have
convinced myself of the fact that NH4HO, which is much
less dissociated than NaOH, also acts physiologically as a
much weaker alkali solution. Hydroxyl ions are also present
in a Na2C(X solution which bring about the specific alkali
effects of this solution. The idea, therefore, suggested
itself that the hydroxyl ions are capable of calling forth
rhythmical contractions in a similar way to Na and other
ions. This seems to be true for the HO ions in even a

i Part II, p. 450.

RHYTHMICAL CONTRACTIONS IN MUSCLE 525

higher degree than in the case of Na ions, since, according
to Gaule, only a trace of NaOH or Na2CO3 favors the
rhythmical activity of the heart. The same favorable effect
of the HO ions shows itself also in the rhythmical contrac-
tions of skeletal muscle.

When a muscle is introduced into a 0.7 per cent. NaCl
solution, the rhythmical contractions of individual fibers or
the entire muscle begin after about sixty to ninety minutes.
If a trace of alkali is added, however, contractions begin
much sooner. I will give an example. In one series of ex-
periments the muscle began to beat rhythmically after
twenty-seven minutes in 100 c.c. of a 0.7 per cent. NaCl
solution to which had been added 2 c.c. of a one- tenth
normal LiOH solution. In 100 c.c. of a NaCl solution of
the same concentration to which had been added 3 c.c. of a
one-tenth normal LiOH solution weak rhythmical contrac-
tions began immediately. In 100 c.c. of a 0.7 per cent.
NaCl solution to which had been added 4 c.c. of the same
LiOH solution strong rhythmical contractions began imme-
diately. In the 0.7 per cent. NaCl solution (to which no
LiOH had been added) it required seventy-two minutes be-
fore rhythmical contractions began. Other series of ex-
periments yielded similar results. This shows without a
doubt that LiOH accelerates the appearance of rhythmical
contractions. That we are, indeed, dealing only with the
action of the hydroxyl ions is shown by the fact that it does
not matter what alkali is added (for example, LiOH, KOH,
NaOH, etc.), as long as the degree of dissociation is the
same.

It might be concluded from this that the hydroxyl ions
indeed belong to that class which of themselves are able to
bring about rhythmical contractions, for the rhythmical con-
tractions which occur immediately can be caused only by the
hydroxyl ions.

526 STUDIES IN GENERAL PHYSIOLOGY

I now made a series of experiments with a glycerin solu-
tion the osmotic pressure of which was 4.91 atmospheres;
which was, therefore, isosmotic with a 0.7 per cent. NaCl
solution. I added to 100 c.c. of this solution one-tenth
normal LiOH in different quantities from 1 to 4 c.c. In no
case did I obtain rhythmical contractions. Similar experi-
ments with sugar solutions having an osmotic pressure of
4.91 atmospheres yielded the same results. These experi-
ments with sugar solutions are, of course, less conclusive,
since a portion of the hydroxyl ions are in this case rendered
inactive. The addition of 5 c.c. of a one- tenth normal
LiOH to 100 c.c. dextrose or cane sugar caused no contrac-
tions. Upon the addition of 10 c.c., however, of a one-tenth
normal LiOH solution a few weak contractions appeared
during the first minute. Beside the experiments with the
glycerin solutions the experiments with distilled water
adduce proof. In no case did hydroxyl ions in distilled
water bring about rhythmical contractions. One might
think perhaps that distilled water reduces the irritability so
rapidly that contractions are no longer possible.1 That is,
however, not the case. In one case in distilled water the
muscle was still irritable after an hour to an induction cur-
rent from the induction coil used in all these experiments
when the distance of the coils was 310mm., while the normal
irritability lay at about 390 mm. Rhythmical contractions
occur in solutions of sodium salts at a still lower irritability.
The addition of 10 c.c. one-tenth normal LiOH to 100 c.c.
of distilled water gave no rhythmical contractions. Even
in a one-fortieth normal or one-tenth normal LiOH solu-
tion no rhythmical contractions occur. Very weak, one-
thousandth to one-hundredth normal LiOH or NaOH solu-

1 It was found in these experiments that a dextrose solution isosmotic with a
0.7 per cent. NaCl solution reduced the irritability of the muscle more rapidly than
the NaCl solution; sugar, therefore, is no indifferent substance. That NaCl is not
has been proved by Locke.

RHYTHMICAL CONTRACTIONS IN MUSCLE 527

tions were also ineffective. Hydroxyl ions, therefore, do not
belong to those ions which are capable of liberating rhythmi-
cal contractions. They only accelerate the production of
rhythmical contractions when added to electrolytes in which
such rhythmical contractions occur without the addition of
the hydroxyl ions.

8. What has been said in the foregoing on the effects of
hydroxyl ions holds also for hydrogen ions. The addition
of hydrogen ions accelerates the appearance of the contrac-
tions in a 0.7 per cent, sodium-chloride solution. If 1, 2, or
3 c.c. of a -jig- normal HNO3 solution are added to 100 c.c.
of a 0.7 per cent. NaCl solution, rhythmical contractions
begin immediately, or at least earlier than in the pure salt
solution. It can, however, again be shown that in non-
electrolytes, such as glycerin and sugar solutions, having an
osmotic pressure of 4.91 atmospheres acids do not produce
this effect, nor do they do it in distilled water, no matter
what the concentration of the acid. That we are indeed
dealing in this case with the action of hydrogen ions is
shown by the fact that inorganic acids produce the same
effects if the same number of hydrogen ions are contained
in the unit volume. Hydrogen ions, therefore, do not
belong to the ions which produce rhythmical contractions.

9. The question now arises how it happens that H and
OH ions accelerate the rhythmical contractions in electro-
lytes. It might be thought that these ions have an etching
effect, and so bring about continually differences in potential
between different portions of the muscle fibers. In this way
the muscle would be stimulated continually through its own
currents and would be kept in a state of rhythmical activity.
It would be in harmony with this view that these contrac-
tions do not occur in distilled water and in non-electrolytes.
Against it, however, stands the fact that not every electro-
lyte brings about rhythmical contractions upon the addition

528 STUDIES IN GENERAL PHYSIOLOGY

of an alkali. If 2 or 3 c.c. of a ^ normal LiOH solution is
added to 100 c.c. of a KC1 solution isosmotic with a 0.7 per
cent. Nad solution, no rhythmical contractions occur.

One might think that acids and alkalies increase the
irritability, and that for this reason their addition would
accelerate the contractions. We find, however, that in cer-
tain solutions periodical contractions occur when the irrita-
bility has been decreased, while in other solutions these
contractions do not occur at all even when the irritability is
normal or above normal. In 100 c.c. KCl solution to which
had been added 2 c.c. of a y1^ normal LiOH solution the
muscle was still irritable after half an hour when the
secondary coil was 420 mm. away from the primary, without
a single contraction occurring, while another muscle the irri-
tability of which had dropped to 300 mm. in a LiCl solution
still gave strong rhythmical beats at the same time. It can
further be easily shown that the addition of an acid which
accelerates the beginning of contractions soon decreases
markedly the irritability of the muscle.

I am much more inclined to go back to the first explana-
tion given, that certain ions — for example, Na ions — bring
about rhythmical contractions in that they enter the muscle
and here form definite compounds. H and OH ions have a
catalytic action; that is, they accelerate the formation of
these compounds. It might also be that they owe their
activity to their power of splitting off the material necessary
for the formation of the compounds with the Na ions.1

10. We have thus far become acquainted, first, with a
group of ions which are able to bring about rhythmical con-
tractions in skeletal muscle (Li, Na, Rb, Os, F, Cl, Br, I);
secondly, we have seen that H and OH ions accelerate the
appearance of the effect of the ions just named. There is a
third class of ions which inhibit rhythmical contractions.

1 It is also possible that through their effect on the lipoids they increase the
permeability of the muscle for Na and other ions or salts. [1903]

KHYTHMICAL CONTRACTIONS IN MUSCLE 529

To these belong first of all the K ions. I prepared a series
of potassium compounds, all of which were isosmotic, with a
0.7 per cent. NaCl solution, namely, KC1, KBr, KI, K2SO4,
and potassium oxalate. Rhythmical contractions did not
occur in any of these solutions. We know, however, that
Br ions have the power of causing rhythmical contractions,
or at least are able to accelerate the appearance of these con-
tractions, for in NaBr the contractions occur much sooner
than in an isosmotic NaCl solution. Potassium must, there-
fore, prevent the contractions.

The sodium compounds corresponding to the potassium
compounds just given are all able to bring about rhythmical
contractions.

Ca also inhibits the contractions; and not only this, but
the entire group, Be, Mg, Ba,1 Sr, and also Mn and Co. No
contractions occur in these solutions.

The behavior of Ca is so characteristic and its r6le in the
phenomena of contractions of such significance that I shall
have to discuss this point in greater detail.

In one series of experiments I used the following solutions :

a) 100 c.c. NaBr isosmotic with a 0.7 per cent. NaCl
solution.

6) The same plus 2 c.c. of an equimolecular CaCl2 solu-
tion (about 1.33 per cent.).

c) The same with 3 c.c. of the CaCl2 solution.

d), e),f} The same with 4, 5, and 6 c.c. respectively of
the CaCl 2 solution.

The percentage of CaCl contained in the various solutions
was, therefore, in order as follows: 0.0265, 0.0387, 0.0511,
0.0633, 0.0744 per cent. A fresh gastrocnemius muscle
was introduced into each of these solutions. The concen-
tration of the CaCl 2 in the second solution was equal to

i It was found later on that Ba salts cause contractions in a low concentration.
This had been noticed before. Why this was at first overlooked by me I cannot telL
[1903]

530 STUDIES IN GENERAL PHYSIOLOGY

that found in the serum of the turtle (according to Greene).
One can scarcely conceive of a more convincing experiment
than the foregoing. In the pure NaBr solution rhythmical
contractions occurred after a minute, and lasted uninter-
ruptedly for twelve hours, when the experiment was brought
to a close. In the second solution, which contained the least
amount of Ca, occasional very weak contractions occurred
which were noticeable 'during the first thirty minutes, but
for the remainder of the day the muscle remained absolutely
quiet. In the other solutions containing greater amounts of
CaCl2 no contractions whatever occurred.

So slight an amount of calcium as 0.026 per cent. — that
is to say, the amount of calcium contained in blood serum — •
is sufficient to render almost entirely impossible the periodi-
cal contractions brought about through Na and Br ions, and
a little more, namely, 0.038 per cent, of CaCl3 , suffices to do
away with the contractions entirely.1

It might be doubted that in these experiments the Ca
acted as the specific inhibiting substance for the muscular
contractions. It might be considered possible that the
CaCl3 had decreased the irritability of the muscle to such an
extent that the liberation of contraction through Na and Br
ions had been rendered impossible ; but that is not the case.
I tested after three hours the faradic irritability of all the
muscles. That contained in the NaBr gave the first con-
tractions when the distance of the secondary coil was 310
mm. ; that in the NaBr plus 0.026 CaCl3 solution had the
same threshold of stimulation; that in the other solutions
was only slightly lower. On the following morning the
muscles contained in the CaCl2 solution were more irritable
than that in the pure NaBr solution, which had contracted
continually. We can, therefore, only be dealing with the
fact that the entrance of Ca ions into the muscle inhibits the

1 We are therefore indebted to the calcium contained in our blood for the fact
that our muscles do not twitch continually.

RHYTHMICAL CONTRACTIONS IN MUSCLE 531

liberation of rhythmical contractions through Na and Br
ions. Because of the importance of this fact, I will briefly
report another experiment. I repeated the same experiment
given above, with this difference, that a 0.7 per cent. NaCl
solution was used instead of NaBr. In this case the periodic
contractions began in the pure NaCl solution in eighty
minutes; in the remaining solutions, which contained a trace
of CaCl2, contractions did not occur at aliquot even in the
solution which contained 0.026 per cent. CaCl2.

It could be shown in these experiments also that the
faradic threshold of stimulation of the muscle contained in
the solution containing Ca was no lower than that of the
muscle which beat rhythmically in the NaCl solution free
from Ca.

In a more concentrated CaCl2 solution — for example, in
a solution isosmotic with a 0.7 per cent. NaCl solution — not
only do no contractions occur, but the faradic irritability is also
rapidly destroyed. If, however, only a small amount of Ca
is present in the NaCl solution — for example, as much as
there is contained in an equal volume of serum — only the
specific inhibiting effect of the Ca ions upon the liberation
of the rhythmical contractions through Na and other ions
appears, while the irritability does not suffer.

We therefore come to this necessary conclusion* that there
are ions the entrance of which into the muscle has a specific
inhibiting effect upon the liberation of rhythmic contractions.
For practical purposes the most important of these ions are
Ca and K

11. An apparent contradiction to what has been said 18
found when the following experiments are made. Two
solutions are prepared, one a 0.7 per cent. NaCl solution,
and a second similar solution to which 2 c.c. of an equimo-
lecular CaCl2 solution have been added to 100 c.c. of NaCl.
The latter solution then contains the amount of CaCl2 found

532 STUDIES IN GENERAL PHYSIOLOGY

in the serum. In the former solution the muscle begins to
twitch after about eighty minutes, and continues to contract
rhythmically from twelve to forty hours. In the second
solution either no contractions whatsoever occur, or the con-
tractions begin later and cease earlier, that is, are decreased
in number. This is only a repetition of what has been said.
If now, after the contractions have ceased in the pure NaCl
solution, the muscle which has been in the NaCl solution
containing Ca, but which has not contracted, is put into the
pure NaCl solution, it begins to contract in this solution,
while the other muscle will contract in neither of the two
solutions.

The two facts can perhaps be explained upon the following
basis: From my observations on the absorption of fluids by
muscle, it follows that Na, Ca, and K ions must be present
in the muscle in combinations in which these three ions can
be exchanged one for the other. If the muscle is contained
in a solution of one or more of these ions, the relative com-
bination of three kinds of ions is governed by the laws of
chemical equilibrium. If only Na ions are present, but no
K or Ca ions, a number of Na ions will take the place of Ca
ions and K ions until the laws of equilibrium have been
fulfilled. In this process the muscle becomes poorer in Ca
compounds, or at least in such Ca compounds in which Ca
exists in the ionic state and in which it can be replaced by
Na ions. (Ca may yet exist in still another form in the
muscle substance, and be able to enter from this form into
the ionic form through metabolic changes.) If, on the other
hand, Ca ions are in the solution then at a low (very low)
concentration of the Ca ions, a substitution of Na ions for
the Ca ions is at once compensated by an equal substitution
of Ca ions for Na ions. If the concentration of the Ca ions
is greater, the Ca ions must crowd out a part of the Na ions
from these combinations. Thus far this is no hypothesis, as

RHYTHMICAL CONTRACTIONS IN MUSCLE 533

it is only the application of simple laws to the muscle. We
now find, when we keep in mind what has been said above,
that the entrance of sodium ions (but also Cl, Br, and other
ions which have been mentioned above or are at present still
unknown) into certain muscle compounds is accompanied by
contractions. We observe, secondly, that the entrance of
Ca ions into the normal muscle inhibits these contractions.
Indeed, one might think that the process of substituting any
ions for Ca ions is adapted in a peculiar way to giving rise
to a contraction, while the reverse process, the substitution
of Ca ions for any ions, alters the muscle substance in an
opposite sense. In this way it might be explained why a
muscle does not contract in the NaCl solutions containing
Ca, while it begins to contract immediately when introduced
into a NaCl solution free from Ca. It also becomes intelli-
gible that the muscle which has been in a pure NaCl solution
for some time no longer contracts. It has no more Ca ions
to exchange. What has been said harmonizes also with the
previous observations of Ringer, that a muscle in a NaCl
solution remains irritable for a longer time when the solution
contains a trace of Ca and K salts. And it also harmonizes
with what has been said, that, according to Howell, a muscle
from which the Ca salts have been removed by washing with
oxalates loses its irritability. Our views and observations
do not, however, agree with Howell's claim that NaCl "is of
importance only in so far as it maintains the osmotic pressure
between the tissues and the surrounding fluid." This view
of Howell contradicts also the observations of Locke, who
showed that NaCl is not so indifferent a substance. I am
gradually coming to believe that, strictly speaking, there is
no solution which is merely of osmotic importance for living
tissues. Even an isotonic sugar solution (about 4.91 atmos-
pheres pressure) affects the irritability, and probably also
other properties, of the muscle to a high degree. We must

534 STUDIES IN GENEEAL PHYSIOLOGY

in this not allow the statements of the botanists to lead us
astray. We have in the contractile and rhythmically acting
animal tissues much more sensitive living reagents than the
botanist has in most plants. True has also recently found
that NaCl is not an indifferent substance. He attributes,
however, a purely osmotic effect to the pure sugar solutions.1
This is, however, incorrect for animal tissues at least.

12. Naturally ions which decrease the irritability must
always counteract the effects of an ion capable of calling
forth contractions when both are present in the solution.
That can be best understood when a large number of Na
salts are prepared which are equimolecular with a 0.7 per
cent. NaCl solution. In nearly all of these solutions
rhythmical contractions occur at first. It is dependent
only upon the anion how long these contractions continue.
While the contractions in a NaCl or NaBr solution may
last from twenty-four to forty-eight hours, they cease in an
isotonic solution of sodium acetate, butyrate, oxalate, tar-
trate, or citrate in one-half to one and a half hours. In a
NagSO4 solution the contractions cease in less than two
hours. The anions of the given salts diminish very markedly
the irritability. In Na3PO4 no contractions whatsoever
occur, and here the irritability is reduced very rapidly.
The same holds also for Na2CO3 equimolecular with a 0.7
per cent. NaCl solution. Hydrogen and hydroxyl ions also
decrease the irritability, even in very dilute solutions. The
same is true of NH4 ions.

13. I believe that these and similar facts will help us to
understand the phenomena of contraction, and possibly also
the phenomena of irritability in general. I will only show
in this paper that the conclusions which we have drawn hold
also for heart muscle. Aubert has found that the ventricle
will beat in a physiological NaCl solution, but that it is not

IR.H.TEUE, Botanical Gazette, Vol. XXVI (1898).

RHYTHMICAL CONTRACTIONS IN MUSCLE 535

able to beat rhythmically in the serum of blood containing
Ca. Greene has recently shown that when CaCl2 is added
to a physiological NaCl solution in a concentration equal to
that in which it exists in the serum, the pulsations of the
ventricle cease, while rhythmical irritability continues. That
is the same behavior, therefore, as is the case with the
rhythmical contractions of the gastrocnemius, and we may
therefore assume that for the heart-beat the same circum-
stances come into consideration as for the rhythmical con-
tractions of skeletal muscle; with the exception, of course,
'of the conditions for the conduction of the stimulus which
we have discussed under paragraph 2. The role of the
hydroxyl ions for the heart activity might also be the same
as that we have found to be the case for the rhythmical con-
tractions of skeletal muscle.1 The further we carry the
analysis of the two processes, the more complete, I believe,
we shall find their identity, with the exception, of course, of
the conditions of conductivity of the stimulus. I find only
two contradictory facts. One is originated by Howell — that
NaCl is only of osmotic significance in the activity of the
heart. So far as the ventricle is concerned, this statement
of Howell's is contradicted by the observations of Aubert.
The second assertion comes from Howell's pupil, Greene.2
Greene has observed very correctly that CaCl2 in the same
concentration as it exists in the serum prevents the rhythmi-
cal contractions of the ventricle. Then, however, he adds
the following assertion, that "when the amount of CaCl2 in
the serum is slightly increased, regular contraction begins."

i 1 have recently tried to determine how many free hydroxyl ions the blood con-
. tains in the unit volume. I find that it cannot possibly be more, and is probably
less, than those present in the same volume of a one-thousandth normal NaOH solu-
tion. Until now we have always made the alkalinity determination of the blood by
titration. For the specific alkalinity of the blood, however, only the active alkalin-
ity— that is, the concentration of the free hydroxyl ions in the blood — comes into
consideration. It has since been found by Friedenthal, Fraenckel, Farkas, and
Hoeber that blood has a neutral reaction. [1904]

2 GREENE, American Journal of Physiology, Vol. II (1898).

536 STUDIES IN GENERAL PHYSIOLOGY

The concentration with which we have to deal in this case
is, according to him, 0.04 per cent, instead of 0.026 per cent,
of the serum. So far as the rhythmical contractions of the
muscle are concerned, the claim of Greene is incorrect.
When a muscle does not contract rhythmically in a NaCl
solution containing Ca, when the concentration of the CaCl
solution is 0.026 per cent., then no contractions occur at all
at a concentration of 0.04 per cent., or higher. For the
apex of the heart the relations are no different, and the claim
of Greene to the contrary rests, so far as I can see from his
paper, upon a misunderstanding.

In observing the "stimulating" effect of the 0.04 per
cent. CaCl 2 solution the strip of heart muscle did not lie in
such solution, but hung in a moist chamber, and was moistened
with two or three drops of the CaCl2 solution named. It
then began to beat rhythmically. If we assume that cal-
cium did indeed have something to do with this, we are still
not dealing in this case with the same condition of affairs
that we have when heart muscle is immersed in a large quan-
tity, about 100 to 200 c.c., of such solution. In the latter
case a larger number of Ca ions would enter the muscle in
the unit of time. By moistening the muscle with two or
three drops of a 0.7 per cent. NaCl solution containing
0.04 per cent. CaCl2 the larger amount of the fluid runs off,
and the few Ca ions which enter the muscle are perhaps in
actual number equal to those which enter from a NaCl solu-
tion containing a very small amount, say 0.001 per cent.
CaCl g . Greene and Ho well have overlooked the fact that
the concentration of the solution is not the determining
factor in the latter case, but the number of molecules which
are driven into the muscle (through osmotic pressure).
When it is found that 100 c.c. of morphine of a certain
concentration kill a dog, and when it is further observed
that y-J-^ c.c. oe n morphine solution two or three times as

KHYTHMICAL CONTKACTIONS IN MUSCLE 537

strong does not injure the dog, the conclusion cannot be
drawn from this that a morphine solution is more harmless
the greater its concentration, I do not doubt but that,
when a ventricle which does not beat in a 0.7 per cent. NaCl
solution containing 0.026 per cent. CaCl2 is introduced in-
to a 0.7 per cent, solution containing 0.04 per cent. CaCl2,
and the quantity of the solution is the same in both cases,
the ventricle will not beat in the latter case either. Whether
a very dilute CaCl2 solution, the concentration of which lies
far below that of the serum (that is, far below 0.026 per cent.),
is able to augment the stimulating effect of a NaCl solution
is yet to be tested, and might easily be made to harmonize
with what has been said above.

14. Conclusions.

a) There are certain ions — for example, Na, 01, Li, F,
Br, I, and others — which (in solutions having an osmotic
pressure of 4.91 atmospheres) are capable of calling forth
rhythmical contractions in muscles. These contractions are
not brought about in that the given ions increase the irrita-
bility, since, first, these contractions may continue even when
the irritability has been diminished; and, secondly, since in
solutions of non-electrolytes (for example, glycerin and
sugar) of the same osmotic pressure these contractions do
not occur even when the tissues have their normal irritability.
It is to be believed rather that the entrance of the ions
mentioned above into definite compounds in the muscle is
the cause of these contractions.

6) There are ions which inhibit the liberation of rhyth-
mical contractions of the normal muscle ; for example, Ca, K,
Mg, Be, Sr, Co, and Mn. The inhibiting effect of Ca, K,
and possibly also the other ions, does not rest upon the fact
that they reduce the irritability of the muscle ; for through
the addition of only a small amount of Ca012 to a physi-
ological salt solution the contractions are prevented, while

538 STUDIES IN GENERAL PHYSIOLOGY

the irritability of the muscle suffers less and is maintained
for a longer time than in the pure physiological salt solu-
tion in which these rhythmical contractions take place. It
is rather to be believed that the entrance of Ca (and K)
into definite compounds in the muscle renders difficult or
impossible the rhythmical contractions.

c) Hydroxyl and hydrogen ions accelerate the beginning
of rhythmical contractions when added in a sufficient dilu-
tion to the solutions of the ions mentioned under a). If,
however, they are added to solutions of non-electrolytes or
ions which inhibit contractions, they do not have these
effects. They have, therefore, a catalytic effect in the start-
ing of rhythmical contractions by other substances with-
out, however, being able to call forth these rhythmical
contractions directly.

d) According to our present state of knowledge, which is
still limited, we dare say, that only ions, and not non-electro-
lytes, are able to call forth rhythmical contractions in skeletal
muscle.

e) The laws governing the periodic contractions of the
ventricle of the heart seem to be the same as those which
have shown themselves to govern voluntary muscle.

XXVI

ON THE NATURE OF THE PROCESS OF FERTILIZA-
TION AND THE ARTIFICIAL PRODUCTION OF NOR-
MAL LARV.E (PLUTEI) FROM THE UNFERTILIZED
EGGS OF THE SEA-URCHIN l

1. FORMER researches had led me to suspect that changes
in the state of matter (liquefactions and solidifications) might
play an important role in the mechanics of life-phenomena.
While studying the absorption of liquids by muscle I found
that, to all appearances, a | solution2 of CaCl2 favors the
formation of solid compounds in the muscle, while an equi-
molecular solution of KC1 favors the formation of more
liquid compounds. Na ions rank between the K and Ca
ions. In these phenomena, however, much depends upon
the concentration of the salts. We know that the enzymes
of coagulation and liquefaction are greatly influenced in
their action by the Ca, Na, K, and Mg ions. Ca favors
coagulation, and Mg does the reverse. Between these come
the two other ions. In this case also much depends upon
the concentration.

I have made a series of studies on the mechanics of life-
phenomena, which will be published shortly in this Journal.
I wish now to deal only with one part of these studies,
namely, that referring to the nature of the process of fertili-
zation.

I found that in fn solutions3 of CaCl2, NaCl, KC1, and
MgClg the segmentation of fertilized eggs of sea-urchins

1 American Journal of Physiology, Vol. Ill (October 1, 1899), p. 135.

2 I propose to substitute in the future the -5- solution of NaCl for the 0.7 per

O

cent, solution. It is time that we were rid of percentage solutions in physiology.

3 Approximately the concentration of sea-water.

539

540 STUDIES IN GENEEAL PHYSIOLOGY

(Arbacia) proceeded best in MgCl3, next best in KC1, while
CaClg proved to be the most injurious in the series.

Seven years ago I, and later Norman, found that if the
concentration of sea-water be raised sufficiently by the addi-
tion of certain salts, a segmentation of the nucleus takes
place without any segmentation of the protoplasm. Such
eggs, however, when brought back into normal sea-water,
divide into as many cells as there are preformed nuclei.
This year I tried the effects of equimolecular solutions of
MgCl2, KC1, NaCl, and CaCl2 upon this process of nuclear
division (in which the nuclear membrane is apparently
liquefied), and found that the influence of the four salts
(or rather kations) followed the order mentioned above.

We know that enzymes as a rule require a slight degree
of acidity or alkalinity for their action. I showed last year
that the addition of a small amount of H ions to sea-water
retards or prevents segmentation, while a small amount of
HO ions favors and accelerates the development of the Arbacia

egg-

2. It has been known for some time that the unfertilized

eggs of echinoderms, worms, and arthropods begin to seg-
ment when left for a comparatively long time in sea-water.
This has generally been considered a pathological phenom-
enon. Mead succeded in causing a segmentation of the
unfertilized egg of a marine worm, Chsetopterus, by the addi-
tion of a very small amount of KC1 to sea-water. Morgan
tried the effect of more concentrated sea-water on the un-
fertilized egg of sea-urchins, with results similar to those
obtained by me previously with the same methods in fer-
tilized eggs. If the unfertilized eggs are brought back from
the more concentrated sea-water into normal sea-water, they
break up into as many cells as there are nuclear masses pre-
formed in the more concentrated solution. But in none of
these cases did the cell-divisions of the unfertilized eggs lead

ARTIFICIAL PRODUCTION OF NORMAL LARV^J 541

to the formation of a blastula. A heap of cells, at the best
about sixty, were formed, and then everything stopped.
We cannot utilize these observations for the theory of
fertilization, for the simple reason that the essential element
of the process of fertilization, namely, the formation of an
embryo, was lacking. In the case of tumors or galls we have
cell-division and even growth, and yet these cell-divisions do
not result in the formation of an embryo.

3. Some recent observations suggested to me that some-
thing in the constitution of the sea-water prevented the
unfertilized eggs of marine animals from developing parthen-
ogenetically. Last year I found that the striped muscles
of a frog beat rhythmically (like the heart) if put into a ^
NaCl or NaBr solution. It is only the presence of K and
Ca ions in the blood that prevents striated muscles from con-
tracting rhythmically in the body. Romanes had observed
that if the margin (with the nerve ring) in Hydromedusse
be cut off, the center no longer contracts rhythmically. I
found this summer that this is due solely to the presence of
K and Ca ions in the sea-water. In a ^n solution of NaCl,
or still better of NaBr, the center continues to beat spon-
taneously. In applying this and my more recent observa-
tions on the relative influence of the various ions upon seg-
mentation to the problem of artificial parthenogenesis it
seemed to me that by making two changes in the constitu-
tion of sea-water the eggs of the sea-urchin might be able to
produce perfect embryos without being fertilized. These
changes were either a reduction of the Na and Ca ions or an
increase in the Mg (or K) ions or both. I think that a great
number of variations in this sense might bring about the
desired effect, but the end of the season allowed me to try
only a limited number of variations. Without going into
details (which may be reserved for the full report) I will
state briefly that the mixture of about 50 per cent. 2^n

542 STUDIES IN GENERAL PHYSIOLOGY

MgCl2 with about 50 per cent, of sea- water was able to
bring about the same effect as the entrance of a sper-
matozoon.1 The unfertilized eggs were left in such a solu-
tion for about two hours. When brought back into normal
sea-water they began to segment and form blastulse, gastrulae,
and plutei, which were normal in every respect. The only
difference was that fewer eggs developed, and that their
development was slower than in the case of the normal
development of fertilized eggs. With each experiment a
series of control experiments was made to guard against the
possible presence of spermatozoa in the sea-water. Un-
fertilized eggs of the same female were brought into normal
sea- water, and in solutions with too little MgCl2. Neither
in the normal sea-water nor in any of these solutions with
too little MgCl3 did one single egg develop into a blastula
or show anything more than the beginning of a segmenta-
tion after a long time.

4. From these experiments it follows that the unfertilized
egg of the sea-urchin contains all the essential elements for
the production of a perfect pluteus. The only reason that
prevents the sea-urchin from developing parthenogenetically
under normal conditions is the constitution of the sea-water.
The latter either lacks the presence of a sufficient amount of
the ions that are necessary for the mechanics of cell-division
(Mg, K, HO, or others), or it contains too large a quantity
of ions that are unfavorable to this process (Ca, Na, or
others), or both. All the spermatozoon needs to carry into
the egg for the process of fertilization are ions to supple-
ment the lack of the one or counteract the effects of the
other class of ions * in the sea- water, or both. The sper-
matozoon may, however, carry in addition a number of

1 The subsequent experiments proved that the increase in the concentration of
the sea-water caused the development of the eggs. I was aware of this possibility
and was looking for it, but was misled through an error in the preparation of the
solutions (which I had intrusted to others). This error was afterward discovered
and corrected. [1903]

ARTIFICIAL PRODUCTION OF NORMAL LARV^: 543

enzymes or other material. The ions and not the nncleins
in the spermatozoon are essential to the process of fertiliza-
tion (which may interest those who believe with me that
physiologists ought to pay a little more attention to inorganic
chemistry). I have no doubt that the same principles hold
good for the process of fertilization of other, if not all, the
marine animals, although the ions involved will probably
differ in various species.

Finally we may ask the question, whether we may expect
to produce artificial parthenogenesis in mammalians. Jan6sik
has found segmentation in the unfertilized eggs of mam-
malians. This is similar to the fact mentioned above, that
the unfertilized eggs of sea-urchins may show a segmenta-
tion if they stay long enough in the sea-water. I consider
it possible that only the ions of the blood prevent the
parthenogenetic origin of embryos in mammalians, and I
think it further not impossible that a transitory change in
the ions of the blood may also allow complete partheno-
genesis in mammalians.

XXVII

ON ION-PROTEID COMPOUNDS AND THEIR ROLE IN
THE MECHANICS OF LIFE-PHENOMENA. — THE
POISONOUS CHARACTER OF A PURE NaCl SOLU-
TION1

I. INTRODUCTORY REMARKS ON ION-PROTEID COMPOUNDS

IN this series of articles I intend to publish some new
facts and ideas concerning the constitution of living matter,
and to apply these facts to a number of life-phenomena.
The new facts concerning the constitution of living matter
are chiefly as follows : The salts or electrolytes in general do
not exist in living tissues as such exclusively, but are partly
in combination with proteids. The salt or electrolyte mol-
ecules do not enter into this combination as a whole, but
through their ions. The great importance of these ion-proteid
compounds lies in the fact that by the substitution of one
ion for another the physical properties of the proteid com-
pounds change (for instance, their power to absorb water
and their state of matter). We thus possess in these ion-
proteid compounds essential constituents of living matter
which can be modified at desire, and hence enable us to vary
and control the life-phenomena themselves.

By making experiments on the effects of ions upon the
absorption of water by muscle I found that a muscle does
not take up the same amount of water in equimolecular
solutions of various chlorides.2 The differences were very
striking. While in a 0.7 per cent. NaCl solution the muscle
absorbed about 7 per cent, of its own weight of water within
eighteen hours, it absorbed about 40-50 per cent, of its

1 American Journal of Physiology, Vol. Ill (1900), p. 327.

2 Part II, p. 510.

544

ION-PROTEID COMPOUNDS 545

weight of water in an equimolecular KC1 solution. In an
equimolecular CaCl2 solution it lost about 20 per cent, of
water. In a LiCl solution it neither lost nor absorbed any
water. The same was true for the bromides and iodides of
the same metals. Even organic compounds of these metals
showed somewhat the same difference, although the effects
of the anion in these cases modified the results quantita-
tively. This difference between the effects- of the various
metal ions upon the absorption of water by the muscle
shows a remarkable parallelism with the influence of the
same ions upon the absorption of water in soaps. There are
Na, K, Ca and other soaps. While K soaps absorb enor-
mous quantities of water the Na soaps absorb much less
and the Ca soaps still less than the Na soaps. If in the Na
soap we substitute K ions for the Na ions, the soap takes up
quantities of water. If we substitute Ca ions for Na ions,
the soap loses water. From this I concluded that in the
muscle the various metal ions exist in combinations in which
they can as easily be substituted for each other as in the
soap compounds. These compounds are similar to the soap
compounds in one physical quality, namely, the absorption
of water. I expressed the opinion that the ions in muscle
must be in combination with proteids. If a muscle be put
into a KC1 solution, the K ions of the solution enter the
muscle and gradually take the place of the Na and Ca ions
in these metal proteids. The K proteids are able to bind
more water than the Na or Ca proteids. If the muscle be
put into a solution of CaCl2, Ca ions will take the place of
the Na and K ions in the proteid compounds of the muscle,
and the muscle must lose water. I have carried these ex-
periments farther and may publish some of the more recent
results in this series of articles. -

I next applied this conception of ion -proteid compounds
to a phenomenon which had hitherto been observed only

546 STUDIES IN GENEKAL PHYSIOLOGY

occasionally, namely, rhythmical contractions of the muscles
of the skeleton.1 I found that such rhythmical contractions
occur only in solutions of electrolytes, i. e., in compounds
which are capable of ionization. In solutions of non-
conductors (urea, various sugars, and glycerin) these
rhythmical contractions are impossible. This is an indica-
tion that they are a function of the ion-proteids. But only
in certain ion solutions are such rhythmical contractions
possible. All the solutions of Na salts are able to produce
them, but in a 0.7 per cent. Nad solution contractions be-
gin later and are less powerful than in an equimolecular
NaBr solution. This indicates that not only the metal ion
influences the physical qualities of the ion-proteids, but that
the anion does so as well. This might be understood from
the assumption that anions as well as kations may combine
with proteids. This forces us to raise the question whether
both ions may not combine with the same proteid molecule,
only with the difference that the two different ions be added
at different places in the molecule. My colleague, Professor
Stieglitz, with whom I discussed this question, called my
attention to the behavior of amido-acetic acid, which indeed
acts in a similar way. From all we know concerning the
constitution of proteids it seems justifiable to assume that
some of them may very well share certain peculiarities of
the amido acids.2

The experiments on the rhythmical contractions of the
muscles of the skeleton, however, led to some other data
concerning the ion proteids. Solutions of Na ions produce
rhythmical contractions only if the muscle cells contain Ca
ions in sufficient numbers. As soon as there is a lack of Ca
ions in the tissues the Na ions are no longer able to cause
rhythmical contractions. On the other hand, if we add Ca

1 Part II, p. 518.

2 SPIKO, Zeitschrifi fiir physiologische Chemie, Vol. XXVIII (1899), p. 174

ION-PROTEID COMPOUNDS 547

or K solutions to the NaCl solution, it will no longer cause
rhythmical contractions in a fresh muscle. It therefore
looks as if the substitution of certain quantities of Na ions
for Ca ions caused contractions ; but if this substitution goes
too far, the muscle loses its irritability. On the other
hand, the presence of Ca ions in the NaCl solution prevents
the substitution of a sufficient number of Na ions for Ca
ions, and in the muscle thus prevents rhythmical contrac-
tions. It is due to the presence of Ca (and K) ions in our
blood that our muscles do not contract rhythmically.

These facts received further support when I tried to
determine the active alkalinity of the blood. I had found
that a slight decrease in the active alkalinity of sea-water
retarded the development and growth of young sea-urchins,
while a slight increase in the number of hydroxyl ions
accelerated both processes. It seemed to me that the main
physiological interest in the alkalinity lay in the osmotic
pressure of the free HO ions in the blood or serum, as
only the free HO ions can have any physiological effects.
In the course of my experiments I found that the quantity
of free HO ions in the blood is neither increased by a
considerable addition of NaHO nor decreased by a con-
siderable addition of HC1.1 Experiments proved that this
is not due to the salts of the blood or serum, but to the
proteids. It is evident that the latter have the power of
combining with H and HO ions. Spiro came to a similar
result by starting from a different point of view.2 Here
again we have to deal with ion proteids.

If we look at this phenomena from a biotechnical view-

1 SPIRO AND PEMSEL, Zeitschrift far physiologische Chemie, Vol. XXVI (1898),
p. 233.

2 The experiments consisted in this, that I added various quantities of acid or
alkali to ox blood and examined how long the muscle of a frog remained alive in
such a solution. While the addition of a small amount of acid or alkali to a
physiological salt solution renders the latter toxic for the muscle, a very much larger
quantity of acid or alkali is required to make the blood toxic for the muscle.
[1903]

548 STUDIES IN GENERAL PHYSIOLOGY

point, they gain in significance. They teach us that we can
impart to a tissue new properties by changing the quality
and the relative proportions of the ions in combination with
the proteids. The characteristic qualities of every tissue
are partly due to the fact that its ion proteids contain certain
ions in definite proportions. Any change in this proportion
is accompanied by a change in the properties of the tissue.

Ten years ago I started upon experiments in which I
substituted at desire one organ for another or transformed
one organ into another (heteromorphosis). The agents I
used for this purpose were various forms of contact, gravita-
tion, and light. But the number of animals in which the
phenomena of heteromorphosis could be controlled was
rather limited. I concluded at that time that it would be
necessary for the development of this technical or con-
structive side of biology to find a more elementary point of
attack. The ion proteids, on account of the ease with which
their properties can be changed by a change of their ions,
seemed to offer the desired opportunity. I therefore decided
to devote last summer at Woods Hole entirely to experiments
in this direction. Marine animals which live in a medium
having a high concentration of ions seemed to offer better
opportunities than fresh-water or land animals.

A brief report of one part of the summer's work was
published in this periodical.1 Since then Dr. W. Pauli, of
Vienna, has published an address in which he reaches similar
conclusions on ion proteids independently.2 His conceptions
are based on experiments upon the physical qualities of
proteids. His address appeared too late to influence me in
my work or my ideas, but the clearness of his results and
statements was a very welcome support. He speaks in his

1 American Journal of Physiology, Vol. Ill, p. 135.

2 W. PAULI, Ueber physikalisch-chemische Methoden und Probleme in der
Medizin (Wien, 1900); and "Ueber die physikalischen Zustandanderungen der
Eiweisskorper," Wiener akademischer Anzeiger, October 12, 1899.

ION-PROTEID COMPOUNDS 549

address of ion-proteid compounds, and I only need to quote
the following sentence in order to show how far his concep-
tions and mine agree. "We cannot doubt the general
existence of ion-proteid compounds in the living organism.
We have even urgent reasons for assuming that all the pro-
teids of the protoplasm exist there only in combination with
ions." I shall have a chance to discuss his views further
when the full account he promises of his experiments appears.
These introductory remarks may suffice for the present.
I will now report on a series of experiments which were
undertaken on the assumption of the existence of ion pro-
teids, and the possibility of changing the qualities of tissues
by changing the relative proportions of ions in the tissues.

II. EXPERIMENTS ON FISH

If it be true that life-phenomena depend upon the pres-
ence of a number of various metal proteids (Na, Ca, K, and
Mg) in definite proportions, it follows that solutions which
contain only one class of metal ions must act as a poison.
The reason for this is that the one class of metal ions will
gradually take the place of the other metal ions in the ion
proteids of the tissues. Even a pure NaCl solution must
thus be poisonous, although this salt permeates all our tissues
and is the main constituent of the inorganic matter of the
ocean. I have looked through the literature in vain to find
facts which corroborate this view. I found only the follow-
ing data: Ringer and Locke1 mention the fact that in NaCl
solutions the contraction curve of a muscle may show slight
variations, and Locke adds that the same is true for certain
electric phenomena in the nerve. True has added another
observation in this direction. It has been known for many
years that if we put plant cells, for instance Spyrogyra, into
a very concentrated solution of salts or sugars, the cells
lose water and cease to grow. True found that the osmotic

i LOCKE, Archivftir die gesammte Physiologie, Vol. LIT (1893), p. 501.

550

STUDIES IN GENERAL PHYSIOLOGY

pressure of a NaNO3 and KNO3 solution which is just able
to prevent growth in Spyrogyra is lower than that of a sugar
solution which renders growth impossible. Hence he con-
cludes that NaNO 3 is not harmless for Spyrogyra.1 In this
case, of course, the concentration of Na ions was much
greater than in the blood or in sea-water. It is, however,
easy to give striking illustrations of the fact that a pure
NaCl solution is a strong poison. In a former paper I have
shown that Fundulus, a marine fish, can endure an astonish-
ing increase in the concentration of sea- water. An addition
of 5 per cent. NaCl to sea- water does not injure the animal.2
I selected it for testing my conceptions concerning the metal
proteids. I found that it dies in a short time in a pure NaCl
solution of the same concentration as that in which this salt
exists in the sea-water.3 In %n solutions of NaCl which
were diluted with various quantities of distilled water, the
animals lived the longer the greater the dilution was, and in
distilled water the young fish lived indefinitely. The
following table shows the duration of life of these fish in
pure NaCl solutions of various concentrations:

TABLE I

AVEEAGB DURATION OF LIFE OF YOUNG FUNDULUS IN PURE NaCl SOLUTIONS OF
DIFFERENT CONCENTRATION

Nature of the Solution

Duration of Life

100 C -n !. 5'

ft NaCl

Less than 12 hours

90

M

" + 10 c.c. distilled water

About 24 hours

80

u

" + 20

" 30 "

50

U

" +50

". 40 "

20

it

" +80

" 60 "

10

U

" +90

« 72 "

0

u

" +100

Still alive after 10 days

1TRUE, Botanical Gazette, 1898, p. 407.

2LOEB, Archivfiir die gesammte Physiologic, Vol. LV (1894), p. 530.

3 For these experiments the young fish which had just hatched were used. As
they were less than a centimeter long, their mass was small compared with the
volume of the solution.

ION-PROTEID COMPOUNDS 551

This result agrees with our theory. But in order to
make the proof complete we must be able to show that an
addition of certain other ions annihilates the poisonous
effects of a pure NaCl solution. I tried the following solu-
tions upon Fundulus:

(1) 96 c.c. f n NaCl + 4 c.c. V n MgCl 2

(2) " " " +4 c.c. 1-nKCl

(3) " « " +4 c.c. ^

In each of these solutions the fish died in less than or in
about twenty-four hours. After this experiment two salts
were tried in combination with NaCl, and the following
solutions were prepared:

(1) 96 c.c. | n NaCl + 2 c.c. V- n MgCl2 + 2 c.c. ^ n CaCl2

(2) " " " + " u H- " I n KC1

(3) " " " 4- " Y

This time the result was very striking. In the first of
these three solutions the animals lived less than thirty hours,
in the second a few hours longer; in the third they were
still alive ten days later when I discontinued the experiment.
Thus we see that the poisonous effects of the NaCl solution
really disappear if we add a small amount of K and Ca ions,
which makes the proof of our theory complete.

In the pure NaCl solutions we have to deal with two ions,
Na and Cl ions. Are both equally responsible for the
poisonous effect? The fact that KC1 and CaCl3 prevent the
poisonous effects of the NaCl solution proves that the metal
ions are of greater importance than the Cl ions.

I stated above that Fundulus stands the addition of rather
large quantities of NaCl to sea-water. I tried to determine
whether K and Ca ions were able to counteract even larger
doses of NaCl than are contained in a |w NaCl solution. A
number of young Funduli were put into the following solu-
tions :

552 STUDIES IN GENERAL PHYSIOLOGY

(1) 100 c.c.

(2) 96 " " +4 c.c.

(3) 96 " « +4 "

(4) 96 " « + 2 " VwCaCla+2c.c.inKCl

(5) 93 " " +5 " VwCaCl2+2c.c. fnKCl

In solutions 1 and 2 the animals died in less than two hours.
In solution 3 the animals were found dead the next morning.
In solution 4 the animals died within three days; and in
solution 5 one animal was still alive at the end of the third
day when the experiment was discontinued. The presence
of small amounts of K and Ca ions prevents or weakens the
poisonous effects of even large quantities of NaCl.

I will now consider some possible objections to our theory.
One might think that with the CaCl3 a small amount of HO
ions might possibly be introduced in cases in which the
CaCl2 was heated before it was dissolved. One might think
that these HO ions were the essential constituent that
prolonged the life of these fish. The K ions were only
needed to overcome certain effects of the Ca ions. There is
indeed an antagonism between K and Ca ions, as shown by
Ringer's experiments. But the fact that Fundulus lives
indefinitely in distilled water proves that HO ions are not
necessary to maintain its life. The second objection might
be that NaCl used contained impurities. But this objection
may be discarded at once. The NaCl was obtained from
several leading factories and was chemically pure. The only
impurity possible could have been a trace of K. But as a
further addition of K made the NaCl more harmless, it is out
of the question that the trace of KC1 which the Nad might
have contained could have had anything to do with the
poisonous effects. A third possible objection might be that
these experiments only prove the necessity of K and Ca ions
for Fundulus. But this idea is refuted by the fact that
Fundulus can live indefinitely in distilled water, it is, per-
haps, worthy of mention that the positive proof for the

ION-PROTEID COMPOUNDS 553

poisonous character of a pure NaCl solution would not have
been possible except in a marine animal like Fundulus for
which distilled water is not poisonous. Another possibility
might have been the presence of a trace of acid. My
colleague, Professor Stieglitz, was kind enough to test the
NaCl used, for acids, but it was found to be absolutely free
from acids. Hence I do not see any other possible explana-
tion of the results than the theory from which we started.

What is true for pure NaCl solutions is, of course, still
more true for equimolecular pure solutions of KC1 and
CaClg. They act like poisons. I have not yet been able to
convince myself that their poisonous effect can be prevented
by the addition of small amounts of other metal ions.

The fact that Fundulus can be thrown from sea-water
into distilled water without any considerable swelling, or
without any visible injurious effects, may find its explanation
through the influence that various ions have upon the ab-
sorption of liquids. The above-mentioned experiments on
the absorption of liquids by the muscle have shown that the
simple osmotic theory of absorption which has been accepted
by botanists cannot possibly be correct. I shall deal with
this problem in another paper.

III. EXPERIMENTS ON JELLYFISH (GONIONEMUS)

The locomotion of Medusae is due to rhythmical con-
tractions of their swimming-bell. I experimented on a
form which is very abundant at Woods Hole, Gonionemus.
If we put a Gonionemus into a |- n solution of NaCl, it soon
stops contracting rhythmically. Too many Na ions take the
place of Ca and K ions, and this alters the physical properties
of the tissues to such an extent that no more contractions are
possible. If such a Medusa is brought back into normal
sea-water, it begins to beat again after a short time. In
this case Ca and K ions take the place of some of the Na ions

554 STUDIES IN GENERAL PHYSIOLOGY

in the ion proteids, and this restores the irritability (or con-
tractility) of the Medusa. We thus see again that the Na ions
in a pure NaCl solution are poisonous. If this idea were
correct, we should expect that in a more diluted NaCl solu-
tion the Medusa would be able to contract much longer.
This is indeed the case. I tried the following solutions :

90 cc. | n NaCl + 10 c.c. distilled water

80 " " +20 "

70 " " +30 " ' etc.

The result of the experiments was that the Gonionemus con-
tracts longest in a mixture of equal parts of a -| n NaCl solu-
tion and distilled water. The case is parallel to that of
Fundulus, with the exception that the Gonionemus is not
able to stand distilled water. Otherwise the NaCl is the less
poisonous for Gonionemus the more dilute it is. We are
forced to conclude that if we add certain other metal ions to
the NaCl solution, its poisonous effects must disappear.
We tried the following solutions:

(1) 96 c.c. in NaCl + 4 c.c. *f-n MgCl2

(2) 96 " " +4 c.c. InKCl

(3) 96 u " +4 c.c. V-nCaCla

In the first two solutions the Medusa3 make a few contractions
during the first minute, and then stop. In the third solution
the rhythmical contractions may go on for an hour. If we
take 2 c.c. of the CaCl2, KC1, and MgCl3 solution (instead
of 4 c.c.), the results are practically the same. After this
the following solutions were tried:

(1) 96 fn NaCl + 2 c.c. *£n MgCl2 + 2 c.c. f n KC1

(2) " <* + " " +2 c.c. YwCaCla

(3) " " + " f n KC1 +2c.c.Y^CaCl2

In solution 1 no contractions occurred, while in solutions 2
and 3 regular contractions set in, whose period was almost
normal. They lasted an hour or more. Mg ions act in this
case more like K ions. We thus see again that for the

ION-PKOTEID COMPOUNDS 555

Medusa the same is true as for Fundulus. Na ions are poi-
sonous in a pure NaCl solution, while the same solution is
harmless if a certain amount of K and Ca ions are present.
Our theory that the irritability of tissues depends upon the
presence in definite proportions of Na, K, and Ca ions is
once more verified. It goes without saying that a pure -|n
KC1 and a pure y n CaCl2 solution was still more poisonous
than a -| n NaCl solution.

IV. EXPERIMENTS ON CILIARY MOTION

The conditions for ciliary movements were studied in the
young larvae (blastula, gastrula, and pluteus) of the sea-
urchin. The movements of these larvae are due to cilia
which are incessantly active. I found that these larvae do
not die rapidly in a f n NaCl solution. They may live
twenty-four hours. But if we add a small amount of f n
KC1 and *£-n CaCl2, they may be kept alive and in motion
for ten days or more. In the latter solution their develop-
ment can continue, while in the pure NaCl solution this is
not possible.

I was, however, surprised to find that this ciliary motion
continued in solutions in which no muscular contractions of
Fundulus or Gonionemus were possible. Larvae which were
twenty hours old were able to swim for about forty-eight hours
in the following solutions:

90 c.c. V n MgCl2 + 10 c.c. V n CaCl2
80 « +20

50 " +50

The reader will notice that these solutions contain no NaCl.
I tried the effects of these solutions on Gonionemus. As
was to be expected, not one contraction was possible in these
solutions. The following observation is equally astonishing.
Combinations of NaCl and KC1 were tried:

(1) 80 c.c. in NaCl + 20 cc. f n KC1

(2) 20 c.c. " +80

556 STUDIES IN GENEBAL PHYSIOLOGY

In both solutions the ciliary motion continued for more than
six, but less than eighteen, hours. The first solution seemed
to be a little more harmless than the second. Anybody who
has had experience with the effects of K ions on muscular
contraction will realize that it is out of the question to
expect a muscle to keep its contractility for over six hours
in any of these solutions. Neither was Gronionemus able to
contract in these solutions. These experiments certainly
warn us against taking it for granted that the mechanics of
protoplasmic motions is the same everywhere, although there
may be some identity up to a certain point. In the case of
the blastula we have to deal with very young embryonic
tissue, and we shall see in one of the subsequent publi-
cations that embryonic tissue, or rather the egg-cells, differ
radically from the muscles and the ganglia, as far as the
effects of ions are concerned.

V. ABE THE NA IONS OF OUB BLOOD AN INDIFFEBENT SUBSTANCE ?

A pure solution of NaCl (of about 0.7 per cent.) has been
called the physiological salt solution, inasmuch as the tissues
of a frog may live in such a solution for forty-eight hours. Th e
NaCl in our blood is considered to play chiefly the role of
preventing the tissues from losing or taking up any water.1
According to our opinion, the Na ions of the blood as well
as of the sea-water are essential for the maintenance of life-
phenomena.2 A reduction of the Na ions in the blood would
lead to a loss of Na ions and a substitution of other ions in
their place in the ion proteids of the tissues. But does
the fact that a frog's muscle can live for about forty-eight
hours in a ^ n NaQl solution without being poisoned indicate
that a pure NaCl solution is harmless for the muscle? A

1 HOWELL, American Journal of Physiology, Vol. II (1898), p. 47.

2 Very recently Overton has published a similar idea, but without realizing that
the same idea had already been expressed and verified by me in a number of papers.
[1903]

ION-PROTEID COMPOUNDS 557

^n NaCl solution is certainly not so poisonous as a|-n solu-
tion of the same salt. A Fundulus that would be killed by a
In NaCl solution in twelve hours is able to live in a ±n

o o

NaCl solution two or three days, which is as long as or a little
longer than, the frog's muscle lives at the same (summer)
temperature in a solution of the same concentration. That
the effects of a pure NaCl solution upon a frog's muscle are
in no way different from those on Fundulus is proved, more-
over, by the fact that the muscle lives longer in a NaCl solu-
tion if small amounts of KC1 and CaCl2 are added. This
explains the superiority of Ringer's solution over a physio-
logical salt solution. Ringer's solution prevents the Na
ions of the physiological salt solution from taking the place
of the Ca and K ions in the tissues. Contractility is pos-
sible only if the Na, Ca, and K ions exist in definite propor-
tions in the ion proteids. Hence there is no reason for sup-
posing that what we have proved for marine animals does
not hold good for other animals.

VI. SUMMARY

The main results of this paper are as follows:

1. A pure solution of NaCl of the same concentration as
sea-water is a strong poison for many (if not all) marine
animals. The poisonous effects of this solution are due to
the Na ions. The same is true for pure equimolecular solu-
tions of CaCl2 and KCL

2. The poisonous effects of the Na ions are antagonized by
the addition of a small amount of Ca and K ions. Through
the presence of these two ions the Na ions in the ocean lose
their poisonous effect.

3. The Na ions of the blood would not allow the tissues
to live. The presence of Ca, K, and possibly other ions
counteracts the poisonous effects of Na ions in the blood.
This is the reason why tissues live longer in Ringer's solu-
tion than in a physiological salt solution.

558 STUDIES IN GENERAL PHYSIOLOGY

4. The reason for all these peculiar effects of the Na,
Ca, and K ions is that these (and other) ions form combina-
tions with the proteids of the protoplasm. The various
metal-proteids show various physical qualities. Muscles are
only contractile as long as they contain all three classes of
ions in a certain proportion, which, however, may vary within
certain limits. In a pure solution of NaCl Na ions will
gradually take the place of the Ca and K ions in the ion
proteids of the tissues, and this leads to a loss of contractility
or irritability. This is the reason why a pure NaCl solution
is poisonous. For the same reason pure equimolecular solu-
tions of other chlorides are also poisonous.

5. The conditions for the ciliary motion of the larvse of
the sea-urchin are in various respects different from those
mentioned above. The ciliary motion of these organisms
can continue for several days in a solution of MgCl8 and
CaCl2 which is free from Na ions.

XXVIII

ON THE DIFFERENT EFFECTS OF IONS UPON MYO-
GENIC AND NEUROGENIC RHYTHMICAL CON-
TRACTIONS AND UPON EMBRYONIC AND MUSCULAR
TISSUE 1

I. ON THE DIFFERENT EFFECT OF IONS UPON THE MARGIN
AND THE CENTER OF A HYDROMEDUSA (GONIONEMUS)

IN a preceding paper2 I gave a number of facts which
force us to assume that not the salts themselves, but their
ions, are in combination with the proteids, and that the
physical qualities of the various ion proteids are different.
This being true, a pure solution of an electrolyte ought to be
poisonous, and I have been able to prove that a pure NaCl
solution of the strength in which marine animals live kills
them in a comparatively short time. The addition of a small
amount of certain other metal ions (K and Ca) renders the
solution harmless. This supports the assumption that irri-
tability depends upon the various ions, especially the metal
ions (Na> Ca, jfiT, and Mg) existing in definite proportions
in the tissues. But as each tissue has its own specific irri-
tability, it would follow that various tissues must possess the
various ions in different proportions. This paper contains
the result of a series of investigations on this subject.

Gonionemus propels itself by rhythmical contractions of
its swimming-bell. The swimming-bell, however, does not
contract continuously, like the heart, but in groups of
rhythmical contractions, followed by longer pauses. The
swimming-bell of the Medusa may be divided into two
regions, a marginal region containing the double nerve ring

1 American Journal of Physiology, Vol. Ill (1900), p. 383.

2 Part II, p. 544.

559

560 STUDIES IN GENERAL PHYSIOLOGY

and its ganglia, and the central region which has no ganglia,
but is said to possess scattered ganglion cells. The case is
similar to that of the heart, which has ganglia in the auricles
and sinus vinosus, whose ventricle, however, is free from
ganglia, but contains scattered ganglion cells.

Romanes first stated that if we cut a Hydromedusa in
two, the marginal part with the ganglia will continue to beat
rhythmically very much like the whole Medusa, while the
center ceases to beat. These results have been confirmed by
several authors, but I have found that the statement of
Romanes is only correct for sea-water. If the center of a
Hydro-medusa be put into a pure ^n NaCl or ^n NaBr solu-
tion, it begins to beat rhythmically for an hour immediately
after the operation. Hence the center as well as the margin
is capable of spontaneous contractions. But why does the
center not beat rhythmically in sea-water? If it be put in-
to a solution of 98 c.c. -|w NaCl + 2 c.c. y n CaCl2, it no
longer beats rhythmically. The same is true for a solution
of 98 c.c. |n NaCl + 2 c.c. |n KC1, or a solution of 96
NaCl -f 2 CaCl2 + 2 KCL Hence the Ca and K ions of the
sea- water prevent the center from beating rhythmically.

This harmonizes with my previous experiments on the
muscles of the skeleton.1 The latter are able to beat rhyth-
mically in a pure NaCl or a NaBr solution or any solution
with Na ions. But a small addition of Ca ions or K ions, or
both, prevents rhythmical contractions. We owe it to the
presence of these ions in our blood that our muscles do not
contract rhythmically like the heart.

Thus we see that there is a typical difference between the
effects of ions on rhythmical contractions originating in the
muscles directly and those originating in parts which con-
tain ganglia or which originate in the latter themselves.
Inasmuch as the whole Gonionemus beats in the rhythm of

1 Part II, p. 518.

EFFECT OF IONS UPON TISSUE 561

the margin, and inasmuch as the whole Gonionemus is just
as immune against the Ca and K ions of the sea-water as
the margin, it follows that the normal contractions of the
Gonionemus originate in the part which contains the ganglia.
It is probable, moreover, that the margin and the center
must contain the three metal ions (Na, Ca, and K) in differ-
ent proportions. That this is only a difference in degree,
however, is proved by the fact that an increase in the amount
of K and Ca ions above that of the sea-water will finally
stop the rhythmical contractions of the margin. On the other
hand, it is probable that a very small amount of K and Ca
ions, smaller than that in the sea- water, allows the center to
beat rhythmically.

This difference between the margin and the center is not
the same in all Medusae. , If we cut off the margin in an
Acalepha (for instance, Aurelia aurita), the center begins to
beat in sea-water a short time after the operation. It is
possible that a comparative study of the heart-beat would
reveal similar facts.

We have thus far shown that the center of a Gonionemus
is able to beat for about an hour in a pure NaCl solution ,
while the whole Gonionemus or the margin is able to beat in
a NaCl solution containing in addition a small amount of K
and Ca ions. How does a whole Gonionemus behave in a
pure NaCl solution? As stated above, the contractions of
Gonionemus occur in sea- water in groups followed by long
pauses. If a Gonionemus be put into a f n NaCl solution,
the swimming-bell contracts without interruption and the
rate of contraction increases considerably. It may within
two minutes reach a rate of 200 contractions per minute, but
soon ceases to beat. If the f n NaCl solution be diluted
with distilled water, the increase in the rate of contractions
occurs more slowly and the contractions continue longer. If
we use a solution of 98 c.c. f n NaCl + 2 c.c, ^° n CaCl2, it

562 STUDIES IN GENERAL PHYSIOLOGY

contracts much more slowly, but the contractions last longer.
In a solution of 98 c.c. |n NaCl + 2 c.c. f n KG1 it does
not beat at all, with the exception of a few contractions at
the beginning. In a solution of 96 c.c. J- n NaCl -f 2 c.c.
-f- n KC1 + 2 c.c. l-£n CaCl2 it beats very slowly, but much
longer than in any other of the solutions mentioned. In
pure ^n KC1 or *-/ n CaCl3 solutions no contractions
occur.

The explanation of all these facts seems to me to be as
follows: If a Gonionemus be put into a pure NaCl solution,
Na ions begin to enter the tissues. As soon as they contain
a certain number of Na ions, any further increase of the Na
ions raises the rate of contractions. On the other hand,
the substitution of Ca and K ions for Na ions has the
opposite effect (as long as not too many Na proteids are
formed). If too many Na ions have entered into combina-
tion with the proteids, the irritability ceases. We shall see
later that in this case the substitution of Ca or K ions for
Na ions restores the irritability.

Thus the Na ions play an important rdle in the rhythmical
contractions. It is just as necessary that a certain number
of Na proteids exist in the tissues of the Gonionemus as
that a certain number of Ca and K proteids be present.
The proportion of these three proteids is, however, apparently
different in the margin and in the center. In both kinds of
tissue the relative number of Na proteids is greater than
that of the other proteids.

The view differs from the one generally held in connec-
tion with the heart-beat, that the NaCl in the blood serves
mainly the purpose of preventing the tissues from losing or
taking up water,1 while the Ca salts are considered as the
cause of the systole and the KC1 is said to favor the diastole
of the heart. The fact that a Medusa not only contracts

1 HOWELL, American Journal of Physiology, Vol. II (1898), p. 47.

EFFECT OF IONS UPON TISSUE 563

rhythmically in a pure NaCl solution, but beats much more
rapidly in such a solution than in sea-water, shows that
neither the Ca nor K ions of the surrounding medium are
directly necessary for the systole or diastole. If they have
any effect, they only diminish the rate of contraction (besides
maintaining the contractility much longer). But the above-
mentioned erroneous conception concerning the role of the
three ions can be disproved in another way. .1 had solutions
of cane sugar and glycerin prepared which were isosmotic
with a -f n NaCl solution. The following solutions were
tried :

(1) 96 c.c. distilled water + 2 c.c. f n KC1 + 2 c.c. Y n CaCl2

(2) " cane sugar + " + " "

(3) " glycerin -f " + " "

(4) " fwLiCl + " " + " "

(5) " in NaCl + « +

(6) " fnNaBr + " " +

In the first four solutions no rhythmical contractions occurred
after the first minute. In the fifth and sixth solutions the
rhythmical contractions continued for several hours. If it
were true that the NaCl serves only to maintain the osmotic
pressure, while the Ca produces the contractions, we ought
to expect that the Gronionemus would contract just as well in
the glycerin or" sugar or LiCl solution as in the NaCl solu-
tion. I have made, in addition to these, a number of other
experiments, all of which prove that only in solutions of
electrolytes (especially Na salts) is the Gonionemus able to
contract rhythmically. The belief that calcium is the
stimulus that produces the heart-beat is based upon another
observation which I think was first made by Howell and his
pupils.1 When a heart stops beating in Ringer's solution
it begins to beat again (for a little while) in a solution which
contains more Ca. It is easy to confirm this observation for

i Ibid.

564 STUDIES IN GENERAL PHYSIOLOGY

Gonionenms. If a Gonionemus is thrown into a solution of
98 c.c. frc NaCl + 2 c.c. ^n CaCl3 solution or a pure %-n
NaCl solution, it stops contracting after a certain time, but
beats again for a little if thrown into a solution with more
CaCl3 (for instance, 95 c.c. f wNaOl + 5 c.c. y nCaC!2). This
seems to favor the assumption that the Ca ions are the stimulus
for the contraction of the swimming-bell of a Medusa. But
a simple control experiment shows that this assumption is
erroneous. If we throw a Gonionemus first into the stronger
solution (for instance of 95 c.c. f n NaCl -f 5 c.c. -|w CaClg),
and wait until it stops contracting, it will begin to contract
again if we put it back either into the solution with less
CaCl2 (for instance, 98 c.c. |n NaCl + 2 c.c. ™n CaCl2)
or into a pure NaCl solution. The true explanation of this
phenomenon is, I believe, as follows : In the pure NaCl
solution or the solution with little CaCl2, too many Na ions
combine with the proteids, and this leads to a loss of irrita-
bility. If the Gonionemus be brought into a solution
with more Ca and less Na ions, some Ca ions will take the
place of Na ions in the tissues, and this restores the irrita-
bility. But finally too many Ca ions enter, and the physical
qualities are changed again, thus making the Gonionemus
inirritable. If the same Gonionemus then be put into a
pure NaCl solution or into a NaCl solution with fewer Ca
ions, the Na ions will take the place of some of the Ca ions,
and this will restore the irritability.

We thus arrive at the conclusion that the rhythmical
contractions of Gonionemus depend upon the presence of
Na, Ca, and K ions in definite proportions in the ion pro-
teids of the tissues. , These proportions evidently differ in
various kinds of tissues. My o genie contractions are pre-
vented by a smaller amount of K and Ca ions in the sur-
rounding NaCl solutions than neurogenic contractions or
contractions originating in parts containing ganglia.

EFFECT OF IONS UPON TISSUE 565

II. ON THE DIFFERENT EFFECTS OF IONS UPON UNDIFFER-
ENTIATED EMBRYONIC TISSUE AND UPON MUSCLE

While the method established in the preceding section
may be successfully applied to all kinds of tissues, I was
most interested to know whether there is a marked difference
between undifferentiated embryonic and differentiated older
tissue. By embryonic protoplasm or tissue I mean the early
egg-cells, the growing regions in plants and animals, rapidly
growing tumors, regenerating parts or organs, in short, cells
which are characterized by rapid multiplication. If we are
ever to build up a technical or constructive, in the place of
a merely analytical, biology, we shall be able to do it on the
basis of a more thorough knowledge of the character of
embryonic matter. I tried to find out whether the various
metal ions have the same effect upon the undifferentiated
egg-cells as upon muscle. These experiments throw some
light upon another problem. The karyokinetic cell-division
has been identified with phenomena of muscular contraction.
We shall see incidentally how far such an idea is justifiable.

In my former experiments on development I was guided
by the idea that the various morphological stages were
preceded by chemical changes. In order to see how much
justification there is for this idea I tried to discover whether
lack of oxygen or an increase in the concentration of sea-
water affects the embryo differently in different stages of its
development.1 I used for these experiments the eggs of a
marine fish (Fundulus). The eggs of this fish complete their
development in about two weeks (at the proper temperature).
We may discriminate three stages in the development of this
fish. The first consists solely of processes of cell-division
and the expansion of the blastoderm. This stage lasts about
twenty -four hours. It is followed by the formation and
beginning differentiation of the embryo during the second

i Part I, p. 309.

566 STUDIES IN GENERAL PHYSIOLOGY

twenty-four hours. The third stage of development begins
with the establishment of muscular activity, especially the
heart-beat, about seventy-two hours after fertilization.

If newly fertilized eggs be exposed to a partial oxygen
vacuum, the development may go on for some time (about
twenty-four hours). The eggs, however, may remain alive
in the oxygen vacuum for four days. If after that time
they are put back into normal sea-water, they develop into
normal fish, which hatch in due time. If we put an embryo
which is three days old into the same oxygen vacuum, it
loses its power of development within twenty-four hours.
The older the embryo, the more deleterious is the lack of
oxygen. This is comprehensible only on the assumption
that the morphological differentiation is accompanied or
preceded by changes in the chemical constitution of the
embryo.

The same result was obtained in experiments in which
the concentration of sea-water was raised by the addition of
NaCl, but curiously enough in this case the younger embryo
was more sensitive to an addition of NaCl to sea- water than
the older embryo. An addition of 5 g. of NaCl to 100 c.c.
of sea-water did not prevent the development of the Fundulus
egg, but an addition of 10 g. of NaCl to 100 c.c. of sea-
water prevented the formation of an embryo. Newly fer-
tilized eggs began to segment in such a solution, but stopped
very soon and lost their power of development permanently
within from six to ten hours. A germ that was allowed to
develop during the first twenty-four hours in normal sea-
water withstood much better a solution of sea-water to
which 10 per cent. ,of NaCl had been added. In such a
solution it could go on with its development for several days ;
in some cases as long as ten to fourteen days. An embryo
which had been allowed to develop in normal sea-water until
its circulation was established (third or fourth day) was even

EFFECT OF IONS UPON TISSUE 567

able to live for several days in sea-water to which 24 per
cent. NaCl had been added. When I made these experi-
ments I still accepted the common view that NaCl was an
indifferent substance, and that in these experiments it acted
only osmotically. The results of my recent work and of
experiments to be mentioned in this paper, however, prove
that we have to deal with the effects of Na and Cl ions in
these experiments. It therefore follows that, the Na and Cl
ions (especially the former) are more injurious during the
earliest stages of cell-division than during the later stages.

O O O

I made my new experiments on the effects of various ions
upon development on the eggs of the same form.

If the eggs of Fundulus be put into a f n NaCl solution
immediately after fertilization, the development stops in most
cases at an early stage (64 to 128 cells) and only a few eggs
form an embryo. If the development does not stop during
the first twenty-four hours, it continues as a rule normally
for one or more weeks. Hence a pure NaCl solution seems to
be more poisonous during the first twenty-four hours of devel-
opment than during the later stages. Control experiments
verify this assumption. Eggs that were allowed to develop the
first eighteen or twenty-four hours in sea-water, and are then
put into a f n NaCl solution, continue to develop in almost
every case. No embryo is able to hatch in these solutions.

In our previous paper we showed that a young fish died
in a few hours in such a solution. The fact that the egg
lives longer in it may be due either to the fact that the tough
egg membrane does not allow the ions to penetrate so fast
into the embryo, or to the presence of the yolk which to a
certain extent may regulate the proportion of ions in the
embryo.

If we dilute a -§- n NaCl solution with distilled water, we
find that in a ^ n NaCl solution all the newly fertilized eggs
may form an embryo. Some of the embryos even hatch in

568 STUDIES IN GENERAL PHYSIOLOGY

such a solution. Their duration of life after hatching is,
however, very short. The fact that the eggs of Fundulus
are able to develop in sea-water to which as much as 5 per
cent. NaCl has been added shows that other constituents of
the sea-water are able to counteract the poisonous effects of
a pure NaCl solution. In a solution of 98 c.c. ^n NaCl
+ 2 c.c. ^n KC1 only a small number of embryos are formed.
They cannot be kept alive for more than a week. In a solu-
tion of 98 c.c. •§- n NaCl -f- 2 c.c. -f-n CaCl2 every egg devel-
ops, but only in exceptional cases does the fish hatch. Those
that hatch die immediately afterward. The addition of
even as little as ^ c.c. ^n CaCl2 to 100 c.c. f wNaCl causes
all the eggs to develop. A small amount of Ca ions counter-
acts the poisonous effect of a large quantity of Na ions
sufficiently to allow the development to go on, but not enough
to allow the embryos to hatch. In a solution of 96 c.c. -f w
NaCl + 2 c.c. y n CaClg +2 c.c. fra KC1 not only all the
eggs develop, but the young fish hatch and live indefinitely.

In distilled water all the eggs are able to develop, and
the young fish hatch in due time and live indefinitely.
Hence the Ca and K ions of the above-mentioned solution
are not directly necessary for the development of the fish.
They are only indirectly necessary to counteract the poisonous
effects of the Na ions in a solution of a Na salt. In a gly-
cerin solution of the same osmotic pressure as a \n NaCl
solution, no embryo was formed. In mixtures of glycerin
and sea- water embryos formed, but the glycerin acted as a
poison ; the more glycerin the solution contained, the quicker
it killed them.

Thus far our results agree entirely with our previous
results on the poisonous character of a pure NaCl solution.
Such a solution has a poisonous effect on the germ of
Fundulus, and the Na ions are exclusively or mainly
responsible for the poisonous effect. But this poisonous

EFFECT OF IONS UPON TISSUE

569

effect is much more marked during the first twenty-four
hours of development than during the later stages.

Everyone who has had experience with the effects of
KC1 upon the contraction of muscles knows how poisonous a
pure KC1 solution is. I was much surprised to find that a -§• n
KC1 solution is even less harmful to the newly fertilized egg
of Fundulus than a -f- n NaCl solution. More eggs develop
in the former than in the latter solution. .The following
table gives a clear illustration of this condition : Mixtures of
-|n NaCl and %n KC1 solutions were used. Each solution
had about seventy to eighty eggs. The percentage of eggs
that formed embryos is indicated for each solution.

TABLE I

Character of Solution

Percentage
of Eggs That
Formed Embryos

1

98
95
90
60
40
10
5
2
0

c.c. frc NaCl -

.. _

u
tl _

• ;;

*

h 2 c.c. fnKCl
-5 "
- 10
-40
- 60
- 90
- 95
- 98 "
-100

10
25
30
40
33
33
33
38

2

3

4

5

6

7 . .

8

9

It is evident that if the number of K ions is greater than
the number of Na ions, the percentage of eggs which are
able to develop is larger. It is remarkable that in a solu-
tion of 98 NaCl + 2 KC1 fewer eggs from an embryo than
in 95 NaCl -|- 5 KC1 or in 90 NaCl + 10 KC1, although in
these last two solutions the amount of KC1 is far in excess
of that in sea-water. Hence we are forced to conclude that
K ions are less poisonous for the earlier stages of Fundulus
than Na ions. It is better for the egg that K ions enter
into combination with the proteids of the protoplasm than
Na ions.

570

STUDIES IN GENEKAL PHYSIOLOGY

As soon, however, as the heart begins to beat and circu-
lation becomes necessary for the embryo, the K ions become
more poisonous than the Na ions. Only in the first three
solutions is the heart of the embryo able to beat for a few
days. But even in these solutions no embryo lives longer
than about a week. In the other solutions the embryos die
much earlier. This should certainly serve us as a caution in
taking it for granted that the cell-division is due to con-
tractile phenomena of the same order as those occurring in
the muscle.

Ca ions are in small quantities more beneficial, in larger
quantities more injurious, than Na ions. The addition of a
little y n CaCl3 to a pure Nad or a pure KC1 solution
causes all the eggs to develop, but very soon a limit in the
addition of CaCl3 is reached where no more embryos are
able to form. The following table shows this in a very
marked way:

TABLE II

Character of Solution

Percentage
of Eggs That
Formed Embryos

1

100 c.c.
98
95
90
75
60
50

fnKCl-

u
(4

ti _
it _
u
" -

- 0 c.c. V* n CaCl2
- 2
- 5
-10
-25
-40 "
-50 "

15*

100
100
100
100
9
0

2

3

4

5

6 .

7

In none of these solutions was an embryo able to hatch or
to live through the whole period necessary for develop-
ment. The K ions in these solutions caused a cessation of
the heart-beat. The more K ions the solution contained,
the sooner this happened. In mixtures of Na and Ca ions
the limit where Ca ions prevent the formation of an embryo
is still lower. As a rule in a mixture of 75 c.c. \n NaCl -j-
25 c.c. *-£n CaCl2 no embryo is formed.

EFFECT OF IONS UPON TISSUE 571

It has long been maintained that there is an antagonism
between Ca and K ions. The following experiment demon-
strates this relation in a very striking way : If we mix 75 c.c.
of sea-water with 25 c.c. y n CaCl2, as a rule only a very
small percentage of the eggs are able to form an embryo.
But if we add a large quantity of KC1, almost every egg
forms an embryo. In two experiments not a single embryo
was formed in a mixture of 75 c.c. of sea-water -f- 25 c.c. y n
CaCl2, while in 50 c.c. of sea-water + 25 c.c. *•£ n CaCls
4- 25 c.c. |- n KC1 every egg formed an embryo. I have
repeated this experiment very often, and in each case ob-
tained similar results. But while the K ions antagonized
the poisonous effects of the Ca ions upon the formation of the
embryo, the poisonous effects of the K ions upon the heart-
beat were not counteracted by the Ca ions. In none of the
embryos formed in this solution was the heart able to beat
sufficiently long to enable the embryo to complete its devel-
opment. As a rule, on the sixth day every egg was dead.

In a pure y n CaCl2 solution the germ died in the early
stages of segmentation (four- to eight-cell stages). It ap-
peared to be coagulated. Not even in a mixture of equal
parts of such a solution with distilled water was a single
embryo formed. In a solution of 25 c.c. y n CaCl2 with 75 c.c.
of distilled water embryos could be formed. In one case
as many as 80 per cent, of the eggs contained embryos.
This is the more remarkable as in a mixture of 75 c.c. of
sea- water -f- 25 c.c. l^n CaCl3 a much smaller percentage of
eggs formed embryos. In a ^n CaCl2 solution all the eggs
formed embryos whose development was normal.

We should expect that if we put eggs immediately after
fertilization into a pure solution of a Na salt whose anion
precipitates calcium, the fatal effects of the Na ions upon the
development would be still more obvious than in a pure NaCl
solution. The precipitation of Ca ions in the protoplasm

572 STUDIES IN GENERAL PHYSIOLOGY

would accelerate the disproportion between the Na and Ca
ions of the protoplasm. I tried y n NagSO4 solution. In
no experiment was a single embryo formed, and in each case
the development of the germ stopped in an earlier stage
than in the pure NaCl solution. This corroborates our
view that the poisonous character of the pure NaCl solution
is due to the fact that for the development of the egg the Na,
Ca, and K ions must exist in definite proportions in the
protoplasm.

In a pure y n MgCl2 solution no egg can develop. Even
in equal parts of y n MgCl3 and distilled water only a small
proportion of eggs (20 per cent.) were able to form embryos,
none of which hatched. Mg ions behave toward the egg of
Funduhis very much like Ca and unlike K ions. In a solu-
tion of 98 c.c. -|tt NaCl + 2 c.c.y n MgCl2 all the eggs form
embryos, although no fish hatch. But in larger quantities
the Mg ions are not so poisonous as the Ca ions. Even in a
mixture of equal parts of |n NaCl+ ^ n MgCl2 as many as
75 per cent, of the eggs form embryos (although none of
the latter hatch). This behavior of the Mg ions is similar to
the one described in my paper oii-the absorption of liquids.
The above-mentioned experiments on the effects of K ions
show very clearly that the effect of these ions upon cell-
division is altogether different from their effect upon the
rhythmical contractions. This is not only true for the cells
of Fundulus, but also for the egg-cells of the sea-urchin. I
intend to discuss the effect of ions upon the cell-division in
the eggs of sea-urchins in the next paper.

III. SOME GENERAL CONCLUSIONS

1. The results of this paper bear upon several other
problems which we have thus far had no chance to discuss
sufficiently. There has been a controversy as to whether
the contractions of the heart are myogenic or neurogenic.

EFFECT OF IONS UPON TISSUE 573

The problem is the same as for Gonionemus. In the latter it
(S certain that under ordinary circumstances (i. e., in the
presence of the K and Ca ions of the sea- water), the impulses
for the rhythmical contractions originate in the nervous
system, or at least in the margin. It is, however, not the
histological or morphological structure of the ganglia which
allows them to be so important, but their chemical constitution.
The center of Gonionemus is able to beat rhythmically in a
pure NaCl or NaBr solution. It is true that the center of
Gonionemus (and the apex of the heart) contain single scat-
tered ganglion cells. One might think that these latter are
responsible for the rhythmical contractions of the center
which occur in pure NaCl or NaBr solutions. But the mus-
cles of the skeleton (even if curarized) show rhythmical con-
tractions in the same pure NaCl or NaBr solutions, provided
the latter do not contain any K or Ca ions.

2. It would be unwarranted to say that Ca or any other
ions are the cause of, or the stimulus for, the rhythmical con-
tractions in Gonionemus, or the heart, or any other organ. It
would be much nearer the truth to assume that for the possi-
bility of rhythmical contractions the Na, Ca, and K ions must
exist in definite proportions in the tissue which is expected to
show rhythmical activity. Only so long as these propor-
tions are preserved does the tissue possess such physical
properties and such labile equilibrium as to be capable of
rhythmical processes or contractions. If the tissue has per-
manently or temporarily more Ca and fewer Na ions than
are required for the above-mentioned physical properties
and condition of equilibrium, an increase of Na ions in the
tissue will cause rhythmical contraction. In such a case the
tissue will begin to contract rhythmically or beat at an
increased rate in a pure NaCl solution. If the tissue, how-
ever, contains too many Na and too few Ca ions, a further
increase of the latter in the tissue will cause the beginning

574 STUDIES IN GENERAL PHYSIOLOGY

of rhythmical contractions. In this case the addition of Ca
ions to a pure NaCl solution will produce rhythmical activity.
In a former paper I have shown that skeletal muscle can be
caused to beat rhythmically if we increase the number of its
Na ions without increasing the number of its Ca ions. In
one of the next papers it will be proved that the same result
can be obtained more rapidly if we decrease the number of
Ca ions in the muscle by precipitating them.

3. The phenomena of muscular contractility and the phe-
nomena of cell-division are considered by many authors as
being of the same order. The rays of the astrosphere are
said to be contractile fibrils which pull the chromosomes
apart and accomplish the division of the mother-cell into
two daughter-cells. I do not see how we can harmonize
this hypothesis with the fact that enormous quantities of K
ions in no way interfere with the process of karyokinesis,
while even a much smaller amount of K ions annihilates
muscular activity in a very short time. In the preceding
paper I mentioned the fact that the ciliary motion of the
blastulse of the sea-urchins continues in the presence of enor-
mous quantities of K ions. The riddle of contractility is
still unsolved. It yet remains to be proved that the ciliary
motion and cell-division are due to contractile processes
identical with those in the muscle. Our experiments on the
effects of K ions should warn us against taking such an
identity for granted.

4. While a solution of NaCl with a small amount of K and
Ca ions allows all the various vital processes to go on (except
such special phenomena as the formation of the skeleton,
with which we shall deal in the next paper), we find other
combinations of ions which enhance some of the vital pro-
cesses, while they prevent others. The most important com-
bination in this direction is the mixture of ^n KC1 with a
small amount of \°n CaCL. In such a solution the first

EFFECT OF IONS UPON TISSUE 575

stages of the development of the Funduhis egg occur in a
normal way. The fact that such a solution does not contain
any Na ions raises the question whether the main importance
of ions in these phenomena does not lie in the influence they
have upon the physical qualities of the protoplasm (absorp-
tion of liquids, state of matter, etc.) If this were the case,
we might easily understand that various mixtures of ions
might bring about the same effect upon -tissues, provided
that they affect the physical qualities of the protoplasm in
the same manner. In the next paper we shall show that the
esffifs of the sea-urchin can reach the blastula stage in a

oo o

mixture of l^n MgCl2 and *g-n CaCl2. But each of these
vicarious mixtures serves only for a certain class of vital
processes, while a mixture of NaCl with a small amount of
Ca and K ions allows the whole cycle of life phenomena
(with certain exceptions) to be completed.

5. Herbst1 has tried to prove that practically every sub-
stance contained in the sea-water is necessary for the devel-
opment of the egg of the sea-urchin. His proof consisted
chiefly in removing one of the constituents of the sea- water,
and showing that in such modified sea-water the eggs were
not able to develop. This method does not warrant the con-
clusions Herbst has drawn from them. In a solution of 96
c.c. fn NaCl + 2 c.c. Y CaCl2 + 2 c.c. f-ra KC1 all the Fun-
dulus eggs develop and hatch. If we remove the Ca ions,
the majority of Fundulus eggs cannot develop, and of the
few that develop none hatch. According to Herbst it would
follow that the surrounding medium must contain Ca ions
for the development and hatching of the Fundulus eggs.
Yet we have seen that the Fundulus egg develops and
hatches in distilled water. Ca ions become a necessity only
if the surrounding solution contains Na ions in excessive
quantities.

1 HEKBST, Archivfiir Entwickelungsmechanik, Vol. V (1897), p. 649.

XXIX

ON THE ARTIFICIAL PRODUCTION OF NORMAL LAR-
VJE FROM THE UNFERTILIZED EGGS OF THE SEA-
URCHIN (ARBACIA)1

I. INTRODUCTOEY REMARKS

EIGHT years ago I published the results of some experi-
ments on the effects of an increase in the concentration of
sea-water upon the segmentation of the egg. I had found
that the addition of a small quantity of NaCl to sea-water
retarded segmentation in the egg of the sea-urchin. By
increasing the concentration a point was soon reached where
no further segmentation occurred. If one carefully selects
the minimum increase in the concentration which is able to
prevent the segmentation of the egg, and the eggs be kept
in this solution for one or more hours, a peculiar phenome-
non occurs. When put back into normal sea-water the eggs
do not segment into two, four, eight cells, and so on, succes-
sively, but begin to divide into more than two cells very
soon after being brought back into the sea-water. The
longer the egg is kept in concentrated sea- water, the greater
is the number of cells into which it breaks up at once. I
repeatedly saw an undivided egg go into a morula stage
within fifteen minutes after it was put back into the normal
sea-water. I did not make a thorough histological examina-
tion of these eggs. Dr. Conklin was kind enough to stain a
lot of eggs that had been in concentrated sea-water and
which showed no trace of segmentation. "Some of these
eggs showed very distinctly from four to about thirty nuclei ;
in other eggs the segmentation of the nucleus was not so
perfect. The nucleus, extremely large, seemed to consist of

i American Journal of Physiology, Vol. Ill (April 1, 1900), p. 434.

576

ARTIFICIAL PRODUCTION OF NORMAL LARV^ 577

several parts, which, however, were still connected." These
histological examinations were not thorough enough, and it
was my intention to have them continued. The explana-
tion I gave for this phenomenon was as follows: "The
segmentation of the protoplasm is the effect of a stimulus
which the nucleus applies to the protoplasm and which
makes the protoplasm close around the nucleus." On the
other hand, if we put an egg into sea-water whose concentra-
tion has been raised by the addition of certain salts, the
protoplasm loses water, and this loss of water brings about
a loss of irritability. There is a certain concentration at
which the nucleus is still able to divide, while the protoplasm
loses its ability to respond to the stimuli emanating from
the nuclei. This, it seemed to me,, was what happened in
the more concentrated sea-water. The nucleus divided, but
the protoplasm had lost its irritability on account of the loss
of water. Hence there existed a segmentation of the nucleus
without a segmentation of the protoplasm. But as soon as
such an egg was put back into normal sea-water the proto-
plasm began to take up more water and respond to the
stimuli of the nucleus (these stimuli I considered to be
chemical). Hence the protoplasm divided at once into as
many cells as there were nuclei preformed.1

The following year Morgan stated that he had repeated
my experiments and confirmed them, but was unable to
agree with me as regards the nuclear division.2 He found
only one nucleus in the egg and concluded that no segmenta-
tion of the nucleus occurs in the concentrated sea-water, but
that a rapid division of the nucleus occurs when the eggs
are put back into normal sea-water. As he had made only
four experiments in all, I asked the late Professor Norman,
who worked in my laboratory, to make a larger number of

ILOEB, Journal of Morphology, Vol. VII (1892), p. 253.

2T. H. MOEGAN, Anatotnischer Anzeiger, Vol. IX (1894), p. 141.

578 STUDIES IN GENERAL PHYSIOLOGY

experiments in order to find out whether there was a division
of the nucleus without a segmentation of the protoplasm,
and whether this division was mitotic. Norman found that
by carefully selecting the concentration of the sea- water a
division of the nucleus without a segmentation of the proto-
plasm occurred, and, moreover, that the division was mitotic.1
The number of cells into which the egg divides at once when
brought back into normal sea-water is often larger than the
number of the nuclei preformed in the concentrated sea-
water. It therefore seems as if a further division of the
nuclear matter occurs immediately after the eggs are put
back into normal sea- water. The addition of sodium chlo-
ride seemed to injure the eggs, and I asked Mr. Norman to
try the effects of other chlorides. He found that an increase
in the concentration of sea- water by the addition of MgCl2
is less harmful than that of any other chloride.

It seems to me that it is necessary to discriminate in these
experiments between two different effects produced by the
addition of salts (or the increase of the concentration of sea-
water). The one effect is that produced on the nucleus and
consists of a destruction (liquefaction?) of the nuclear mem-
brane, and possibly a dissolution of the substance which
binds the chromosomes together. This effect seems within
certain limits to increase with the concentration of the sea-
water. The other effect consists in the gradual suppression
of the motility of the protoplasm. This may possibly be
due to a decrease in the fluidity of the protoplasm (water
rigor). This effect also becomes stronger with the increase
in the concentration of the sea-water. At a certain point in
the increase of the concentration the nuclear membrane will
be dissolved and the chromosomes scattered (through proto-
plasmic motions), while the protoplasm is no longer able to
undergo segmentation. This was observed by Norman. If

1W. W. NORMAN, Archiv fur Entwickelungsmechanik, Vol. Ill (1896), p. 106.

ARTIFICIAL PRODUCTION or NORMAL LARV.E 579

the concentration is a little higher, the dissolution of the
nuclear membrane occurs, but the protoplasm on account of
its rigor is unable to scatter the chromosomes and to seg-
ment. If such eggs be put back into normal sea-water, the
protoplasm gradually loses its condition of rigor. The
motions that lead to the scattering of chromosomes return
sooner than the ability to segment. In such cases the pro-
cess probably occurs in the form in which Morgan observed
it. There may be intermediate stages and variations.

I mention these experiments mainly for the reason that
they led Morgan to a very important step, namely, to try the
effect of an increase in the concentration of sea-water upon
unfertilized eggs. He found that eggs that were put into
sea-water whose concentration had been raised by the addi-
tion of 1^ per cent. NaCl or 3^ per cent. MgCl3 began to
segment into two or more cells when put back into normal sea-
water. This segmentation went in some cases about as far as
the sixty-four-cell stage, but then the development stopped.1

Meade made the observation that the unfertilized eggs of
Chsetopterus could be caused to throw out the polar bodies
by the addition of a small amount of KC1 to sea-water.
The addition of NaCl had no such effect.2 Last year Dr.
Mathews made an experiment with rennet ferment which he
did not publish. In a previous paper on the origin of
fibrinogen he had expressed the idea that the origin of
the astrospheres in a cell was due to a process of coagu-
lation. He tried the effect of rennet ferment upon unfer-
tilized egffifs of the sea-urchin to see whether he could in this

oo

way cause the egg to develop. The eggs were put into a
solution of rennet tablet and when taken out began to seg-
ment, but the development did not go beyond the division
into a comparatively small number of cells. The phenomenon

1 T. H. MORGAN, Archiv fur Entwickelungsmechanik, Vol. VIII (1899), p. 448.

2 MEAD, Lectures delivered at Woods Hole, 1898 (Boston : Ginn & Co.).

580 STUDIES IN GENEKAL PHYSIOLOGY

resembled the one described by Morgan to such an extent
that Mathews came to the conclusion that it was not the ren-
net which acted in his experiments, but the salts in the ren-
net tablets. In other words, it was practically the increase
in the concentration of the sea-water which brought about
the segmentation of the unfertilized egg, just as in Mor-
gan's experiment.

There are some earlier observations concerning the fact that
unfertilized eggs may show the beginnings of segmentation.
Hertwig mentions,1 that the eggs of Arthropods, Echino-
derms, and Annelids show a beginning of segmentation
when left in sea-water for a long time (about twenty
hours). Tichomirof is quoted as having produced arti-
ficially a beginning of development in the eggs of Bombyx.
But these eggs are naturally parthenogenetic. Nussbaum2
has repeated these experiments, and, as far as I can see, the
unfertilized eggs of Bombyx seem to develop naturally just
as well as with the treatment given them by Tichomirof.
There is a statement by Dewitz3 that treatment with cor-
rosive sublimate causes the eggs of a frog to show a begin-
ning of segmentation, but, if, I am not mistaken, Dewitz
made no sections through these eggs, and he himself ex-
pressed his doubts to me as to whether there was a real
segmentation, or whether the surface of the egg simply
resembled that of segmented eggs.

Kulagin recently made the following statement: "I ex-
posed unfertilized eggs of fish and amphibians to diphtheria
antitoxin, and noticed in many the process of segmentation."4
As this one sentence is all he has published about his ex-
periments, it is impossible to express an opinion concerning
them. If there was a real segmentation, it still remains an

1 O. HERTWIG, Die Zelle und die Gewebe, Vol. I, p. 239.

2 M. NUSSBAUM, Archiv fur mikroskopische Anatomic, Vol. LIII (1899), p. 444.

3 J. DEWITZ, Biologisches Centralblatt, Vol. VII (1887), p. 93.
* KULAGIN, Zoologischer Anzeiger, Vol. XXI (1898), p. 653.

ARTIFICIAL PRODUCTION or NORMAL LARV^: 581

open question whether it was not caused by the salts of the
serum. This constitutes about all the data existing at the
time I started my experiments.1

I had in the meantime made my experiments on the
effects of ions upon the rhythmical contractions of muscle,
and reached the conclusion that by changing the ions con-
tained in a tissue we can impart to it qualities which it does
not ordinarily possess.2 I concluded that it might be pos-
sible to produce blastulse, or even plutei, from an unfertilized
egg by merely changing the ions in the egg. Such changes
were possible in three ways: first, by altering the qualitative
constitution of the sea- water without altering its total osmotic
pressure; second, by altering its osmotic pressure by the
addition of certain salts; and, third, by combining both
methods. The last way led to positive results.

1 began my experiments with a study of the effects of
various ions on the development of the fertilized egg.

II. THE EFFECTS OF VARIOUS IONS UPON THE FERTILIZED
EGGS OF ARBACIA

The eggs were fertilized in normal sea- water, and after
five minutes were put into the various solutions. The
greatest care was used with the eggs, and as little sea- water
as possible was added to the artificial solution to be tested.
The eggs were collected in vessels in such a way as to form
a thick layer. One or two drops from a pipette gave all the
eggs needed for an experiment. These two drops consisted
chiefly of eggs with the minimum amount of sea-water. The
volume of each of the artificial solutions was 100 c.c.

One chloride in solution. — In a ^n NaCl solution 10,
20, and in one case 50 per cent, of the eggs began to segment.
They very rarely reached the sixteen-cell stage. The majority

i 1 should have mentioned also the observations made by R. Hertwig, that by
adding strychnin to sea-water the eggs of the sea-urchin can be caused to show
the first segmentation. [1903]

2 Part II, p. 518.

582 STUDIES IN GENERAL PHYSIOLOGY

of the eggs stopped developing at the two-cell stage. The
size of the cells was as a rule unequal from the beginning.

In a mixture of 90 c.c. of this solution with 10 c.c. of
distilled water about 80 per cent, of the eggs developed, some
of which even reached the thirty-two-cell stage. In making
more dilute solutions fewer eggs segmented, and in solutions
that were more dilute than a mixture of 70 c.c. -| nNaCl -f- 40
c.c. distilled water as a rule no eggs segmented. This was
not due to the reduction in the osmotic pressure, for in a
solution of 70 c.c. -f n NaCl + 30 c.c. of cane sugar of the
same osmotic pressure but very few eggs began to segment.
They did not develop beyond the four-cell stage. In equal
parts of the NaCl and the sugar solution not an egg segmented.

In a ^n KOI solution about 70—80 per cent, of the eggs
segmented, and many reached the eight-cell stage. A slight
dilution of the KC1 allowed the eggs to reach the thirty-two-
cell stage. Even in a mixture of 60 c.c. KC1 and 40 c.c.

o

distilled water about 5 per cent, of the eggs began to seg-
ment, but reached only the two- or four-cell stage. In
more dilute solutions no segmentation occurred. The
cleavage cells were more equal in size than in the NaCl
solutions. It is obvious that a pure KC1 solution is more
favorable for segmentation than the pure equimolecular NaCl
solutions. In a former paper I published a similar observa-
tion on the Fundulus egg.

In a ^n MgCl3 solution the eggs reached the thirty-
two-cell stage, and in more diluted solutions, for instance
50 c.c. MgCls + 50 c.c. of distilled water, the development
went even farther (sixty-four cells or more). But the various
experiments with, a pure MgCl2 solution varied somewhat in
their results. On the whole, the MgCl2 was more favorable
than the KC1 or NaCl.

In a l-g n CaCl2 solution there was at the best only the
beginning of a segmentation. In a mixture of 90 c.c.

ARTIFICIAL PRODUCTION OF NORMAL LARVAE 583

yn CaCl3 with 10 c.c. of distilled water I occasionally saw
an egg in the two-cell stage. In more dilute CaCl2
solutions no trace of a segmentation occurred. Hence Mg
and K were more favorable than Na and Ca ions for the con-
centration used in the experiments. It is very evident from
these experiments that the optimum concentration for each
of these four chlorides is different.

In a f n LiCl solution the majority of eggs remained un-
segmented, and only very few reached the two-cell stage.
Mixtures of LiCl with sugar were no more advantageous.
In pure glycerin and sugar solutions of the same osmotic
pressure as that of a -|n NaCl solution no egg segmented.
It is evident that the quality of the ions is of more impor-
tance in these experiments than the osmotic pressure, and
that NaCl is not an indifferent substance.

Two chlorides in solution. — In a solution of one chloride
the eggs of Arbacia cannot reach the blastula stage. Are
mixtures of two chlorides more favorable for segmentation?
Among the possible mixtures of the two chloride solutions
of the same osmotic pressure as the sea-water I found those
between *-£ n MgCl2 and J¥° n CaCl2 the most favorable.
The following twelve mixtures were prepared : !

(1) lOOc.c. V^MgCl2+ Oc.c. J^wCaCla

(2) 95 " +5

(3) 90 " +10

(4) 80 " +20 "

(5) 70 " + 30 "

(6) 60 " + 40

(7) 50 " + 50

(8) 40 " + 60

(9) 30 " + 70 "

(10) 20 " + 80

(11) 10 " + 90

(12) 0 " +100

1 It will save repeating these figures if I may state here that the same twelve
proportions were used in all the following experiments with two chlorides in
solution.

584 STUDIES IN GENEEAL PHYSIOLOGY

In the first solution the eggs reached the thirty-two-cell
stage. In the second, third, and fourth solutions they formed
blastulae, which, however, did not move. I first thought
that for the motility of the cilia the presence of other ions
might be required, but I found that blastulse that had devel-
oped in normal sea-water continued their motion for two
days in a solution of 80 c.c. of l-g n MgCl2 +20 c.c. of sea-
water. It is possible, however, that in such a solution cilia
cannot be formed. I placed a lot of these eggs that had
reached the blastula stage in a mixture of MgCl2 and CaCl2
in normal sea-water. The next morning they moved about
in the most lively manner. It is certainly contrary to the
current ideas concerning adaptation that the egg of Arbacia
should reach the blastula stage in a solution which is practi-
cally free from Na ions.

In the fifth solution only very few eggs segmented
and reached the eight-cell stage, while the other solutions
were still worse. The segmentation was more regular the
more Mg the solution contained, and became more irregular
the more the Ca ions predominated. One of the chief features
of this irregularity was the unequal size of the cleavage
cells. As in certain eggs the unequal size of the cleavage
cells is a characteristic feature which plays a great r6le in
the theories of development, it is of interest that such differ-
ences can be brought about through the presence of a cer-
tain quantity of definite ions, especially of Ca and Na ions.

In the mixtures of -|n NaCl with ^ n MgCl2 the results
were not so good. No swimming blastula3 were formed. In
solutions of 90 to 30 c.c. of MgCl2 with 10 to 70 c.c. of
NaCl a morula stage was reached.

Mixtures of y n MgCl2 with -|w KC1 were still less favor-
able. The solutions with more MgCl2 than KC1 reached
the thirty-two-cell stage, or even went a little farther in
their development.

ARTIFICIAL PRODUCTION OF NORMAL LARV.E 585

Mixtures of f n KC1 with y CaCl3 allowed only the be-
ginning of the segmentation, and this only as long as CaCl 2
was used in very small quantities. In 96 c.c. K£l-|-4 c.c.
CaCl 2 one egg in a thousand went into the two-cell stage, or
formed two large cells with two micromeres; but in 90 c.c.
KC1-J-10 c.c. CaCl3 not one egg segmented, and the solu-
tions with more Ca were not more favorable.

A combination of |-ra NaCl with y n CaCl.2 was equally
poisonous. Even in 98 c.c. NaCl-f 2 c.c. CaCl3 the eggs
did not go beyond the beginning of the segmentation, and in
96 c.c. NaCl + 4 c.c. CaCl3 the eggs died in the four-cell
stage. It is worthy of mention that in these solutions the
cleavage cells were very unequal in size.

Mixtures of ^n KC1 and -f- n NaCl were, on the other
hand, almost as favorable as the MgCl2 solutions. In 98 c.c.
NaCl + 2 c.c. KC1 the eggs reached the sixty-four-cell
stage or went even beyond this. It was the same in 96 c.c.
NaCl -f 4 c.c. KC1 for almost every egg divided. With
more KC1 and less NaCl the results were less favorable.
In a former paper we pointed out that the comparative
harmlessness of K ions for the phenomena of cell-division
is in striking contrast with the harmfulness of the same
ions for the phenomena of muscular contraction. We thus
see that the following two combinations of two chlorides
in solution are the most favorable for development: (1)
90 c.c. yn MgCl 4- 10 c.c. ^n CaCl3; and (2) 98 c.c.
fn NaCl + 2 c.c. f n KC1.

Three chlorides in solution. — Neither with one nor with
two chlorides in solution was it possible to obtain swimming
blastulse. From the experience with Fundulus1 I expected
that a combination of three metal ions (especially NaCl,
with small amounts of KC1 and CaCl3) would allow the
eggs of the sea-urchin to complete their development.

i Part II, p. 544.

586

STUDIES IN GENERAL PHYSIOLOGY

This was indeed the case. In a mixture of 96 c.c. J- n Nad
-f 2 c.c. -| n CaCl2 + 2 c.c. -§- n KC1 the eggs not only reached
the blastula stage and swam around in the most lively way,
but they reached the gastrula and even pluteus stage, with
the exception, however, that practically no skeleton was
formed. Such larvae lived for about ten days in this solution !
We might think that the NaCl is an indifferent substance,
and that the Ca and K ions are responsible for the effect.
From what has been shown in the foregoing papers of this
series it follows that this assumption is erroneous. The
same can be proved again directly for this case. I had cane-
sugar and glycerin solutions prepared of the some osmotic
pressure as a ||n NaCl solution. Table I gives the results of
a series of experiments.

TABLE I

Character

of Solution

How Far the Eggs
Developed

1

96 c.c. | n NaCl + 2

|wKCl + 2V»nCaCl,

Pluteus (without

skeleton).

2..

96 c c. V 7i MgCl. -4-

" + "

Unsegmented.

3

96 c.c. fnLiCl +

tt [ tt

Mostly unseg-

mented, few

reach the 2-cell

stage.

4
5

96 c.c. cane sugar -f-
96 c.c. glycerin -j-

<( i u
u \ t(

Unsegmented.
Unsegmented.

The results could not be more striking. MgCl2 is more
favorable for the segmenting egg than NaCl, but still with
the addition of Ca and K ions not an egg segments ! These
experiments prove once more that the conception formed in
the previous papers is correct, namely, that a pure NaCl so-
lution is poisonous, and that it requires a small amount of
both Ca and K ions to antagonize the poisonous effect of a
NaCl solution. It seems that for the egg of a sea-urchin the
three metal ions in the above-mentioned proportion give the
colloids those physical properties which allow them to go

ARTIFICIAL PRODUCTION OF NORMAL LARV.E 587

through the changes of cell-division and assimilation re-
quired for the process of development. I next tried whether
it was necessary that the Ca and K ions be added in equal
proportions to the NaCl. Table II gives the results of such
experiments.

It is obvious that the proportion of K and Ca ions may
vary within certain limits as long as they are present in
sufficient quantity. I will add that I have not yet found any
other combination of three chlorides that yields swimming
blastulse. In the foregoing papers I mentioned that the
anions are not indifferent, and that in a NaBr solution the
rhythmical contractions of a muscle begin even sooner than
in an equimolecular NaCl solution. I made some experi-
ments on the effect of bromides on development. In a solu-
tion of 96 c.c. %n NaBr + 2 c.c. ^n KCl + 2 c.c. ^n KCl
the eggs developed into normal blastulce. In a solution of
96 c.c. LiBr + 2 c.c. CaCl2 + 2 c.c. KCl the eggs reached
the sixteen-cell stage, while in the corresponding LiCl solu-
tion practically no segmentation took place. All these ex-

TABLE II

Nature of the Solution Used

Stage of Development
Reached

1..

2..

96 c.c. | n NaCI +- 2 c.c. f n KCl + 2 c.c. V n CaCl2

+ 2 " +1

Normal blastula.
Normal blastula.

3..

" 2 " | i «

Normal blastula.

4..

" -j-l " --1 "

Normal blastula.

5..

+ | " +2

Normal blastula.

6..

" +1 " +1 "

Normal blastula

developed a

little more

slowly.

7..

+ 1 " +*

No blastulae.

Stopped at 8-

32-cell stage.

periments together give us the impression that different com-
binations of ions may exist which all have the same effect.

588

STUDIES IN GENERAL PHYSIOLOGY

It seems as if the physical condition of the colloids was the
essential point, and that this might be affected by various
ion combinations in the same way.

Solutions which allow the formation of a skeleton. — The
next question was what ions should be added to the above-
mentioned solutions in order to obtain plutei with a skeleton.

1.

FIG. 147

I found that a trace of Na2CO3

tion of 95 c.c. w NaCl

has that effect. In a solu-
2 c.c. y n CaCl2 + 1 c.c. f w KC1

+ 1 c.c. ^n NaCO3 a skeleton was formed within three days.
This skeleton was not quite normal. It showed a formation of
knobs and spheres which I never saw in the skeleton formed
in normal sea-water (Fig. 147 B). I was anxious to obtain
plutei with normal skeletons, and succeeded in doing so by
adding a trace of- MgCl2 to the above-mentioned solution.
The solution which yielded plutei with a normal skeleton con-
sisted of 95 c.c. f n NaCl + 1 c.c. i*n M^018 + 1 c.c. gn

KC1 -f- 2 c.c.

CaCl

c.c.

Na2CO3.

The skele-

ton is sketched in Fig. 147, A. It was therefore evident that

ARTIFICIAL PRODUCTION OF NORMAL LARV^I 589

a small amount of Na2CO3 allowed the formation of a skel-
eton. The addition of Na2CO3 causes an addition of HO
ions as well as of CO3 ions. Which of the two are respon-
sible for the formation of a skeleton ? The substitution of
KHO for the Na2CO3 did not allow the formation of a
skeleton. We must therefore conclude that it is the CO3
ion which is essential.1 .

Conclusions. — We thus see that a mixture of 96 c.c. ^n
NaCl (or NaBr) + 2 c.c. f n KC1 + 2 c.c. y n CaCl is suffi-
cient to allow the fertilized egg of Arbacia to develop into
the gastrula stage. But does this force us to conclude that
the three kations Na, K, and Ca are utilized by the egg for
the process of development? I think that our previous ex-
periments on Fundulus2 may serve as a criterion in answer-
ing the question. In a pure NaCl solution the young fish
died rapidly, while in the above-mentioned mixture they re-
mained alive. And yet this same fish could live indefinitely
in distilled water. This proves that it does not require any
ions from the surrounding medium. It might be possible
that only Na ions were needed for the development of the
sea-urchin egg. In this case the K and Ca ions would have
to be present in order to prevent the poisonous effects which
a pure NaCl solution would produce. On the other hand,
we found that in a mixture of MgCl2 and CaCl2 which is
practically free from Na ions the eggs can develop and al-
most reach the blastula stage. This makes it still more dif-
ficult to state positively that the Na ions of the surrounding
medium are needed for the development of the sea-urchin
egg. Perhaps it is safest to assume that for the process of
cell-division and development a certain physical condition—
a certain labile equilibrium — of the protoplasm of the col-

iFor further facts concerning the formation of a skeleton in sea-urchins see
HEEBST, Archiv filr Entwickelungsmechanik, Vol. II (1896), p. 455; and DRIESCH.
ibid., Vol. IX (1899), p. 137.

2 Part II, p. 544.

590 STUDIES IN GENEEAL PHYSIOLOGY

loids has to be maintained. This requires certain ions in
definite proportions, either Na, K, and Ca (or other combi-
nations, for instance Mg and Ca). Distilled water is a poison
for the eggs of Arbacia. Hence, if Na be one of the ions of
the surrounding solution, Ca and K ions are likewise required.

My results differ somewhat from those obtained by
Herbst.1 But I pointed out in my last paper that Herbst
was misled by his method.2 This method consisted in
making a solution of the same complication as the sea-
water, in which, however, one constituent was omitted. In
case the eggs did not develop in such a solution, this was
considered a proof that the constituent omitted was neces-
sary for the development of the eggs. My experiments
show that this conclusion is not correct. It is quite possible
that the substance which was omitted or removed was not
directly necessary for the egg, but only indirectly, inasmuch
as it served to counteract the poisonous effects of another
constituent of the solution.

It seems to me that my experiments necessitate the intro-
duction of a new conception, namely, that of physiologically
balanced salt solutions. By this I mean salt solutions which
contain such ions and in such proportions as completely to
annihilate the poisonous effects which each constituent would
have if it were alone in solution. Sea-water and blood (and
approximately a mixture of 96 c.c. fn NaCl-f-2 c.c. l-£n
CaCl2 -f 2 c.c. f n KC1) are physiologically balanced salt
solutions.

It will be necessary to investigate how far the conclusions
of pharmacologists, botanists, and bacteriologists concerning
the effects of various salts require a correction on the basis
of these new facts and conceptions. Their consideration
might even prove of use in problems of immunity and

adaptation.

i

i HERBST, loc. cit., Vol. V (1897) , p. 649. 2 part II, p. 559.

ARTIFICIAL PRODUCTION OF NORMAL LARV.E 591

III. IS IT POSSIBLE TO PRODUCE BLASTUL^ FROM UNFERTI-
LIZED EGGS WITHOUT RAISING THE CONCENTRATION OF

THE SEA-WATER?

The early experiments with which I started had indicated
that an increase in the concentration of the sea- water caused the
segmentation of the nucleus in the fertilized egg. Morgan's
experiments had shown that the same influence may cause
the unfertilized egg to show a beginning of -segmentation.
In Mead's experiments, however, there was practically no
increase in the osmotic pressure of the sea-water, while the
nature of the ions seemed to determine the result. I wished
to find out whether a blastula could be secured from an
unfertilized egg without raising the concentration (the total
osmotic pressure) of the sea-water. All my experiments
thus far have yielded the result that this is impossible, and
that by this method only a beginning of a segmentation can
be produced in an unfertilized egg. It goes without saying
that in these experiments bacteriological precautions are
necessary to guard against the possibility of the introduction
of spermatozoa by the instruments, or of their presence in
the sea- water. One has a pretty reliable criterion for the
entrance of a spermatozoon into the egg of Abacia in the
formation of the egg membrane. An unfertilized egg has
no distinct membrane, but immediately after the fertilization
a very distinct and rather thick membrane is formed. As
none of the eggs in the following experiment formed a mem-
brane or showed more than the beginning of a segmentation,
we may reserve the discussion of our methods of protection
against fertilization for the next section. Unfertilized eggs
of the same female were divided into three lots. One was
put into a solution of 96 c.c. fn NaCl -f 2 c.c. ^n KCl-j-
2 c.c. y n CaCl2. After three and one-half hours a few of
the eggs showed a beginning of segmentation. After eight
hours a majority of the eggs had divided into from 2 to 4

592 STUDIES IN GENERAL PHYSIOLOGY

cells. Some had even gone a little farther.1 But then the
segmentation stopped. No egg had a membrane. A second
lot was put into normal sea- water. Eight hours later none
of these eggs had segmented or had a membrane. This
indicates that a mere change in the constitution of the sea-
water without any change in the osmotic pressure may cause
the beginning of a segmentation of the egg. A third lot was
put into a solution of 96 c.c. V° n MgCl2 + 2 c.c. V° n CaCl3 +
2 c.c. -|n KC1. No egg segmented. One lot of these eggs
was put back into normal sea- water five hours later. A few
eggs now went into the two-cell stage, but developed no farther.

In a mixture of 75 c.c. l-g n MgCl3 with 25 c.c. distilled
water a small number of unfertilized eggs segmented. In
equal parts of y* n MgCl2 and %n NaCl no eggs segmented.

In Mead's experiments it was KC1 that caused the eggs
of Chsetopterus to throw out their polar bodies. I put
unfertilized eggs of Arbacia first into sea- water for five
hours. No eggs showed a trace of beginning segmentation.
After this the eggs were put for two hours into a mixture of
90 c.c. sea-water and 10 c.c. -|n KC1. When put back into
normal sea-water, in fifteen minutes almost every other egg
began to divide, but the segmentation never went beyond the
sixteen-cell stage at the best. Neither these nor the above-
mentioned experiments gave constant results. The greatest
differences existed in the proportion of eggs that showed a
segmentation. In a former paper I had proved that the
addition of a small amount of -fan NaHO caused an increase
in the rate of development and growth of the unfertilized
Arbacia egg, while the equally small addition of an acid
(HC1) produced the opposite effect. This summer I tried the
effect of HO and H ions upon the unfertilized egg. The
following solutions were prepared:

1 1 am now inclined to believe that the normal concentration of the sea- water
was slightly less than that of a I n NaCl solution, and that this beginning of a
i arthenogenetic development was due to the fact that the solution used was slightly
hypertonic. [1903J

ARTIFICIAL PRODUCTION OF NORMAL LARV^ 593

(1) 99 c.c.

sea- water +1 c.c.

-fan KHO

(2) 98

«

+2

a

(3) 99

M

+ lc.c.

TO % HC1

(4) 98

M

+2

u

(5) 97

ti

+3

a

In solution 1 almost every egg was in segmentation five
hours later, but the segmentation was very irregular and
often incomplete, and the egg showed very lively amoeboid
motions. Never more than 10 cells were formed. In solu-
tion 2 the effects were similar, but fewer eggs segmented.
The segmentation did not go any farther. In solutions 3,
4, and 5 not an egg showed any trace of segmentation, nor
did any egg in the normal sea-water segment.

Some of the eggs that were put into solutions 4 and 5
were left there only ten minutes, and then brought back
into normal sea- water. Five hours later many of these eggs
had begun to segment. The segmentation did not go beyond
the first cell-division. It should be said that the sea-water
naturally contains some free HO ions. After a short treat-
ment with acid the HO ions in the sea-water were able to
produce an effect which they could not have had if the acid
treatment had not been applied. ,

None of these experiments, however, led to the formation
of a blastula, nor did they offer any promise of the possi-
bility of producing blastulse in an artificial way. The ex-
periments were made at various periods of the spawning
season. After these and some other unpromising attempts
I tried whether an increase in the concentration of sea- water
would yield better results than a mere change in the propor-
tion of the ions. Instead of using ^n NaCl and KC1 and
y n CaCl2 and MgCl3 solutions I now tried y n NaCl and
KC1 and ^° n MgCl3 and CaCl2 solutions. I do not wish
to give an account of all the experiments I made in this
direction, but prefer to confine myself to an account of one

594 STUDIES IN GENERAL PHYSIOLOGY

experiment which led me in the right direction. In this ex-
periment the following four mixtures were used:

(1) 60 c.c. \°-n MgCl2 +40 c.c. of sea-water

(2) 60c.c.

(3) 60 c.c.

(4) 60 c.c.

A lot of unfertilized eggs were distributed in these four
solutions, and remained in the same for one hour and fifty
minutes. They were then brought back into normal sea-
water. The eggs that had been in the first (MgCl2) solution
began to show an amoeboid change of form (indicative of a
segmentation) in about fifteen minutes after they were
brought back into normal sea- water. About one in a thousand
eggs were in this amoeboid stage. One does not see such
changes in the normal egg where the membrane limits the
amoeboid motions. Fifty minutes later one in a thousand
eggs divided into 2 or 3 cells. The cells were of about equal
size. About two and one-half hours after the eggs had been
put back into normal sea-water about one egg in five hundred
had segmented, and the segmentation had proceeded to the
eight-cell stage, although not all the eggs had reached this
stage. But then the development stopped. The next morn-
ing the eggs were still without any membrane. All looked
normal and healthy. In this experiment the volume of nor-
mal sea- water into which the eggs were put after they had
been in the MgCl2 solution was small (only a watchglass full).
Hence the few drops of the MgCl3 solution which were
transferred with the eggs modified the sea-water, and I
think interfered with their development. It seemed to me
that by avoiding tjiis source of error, and by using large
dishes with several hundred cubic centimeters of sea-water
instead of the watchglass, it might perhaps be possible to
see the eggs develop further.

The eggs that had been in solution 2 (60 c.c. KCl-f-40

ARTIFICIAL PRODUCTION OF NORMAL LARVAE 595

c.c. sea-water) also began to segment. The next morning about
1 per cent, of the eggs were divided into from 2 to 4 cells.
They were all without membranes, but they looked less normal
than the MgCl2 eggs, and soon began to disintegrate.

The third lot of eggs had been in 60 c.c. NaCl -|- 40 c.c.
sea-water. Practically none of these eggs segmented during
the next twenty-four hours, and none formed a membrane.
The fourth lot of eggs had been in 60 c.c. CaCl2 +40 c.c.
sea-water. A few of these showed a beginning of segmen-
tation, but every egg had a membrane. I have found since
that in pure Ca013 solutions of even lower concentration
unfertilized eggs form a membrane. It is possible that the
formation of a membrane consists in a process of coagula-
tion which is favored by Ca ions.1

I, made a parallel series of experiments with fertilized
eggs of the same female. The eggs were, as usual, fer-
tilized in normal sea-water, and five minutes later were put
into the various solutions. The eggs were divided into
four lots, and put into solutions of the same character as in
the above-mentioned experiment with unfertilized eggs.
Like the unfertilized eggs, the fertilized eggs remained in
the solution one hour and fifty minutes. When brought
back into normal sea-water those that had been in solu-
tion 1 (60 c.c. MgCl2 + 40 c.c. sea- water) began to divide in
fifteen minutes. The segmentation was very regular. Two
hours and forty-five minutes later every egg was segmented
into from 8 to 32 cells. Every egg had a membrane. The
next morning a large number of eggs swam about in the
blastula stage, still having a membrane. This observation is
of importance, as it shows that even in eggs that were in a
mixture of 60 c.c. 2^nMgCl2 with 40 c.c. sea-water the

i Hertwig showed that unfertilized eggs form a membrane in water saturated
with chloroform. Herbst found that benzol, toluol, and xylol bring about the same
effect. All these media have a coagulating effect. HEHBST, Biologisches Central-
blatt. Vol. XIII (1892), p. 14.

596 STUDIES IN GENERAL PHYSIOLOGY

existence of a membrane may serve to show whether eggs
were fertilized or not. The fertilized eggs that were put
into the second solution (KC1) did not reach the blastula
stage. They stopped at about the thirty-two- to the sixty-
four-cell stage. Those that had been in the third solution
(NaCl) were in about the same condition, with the exception
perhaps that at first the segmentation was more unequal.
In the fourth solution (CaCl2) no egg segmented, and only
one egg in a thousand showed a beginning of segmentation,
consisting of an incision at one side of the egg.

I finally wished to know how fertilized and unfertilized
eggs behaved if left for eighteen hours in a mixture of
60 c.c. 2-/n MgCl2 +40 c.c. sea-water. The unfertilized
eggs formed no membrane, but a very large part, more
than 50 per cent., of the eggs was divided into from 2 to 8
cells. The fertilized eggs had a membrane. In regard to
segmentation there was little difference between the two lots.
It was especially this circumstance which made me hope
that with a little more care it would be possible to raise liv-
ing larvse from unfertilized eggs by treating them with a
suitable mixture of 2^ n MgCL3 solution and sea-water.

In these and other similar experiments, which I will not
describe, it was moreover evident that after the treatment
with Mg ions the character of the segmentation was much
more normal than after the treatment with K and Na or Ca
ions. The K ions were nearest the Mg ions in their effect.
The Ca ions were the most unfavorable. The former experiments
of Norman had also yielded the result that the Mg ions were
the most harmless for the segmentation of the sea-urchin egg.

IV. THE ARTIFICIAL PRODUCTION OF NORMAL LARV.E (PLU-
TEl) FROM THE UNFERTILIZED EGG OF THE SEA-URCHIN

The most serious danger in experiments with unfertilized
eggs is the possibility that the sea- water or the instruments

ABTIFICIAL PRODUCTION OF NORMAL LARV.E 597

contain spermatozoa. It is imperative to guard against
both possibilities. The sea-urchins have practically died
out in the immediate neighborhood of the Woods Hole
laboratory, and we have to send out the steam launch to
collect them. For this reason even at the height of the
spawning reason there is little danger of the sea-water con-
taining spermatozoa in such quantities as to interfere with
experiments on unfertilized eggs. Moreover, the danger that
the spermatozoa contained in the sea-water of the laboratory
may interfere with experiments on unfertilized eggs is not
very great, even at the height of the breeding season. This
is shown indirectly by the fact that in the experiments
described in the previous chapter not a single egg was fer-
tilized through contamination of the sea-water with sper-
matozoa. The spermatozoa if scattered in sea- water soon lose
the power of impregnating the egg. Gemmill found experi-
mentally that this occurs in less than five hours after the
spermatozoa leave the testicle.1 My experiments were carried
on after the breeding season was practically over, in Septem-
ber, when the majority of sea-urchins contained practically
no more eggs. I had already made up my mind that my
further experiments would have to be postponed a year, when
through the kindness of Professor Bumpus of the Fish Com-
mission I obtained a few dozen sea-urchins that he had col-
lected early in the season and kept in a small pond. It
happened that almost every one of these animals was a female
and full of eggs. Though there was little possibility that the
running water of the marine laboratory could contain any
spermatozoa of sea-urchins which were able to fertilize eggs,
I had no right to take anything for granted in this direction.
I therefore conducted with each experiment a series of con-
trol experiments to guard against the possibility of contam-

i GEMMILL, Journal of Anatomy and Physiology, Vol. XXIV (1900), p. 163. The
results are much better if sterile sea-water is used, as was the case in Fischer's ex-
periments. [1903]

598 STUDIES IN GENEKAL PHYSIOLOGY

ination of the sea-water with spermatozoa. As a rule, I
proceeded in the following way. The unfertilized eggs of
one female were divided into three or more lots. One lot
was put into the artificial solution by which I hoped to cause
the development of the unfertilized eggs. The second lot
was put into normal sea-water to serve as a test or control
for the presence of spermatozoa in the sea-water. The third
lot was put into an artificial solution which as a rule differed
less from the normal sea-water than the solution which
caused the development of the egg. Whenever the eggs of
one lot were put back into normal sea- water, the eggs of
the other lots were put into the same sea-water. Thus all
three lots of eggs were kept in sea- water of exactly the same
degree of contamination. In no case did a single egg of
the three lots form a membrane. No egg of lot 2, which
remained in normal sea-water all the time and served as a
test for the presence of spermatozoa, showed any develop-
ment except a beginning of segmentation (2-3 cells) after
about twenty hours. In no case did any of the eggs of lot
2 or 3 develop into a blastula.

The chief sources of infection in such experiments are
the instruments and the hands of the experimenter if he
opens male and female animals at the same time. The dishes
in my experiments were cleaned with fresh water, in most
cases the evening before the experiment was made. The
instruments which were used had been cleaned in fresh water
and kept dry for twenty -four hours. In case the first animal
opened was a male the instruments were laid aside, the hands
disinfected, and new instruments used for the next animal.
It happened that in almost every one of the following ex-
periments the first animal I opened was a female, and thus
the chief danger of contamination by spermatozoa was
naturally avoided.

But even if the experiments had not been carried on with

ABTIFICIAL PRODUCTION OF NOBMAL LARV.E 599

such precautions, the results obtained were of such a char-
acter as to absolutely exclude in themselves any idea of con-
tamination by spermatozoa. In all the successful experi-
ments the cultures of unfertilized eggs that had been treated
with the right MgCl2 solution were teeming with blastulae
the next day. Twenty per cent. , in some cases even more,
almost 50 per cent, of the eggs, had developed. In former
experiments with unfertilized eggs where no such precautions
were taken, I never noticed that more than perhaps one egg
in a thousand developed. I shall describe each series of ex-
periments independently. TheMgCl2 used in these experi-
ments was chemically pure, but had been dried by heating it.
First series. — Unfertilized eggs of the same female were
divided into four lots and distributed into the following four
solutions :

(1 ) 60 c.c. *$- n MgCl a + 40 c.c. sea-water

(2) 100 c.c. YrcMgda

(3) 100 c.c. y-n CaCl2

(4) 100 c.c. normal sea-water

After the eggs had been in these solutions one and one-
half hours they were carefully examined. Not one had a
membrane and not one was segmented. Twenty minutes
later one part of the eggs of each of these four solutions
was transferred back into normal sea-water. The latter was the
same for all four solutions. This time I took special care to
see that each lot of eggs was given enough normal sea- water
(about 200 c.c.). After they had been back in the normal
sea-water for about two hours and fifty minutes, they were
examined again. The eggs that had been in solution 1
(60 c.c. 2¥°n MgCl2 -f 40 c.c. sea-water) were all without a
membrane. About 20 per cent, of the eggs were segmented
into as many as 32 cells. The eggs that had been in solu-
tion 2 (100 c.c. l-£n MgCl2) were without any membrane
and unsegmented. Many of those that had been in solution

600 STUDIES IN GENERAL PHYSIOLOGY

3 (100 c.c. J¥0n CaClg) had membranes. A few were seg-
mented very irregularly into 2 to 3 cells. All the eggs were
examined again three hours later. Those that had been in
solution 1 were now in a morula stage. As they had no
membranes, their outline was very irregular, and I wondered
what kind of blastula would result if these eggs ever reached
that stage. The eggs that had been in solution 2 (100 c.c.
*-/n MgCl2) were without membranes and unsegmented.
Of the eggs that had been in solution 3 (100 c.c. y n CaCl3)
about 5 per cent, were segmented into from 2 to 4 cells of
very unequal size. The last examination had taken place in
the evening. The next morning the eggs of solution 1 were
teeming with blastulse ; some with regular, the majority, how-
ever, with most fantastic outlines (see Fig. 149). Their size
was very unequal. I expected as much from the irregular
appearance of the morulse of the evening before. In the fer-
tilized egg the membrane prevents any irregularity in the
form of the blastulse. The unfertilized eggs, however, have no
membrane, and hence the cells are only kept together by an
intercellular substance or by adhesion ; but it is very probable
that the processes of cell-division are accompanied by
amoBboid motions (Fig. 148), which have the effect of
making the arrangement of cells irregular. I have noticed
and described this effect of the amoeboid motions of the
cleavage cells in my experiments on eggs whose membrane
I had caused to burst and whose contents partly flowed out
of the egg.1 These extraovates behaved very much like the
unfertilized eggs. In the latter case it was evident from the
size of the blastulse that only in rare cases had the whole
egg developed into qne single blastula. As a rule, each egg
gave rise to several blastulse. Through the amoeboid motions
connected with the process of cell-division groups of cells
became disconnected and developed into dwarf blastulse. I

I T-OEB, Archivfilr Entwickelungsmechanik, Vol. I (1895), p. 453.

ARTIFICIAL PRODUCTION or NORMAL LARV.E 601

shall discuss this point more fully in connection with the
drawings.

I have not yet mentioned one control experiment which I
made in this series. I had one part of the eggs of the same
female fertilized and put into the same four solutions for the
same time as the unfertilized eggs. Every one of the ferti-
lized eggs formed a membrane. The behavior of these eggs
and their larvas differed also in other respects, from the larvae
produced from the unfertilized eggs. While the blastulce
of the fertilized eggs even after the treatment with solution
1 swam at the surface of the sea-water, the parthenogenetic
blastulce were all at the bottom of the dish and unable to
rise. This difference seems to be typical, as I found it in
all my experiments. All the parthenogenetic blastulse in
these experiments died during the day. It goes without
saying that the blastulse which developed from the fertilized
eggs treated with solution 1 did not show the ragged con-
dition of the parthenogenetic larvae that had developed
without a membrane.

Unfertilized eggs that had been in solution 2 for one
hour and fifty minutes were the next day unsegmented and
without membrane. The unfertilized eggs that had been in
solution 3 were all dead. The unfertilized eggs that had
been kept in normal sea-water all the time were without a
membrane and unsegmented. Thus it is evident that the
unfertilized eggs of Arbacia, if put for one hour and fifty
minutes in a solution of 60 c.c. 2¥° n MgCl3 -j-40 c.c. sea-
water are able to develop into blastulae which move about.
But it is also evident from the control experiments that this
cannot be due to the partial pressure or concentration of the
Mg ions alone, for in solution 2 (100 c.c. ^ n MgCl2) the
concentration of the Mg ions was almost the same as in solu-
tion 1, and yet no unfertilized egg was caused to segment by
this solution. That the latter solution is not very poisonous

602 STUDIES IN GENEKAL PHYSIOLOGY

for the Arbacia egg is shown by the fact that the fertilized
eggs of Arbacia develop better in this solution than in 60 c.c.
2¥° n MgCl2 -+- 40 c.c. sea-water. Hence it is evident that
the Mg ions alone were not able to bring about the develop-
ment of the unfertilized Arbacia eggs that had been in solu-
tion 1. Either the presence of other ions, such as are
contained in the 40 c.c. of sea- water or the increased osmotic
pressure in the mixture of 60 c.c. 2-/n MgClg +40 c.c. sea-
water is essential for the development. The osmotic pres-
sure of a *-g n MgCl2 solution is not very different from that
of sea-water.

Second series. — It was, of course, my next task to repeat
this experiment. I now knew which solution must be used
in order to obtain parthenogenetic blastuke, but I wanted to
find out how long the eggs must remain in this solution in
order to develop into a blastula. I put a quantity of
unfertilized eggs of one female into a solution of 60 c.c.
2-£n MgCl2-|-40 c.c. sea- water at ten o'clock. At various
intervals a portion of these eggs was taken out of the solu-
tion and put back into 200 c.c. of normal sea-water. The
first lot was put back into normal sea-water after thirty
minutes. No egg had a membrane and none was seg-
mented. The second lot was put back into normal sea- water
after sixty-five minutes, the third lot after one hour and
forty minutes, the fourth after one hour and fifty minutes,
the fifth after two hours and five minutes, the sixth after
two hours and fifteen minutes, lot 7 after two hours and
thirty minutes, and the eighth lot after three hours and
fifteen minutes. The first, second, and eighth lots differed
much in regard to the time they were exposed to the artificial
solution from lots 3, 4, 5, 6, and 7, which were taken out of
the solution at much shorter intervals. No egg in any of
these solutions had any membrane or showed any trace of
segmentation at the time they were put back into normal

ARTIFICIAL PRODUCTION OF NORMAL LARV^J 603

sea- water. At 1:15 all the eggs were examined again. In
lots 3 to 7 many eggs were segmented into from 2 to 16
cells. The sixteen-cell stages were only found in lot 4. The
rest had not gone beyond the eight-cell stage. In lot 2 very
few eggs had segmented; in lot 1 all the eggs were seg-
mented. No egg had a membrane. Another examination
of the eggs was made at 3:05. In lot 1 about 1 per cent,
of the eggs was segmented in 2 cells ; in lot 2 about 5 per
cent, of the eggs were divided, most of them into 2, some of
them, however, into 8 or even 16 cells. In lot 3 about 20
per cent, segmented. Some had reached the thirty-two-cell
stage. In lot 4 more than 20 per cent, were segmented,
some as far as into 32 cells. In lot 5 almost every second
egg was segmented. Some had reached about the thirty-
two-cell stage. The same was true of lots 6 and 7. In lot
8 many eggs had segmented, but they were far behind in
their development. No egg had a membrane. The single
cells did not stick as closely together as they did in the
fertilized egg with the membrane. I was afraid from the
appearance of the eggs, that they would not give rise to
blastulse, inasmuch as it seemed as though the cleavage cells
would all fall apart.

The eggs were not examined until the next morning. In
lots 1 and 8 there were practically no blastulse in motion,
or if there were any they escaped my observation. In lot 7
I found a small number of blastulse. In lots 3, 4, 5, and 6
the water at the bottom of the dish was teeming with blastulse,
which with their irregular outlines and the variation in size
betrayed clearly that they had developed from eggs without
a membrane.

In the afternoon I found living larvse only in lots 3, 4,
5, and 6. Some larvse seemed to be in a gastrula stage, and
some even in the transitional stage to the pluteus form.
Many had died, and this accounted perhaps for the fact that

604 STUDIES IN GENERAL PHYSIOLOGY

no blastulse were left in lots 2 and 7, where they had at
most been very scarce. The next morning only a few larvse
were left in lots 3 to 6, and these died during the day.

I stated at the beginning of my experiments that only a
part of the eggs of one female were put into the MgClg
solution. The others were left in normal sea-water to serve
as control material. None of these eggs which were in the
same sea-water as that used for the eggs treated with the
MgCl2 solution formed any membrane. After twenty-four
hours a few eggs were found divided into 2 cells. No egg
developed beyond this stage.

This experiment shows that the time during which
unfertilized eggs must remain in contact with a mixture of
60 c.c. 2-£n MgCl2 +40 c.c. sea- water in order to give rise
to blastulse is limited in two directions. If the eggs remain
only thirty minutes in such a solution, a few of them may
begin to develop, but none will reach the blastula stage. But
if the unfertilized eggs remain in this solution from one and
one-half to two hours, more than 20 per cent., and as many
as 50 per cent., may develop, and the solution then teems
with moving blastulse, which however remain at the bottom
of the dish. On the other hand the time limit will be
exceeded if the eggs are left in the solution more than two
and one-half or three hours.

I tried another experiment in this series to see how soon
the unfertilized eggs would lose their power of being affected
by the MgCl2 solution. The eggs were left in normal sea-
water for eighty minutes, then put in a solution of 60 c.c.
2^n MgCl2 +40 c.c. sea-water for two hours. Two hours
later they were put back into normal sea-water. A large
number of eggs began to segment, but I did not find any
blastulse the next day.

Third series. — Thus far I had found the right solution
for producing blastulse from unfertilized eggs, and had found

ARTIFICIAL PRODUCTION OF NORMAL LARV^ 605

about how long the eggs must remain in this solution. I
now desired to verify these results and in addition find out
accurately how far the proportions between sea-water and
the 2¥° n MgCl2 solution might vary without interfering with
the results. The unfertilized eggs of one female were dis-
tributed in the following solutions :

(1) 60 c.c. Yn MgCl2 +40 c.c. sea-water

(2) 30 " +70

(3) Normal sea-water

At five different periods (one hour, one hour and forty
minutes, one hour and fifty-five minutes, two hours and
twenty minutes, two hours and forty-five minutes) portions
of the eggs in solutions 1 and 2 were brought back into
normal sea-water. After all that has been said, it seems
superfluous to give all the details as explicitly as in the pre-
ceding experiments, and I shall therefore confine myself to
a description of the main results as they appeared next
morning. The eggs in solution 3 (normal sea- water) had no
membranes nor had any egg segmented. It is obvious that
unfertilized eggs do not always undergo a beginning of seg-
mentation in normal sea- water after twenty or twenty-four
hours. Of the eggs that had been in solution 1 for one
hour and fifty-five minutes, about 25 per cent, had developed
into a blastula which swam about. About the same result
was obtained in the lot that had been for two hours and
twenty minutes in solution 1. The appearance of these
blastula was the same as in the previous experiments. Most
of them were only fractions of one egg, and it was not
uncommon to see four smaller blastulse swim together, each
apparently having developed from one of the blastomeres of
the four-cell stage. In the other lots which were taken from
solution 1 a few blastulae were formed. The eggs that had
been in this solution for one hour were practically all
undivided, except that one in a thousand had segmented into

606 STUDIES IN GENERAL PHYSIOLOGY

2 to 3 cells. It was not much better in the lot of eggs that
had been in this same solution for one hour and forty minutes.
The lot that had been in the solution two hours and forty-five
minutes had living blastula, but not so many as the two lots
mentioned above. It is therefore obvious that the eggs of
different females show slight variations in the time required
for the eggs to remain in the mixture of 60 c.c. 2¥°n MgCl2
+ 40 c.c. sea-water in order to reach the blastula stage.

I have thus far only spoken of eggs that had been in
solution 1. Of the eggs that had been in solution 2 not one
developed into a blastula. Those that had been in this
solution for two hours had not even segmented. Only the
eggs that had remained in that solution for two hours and
forty-five minutes showed a beginning of segmentation (2
cells), but only one in a thousand had segmented. It is
evident that either the amount of Mg ions or the total
osmotic pressure of the solution was too small to cause the
unfertilized eggs to develop. These experiments with
negative results are however very valuable as control experi-
ments against the possible contamination of the sea-water
with spermatozoa. If in such cases contamination had
happened, the eggs that had been in solution 2 ought to have
developed equally as well as, or better than, those that had
been in solution 1. The same remark might apply to the
preceding and following experiments. None of the eggs in
any of these solutions formed a membrane.

Fourth series. — In all the experiments in which blastulse
were produced from the unfertilized eggs three conditions
were united: (1) the total osmotic pressure of the artificial
solution was higher, than that of sea- water ; (2) the amount
of the Mg ions was increased; (3) the absolute amount of
the other ions normally present in sea-water was reduced.
In this series I desired to find out whether the third condi-'
tion was essential, and whether the mere increase in the

ARTIFICIAL PRODUCTION or NORMAL LARV.E 607

osmotic pressure was not sufficient. Moreover I wished
once more to repeat the former experiments. The unfer-
tilized eggs of one female were distributed in the following
solutions ;

(1) 60 c.c. *g-n MgCl2 +40 c.c. sea-water

(2) 100 c.c. sea-water + 31 gr. (wet) MgCl2
(8) 100 c.c. sea-water + 8 gr. (wet) MgCl2
(4) Normal sea-water

At various intervals a lot of the eggs were taken out of
each of the four solutions and put into normal sea-water.
The eggs that had been in solution 1 from one and one-half
to two hours had developed into blastula3 the next norning.
The number of blastulse was comparatively larger than in
any of my previous experiments. The eggs that had been
in solutions 2 and 3 contained no blastulae. Solution 2 is, by
the way, the one Norman and Morgan had used in their ex-
periments. In solution 4 no egg was even segmented the
next day. In none of the four solutions had any egg formed
a membrane. These experiments show that the substitution
of a number of Mg ions for one-half of the ions naturally
contained in the sea-water is either necessary or more favor-
able than the mere addition of Mg ions. This experiment
explains why Morgan did not succeed in getting live larvse,
having treated the eggs with solution 2. But I intend to
determine in my future experiments whether the addition
of a little more than 3^ gr. of MgCl2 and a little less than
8 gr. of the solution to 100 c.c. of sea-water may not give
more favorable results.

Fifth series. — I next wished to try whether it would not
be possible to carry the artificial development of the unfer-
tilized egg one step farther. The blastulse thus far obtained
were by no means healthy, and although some of them looked
normal, they died before they had time to reach the pluteus
stage. This latter result I was inclined to ascribe to the

608

STUDIES IN GENERAL PHYSIOLOGY

poisonous effect of the Mg ions, and it seemed possible to me
that a decrease in the amount of MgCl2 and a slight increase
in the amount of sea-water might allow the eggs to reach
the pluteus stage. The eggs of two females were distributed
in the following solution:

(1) 60 c.c. V n MgCl2 +40 c.c. sea-water

(2) 50 c.c. " +50

(3) Normal sea-water

The eggs were brought back into normal sea- water after
one hour and five minutes, one hour and thirty minutes, and

1:55

£.04

2:07

2:15

FIG. 148

one hour and forty minutes. Only the eggs of the last lot
that had been in the solution one hour and forty minutes
showed the beginning of a development. I believe that I
took out the eggs too soon. In some cases such eggs are
able to develop, but in others they are not, and I think it
probable that if the eggs had been left a little longer in so-
lution 1 or 2 they would have developed further. I made
some camera drawings of the way in which the eggs were
segmented (Fig. 148). The successive stages of the segmen-
tation of one and the same egg up to the six-cell stage
were drawn. The reader will see from the drawings that the
egg went within twenty minutes from practically an undivided

ARTIFICIAL PRODUCTION OF NORMAL LARV.E 609

egg into a six-cell stage. It is obvious that these cell-divi-
sions are accompanied by most striking amoeboid motions,
which are characteristic of all the eggs without a membrane.
I believe that these amoeboid motions exist in the fertilized
eggs just as well, but the membrane prevents them from
becoming so conspicuous as in the unfertilized eggs where
there is no membrane. In the normal eggs these amoeboid
motions are more symmetrical, and this is another reason
why they escape our observation. When I made my first
experiments on the effect of more concentrated sea-water
upon the segmentation of fertilized eggs, the idea struck me
that the segmentation by budding (Knospenfurchung) was
the outcome of amoeboid motions, and I soon afterward ex-
pressed the idea that the same is true for the process of cell-
division in general.1 The two nuclei of the mother cell are
the centers around which the protoplasm streams and flows.
These amoeboid motions are only one episode in the process
of cell-division, for whose full explanation other phenomena
of an entirely different character must be taken into con-
sideration.

Sixth series. — The preceding experiment was repeated,
but this time with due consideration of the fact that the
eggs must remain long enough (two hours) in the artificial
solution. The eggs of two females were distributed in three
solutions :

(1) 60 c.c. *$-n MgCl2 +40 c.c. sea-water

(2) 50 c.c. " +50 c.c.

(3) Normal sea-water

None of the eggs formed a membrane. Some of those
that had remained in normal sea-water segmented after
twenty hours. They divided into from 2 to 3 cells and not
further. I have already mentioned the fact that the unfer-
tilized esrsfs of various females differ somewhat in their

1 LOEB, Archiv fur Entwickelungsmechanik, Vol. I (1895), p. 453.

610

STUDIES IN GENERAL PHYSIOLOGY

tendency to segment in normal sea-water. It may be pos-
sible that these variations enhance or diminish the effects of
artificial solutions upon the development of unfertilized eggs.
The eggs that had been for two hours in solution 1 had the

1. ^mrnn^ 2,. x$"M^ o.

9.

FIG. 149

next day developed into the characteristic blastulse some of
which are represented in Fig. 149. Some of these blastulse
originated possibly from the whole mass of one egg, for
instance 1, 3, and 4. But even here the irregular outline
betrays clearly that the blastulaB originated from eggs with-
out a membrane. As I said in an earlier experiment, the
outlines of the eggs became irregular through the amoeboid
motions of the blastomeres, and in the blastulse the outline

ARTIFICIAL PRODUCTION OF NORMAL LARV.E 611

of the irregular morula stage is preserved. This is intel-
ligible if we remember that the blastula originates through
the cleavage cells moving or sticking to the periphery of the
egg. The other blastulse represented only smaller pieces of
a single egg. In some cases one part of the egg disintegrated
and formed debris at-
tached to the other part
which reached the bias-
tula stage (5, 6, and 7).
Each one of these blas-
tulse was moving and
had to be immobilized
to make the camera draw-
ing. It is impossible to
give a fair idea of the
variety of forms of blas-
tulse one sees in such ex-
periments. No egg of
this lot (solution 1)
reached the pluteus
stage. All died the
second day. The eggs
that had been in solu-
tion 2 (equal parts of
2¥°nMgCl2 and sea- water) looked very different from the
preceding lot (Fig. 150). After twenty-four hours many
of them had developed into blastulse. These blastulse left
no doubt that they came from eggs without a membrane, in-
asmuch as in the majority of cases several blastulse originated
from one egg. Quadruplets were especially frequent (Fig
150, IV and v), but twins and triplets were also quite com-
mon. I watched their development, and am thus quite cer-
tain that these multiple embryos sticking together came from
one egg. The feature that distinguished these embryos

FIG. 150

612

STUDIES IN GENERAL PHYSIOLOGY

from those that had been treated with the stronger MgCl2
solution, however, was the fact that the former all had regular
and sharp outlines and were free from debris. The outlines
of the blastulse were much more spherical. These blastulse
had greater vitality than the others and kept alive during the

next thirty-six hours.
The next morning a
number of them had
reached the pluteus
stage with a perfectly
normal skeleton and
intestine, but they
died the following day
(Fig. 151). They had

lived more than two
$\

days. Their develop-
ment was slower than
in the case of fertil-
ized eggs.

All these blastulse
and plutei swam about on the bottom of the dish, not rising
to the surface like the larvae from fertilized eggs.

The control eggs that had been left in the normal sea-
water remained unsegmented, with the exception of a few
which on the second day were found divided into 2 or 3 cells.
The latter, of course, segmented no further. None of these
eggs had a membrane.

Seventh series. — The preceding series had shown that a
mixture of equal parts of 2-$n MgCl3 and sea- water is more
favorable for the development of the eggs than a mixture
with more MgCl3 and less sea- water, for instance 60 c.c.
MgCl2 and 40 c.c. sea- water. In the latter mixture the eggs
seemed to suffer more. It must, however, be stated that as
far as the comparative number of eggs is concerned that

FIG. 151

ARTIFICIAL PRODUCTION OF NORMAL LARV.E 613

undergo development, the solution with 60 c.c. MgCl2 and
40 c.c. sea- water is equally good or even better than the
mixture of equal parts of both. I now tried whether a
mixture with less MgCl2 would still be favorable. A mix-
ture of 40 c.c. ^° n MgCl2 + 60 c.c. sea-water was found
ineffective. The eggs remained two hours in this solution,
and a few of them segmented afterwards, but as the number
was comparatively small I did not follow up this experiment.
It is possible that a mixture of 40 c.c. 2¥° n MgCl2 -\- 60 c.c.
sea-water is too weak to bring about artificial partheno-
genesis of the egg of Arbacia. In one of the preceding
experiments we found that by treating the eggs with a
mixture of 30 c.c. 2¥°n MgCl2 +60 c.c. sea-water we were
not able to bring about parthenogenesis.

Eighth series. — It was evident that in order to produce
plutei from the unfertilized egg of Arbacia we must confine
ourselves to solutions which contain less than 60 and more
than 40 per cent, of 2¥° n MgCl2. In the next experiments
the following four solutions were tried:

(1) 55 c.c. ^-n MgCl + 45 c.c. sea- water

(2) 50 +50

(3) 45 " + 55

(4) Normal sea-water

At three different intervals (two hours, two hours and
ten minutes, two hours and twenty minutes) portions of the
eggs were taken out of these four solutions and put back
into normal sea-water. Two hours later in each of the lots
that had been in the first three solutions about 50 per cent,
of the eggs were segmented into from 2 to 16 cells. None
of them had a membrane. No egg in solution 4 (normal
sea-water) was segmented or had a membrane. The next
morning the eggs that had been in solution 1 were teeming
with blastulse. Many of them resembled the blastulae of
Fig. 149, but the majority were clean and free from debris.

614 STUDIES IN GENERAL PHYSIOLOGY

The eggs that had been in solution 2 had a large number of
blastulse and gastrulse. They were free from d6bris and
looked very much like those drawn in Fig. 150. The eggs
taken from solution 3 had very few blastulse. The latter,
however, were perfect, except that the single egg as a rule
produced more than one embryo. The majority of the eggs
were still in the morula stage. The next morning, forty-
eight hours after the treatment with the MgCl2 solution,
each one of the three dishes contained perfect plutei.
Many eggs of solution 3 which the previous day were still
in the morula stage had in the mean time developed into
blastulse or plutei. This time the plutei were still alive on
the following day (seventy-two hours after the treatment
with the artificial solution). Their vitality was not much
less than that of the normal plutei which often died just as
early. I mentioned that I had put back the eggs from the
MgCl2 solutions into normal sea-water at three different
intervals. Those taken out last gave the best results. It is
very obvious that the unfertilized eggs develop much more
slowly than the fertilized eggs. The latter reach the pluteus
stage at the proper temperature within twenty-four hours or
little more, while the unfertilized eggs reach the pluteus
stage after forty-eight hours at the same temperature. I
had the same experience in all my experiments with unfer-
tilized eggs. The eggs that had been left in normal sea-
water remained undeveloped and not one egg had a mem-
brane. One egg in a hundred was segmented after twenty-
four hours in 2 or 3 cells, but none developed further.

Ninth series. — This time I intended once more to repeat
my experiments ai?d at the same time make control experi-
ments of an altogether different character. I will first speak
of the repetition of the old experiments. The unfertilized
eggs of one female were put into the following two solu-
tions :

ARTIFICIAL PRODUCTION OF NORMAL LARVAE 615

(1) 50c.c. *£n MgCl2+50c.c. sea-water

(2) Normal sea-water

Two hours later the eggs from solution 1 were put back
into normal sea-water. Three and a half hours later about
50 per cent, of the eggs that had been in solution 1 were
divided into from 2 to 16 cells, but not an egg had a mem-
brane. The control eggs that had been in normal sea-water
all the time were all without membrane and absolutely unseg-
mented. Millions of eggs were examined under the micro-
scope. The next morning the eggs that had been in solution
1 had reached the blastula stage and were swimming about.
A small number were in a gastrula stage and even beginning
to assume a pyramidal form. In the control eggs not one
had developed. Perhaps one in a hundred had amoeboid
forms such as precede segmentation in unfertilized eggs, but
not an egg was segmented, and not one had a membrane.
The next day some of the blastulse of the other lot had
reached the pluteus stage. The control experiments will be
discussed in the next paragraph.

Possible sources of error and objections. — From all these
experiments I draw the conclusion that by putting the unfer-
tilized eggs of Arbacia for two hours into a solution of 60 c.c.
^° n MgCl2 -j- 40 c.c. sea- water the eggs develop into blastulse
if brought back into normal sea- water. If we put the unfer-
tilized eggs for about two hours into a solution of equal parts
of 2¥°n MgCl2 and sea-water, the eggs may reach the pluteus
stage. The possible objection might be that the eggs were
fertilized. Such fertilization could only have been caused
by the instruments or hands of the experimenter having
been in contact with spermatozoa, or by the sea-water con-
taining spermatozoa. The first possibility was absolutely
excluded through the above mentioned precautions. The
second possibility was rendered practically impossible, as,
first, the spawning season was practically over, and, second,

616 STUDIES IN GENERAL PHYSIOLOGY

the spermatozoa lose their power of fertilizing eggs in a very
short time (in about five hours). But that it was absolutely
excluded is proven by the following facts:

1. None of the unfertilized eggs kept in normal sea- water
developed or formed a membrane. I examined millions of
eggs in each experiment. Not one was found that was fer-
tilized. The sea- water used in this case was the same as
that used for the unfertilized eggs that did develop. If the
sea-water had contained spermatozoa, the unfertilized eggs
kept in normal sea- water all the time should have been fer-
tilized.

2. None of the eggs which developed after treatment
with MgCl2 solution ever had a membrane. Fertilized eggs
which were put immediately after fertilization into a mixture
of equal parts of 2^° n MgCl2 and sea- water and kept there
for two hours did not lose their membrane. In the ninth
series I made the following control experiments : Unfertilized
eggs that had been in the above-mentioned 2¥° n MgCl3 solu-
tion for two hours were put into normal sea- water to which
fresh sperm was added. In this case a number of eggs
formed membranes.

3. No blastula originated from an egg that had been kept
for some time in one of the following solutions:

(1) 100 c.c. J^nMgCla

(2) 30 c.c. *£n " + 70 c.c. sea- water

(3) 40 " " +60

(4) 100 c.c. sea-water 4- 3J gr. (wet) MgCl2

and the solutions mentioned in chap. iii. Yet eggs of the
same female that had been kept for some time in a mixture
of 50 or 60 c.c. 3¥°,n MgCl2 and 50 or 40 c.c. of sea- water
developed into blastulas or plutei. This happened in spite
of the fact that the vitality of the latter eggs had suffered
more than that of those in the above-mentioned solutions
with more sea-water and less MgCl2. Moreover the water

ARTIFICIAL PRODUCTION OF NORMAL LARV.E 617

was always changed in both classes of eggs simultaneously,
and the chances for fertilization of the eggs from sperma-
tozoa contained in the sea- water were equal for both. If the
sea-water had contained any spermatozoa capable of impreg-
nating the eggs, those eggs that had been in solutions with
less MgCl2 should have been fertilized first.

4. In almost all the experiments eggs were taken out
of the mixture of 60 to 50 c.c. 2¥° n MgCl3 +40 to 50 c.c.
sea-water at different periods. In no case did a single egg
develop into a blastula that had been in this solution for less
than one half-hour, and generally only those eggs yielded
blastulse that had been in this solution for about two hours.
If the sea-water had contained spermatozoa, the latter should
have fertilized those eggs first which had been a shorter time
in the artificial solution. On the other hand, the eggs that
had been left in the artificial solution more than two and
one-half hours as a rule yielded fewer or no blastulse.

5. I stated above that even at the height of the spawning
season eggs are rarely fertilized by spermatozoa contained in
the running sea-water. I do not think one would be likely
to see more than one egg in a thousand undergo develop-
ment under such conditions, provided that no contamination
through the instruments occurred. In our experiments which
were made at the end of the spawning season about 20 to 50
per cent, of the eggs that had been kept in the right solution
developed. It is out of the question to attribute such a
result to spermatozoa contained in the sea- water.

6. As far as I can see, there is only one possible source
of error left. It might be that the sea-water contained
spermatozoa, but that these spermatozoa were not able to
fertilize normal eggs, while a treatment of the egg with
the mixture of 60 c.c. 2¥° n MgCl3 + 40 c.c. sea-water in-
creased its susceptibility to impregnation, or a treatment
of the spermatozoon with the same solution increased the

618 STUDIES IN GENERAL PHYSIOLOGY

fertilizing power of the spermatozoon. Both possibilities
must, however, be discarded. As far as the liability of the
egg to impregnation is concerned, I made the following
experiments in the last series. Unfertilized eggs were put
into a solution of 50 c.c. 2^° n MgCl3 -f~ 50 c.c. sea-water
and left in this solution for two hours. They were then
taken out and fertilized with fresh spermatozoa. At the
same time another lot of the eggs of the same female which
had been kept for two hours in normal sea-water were fer-
tilized with sperm of the same male. Practically every egg
of the latter lot developed into a blastula, while only about
50 per cent, of those eggs that had been in the MgCl2 solu-
tion reached the blastula stage. Hence the treatment with
MgCl2 diminishes the power of development of eggs, but
does not increase it. As far as the spermatozoa are con-
cerned, former experiments by Norman, Morgan, and myself
showed that a slight increase in the concentration of the
sea-water destroys the fertilizing power of spermatozoa very
rapidly. In my experiments I added 2 gr. of NaCI to 100
c.c. of sea- water.

The spermatozoa which had been in this solution for only a few
hours, when brought back into normal sea-water, fertilized only
a thousandth part or less of the normal eggs, while the spermatozoa
of the same animal which had remained in normal sea-water
fertilized at the same time almost all the eggs.1

Morgan repeated my experiments, obtaining the same result.2
Norman tried the effects of a slight increase of MgCl2 upon
spermatozoa.3 I repeat his statement:

I put sperm at 8:30 into MgCl2 solution 2J gr. to 100 c.c. of
sea-water. At 8:30 some of the sperm was mixed with normal
unfertilized eggs, and 'within three minutes the eggs were fertilized.
At 8:42 eggs and sperm were again mixed. In two minutes egg
membranes began to become visible, showing normal fertilization,

1 LOEB, Journal of Morphology, Vol. VII (1892), p. 253.

2 MORGAN, Anato.nischer Anzeiger, Vol. IX (1894), p. 141.

3 NORMAN, Archivfiir Entwickelungsmechanik, Vol. Ill (1896), p. 106.

ARTIFICIAL PRODUCTION OF NORMAL LARVAE 619

and within another minute all the eggs were fertilized. At 8:52
another test was made, but at this time the egg membrane did not
appear, showing that fertilization did not take place. At 9 o'clock
about one egg in every 100 was fertilized.

Norman repeated these experiments several times with the
same result. They prove that even a small addition of
MgCl3 to sea-water, much smaller than in any of our
experiments, suffices to annihilate the power of impregnation
in the spermatozoa in a very short time. In my own experi-
ments the increase in the osmotic pressure of the sea-water
was much greater than in Norman's experiments. I made
another control experiment in the ninth series which bears
on the same question. Unfertilized eggs were left in a
solution of equal parts of 2^° n MgCl 3 and sea-water for two
hours. At the end of that time they were put back into
normal sea- water to which sperm was added which had also
been in a solution of equal parts of 2¥°n MgCl3 and sea-
water for two hours. Only very few of the eggs formed a
membrane.

There is, as we saw, a typical difference between the
blastulse and plutei which develop from fertilized and
unfertilized eggs. The former rise to the surface, the latter
swim at the bottom of the dish. If eggs be kept for two hours
in the MgCl3 solution and then fertilized with normal sperm,
the blastulae rise to the surface. If they be fertilized with
sperm that had been in MgCl3 solution for two hours, they
remain at the bottom of the dish like the unfertilized eggs.
It is thus clear, I think, that even this last possible objection
that the treatment with the MgCl3 solution increases the
impregnating power of the spermatozoa, or the impregna-
bility of the egg must be discarded. Hence I draw the
conclusion that the unfertilized eggs that had been treated
with equal parts of ?g°n MgCl3 and sea- water developed
parthenogenetically.

620 STUDIES IN GENERAL PHYSIOLOGY

V. SOME REMARKS CONCERNING THE NATURE OF THE PROCESS
OF FERTILIZATION

The facts of the preceding section force us to transfer the
problem of fertilization from the realm of morphology into
that of physical chemistry. There is certainly no reason
left for defining the process of fertilization as a morphological
process. The morphology of the spermatozoon itself becomes
of secondary importance as far as the process of fertilization
is concerned.

The spermatozoon not only starts the development of non-
parthenogenetic eggs, but it is also the bearer of the heredi-
tary qualities of the male. From our experiments it becomes
evident that these two functions of the spermatozoon are
not necessarily bound together, for nobody would assume for
an instant that the hereditary qualities that are carried by
the spermatozoon could be imparted to the egg by a change
in the inorganic constituents of the sea-water. We have
learned to attribute the different activities of a cell to
different enzymes. We must in future consider the possible
or probable separation of the fertilizing qualities of the
spermatozoon from the transmission of hereditary qualities
through the same.1

The plutei produced from the unfertilized egg resemble
closely in every regard those produced from the fertilized
egg. The latter in many cases live longer than the former,
but even this was not so in every case, and it is not impos-
sible that in further experiments parthenogenetic plutei with
a greater duration of life will be produced. The only
difference between parthenogenetic and normal blastulae is
that the latter rise to the surface of the water, while the
former do not. One might think that this was due to the
influence of the MgCl2 solution on the egg. This is, how-
ever, not the case. Eggs that had been in such a solution

i LOEB, Biological Lectures, Woods Hole, 1899, Ginn & Co., Boston.

AKTIFICIAL PKODUCTION or NORMAL LARV.E 621

and were fertilized afterward rose to the surface. Even
this difference might be caused to disappear by further
experimentation.

An agency which causes the egg to go through only the
first stages of segmentation, which lead, for instance, to a
division of the egg into 2, 4, or 8 cells, need not necessarily
have much in common with those agencies in the sperma-
tozoon that cause the development of the fertilized egg. But
if the egg can be caused through an artificial influence to
reach the blastula stage and swim about, the artificial cause
must have more in common with the effective element in
the spermatozoon. If however the artificial influences cause
the egg to reach the pluteus stage, or in other words cause
the egg to develop as far as the fertilized egg can be
developed at present in our laboratory, I think the two pro-
cesses of artificial and natural development must be pretty
closely allied.

It is in harmony with our statement that a very large
number of conditions cause an unfertilized egg to reach a
two- or four-cell stage. It suffices to leave the eggs for some
time in sea- water (about twenty-four hours). A slight
increase in the alkalinity of the sea-water causes the begin-
ning of a segmentation much sooner. A short treatment
with sea-water that is faintly acid has the same effect. An
increase in the concentration of the sea-water which probably
causes a loss of water in the egg has the same effect (Morgan).
Morgan found more recently that treatment with a solution
of strychnia salts may lead to a beginning of segmentation.1
Possibly in this case the alkalinity of the sea-water was
modified. But none of these or the other methods mentioned
above has yielded blastulse, gastrulae, or plutei.

There is at present only one way known by which the

i MORGAN, Science, Vol. XI (1900), N. S., p. 176. R. Hertwig had found this many
years ago.

622 STUDIES IN GENERAL PHYSIOLOGY

unfertilized egg of Arbacia can be caused to develop into a
pluteus.1 This consists in treating the unfertilized egg for
two hours with a mixture of about equal parts of a 2-£ n
MgClg solution and sea-water. It is of theoretical interest
to find how this treatment may possibly affect the egg sub-
stance. The bulk of our protoplasm consists of proteids,
which according to their physical behavior belong to the
colloidal substances. The proteids are characterized by two
qualities which are of the utmost importance in the analysis
of life phenomena. The proteids change their state very
easily, and readily take up or lose water. It is more than
probable that one or both of these qualities may account
for muscular contractility and protoplasmic motion. The
agencies which affect these two variable qualities of the
protoplasm most powerfully are, first of all, certain enzymes
(for instance, plasmase, trypsine, etc.). Almost equally
powerful are ions in certain concentrations. As I have
dwelt upon this point in my three preceding publications,2
it need not be repeated here. But I wish to call attention
to a most interesting paper by Dr. E. Pauli, which has
recently appeared and which throws more light on this sub-
ject.3 The third agency is temperature.

In our experiments it was evidently the second factor
which affected the condition of the colloids. The transitory
treatment of the unfertilized eggs with a mixture of equal
parts of a 2¥° n MgClg solution and sea-water brings about a
change in the physical conditions of certain colloids which
is not reversed by putting them back into normal sea-water,
and which allows them to develop into normal plutei.

As far as the spermatozoon is concerned, it may bring
about the same change in the condition of the colloids in

1 1 have not been able to raise the fertilized eggs of Arbacia beyond the pluteus
stage in the laboratory.

2 Part II, pp. 539, 544, and 559.

3 PAULI, Archivfilr die gesammte Physiologic, Vol. LXXVIII (1899), p. 315.

AKTIFICIAL PRODUCTION OF NORMAL LARV^ 623

the egg, either by its carrying specific ions into the egg or
by carrying enzymes, or in some other way which is as yet
unknown to us. It is certainly remarkable that the sperma-
tozoa contain a large amount of ash (5 per cent., according
to Hammarsten). In the parthenogenetic egg the colloids
are from the beginning in such a condition as to allow the
development to proceed. In other animals it is perhaps
solely the ion constitution of the sea-water or of the blood
which prevents the eggs from developing parthenogenetic-
ally. I shall discuss this point more fully in connection
with further experiments on this subject.

XXX

ON ARTIFICIAL PARTHENOGENESIS IN SEA-URCHINS1

IN the last October number of the American Journal of
Physiology I published a preliminary note on the artificial
production of larvae from the unfertilized eggs of the sea-
urchin. I mentioned that unfertilized eggs were able to
develop into normal plutei after having been in a solution of
equal parts of a 2^° n MgCl2 solution and sea-water for about
two hours. The control experiments by which the possibility
of the fertilization of these eggs through spermatozoa had
been excluded were briefly mentioned. In the April num-
ber of the same journal a full description of my experiments
was published which I believe puts an end to any doubt con-
cerning the possibility of an error. Nevertheless, I decided
to repeat these same experiments with the additional precau-
tion of using sterilized sea-water. Through the kindness
of the board of trustees of the Elizabeth Thompson Fund I
was enabled to make further experiments on artificial par-
thenogenesis at the Pacific coast. These experiments have
led to a number of new results, which will be published in
the American Journal of Physiology. Here I will confine
myself to a description of the precautions which were taken in
these experiments to exclude the possibility of a fertilization
of the eggs through spermatozoa.

The sea- water used for these experiments was heated the
day before, very slowly, to a temperature of from 50 to 70°
C., and was kept at that temperature for about ten minutes
and allowed to cool very slowly. The control experiments
proved that, as was to be expected, the spermatozoa are
killed by this treatment. During the time the water was
heated no sea-urchin was opened in the laboratory or was even

i Science, Vol. XI (April 20, 1900), p. 612.

624

AKTIFICIAL PARTHENOGENESIS 625

kept there. The sterilized sea-water was kept in special
flasks and covered jars which were utilized for this purpose
only. Before we started an experiment we disinfected our
hands thoroughly with soap and brush in the same way as is
customary in a surgical operation. Every sea-urchin before
it was opened was exposed for from two to five minutes to a
powerful stream of fresh water, and care was taken to wash
the whole surface of the animal as thoroughly as possible
with fresh water. The mouth of the sea-urchin was then
cut out with scissors that had been sterilized the day before
in the flame and had been kept dry since. Through the
excision of the mouth the sexual glands were exposed, and
their color allowed to decide whether the animal was a male
or a female. If the first animal that was opened was a
female, the intestine was removed with a sterilized forceps,
and care was taken not to bring the forceps in contact with
the ovaries or with the outside surface of the animal. After
the intestine had been removed and nothing left except the
ovaries, the inside of the animal was repeatedly filled with
fresh water and washed out. Then each of the five ovaries
were taken out in Mo with a sterilized section lifter, and
special pains were taken that the ovaries did not come in
contact with the surface of the sea-urchin or with the hands
of the experimenter. The ovaries were first put into a dish
of fresh water, were washed off carefully, and then put into
sterilized sea-water.

One portion of the eggs was put into sterilized sea-water
to serve as control material. A second portion was put into
a mixture of equal parts of sterilized sea-water and a g¥° n
MgCl2 solution. An hour or two later these eggs were
taken out of this mixture and put into sterilized sea- water-
While of the latter eggs as many as 25 per cent, developed
into blastulse and swam around the next day, not an egg of
the control material even segmented. We spent hours

626 STUDIES IN GENEKAL PHYSIOLOGY

searching the control material for segmented eggs, but were
never able to find a single one.

In addition to these control experiments we made several
others. It was necessary to apply the mixture of equal parts
of the 2-£n MgCl3 solution and sea-water for from one to
two hours to bring about the development of the unfertilized
eggs. We made it a rule to take out one portion of eggs
from this solution much earlier — in some cases after ten min-
utes. In no case did one of these eggs segment or develop.

A third series of control experiments was applied. Solu-
tions with less MgClg and more sea-water were tried. In
solutions of 30 c.c. 2^n MgCl2, and 70 c.c. sea-water not an
egg was able to develop.

If the first animal opened in these experiments happened
to be a male, the instruments were at once laid aside for
disinfection, and the next animal was opened by another
experimenter with the same precautions.

In some experiments we used sea-water that had been
filtered through a new Pasteur filter. Although no sperma-
tozoa are able to pass through such a filter, the eggs treated
with a mixture of equal parts of a 2-/n MgCl3 solution and
filtered sea- water developed, while none of the control eggs
were able to develop.

In one of the former papers I mentioned the fact that
the mixture used for artificial fertilization killed the sperma-
tozoa in a comparatively short time and injured many of
the eggs. Contrary to the common prejudice, it is a fact
that spermatozoa are much more sensitive and are killed
much sooner than the egg.

My experiments at Pacific Grove were carried on with
Strongylocentrotus franc iscanus and S. purpuratus. In both
animals artificial parthenogenesis can easily be accomplished.

In the experiments at Pacific Grove I enjoyed the valu-
able assistance of Mr. W. E. Garrey.

XXXI

ON THE TRANSFORMATION AND REGENERATION OF

ORGANS 1

SEVERAL of the older scientists, for instance, Bonnet,
Spallanzani, and Dalyell had occasionally observed that in
the place of a head a tail may be regenerated in lower
animals.2 These casual observations had been considered as
curiosities or pathological cases, and scientists took no further
notice of them. It occurred to me that it might be possible
to produce the substitution of one organ for another at
desire, and that in this way we might gain an insight into
the physiology of morphological processes. Having tried
in vain to accomplish this result dj^ing the year 1888 in
Kiel, I succeeded the following year at Naples. I found
that if the foot of a Tubularian be cut off and the foot
end of the stem surrounded on all sides by sea-water a head
will be produced instead of a foot, while the same end
produces a foot if it is in contact with some solid body,
like the bottom of the aquarium. This arbitrary substitu-
tion of one organ by another I called heteromorphosis in
contradistinction to the case of regeneration in which the
same organ is reproduced. I succeeded in showing that
phenomena of heteromorphosis can easily be produced in all
kinds of Hydroids and in Tunicates.3

Since then a great number of heteromorphoses in various
classes of animals have been obtained. The most brilliant
accomplishment in this field of science is undoutedly
Herbst's discovery that if in Crustaceans the eye together

1 American Journal of Physiology, Vol. IV (1900), p. 60.

2 Part I, p. 115. 3 LOG. cit.

627

628 STUDIES IN GENERAL PHYSIOLOGY

with the optic ganglion be removed, an antenna will be
produced in the place of the eye, while if the eye alone
is cut off an eye is regenerated. The presence or absence of
the optic ganglion decides whether a regeneration or a hetero-
morphosis will follow.1

I found, very early in my experiments, that in certain
Hydroids a heteromorphosis can be produced without any
organ being cut off or any wound being inflicted upon the
animal. In Antennularia — a Hydroid common at Naples —
the arrangement and orientation of the organ as well as
the direction of growth is dominated by gravitation. The
animal consists of a straight vertical stem, which forms
stolons at its lower end and which carries small branches
with limited growth at regular intervals. On the upper
surface of these branches the polyps are found. If such a
stem be suspended horizontally in the water the lateral
branches which are directed downward and which had
finished growing now begin to grow downward very rapidly.
At the same time the polyps on these branches disappear.
The downward-growing parts no longer resemble the old
side-branches but look like roots. A closer examination
reveals the fact that they not only possess the morphological
appearance of roots but also the physiological reactions of the
latter, inasmuch as they are positively geotropic and stereo-
tropic, while the branches do not show these forms of
irritability. In this case the tissue of the polyps which dis-
appeared seems to have been transformed into the tissue of
roots.2

I made a similar observation shortly afterwards at Woods
Hole in another Hydroid, Margelis. When the uninjured
points of a stem of Margelis are brought in contact with a
solid body the point of the stem assumes the form and

1 HERBST, Archivfttr Entwickelungsmechanik, Vol. IX (1899), p. 215.

2 Part I, p. 191.

TRANSFORMING AND REGENERATING ORGANS 629

reaction of a root. It looks as if the contact with a solid
body brought about a transformation of the stem into root
material which is morphologically and physiologically dif-
ferent from the stem.1 But as neither Antennularia nor
Margelis is sufficiently transparent, it was not possible to
ascertain that a transformation of polyps and stems into
stolons occurs in this case.

Miss Bickford made an observation in my laboratory
which helped in making the assumption of a transformation
of organs more probable. Small pieces were cut from a
stem of a Tubularian. These pieces were smaller in size
than a normal polyp. Miss Bickford found that within
sixteen hours such a piece assumed the form of a polyp.2
Driesch confirmed her observation.3

Last summer I had an opportunity to observe directly
the transformation of organs under the influence of contact.
My observations were made at Woods Hole on a transparent
Hydroid, Campanularia. This Hydroid attaches itself with
stolons to solid bodies. The stem with the polyps grows at
right angles with the solid body to which its stolons are
attached. If these Campanularia be cultivated on a ver-
tical wall all the stems assume an exactly horizontal position
in the water. The stem of a Campanularia is the most per-
fect specimen for negative stereotropism I have ever ob-
served. If a stem be cut off and put on the bottom of a
watchglass filled with sea-water, all the polyps that touch
the glass are transformed into the material of the stem.
This material creeps out of the stem, forming stolons wher-
ever it comes in contact with the glass, giving rise to polyps
on its upper surface which is in contact with sea-water.
The polyps continue growing at right angles toward the

1 LOEB, Woods Hole Biological Lectures, 1893.

2 BICKFOED, Journal of Morphology, Vol. IX (1894), p. 417.

3 DEIESCH, Vierteljahrschrift der Naturforscher-Gesellschaft, Zurich, 1896.

630

STUDIES IN GENERAL PHYSIOLOGY

bottom of the dish. All these processes may occur in less
than a day, and can be observed directly with a lens. I will
try to give a description of these phenomena with the aid of
camera drawings I made while observing them. Fig. 152
shows the condition of a
Campanularia stem that
had been put on the bot-
tom of a watchglass the

a-..

FIG. 152 . previous day. Originally

it had five perfectly de-
veloped polyps. Only

two of these are left (4 and 5); the three others
(1, 2, and 3) have disappeared. At the lower
end, a, of the original stem a new stolon, a 6,
has grown out. What had become of the three polyps
that had disappeared? I watched them very closely and
found that they were transformed into a shapeless mass
and withdrawn into the stem. I will describe this process
of transformation of polyps into the material of the stem
more minutely with the help of Figs. 153, 154, 155. These
are not taken from the same stem, but as the process
occurs almost always in the same form, this makes no
material difference.

The transformation of a polyp into the less differentiated

TRANSFORMING AND ^REGENERATING ORGANS 631

FIG. 153

material of the stem begins with a shortening and folding
together of the tentacles (polyp 1 in Fig. 153). This
process is at the beginning the same as that which occurs
upon any stimulation of the polyp and especially in the act
of taking up food. But while in the
latter case the tentacles unfold again,
in the case of the transformation o,f
the polyp they remain together. Very
soon all the tentacles begin to fuse
into a homogeneous mass. This
process of fusing begins usually at
the peripheral end of the
polyp (polyp 2, Fig. 153).
A little later all the ten-
tacles form an undifferen-
tiated mass of protoplasm
(see polyp 1, Fig. 154).

In the next stage (2, Fig. 154) the
2 original differentiation of the crown of
the polyp into tentacles can no/ longer
be recognized.

At this stage the transformed shape-
less mass of the polyp begins to flow
back into the stem (1, Fig. 155). A little later only
a fraction of the original protoplasm of the polyp is
left in the periderm, the rest having crept back into
the stem (2, Fig. 155). In polyps 3, 2, and 1 (Fig. 152)
we see the further stages of this process of the polyp
material flowing back into the stem.

The transformation of polyps and their creeping into the
stem occurs probably in a similar way in an Antennularia
which is put into the water horizontally. The main difference
between an Antennularia and a Campanularia is that in the
latter this transformation is produced by the polyp coming

FIG. 154

632 STUDIES IN GENERAL PHYSIOLOGY

in contact with a solid body, while, in an Antennularia a
change in the position of the polyp toward the vertical
suffices to bring about this result.1

While these processes are going on, the material of the stem
begins to creep or grow out of the original periderm. It
- seems to me worth while to

call the attention of the
reader to the fact that in this
case the process of growth is
identical with the process of
progressive motion of a pro-
toplasmic mass. In plants
FIG 155 cr^H growth occurs mostly near the

apex of an organ. If we look
at the increase in size of the

stolon from the point of view of growth we notice th at its growing
point is near the apex, just as in plants. But if we look at it
from the point of view of progressive amoeboid motion we notice
that only the foremost point creeps and that the rest of the pro-
toplasm is pulled out more passively. That the protoplasm
of the stem is under a strain will be seen by a glance at
Figs. 152, 153, 154, and 156. The coenosarc or protoplasm
lies in the periderm in the same way as a stretched rubber
thread would lie. Wherever the periderm is bent the proto-
plasm touches it on the concave side. It follows .as nearly
as possible the shortest line in the periderm. It is possible
that the strain under which the coenosarc is kept causes the
protoplasm to flow in the direction of the strain toward the
tip of the stolon. Botanists are inclined toward an ex-
clusively osmotic conception of the process of growth. I
have come more and more to the conclusion that the osmotic
theory of growth is not in harmony with the phenomena

i In former papers I have described the fact that in Eudendrium the polyps
are thrown off when the stems are put into small dishes. Such phenomena may
occur also in Campanularia, but this was not the case here. [1903J

TRANSFORMING AND REGENERATING ORGANS 633

of absorption. I do not consider it
impossible that the phenomena of
protoplasmic motion which we can
actually observe in the growth of a
stolon in Campanularia exist also in
the phenomena of growth of other
organisms, plants as well as animals.
I have already called attention to this
possibility in a former paper.

Before we leave this subject I wish
to describe how the nature of the
contact localizes the development of
polyps from stolons and stems. The
piece, b c, Fig. 156, was cut out from
a fresh Campanularia stem and had
been put into a watchglass filled with
sea-water. This piece had a normal
polyp at i, which was transformed
into a mass of undiff erentiated . pro-
toplasm and began to flow back into
the stem. Simultaneously a new
stolon began to grow out at c, and
very soon reached the considerable
size, c d. Then a new polyp, h, began
to rise on the upper surface of the
stem. It grew at right angles toward
the watchglass, a point which cannot
be rendered accurately in the draw-
ing. A new stolon, a b, began to
grow or creep out simultaneously at
a. Curiously enough, as soon as this
happened the protoplasm began to
flow back from the old stolon, c d.
At the time the drawing was made

FIG. 156

634 STUDIES IN GENERAL PHYSIOLOGY

it had flowed back to the point e. This was on the third
day of the experiment. I have however noticed that the stem
can send out stolons in different directions simultaneously.

The hereditary arrangement of organs in Hydroids is
unequivocally determined by external circumstances, espe-
cially contact. A germ or larva of a Hydroid will form
roots on one side only, namely the side where it touches
solid bodies: on the opposite side where it touches sea-
water it will produce polyps or stems. The negative stere-
otropism of the latter or their positive heliotropism as in the
case of Eudendrium will cause them to continue growing
away from the solid body into the sea-water. Weismann is
therefore wrong in assuming that the hereditary arrange-
ment of the organs in Hydroids is due to a definite arrange-
ment of the elements in the germ.

II

What is the character of the physical or physiological
processes which underlie the transformation of organs ?
Such complicated formations as the polyp in Campanularia
are only possible if certain of the constituents are solid.
The transformation of such a polyp into the more shapeless
flowing or creeping material of the stem can only be due to
a liquefaction of these solid constituents. It is moreover
certain that contact with sea-water favors the formation of
polyps with its more solid elements, while the contact with
solid bodies favors the formation of the more fluid material
of the stem or stolon. Hence it seems as if the nature of
contact in this case determined the state of matter of certain
colloids in the Campanularia.1 Although I had observed the
influence of the nature of contact upon these phenomena for
many years I had not been able to form any definite idea of

1 1 do not need to mention especially that the periderm does not participate in
these liquefactions.

TRANSFORMING AND REGENERATING ORGANS 635

how the nature of the contact could possibly influence these
processes, and I do not think that anyone else has thus far
offered an explanation. While studying the literature on the
coagulation of the blood I came across Duclaux' s account of
this process in his Trait6 de microbiologie,1 and it seemed
to me that if his notions are correct they might also
be applied to our problem of contact-heteromorphosis.
According to Duclaux it is the character of- the contact
applied to the leucocytes which decides whether the enzyme
of coagulation, the plasmase, becomes effective or not. As
long as the leucocyte touches the endothelium of the blood-
vessels the blood remains liquid because the contact of the
leucocytes with the endothelial cells does not allow the fibrin
enzyme to act. If, however, the leucocyte touches a piece of
glass the plasmase becomes active and causes coagulation.
If the glass is covered with a layer of oil coagulation does
not occur. Duclaux assumes that surface tension phenomena
decide the setting free of plasmase on the part of the leuco-
cyte. Whether this latter assumption be correct or not mat-
ters little for our purpose. We only need to carry the
analogy between the influence of contact upon the state of
matter of fibrinogen and the state of matter of certain col-
loids in the Hydroids far enough to assume that both depend
upon definite enzymes becoming active through certain forms
of contact acting upon the cells in which they are formed.
In the case of the blood a solidifying enzyme, in the case of
the polyps a liquefying enzyme is made active if the leuco-
cyte or the polyp come in contact with glass or some other
solid body.

These considerations possibly allow of a wider application
than to the mere case of contact-heteromorphosis. When a
piece of our skin is cut off, the cells of the margin of the
wound begin to multiply and spread out over the gap. We

i DUCLAUX, TraiU de microbiologie, Vol. II, Paris, 1899.

636 STUDIES IN GENERAL PHYSIOLOGY

might say the change in the character of the contact causes
an increase in the cell-divisions. This is still more obvious
where whole organs are produced or regenerated. In one of
my former papers I pointed out a very definite chemical dif-
ference between embryonic tissue and muscle tissue.1 The
former is more immune against K ions and more sensitive
toward Ca ions. It has long been noticed, especially by
botanists, that young tissue contains comparatively more K
than old tissue. I am inclined to assume that this accounts
for the fact that young tissue contains more water or has a
greater degree of turgidity than old tissue. An increase in
K allows the protoplasm to take up more water, an increase
in Ca has the opposite effect.2 Ion effects and the effects of
certain enzymes of liquefaction or solidification are often
similar, or may at least support each other. It is not impos-
sible that the increase in cell-divisions among the cells of
the margin of the wound may be due to the different charac-
ter of the contact to which these cells are exposed during or
after the lesion, inasmuch as this different contact sets free
or throws "into activity certain enzymes which do not act as
long as these cells are in their natural surroundings, e. g., as
long as they are in contact with other cells.

In returning after this digression to our main subject we
must mention that the nature of the contact is not the only
means by which solid elements in living tissues may be
liquefied. Five years ago I proved that lack of oxygen
liquefies the cell walls in the blastomeres of a teleost egg
(Ctenolabrus),3 and Budgett showed in my laboratory that
lack of oxygen produces the same phenomenon in Infusoria.4
This case may find its explanation through the well-known
experiment of Pasteur on the effect of oxygen on yeast cells.
With plenty of oxygen the yeast cells multiply abundantly,

i Part II, p. 559. 2 part II, p. 510. 3 part I, p. 370.

4 BUDGETT, American Journal of Physiology, Vol. I (1898), p. 210.

TRANSFORMING AND KEGENERATING ORGANS 637

but produce comparatively little fermentation; with little
oxygen they multiply less but cause a more abundant devel-
opment of alcohol and CO2. In the liquefaction of the cell-
walls of the blastomeres of Ctenolabrus or of Infusoria we may
have the analogue of the increased fermentation in Pasteur's
experiment. In the latter we have to deal with a special
enzyme, the zymase.

Miescher pointed out that in the salmon a liquefaction of
muscular tissue occurs, and that the liquid products are util-
ized for the formation of sexual cells. Miescher was inclined
to ascribe the liquefaction of the muscle to lack of oxygen.
He noticed that the liquefaction of the muscle was preceded
by a reduction in the blood supply of the muscles.1 My own
and Budgett's observations agree with Miescher's views.*

It is possible that the processes of histolysis in the meta-
morphosis of insects are of a similar character, and some
authors have claimed that the histolysis in this case is
brought about by a process of asphyxiation. Metschnikoff
assumes that a phagocytosis plays an important role in these
phenomena of histolysis. It is certain that in my experi-
ments on Ctenolabrus and in Budgett's experiments on
Infusoria no phagocytes were present, and it is practically
impossible that they played a role in the above-mentioned
phenomena in Campanularia. I do not think that the lique-
faction of colloids requires the presence of phagocytes any
more than the liquefaction of crystals.

1 Die histochemischen und physiologischen Arbeiten von F. Miescher, Leipzig, Vol.
I (1897), pp. 94-100.

2 It is possible that in the case of Campanularia the histolytic phenomena do not
stop with the liquefaction of certain constituents, but that this process is followed by
hydrolysis. [1903]

XXXII

FURTHER EXPERIMENTS ON ARTIFICIAL PARTHENO-
GENESIS AND THE NATURE OF THE PROCESS OF
FERTILIZATION1

1. IN my previous communications on the subject of arti-
ficial parthenogenesis2 I had confined myself to the proof of
the fact that the unfertilized eggs of Arbacia and Strongy-
locentrotus franciscanus and purpuratus, are capable of a
development into the pluteus form if kept for from one to
two hours in a mixture of equal parts of a 2¥°n MgCl2 solu-
tion and sea-water. The above-mentioned solution, which
brings about the artificial development of the egg, differs in
three directions from the constitution of the normal sea-
water. First, the osmotic pressure of the solution is higher
than that of the normal sea-water ; second, one-half of the
salts contained in normal sea-water are removed. It might
be possible that the sea-water contains ions which are in-
jurious to the development, and that the removal of these
ions makes the development of the unfertilized eggs possible.
Third, a considerable amount of MgClg is brought into solu-
tion, and it might be that the Mg ions have a specific "stimu-
lating " effect upon the development. For the determination
of the nature of the process of fertilization it was necessary
to find out which of the three conditions is essential for the
production of artificial parthenogenesis.

2. I had already mentioned in a previous paper that the
mere change in tfye constitution of the sea-water, if not
accompanied by an increase in its osmotic pressure, can only

1 American Journal of Physiology. Vol. IV (August 1, 1900), p. 178. These ex-
periments were carried out with the aid of the Elizabeth Thompson Science Fund.

2 Part II, pp. 539, 576, and 624.

638

ARTIFICIAL PARTHENOGENESIS 639

cause the egg to go through a few segmentations, but cannot
cause the parthenogenetic production of a blastula or a later
stage of development. The increase in the osmotic pressure
of the solution is therefore an essential condition for arti-
ficial parthenogenesis. As the season was at an end, it was
not possible for me to decide last autumn whether the other
two above-mentioned conditions are equally essential.
Through the aid of the Elizabeth Thompson Fund I was
enabled to carry on experiments in co-operation with Dr. W.
E. Garrey at Pacific Grove during the spring,1 and I have
since had a chance to continue this work at Woods Hole.
My new results enable me to give a more definite answer to
the question of the nature of the process of fertilization.
I first tried to ascertain whether the MgCl2 plays a specific
r6le in artificial parthenogenesis, or whether its place may
be taken by some other salt. I found that the latter is the
case.2 A mixture of equal parts of a *-£ n NaCl solution and
sea-water, or of equal parts of a J¥0 n KC1 solution and sea-
water, is just as effective as, if not more so than, a 2-^ n
MgCl3 solution. Unfertilized eggs of Strongylocentrotus,
if left for seventy minutes in any of these solutions, devel-
oped, and some of them reached the pluteus stage. Such
eggs remained alive as long as ten days. Even a mixture
of equal parts of a 2^ n CaCl2 solution and sea-water
brought about the development of the eggs, but it was
necessary to take the eggs out in about forty to fifty
minutes, as otherwise the solution killed them. None of
the eggs treated with the CaCl2 solution developed beyond
the blastula stage, or lived longer than one day.

1 noticed that in these experiments with a y n NaCl or

i 1 wish to express my thanks to Professor Jenkins, of Stanford University, for
kindly allowing me the use of the Hopkins Laboratory.

2 I had been misled in my original experiments of 1899 through the fact that
the solutions which I considered as isosmotic differed in their concentration, owing
to an error in their preparation. When I resumed the experiments in 1900 I dis-
covered the error and corrected it. [1903]

640 STUDIES IN GENEKAL PHYSIOLOGY

KC1 solution only a comparatively small number of eggs
reached the blastula stage, certainly many less than in my
previous experiments with MgCl2 on Arbacia. A further
examination revealed the fact that the MgCl2 solution
which I had used was, through an error or a misunder-
standing of the assistant who made it, weaker than a 2^° n
solution. As soon as I found this out, I started experi-
ments with more diluted NaCl and KC1 solution. Instead
of using equal parts of a y n NaCl or KC1 solution and
sea-water, I used the following mixtures:

20 *$-n NaCl + 30 distilled water + 50 sea-water,
or — 171 V-n NaCl -f 32i distilled water + 50 sea-water.

In both cases more eggs reached the blastula and pluteus
stage than with the original stronger mixture. In one case
unfertilized eggs developed beautifully after having been
for two hours in a solution of equal parts of 15 2^ n NaCl
+ 35 distilled water -j- 50 sea-water. But this was nearly
the lowest limit for artificial parthenogenesis in Arbacia.
As a rule, 25 per cent, or more of the unfertilized Arbacia
eggs reached the blastula stage.

3. It was thus proved that MgCl8 does not play a specific
r6le in the production of artificial parthenogenesis. It
remained to decide whether it is essential to remove one
part of the normal constituents of the sea-water, or whether
the mere increase of the osmotic pressure suffices. I found
that the increase in the osmotic pressure of the sea-water is
all that is needed. In the experiments in which the maximal
number of unfertilized eggs reached the bastula stage about
1 gram NaCl had been added to the sea-water. We can
produce the same Increase in the osmotic pressure of the
sea- water by adding 10 c.c. of the 2^n NaCl or 2^n KC1
solution1 to 90 c.c. of sea- water. In this case the mixture

!My2l/2nNaCl solution contained 146.25 g. in a liter. The 21/2nKCl solution
contained 186.25 g. in a liter.

ARTIFICIAL PARTHENOGENESIS 641

contained practically all the constituents of normal sea-water.
Yet if unfertilized eggs of Arbacia are left in such a solution
for from one and one-half to two hours, as many as 50 per cent,
of the eggs may reach the blastula stage when put back into
normal sea-water. Many of these eggs die in the blastula
stage and only a small number reach the gastrula or pluteus
stage. The blastulse are like those which I described in one
of my former papers.1 In the majority of cases more than
one blastula develops from one egg. I have seen as many
as six moving blastulaB arise from one egg. The tendency
to give rise to more than one embryo is greater in the egg
of Arbacia than in the egg of Strongylocentrotus. This
difference is probably due to the fact that even the unferti-
lized egg of Strongylocentrotus often forms a fine membrane
which is much thinner than the one produced through the
entrance of a spermatozoon, but which is sufficient to keep
the blastomeres together. The addition of NaCl or KC1 to
sea- water favors the formation of this membrane.

4. In all the experiments mentioned thus far the increase
in the osmotic pressure had been brought about by the
addition of electrolytes. This might be considered as an
indication that the electrically charged ions in the sea-water
played an important role in the production of partheno-
genesis. I myself was originally inclined to such an assump-
tion. I have convinced myself, however, that an increase in
the osmotic pressure of the sea-water through the addition
of cane-sugar or urea can produce parthenogenesis. My
stock solution of cane-sugar (rock candy) was Zn and con-
tained 684.3 g. in a liter, while the stock solution of urea
was 2^n and contained 150.31 g. in a liter. I found that
the unfertilized eggs of Arbacia were able to develop after
they had been for from one and one-half to two hours in one
of the following solutions:

1 Part II, p. 576.

642 STUDIES IN GENERAL PHYSIOLOGY

(1.) 100 sea-water + 25 2 n cane-sugar
(2.) 82J sea-water+17i 2$n urea

Both the sugar solution as well as the urea solution injured
the eggs, the urea solution much more than the sugar solu-
tion. I made an attempt to produce parthenogenesis by
submitting unfertilized eggs to a pure cane-sugar solution
whose osmotic pressure was about equal to that of the sea-
water, to 90 c.c. of which 10 c.c. of a 2^n NaCl solution had
been added. When the unfertilized eggs of Arbacia were
put for about two hours into a mixture of 60 2 n cane-sugar
+ 40 distilled water or 55 2n cane-sugar + 45 distilled
water, many of them segmented and a few developed into
swimming blastulse, but they died within the first twenty-
four hours. This proves conclusively that the development
of the unfertilized egg is produced through an increase in
the concentration of the surrounding solution. As it is
immaterial whether the increase in the osmotic pressure is
brought about by electrolytes or non-conductors, there can
be no doubt that the essential feature in this increase in the
osmotic pressure of the surrounding solution is a loss of
water on the part of the egg.

5. Having reached the conclusion that the loss of water,
or rather the loss of a certain amount of water, causes the
parthenogenetic development of the egg, it seemed possible
to take another step in advance. In all the previous experi-
ments the unfertilized eggs had been submitted to a solution
of higher osmotic pressure for from one to two hours, and
were then put back into normal sea-water to develop. If
the initial loss of water on the part of the egg were all that
is required for the . production of artificial parthenogenesis,
it would be possible to find a solution which would not only
take away water from the egg, but which would also allow
development to go on. I remembered from my earlier
experiments on the effects of an increase in the concentration

ARTIFICIAL PARTHENOGENESIS 643

of sea-water upon development1 that so slight an increase in
the concentration of sea-water as is sufficient to induce par-
thenogenesis allowed the development of the eggs to go on
for at least twenty-four hours. / found that if we put
unfertilized eggs into a mixture of 93 sea-water and 7 2\n
NaCl solution, many eggs develop in the solution, and some
of them even reach the blastula stage and swim about. If
we use a mixture of 90 sea-water and 10 2^n NaCl solution,
the development stops earlier, for the simple reason that
such a solution is more injurious. Those facts show clearly
that the function of the artificial solution in the production
of parthenogenesis is that it has to deprive the egg of a
certain amount of water. In the majority of cases the
solutions that produce such an effect are at the same time
too injurious to allow the egg to develop or live long enough
to reach the blastula stage. This is the reason why we have
to take the eggs out of this solution and bring them back
into normal sea-water, if we wish them to develop into nor-
mal Iarva3.

6. A consequence of the loss of water on the part of the
egg is an increase in its osmotic pressure. The osmotic
pressure inside the egg is furnished chiefly or almost ex-
clusively by electrolytes. It is thus not impossible that the
ions in the egg, if their concentration is raised, bring about
that change which causes the egg to develop. If we assume
that the spermatozoon starts the development of the egg in
the same way as in the case of artificial parthenogenesis it fol-
lows that the spermatozoon must possess more salts or a
higher osmotic pressure than the eggs. As I pointed out
in a former paper, this seems to be the case. But there is
no reason why the spermatozoon should not bring about the
same effects that we produce by reducing the amount of
water in the egg in some different way. At present, how-

i Journal of Morphology, Vol. VII (1892), p. 253.

644 STUDIES IN GENERAL PHYSIOLOGY

ever, the only light that can be thrown upon the nature of
the process of fertilization must be expected from an analysis
of the effects of a loss of water upon the egg.

It seems as if the liquefaction of the nuclear membrane
and other constituents of the nucleus were a prerequisite for
cell-division. Norman showed that a certain increase in the
concentration of the sea-water brings about a distribution of
the chromosomes in the egg. Morgan's observations agree
with this. But as all these observations were made with
solutions whose osmotic pressure was considerably higher
than that of the solutions used in my experiments, new ob-
servations will be required to decide this question. Hoppe-
Seyler, in one of his papers, points out that a loss of water
on the part of the protoplasm brings about a diminution
in the processes of oxidation. We know that lack of
oxygen can bring about the liquefaction of solid con-
stituents. I add these remarks for those who enjoy the
speculative side of biology. But at the best a theory can-
not give us anything more than the facts it includes, and
it is therefore clearly our task to supply the lacking ex-
perimental data in this field of biology before we begin
to theorize.

7. I think we should try to discover first of all whether
the process of development can be started by depriving the
egg of water in a few forms only, or whether this is a gen-
eral condition. I have thus far tried among the sea-urchins
Arbacia and Strongylocentrotus f ranciscanus and purpuratus.
Each of these forms is capable of osmotic parthenogenesis.
I am confident that the same is true for all species of sea-
urchins, although the optimal increase in the osmotic pressure
of the surrounding solution may vary for different forms.
But I consider it of more importance that with the same
methods I have been able to produce artificial partheno-
genesis in a starfish (Asterias Forbesii). By putting the

ARTIFICIAL PARTHENOGENESIS 645

unfertilized eggs of this starfish for about two hours into
a mixture of 88 c.c. of sea-water and 12 c.c. of a 2Jn NaCl
solution the eggs can be forced to develop and reach the
blastula stage, if put back afterward into normal sea-water.
I have not yet found the optimal condition for the partheno-
genetic development of Asterias, but the facts thus far ob-
tained suffice to state that a certain increase in the osmotic
pressure of the surrounding solution (and a loss of a certain
amount of water on the part of the egg) causes the egg of
this form to develop parthenogenetically.

I have mentioned in another place1 the precautions and
control experiments used to guard against the presence of
spermatozoa. I do not consider it necessary to repeat these
statements in this paper, but will mention one additional
precaution, for which I am indebted to the collector of the
Marine Biological Laboratory, Mr. Gray. Mr. Gray selects
the females of Arbacia for my experiments, so that in all
these later experiments I have not had one male in the labo-
ratory. Not one egg developed in the control material. All
the sea-water used in these experiments was heated to the
temperature of 70° 0.

CONCLUSIONS

The results of my experiments are as follows:

1. Through a certain increase in the osmotic pressure of
the surrounding solution the unfertilized eggs of some
(probably all) Echinoderms (Arbacia, Strongylocentrotus,
Asterias) can be caused to develop into normal blastulse
or even plutei.

2. This increase in osmotic pressure can be produced by
electrolytes as well as by non-conductors. It is therefore
probable that the parthenogenetic development is caused by
the egg losing a certain amount of water.

i Part II, p. 576.

XXXIII

EXPERIMENTS ON ARTIFICIAL PARTHENOGENESIS IN
ANNELIDS (CH^TOPTERUS) AND THE NATURE OF
THE PROCESS OF FERTILIZATION1

I. INTRODUCTION AND METHODS

MY preceding papers on artificial parthenogenesis2 had
proved that by an increase in the osmotic pressure of the
sea-water the eggs of many, if not all, Echinoderms can be
caused to develop parthenogenetically. Two new problems
presented themselves for immediate consideration. The one
was to raise the parthenogenetic larvae until they were sex-
ually differentiated, in order to decide whether or not they
are of uniform sex. The second problem was to try whether
artificial parthenogenesis is confined to the group of Echino-
derms or whether it is a more general phenomenon. As the
means for the raising of sea-urchins were not available at
Woods Hole this year, the former problem had to be post-
poned. The solution of the second problem, however, was
possible, and yielded the result that the unfertilized eggs of
Chsetopterus, a marine Annelid, can be caused to develop into
swimming ciliated larvae (trochophores). A short preliminary
report of this result has been published in Science?

In experiments on parthenogenesis the greatest precau-
tions are necessary to exclude the possibility of a contamina-
tion of the eggs by spermatozoa. I purposely selected
Chsetopterus for my further experiments on account of the
possibility of discriminating between and separating the
females and males. If the experimenter handles females
and males in the same experiment or with the same instru-

1 American Journal of Physiology, Vol. IV (1901), p. 423.

2 Part II, pp. 539, 576, 624, and 638. * Science, Vol. XII (1900), p. 170.

646

AKTIFICIAL PARTHENOGENESIS IN ANNELIDS 647

ments, it is extremely hard to avoid an infection of the eggs
by sperm. I proceeded as follows in the experiments with
Chsetopterus. As soon as the animals were brought into the
laboratory by the collector, the tubes in which they live were
opened and the worms removed. As soon as the first female
was found it was put into a special dish and thoroughly
washed off with sea-water, the water being renewed from six
to twelve times in succession. The sea-water in the labora-
tory was found to be absolutely free from spermatozoa of
Chsetopterus. (The animals are found on the beach of an
island at some distance from the laboratory.) After the
female had undergone the process of washing, it was exposed
to a current of sea-water over night to remove as far as pos-
sible any spermatozoa that might have been left on the sur-
face. The next day the animal was ready to be used for an
experiment. On that day and before the experiment began,
the experimenter did not bring his hands in contact with
any other Chsetopterus or with the aquarium that contained
such animals. His hands and instruments were sterilized
with fresh water. The posterior part of the animal which
contains the eggs was cut off and thoroughly washed for two
minutes in distilled or fresh water. Had any spermatozoon
been left on the surface of the animal, the distilled water
would have killed it. After this the part containing the eggs
was put into a vessel with sterilized sea-water, washed off
once more and then put into another dish containing steril-
ized sea-water. In this dish the single parapodia were
opened successively, the eggs sucked out from each with a
pipette, and then collected in another dish with sterilized
sea-water. After all the eggs had been collected they were
divided into two lots. The one lot remained in normal
(sterilized) sea-water, to serve as control material. The
other lot was distributed into the various solutions whose effect
I intended to test. In no case did I see a single egg of the

648 STUDIES IN GENERAL PHYSIOLOGY

control material develop into a larva. I noticed only that
after from seven to ten hours some of these eggs may show
a beginning of a segmentation which, however, soon ceases.
This phenomenon seems to be quite common among many
marine animals. I mentioned in a former paper that O.
Hertwig had already noticed that it is a common occurrence
among Arthropods, Worms, and Echinoderms.1 If, however,
no such aseptic measures against spermatozoa were taken, a
number of eggs in the control material usually reached the
trochophore stage. The sea-water used in these experiments
was sterilized by heating it slowly to a temperature of from
60° to 80° C. In a smaller number of experiments I used
sea-water which had gone through a Pasteur (Chamberland)
filter which, of course, is absolutely impermeable to sperma-
tozoa.2 If the eggs of more than one female were used for
an experiment, all the eggs were first gathered in one dish,
thoroughly mixed, and then divided into two lots, one to serve
as control material and one to be distributed into the various
solutions. Thus the control material and the material experi-
mented upon consisted always of the eggs of the same
females. It goes without saying that the same was the case
in all my previous experiments on Echinoderms.

II. ARTIFICIAL PARTHENOGENESIS CAUSED BY AN INCREASE
IN THE OSMOTIC PRESSURE OF THE SEA-WATER

It was natural to try first whether or not the same means
that cause the parthenogenetic development in Echinoderms
are also sufficient to bring about the parthenogenetic devel-
opment of the eggs of Chsetopterus.

First series. — When I received the first material, I at

1 O. HEETWIG, Die Zelle und die Gewebe, Vol. I (1893), p. 239.

2 In almost all the experiments the sea- water used was sterilized. In a few
exceptions this precaution was purposely omitted in order to find out whether or not
the sea-water in the laboratory contained spermatozoa of Cheetopterus. This, how-
ever, was not the case.

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 649

once started an experiment, although I knew that it was
practically impossible to exclude contamination by sperma-
tozoa if I attempted to isolate the eggs immediately after
having handled a male. The female was washed off in steril-
ized sea-water, but of course I was aware that this would
not suffice to get rid of any spermatozoa that might be stick-
ing to the surface of the animal. The eggs, however, were
taken and distributed into the following five solutions:

(1) 6 c.c. 2±n KC1+94 c.c. sea-water

(2) 8 " +92

(3) 10 " +90 "

(4) 12 " +88 "

(5) Normal sea-water (control)

One part of the eggs remained one hour and twenty-five
minutes, the rest one hour and forty minutes in the solu-
tions. The experiment was started in the afternoon. The
next morning1 I found numerous swimming larvse (trocho-
phores) in the material that had been in the first four solu-
tions for one hour and twenty-five minutes. In the second
lot they were less numerous. But even in the control
material I found two swimming trochophores. It followed
that the Chsetopterus were either naturally parthenogenetic or
the precautions against the entrance of spermatozoa had not
been sufficient.

Second series. — From now on I applied the rigid anti-
septic measures against spermatozoa described above in the
introduction. The following solutions were used:

(1) 8 c.c. 2Jw KC1 +92 c.c. sea-water

(2) 10 c.c. 2±nKCl +90

(3) 12c.c.2i^KCl +88

(4) 12 c.c. 2±n NaCl +88

(5) 20 c.c. 2|nMgCl2+80

(6) Normal sea-water (control)

1 1 shall in the following descriptiou of the experiments consider only whether
or not swimming trochophores were formed. The morphological details will be
given in section v. It goes without saying that all the experiments deal with unfertil-
ized eggs, unless the contrary is distinctly stated.

650 STUDIES IN GENERAL PHYSIOLOGY

All the sea- water had been sterilized the previous day by
heating it to a temperature of 80° ; one part (a) of the eggs
remained one hour, a second part (6) one hour and twenty
minutes in these solutions.

The first four solutions yielded numerous swimming
trochophores ; their number was greatest in the first two
solutions. Lot a of the MgCl2 solution yielded no swimming
blastulse, but lot 6 had a few. The control eggs were com-
pletely undeveloped, with the exception that after about ten
hours a few showed the beginning of a segmentation, which
in no case led to the formation of more than from 4 to 6
cells. During the next forty-eight hours no further develop-
ment occurred, and the eggs died and disintegrated. Accord-
ing to this experiment the unfertilized eggs of Chaetopterus
are not able to develop in normal sea-water. They can,
however, be caused to develop into trochophores if exposed
for about an hour to sea-water whose concentration has been
raised through the addition of the right quantity of KC1 or
NaCL

Third series. — The next task was to ascertain how much
the osmotic pressure of the sea-water must be raised in
order to bring about the parthenogenetic development, and
whether the increase in osmotic pressure necessary for this
purpose was the same in each case. The solutions used
were as follows:

(1) 10 c.c. 2±n KC1 +90 c.c. sea-water

(2) 12i c.c. 2fn KC1 +87J

(3) 30 c.c. 2 n cane-sugar + 70 "

(4) 12J c.c. 2±n NaCl +87J "

(5) Normal sea-water (control)

The osmotic pressure in solutions 2, 3, and 4 was about
the same. The eggs remained sixty-five minutes in these
solutions, and were then put back into normal sea-water.
While a great number of the eggs that had been in solutions

AKTIFICIAL PARTHENOGENESIS IN ANNELIDS 651

1 and 2 developed into troehophores, very few of the eggs of
solution 4 and none of solution 3 reached the trochophore
stage. The control eggs remained undeveloped.

Fourth series. — The results were obviously puzzling if
the increase of the osmotic pressure was the only factor that
brought about the development of the unfertilized eggs of
Chsetopterus. But they would be intelligible if there were,
in addition to the effect of an increase in the osmotic pres-
sure, a specific effect of the KC1 or the K ions. In order
to decide this, the unfertilized eggs of a female were dis-
tributed into the following solutions:

(1) 5 c.c. 2J n KC1 +95 c.c. sea-water

(2) 10 " " +90 "

(3) 15 " " +85

(4) 5 " NaCl + 95

(5) 10 « " +90

(6) 15 " " +85 «

(7) Normal sea-water (control)

The eggs remained one hour in these solutions.

The next day the control eggs (7) were undeveloped.
The eggs that had been in the first three solutions were
teeming with troehophores. In lots 4 and 5 not a single
swimming trochophore was found, although many eggs had
begun to develop. The development stopped, however, in
an early stage. Of the eggs that had been in solution 6 a
large number had reached the trochophore stage and were
swimming. These results were as clear as could be desired.
In order to bring about artificial parthenogenesis through
the addition of NaCl, 15 c.c. of the 2^n solution had to be
added, while 5 c.c. of a 2^n KC1 solution were sufficient.

Fifth series. — There was a possibility that the effect pro-
duced by NaCl was a specific Na effect, and not an effect of
the increase in osmotic pressure. An experiment with cane-
sugar could decide this question. My stock solution of

652 STUDIES IN GENERAL PHYSIOLOGY

cane-sugar was a 2n solution, while my NaCl solution was
2 \n. On account of the electrolytic dissociation, more than
30 c.c. of the cane-sugar solution were required to produce
the same increase of osmotic pressure as by 15 c.c. of the
2^n NaCl solution. The following solutions were tried :

(1) 40 c.c. 2n cane-sugar +60 c.c. sea-water

(2) 20 " " +80 "

(3) 10 « " +90

(4) 10 2itiKCl +90 "

(5) Normal sea-water (control)

The eggs remained fifty-five minutes in these solutions.
Eight hours later swimming ciliated trochophores were found
in the eggs that had been in solutions 1 and 4. In 2 and 3
there were no swimming larvae. In the control material all
the eggs were still spherical and unsegmented. The next
morning about 25 per cent, of the eggs that had been in
solution 1 swam about in the most lively manner. A few
trochophores were found among the eggs that had been in
solution 2. But the control eggs and the eggs that had
been in solution 3 had in the best case only reached the
earliest stages of segmentation. This leaves no doubt that
an increase in the osmotic pressure of the sea-water is
sufficient to bring about artificial parthenogenesis in the
eggs of Chsetopterus.

Sixth series. — In order to make this conclusion stronger,
it was necessary to try the effect of an increase in the osmotic
pressure of the sea- water by the addition of still other sub-
stances. The following were tried:

(1) 5, c.c. 5n CaCl2 +95 c.c. sea-water

(2) 10 " " +90 "

(3) 10 2JwMgCl2+90

(4) 20 " " +80

(5) 30 " " +70

(6) Normal sea-water (control)

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 653

The eggs remained in these solutions one hour, and were
then put back into normal sea-water. I neglected to look
at them the same evening. The next morning I found a
small number of swimming larvse among the eggs that had
been in solution 5 (30 c.c. 2^n MgCl2+70 c.c. sea-water).
The control eggs were undeveloped ; the Ca2 eggs had gone to
pieces. The experiment demonstrated only that the increase
of the osmotic pressure through MgCl3 can bring about the
development of the unfertilized eggs of Chsetopterus.

Seventh series. — I suspected that my failure to get swim-
ming trochophores from a mixture of sea- water and a 5rc
CaCl2 solution might have been due to the poisonous effect
of the calcium, having noticed in my previous experiments
on the artificial parthenogenesis in sea-urchins that the par-
thenogenetic larvse produced by the addition of CaCl2 to sea-
water soon died. I therefore started a new experiment early
in the morning and watched the eggs during the day. I
found indeed that an increase in the osmotic pressure of the
sea- water by the addition of CaCl2 leads to the formation of
swimming trochophores from the unfertilized eggs of Chsetop-
terus. The solutions used were as follows:

(1) 2J c.c. 5 n CaCl^ + 97 J c.c. sea- water

(2) 5 " « +95

(3) 10 « " +90

(4) 15 " " +85 "

(5) 20 2JrcMgCla + 80

(6) Normal sea-water (control)

The eggs were exposed to these solutions for fifty minutes.
After nine hours the eggs that had been in solution 3 con-
tained living trochophores which died during the night.
None of the other solutions gave rise to swimming trocho-
phores.

Eighth series. — As there was no more doubt left that the
increase in the osmotic pressure of the sea-water or the loss

654 STUDIES IN GENERAL PHYSIOLOGY

of a certain amount of water on the part of the egg caused
the partheno^enetic development of the eggs of Chsetopterus
in these experiments, it now remained to ascertain how long
the eggs must remain in these solutions in order to develop.
I put the unfertilized eggs of a female into a mixture of
85 c.c. sea- water + 20 c.c. 2^n NaCl. The first lot were taken
out of this mixture after ten minutes, the second after thirty,
the third after sixty, the fourth after ninety, and the fifth
after one hundred and twenty minutes. The same evening
(nine hours later) I found swimming trochophores among
the eggs that had been taken out of the third and fourth
lots. The eggs of the first lot did not show any trace of
development at that time. Of those of the second lot about
one in a hundred had begun to develop. In the fifth lot the
eggs had apparently undergone development, but I found no
swimming larvae. The eggs had possibly been injured by
their long stay in the more concentrated sea-water.

The next morning about 20 to 40 per cent, of the eggs
of the third and fourth lots were swimming about in the
trochophore stage. The rest did not contain any living
larvse, although some of the eggs were in the early segmen-
tation stages.

It is therefore necessary to leave the unfertilized eggs of
Chsetopterus more than thirty and less than one hundred and
twenty minutes in a mixture of 85 c.c. sea- water -+- 20 c.c. 2^n
NaCl in order to cause them to reach the trochophore stage.

Conclusions. — From these experiments we are allowed to
draw the following conclusions:

1. The unfertilized eggs of Chsetopterus do not reach the
trochophore stage • if left in normal sea- water, provided the
proper precautions are taken against contamination by sper-
matozoa. Such eggs show no change during the first seven
to nine hours, but may begin to segment after that time. In
such cases the segmentation as a rule does not proceed beyond

AKTIFICIAL PAKTHENOGENESIS IN ANNELIDS 655

the two- to four-cell stages, but may in exceptional cases go
as far as the twelve- to sixteen-cell stages. We may say that
Chsetopterus possesses a higher degree of parthenogenetic
tendency than the Arbacia egg, which begins to segment
later, after about twenty hours, and does not proceed beyond
the two- to four-cell stage.

2. The unfertilized eggs of ChaBtopterus are able to
develop into swimming trochophores if they are put for about
one hour into one of the following solutions and then put
back into normal sea-water:

(1) 15-20 c.c. 2Jn NaCl +85 c.c. sea-water .

(2) 40 2 n cane-sugar +60 "

(3) 30 2Jn MgCl2 +70 "

(4) 10 5w CaCl2 +90

All these solutions have one element in common, namely,
the about equal increase of the osmotic pressure. It seems
therefore justifiable to assume that the increase in the
osmotic pressure or the loss of water on the part of the egg
is the cause of the parthenogenetic development of these
eggs.

8. KC1 or perhaps the K ions seem to possess a specific
effect upon the eggs of Chaetopterus. We shall discuss this
fact more fully in the next section.

Objections considered. — The possible objection that the
eggs of ChaBtopterus are naturally parthenogenetic in normal
sea-water or that spermatozoa had contaminated the sea-
water is rendered impossible through the behavior of the
control eggs and the antiseptic precautions taken. As far
as I can see, there is only one objection left, which, however,
although far-fetched and highly improbable, shall be consid-
ered. It might be argued that Cha3topterus is hermaphro-
ditic, but that the eggs and spermatozoa do not mature
simultaneously. This prevents fertilization of eggs in nor-
mal sea-water. But the increase in the osmotic pressure of

656 STUDIES IN GENERAL PHYSIOLOGY

sea-water might increase the motility or fertilizing power of
the spermatozoa. The contrary is, however, true. The
eggs of the same female were divided into two lots. The
one was put into normal sea-water, the other was exposed for
fifty minutes to a mixture of 70 c.c. sea-water + 30 c.c. 2^n
MgCl2. At about the same time the sperm of one male was
distributed into two solutions of exactly the same character.

After fifty minutes the eggs that had been in normal sea-
water were divided into three portions. To the first portion
was added sperm from the normal sea- water; to the second
was added sperm that had been for fifty minutes in a mix-
ture of 30 c.c. 2^n MgCl2 + 70 c.c. sea-water. To the third
portion no sperm was added; it was intended to serve as
control material. The result was as striking as could be
desired. While the eggs to which the sperm from the nor-
mal sea-water had been added developed without exception
into trochophores, not one egg developed in lot 2, to which
the MgCl2 sperm had been added. The control eggs remained
likewise undeveloped.

A number of the unfertilized eggs that had been in the
MgCl3 for fifty minutes reached the trochophore stage.

This experiment proves conclusively that the MgCl2 solu-
tion annihilates or certainly diminishes the fertilizing power
of spermatozoa. In a previous series of experiments I had been
able to show that the same is true for the eggs of sea-urchins.

In addition I convinced myself through microscopic ex-
aminations that the females used were not hermaphroditic.

III. THE SPECIFIC EFFECT OF K IONS ON THE DEVELOPMENT
OF THE UNFERTILIZED EGGS OF CH^TOPTERUS

The preceding experiments seemed to indicate that KC1
has a specific effect upon the development of the unfertilized
eggs of Chsetopterus. Mead had already found1 that if -| per

i MEAD, Biological Lectures, Woods Hole, 1898 (Boston : Ginn & Co.).

AKTIFICIAL PARTHENOGENESIS IN ANNELIDS 657

cent. KC1 is added to sea-water the unfertilized eggs of
Chaetopterus throw out their polar bodies, while the addition
of \ per cent. Nad to sea- water produces no such effect.
It seemed of interest to find out whether the K ions were
possibly able to cause the parthenogenetic development of
Chaetopterus larvae without the osmotic pressure of the sea-
water being raised.

Ninth series. — The following mixtures were prepared:

(1) 10 c.c. 2Jn KC1 + 90 c.c. sea-water

(2) 5 " " +95

(3) 2J " " +98

(4) Normal sea-water (control)

The eggs remained in the solutions one hour. The sea-
water used had been sterilized by heating it to a temperature
of 80° C., as in all the previous experiments.

The next morning each of the first three lots contained a
large number of free swimming larvae, while the control
material contained none.

Tenth series. — I intended to find out the minimum amount
of KC1 necessary to bring about artificial parthenogenesis.
Moreover, I wished to know whether the addition of KC1 to
sea-water did not act more quickly upon the eggs than an
increase in the osmotic pressure by some other substance*
Seven solutions were used:

(1) J c.c. 2Jn KC1 +99i c.c. sea-water

(2) 1 " " +99

(3) 2 " " +98

(4) 10 " " +90

(5) 1 " NaCl+99

(6) 2 " « +98

(7) Normal sea-water (control)

One lot of eggs remained in these solutions from five to
ten minutes, the others from sixty to seventy minutes.

The results were as follows : None of the two lots that had

658 STUDIES IN GENERAL PHYSIOLOGY

been in solution 1 reached the trochophore stage. None of
the eggs that had been only five minutes in the second solu-
tion reached the trochophore stage. But the lot of eggs that
had remained one hour in the second solution yielded a small
number of swimming trochophores. The eggs that had
been in solutions 3 and 4 differed widely from the preceding
lots. They were teeming with swimming trochophores, those
that had been in these solutions five minutes as well as those
that had been in the solutions one hour.

The control eggs and the eggs of lots 5 and 6 did not
develop, although a number went through the first stages of
segmentation.

I had observed in my former experiments that the eggs
of sea-urchins can develop parthenogenetically if left per-
manently in sea-water whose concentration is raised but
little. If the eggs of sea-urchins are put for two hours into
a mixture of 92 c.c. sea-water + 8 c.c. 2^n NaCl, they will not
develop into blastulse when put back into normal sea-water;
but if left for some time or permanently in such a solution,
a small number of blastulse may be formed. A number of
the unfertilized Cha3topterus eggs were left permanently in
solutions 1-6. The next morning the eggs that had been
left in solution 2 (1 KC1 + 99 c.c. sea- water) had swimming
trochophores. The eggs in solution 1 did not reach the
trochophore stage. In the other solutions everything was dead.

Eleventh series. — This series was practically a repetition
of the preceding one, with the exception that the eggs
remained from twenty to thirty minutes in the solutions,
which were as follows-

(1) 1 c:c. 2Jn KC1+99 c.c. sea-water

(2) 1J " « + 98J

(3) 2 " " +98 ' "

(4) 10 " " +90

(5) Normal sea-water (control)

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 659

The eggs that had been in solution 1 had very few swim-
ming trochophores, those that had been in solutions 2 and 3
had many, and those that had been in solution 4 still more.
The control eggs were mostly undeveloped; a small number
were segmented into from 2 to 16 cells.

Twelfth series. — In the experiments thus far mentioned
a 2^n KC1 solution had been added to normal sea- water.
As the osmotic pressure of the sea- water is about equal to
that of a H n KC1 solution, in all these experiments with KC1
there was a rise in the osmotic pressure of the sea-water. I
now wished to try whether this increase in osmotic pressure
is essential for the KC1 effect, or whether a mere increase in
the number of K ions without an increase in the osmotic
pressure of the sea-water is able to bring about the par-
thenogenetic development of the eggs of Chsetopterus. The
solutions used were as follows:

(1) 2 c.c. 2%n KC1 +91 c.c. sea-water + 7 c.c. distilled water

(2)3 « " +86 " +11

(3) 2 " « +98

(4) 3 " " +97

(5) 5 " MgCl2-+95

(6) Normal sea-water (control)

The eggs remained in the solution fifty-five minutes.
Nine hours later (the same evening) swimming trochophores
were found in those that had been in the first four solutions.
The eggs that had been in the other two solutions were
entirely undeveloped. Some had been left permanently in
these solutions. Some of those left in solutions 1 and 2 had
reached the trochophore stage and were swimming about.

The next morning these results were confirmed. Fully
one-third of all the eggs that had been fifty-five minutes in
solutions 1-4 swam about as trochophores. Those that had
been in solutions 5 and 6 had not reached the trochophore
stage; only a few eggs had begun to segment.

660 STUDIES IN GENERAL PHYSIOLOGY

Thirteenth series. — It was evident that the KC1 brought
about artificial parthenogenesis, even if the osmotic pressure
of the sea-water was not raised. I now tried whether a pure
KC1 solution was able to cause artificial parthenogenesis,
and whether this was possible when the osmotic pressure of
such a solution was lower than that of sea-water. The solu-
tions used were as follows:

(1) 10 c.c. 2Jn KC1 + 90 c.c. distilled water

(2) 20 " " -f 80 " "

(3) 25 " " +75 " "

(4) 2 " " +98 sea-water

(5) Normal sea-water (control)

The osmotic pressure of solutions 1 and 2 was smaller
than that of normal sea-water. One portion was left thirteen,
the other fifty minutes in these solutions.

The next morning a large number of swimming trocho-
phores was found in every one of the dishes that contained
eggs taken from the first four solutions. The control solu-
tions were absolutely free from trochophores.

Fourteenth series. — The experiment was so surprising
that I wished to repeat it. The following solutions were
prepared :

(1) 10 c.c. 2Jw KC1 + 90 c.c. distilled water

(2) 2 c.c. " u + 98 c.c. sea-water

(3) Normal sea-water (control)

The eggs were left in the solutions fifty minutes. The
water was sterilized. The next morning the eggs that had
been in solutions 1 and 2 contained living larvae, while the
eggs in the normal. sea- water were undeveloped.

Fifteenth series. — I wished next to know whether the KC1
solution might be still more diluted without annihilating its
effect upon the unfertilized Chsetopterus eggs. Four solu-
tions were used:

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 661

(1) 5 c.c. 2%n KC1 + 95 c.c. distilled water

(2) 10 " " +90 " "

(3) 15 " " +85

(4) Normal sea-water (control)

The eggs were left in these solutions seven minutes and
were then put back into sterilized sea-water. The next
morning about 1 per cent, of the eggs that had been in
solution 1 were swimming about. The eggs that had been
in solution 2 had practically all reached the larva stage,
although not all of them were swimming. The eggs that
had been in the third solution contained swimming larvae,
but fewer than the other two lots. The control eggs had
remained absolutely unsegmented during the first nine to
ten hours. They showed, however, a beginning of segmen-
tation (2—3 cells) the next morning.

Sixteenth series. — There was no longer any doubt
concerning the fact that KC1 is able to bring about the
development of the unfertilized eggs of Chsetopterus. It
was, moreover, apparent from experiment 10 that if only
^c.c. 2^riKCl is added to 99^c.c. of sea- water, no trocho-
phores are formed. It was natural to conclude from this
that a certain minimal amount of K ions must enter the egg
in order to make it reach the trochophore stage. In order to
decide this the following experiment was tried. The eggs
of one female were put into a solution of 2 c.c. ^\n KC1 +
98 c.c sea- water, and put back into normal sea- water at
various intervals, viz., after one minute, three minutes, seven
minutes, nine minutes, thirteen minutes, twenty minutes,
forty minutes. The results of this experiment were as defi-
nite as could be desired.

After from nine to ten hours the eggs of the first lot (that
had been in the KC1 sea- water for one minute only) were
absolutely unsegmented. In the second lot (three minutes)
a few eggs were segmented, but no trochophore was formed.

662 STUDIES IN GENERAL PHYSIOLOGY

Lot 5 (thirteen minutes) had trochophores which did not
yet move, and in lot 6 (twenty minutes) and lot 7 (forty
minutes) trochophores were found that were just beginning
to move. The control eggs were absolutely unsegmented.

The next morning I found no trochophores in the first
lot (one minute), but many eggs in a two- to eight-cell stage.
In lot 2 (three minutes) about 1 per cent, of the eggs were
swimming about as trochophores. In lot 4 about 10 per
cent, of all the eggs were swimming about as trochophores ; in
lot 5 (13 minutes) it was about the same. In lot 6, whose
eggs had been for twenty minutes in the KC1 sea-water,
about 50 per cent, swam about in the trochophore stage.
Lot 7 seemed to contain not quite so many trochophores.

It is therefore necessary that the unfertilized eggs re-
main more than one minute in a mixture of 2 c.c. 2^ n KC1
-f- 98 c.c. sea- water in order to develop; three minutes (or
possibly a little less) is sufficient. This indicates clearly
that a certain quantity of K or KC1 must enter the egg in
order to bring about the development. This quantity is
very small. It seems to vary, however, for the individual
eggs, inasmuch as the number of eggs that developed was
greater the longer the eggs remained in the KC1 solution.
If they remain too long in such a solution, the KC1 acts like
a poison. From twenty to sixty minutes seems to be the
optimal time.

Seventeenth series. — I wished to determine once more
what was the smallest amount of KC1 that must be added to
sea-water in order to bring about artificial parthenogenesis
of the ChaBtopterus eggs. The following solutions were used:

(1) Jc.e. 2Jn KC1 + 99J c.c. sea-water

(2) 1 " " +99 "

(3) 1} " « +98J

(4) Normal sea -water (control)

The eggs were left in these solutions over night. The

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 663

next morning, after they had been in these solutions for
twenty-four hours, the first solution contained many eggs
in the beginning stages of segmentation but not one swim-
ming larva could be discovered. The second and third
solutions contained a large amount of swimming larvaB; in
the second they were more numerous than in the third. In
the control material only a few eggs began to segment ; no
swimming larvae were to be found. This confirms our former
observation that an addition of ^ c.c. 2^n KC1 to 99^ c.c.
sea-water is insufficient to produce parthenogenesis, while
the addition of 1 KC1 is sufficient.

Eighteenth series. — There is something paradoxical in
the fact that the addition of 2 c.c. 2^ n KC1 to 98 c.c. sea-
water can produce parthenogenesis in three minutes, while
the addition of ^c.c. 2^n KC1 to 99^ c.c. sea- water cannot
accomplish the same result in twenty-four hours. Before I
accepted this as a fact I wished to see it confirmed once
more. The same solutions were applied as before:

(1) \ c.c. 2J n KC1 + 99J c.c. sea-water

(2) 1 " " +99

(3) H • " " +98i

(4) Normal sea-water (control)

Part of the eggs were put back into normal (sterilized)
sea- water after thirty minutes, while the others remained in
the solution during the next twenty-four hours. As far as
the latter are concerned the results were exactly like those
described in the preceding experiment. The eggs that had
remained in solution 1 over night had not developed beyond
the early cleavage stages. No egg had reached the trocho-
phore stage. In the second solution a large number of swim-
ming Iarva3 were found, and in the third solution they were
numerous. About 75 per cent, of all the eggs were in the
trochophore stage, and many of these were swimming about.

The eggs that had remained in these solutions only thirty

664 STUDIES IN GENERAL PHYSIOLOGY

minutes showed the following condition the next morning:
Those that had been for thirty minutes in the first solution
had no trochophores ; only a few had begun to segment.
The eggs that had been taken out of solution 2 after thirty
minutes had formed many larvae, but fewer than the eggs
that had remained in the solution. Those that had been
taken out of solution 3 after thirty minutes had formed
many swimming larvse. The control eggs were undeveloped,
save a few that had begun to segment.

While a stay of thirty minutes in a mixture of 1 c.c. 2-|
KC1 + 99 c.c. sea- water suffices to cause the eggs to develop
parthenogenetically, a stay of thirty hours in a solution of
^ c.c. 2^w KC1 + 99^ c.c. sea-water remains without any
effect. This may mean that a minimal quantity of K or KC1
must enter the eggs in a certain minimal time or rather sud-
denly. It may, however, find a different explanation.

Nineteenth series. — Is the fertilizing power of the KC1
due to the K ions or to the KC1 molecules? The unfertil-
ized eggs of one female were distributed into the following
solutions :

(1) 1 c.c. 2^n KBr + 97 c.c. sea-water

(2) 2 " " +98 "

(3) 1 " KNOs+99

(4) 2 " " +98 "

(5) 3 1.2nK2SO4+97 "

(6) Normal sea-water (control)

The eggs remained in these solutions thirty minutes, and
were then put back into normal sea- water. The eggs that
had been put in solutions 2, 4, and 5 formed a large number
of swimming larvae, .the others remained undeveloped. This
experiment proves that the K ions and not the KC1 mole-
cules produce the parthenogenetic development of the eggs
of Chaetopterus.

Conclusions. — These experiments confirm the conclusion

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 665

drawn above, that the unfertilized eggs of Chaetopterus can-
not develop into a trochophore if left in normal sea-water.
A small number of K ions, however, is able to cause them
to develop parthenogenetically. If the eggs are put for
three minutes into a mixture of 2 c.c. 2|n KC1 -f 98 c.c. sea-
water, they are able to develop parthenogenetically. If the
sea-water contains fewer K ions, e. g., if we add 1 c.c. 2 ^n
KC1 to 99 c.c. sea- water, the eggs must remain longer in the
solution. Finally, if we add only ^ c.c. 2^n KC1 to 99 J c.c.
sea- water, the eggs are not able to develop parthenogeneti-
cally, no matter how long they are left in such a solution.
They can be caused to reach the trochophore stage by a pure
KC1 solution of considerably lower osmotic pressure than
that of sea-water. If the sea-water contained only a slightly
greater proportion of K, we should find that Chsetopterus
was "normally" parthenogenetic.

IV. ARTIFICIAL PARTHENOGENESIS PRODUCED BY A SLIGHT
ADDITION OF HCL TO SEA-WATER

In my experiments on Echinoderms, I had found that
the addition of a small quantity of acid or alkali causes the
unfertilized eggs of sea-urchins to segment much more
quickly than is the case in normal sea-water. I intended to
try the effects of the same agencies on the eggs of Chaetop-
terus. The sea-water is slightly alkaline, i. e., has a small
quantity of free hydroxl ions in solution.1 If we add more
alkali, the number of the hydroxyl ions is but slightly
increased, inasmuch as a precipitate of Mg(HO)3 is formed.
With acids it is different. If we add a certain small amount,
the sea- water becomes neutral; and if we add more, it
becomes acid according to the amount and degree of disso-
ciation of the acid used. All the sea-water in these experi-
ments was sterilized.

1 This was the common view held at that time. I have since found that sea-water
is neutral. [1903]

STUDIES IN GENERAL PHYSIOLOGY

Twentieth series. — The following solutions were used:

(1) 100 c.c. sea-water + 2 c.c. -fan NaHO

(2) 100 " +2 " KHO

(3) 96 + 4 \n Na2CO3

(4) 100 " +2 -iV^HCl

(5) 100 +3 " "

(6) Normal sea-water (control)

The eggs of one female were distributed in these solu-
tions. One portion of the eggs was taken out of these solu-
tions and put back into normal sea-water ; the others remained
permanently in these solutions. Twenty -four hours later
the results appeared to be as follows: Of the eggs that had
remained in the solutions for twenty-four hours, those in
solutions 2 and 4 had well-developed trochophores that swam
about. In solution 1 several eggs seemed to have developed,
but I was unable to find one swimming or with cilia. Those
in the other solutions were undeveloped. The eggs that had
been in solutions 2 and 4 for only five to ten minutes had a
few trochophores ; the others were undeveloped.

It is evident that KHO is more effective than NaHO, and
it is natural that the effect of the K ions should have been
added to the effect of the HO ions. But the fact is very
striking that the addition of a small amount of HC1 to the
sea-water caused the parthenogenetic development of the
Chsetopterus eggs.

Twenty -first series. — The unfertilized eggs of a Chsetop-
terus were distributed in the following solutions:

(1) 100 c.c. sea-water + 1 c.c. -fan HC1

(2) 100 "+2 " "

(3) 100 "4-3 " "

(4) Normal sea-water (control)

One portion of eggs remained in the solutions five minutes,
the others permanently. The eggs that were taken from the
solutions after five minutes were undeveloped, with the

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 667

exception of a few that had been in solution 2 and which
reached the trochophore stage. The eggs that remained
permanently in solutions 1 and 2 formed a large number of
swimming larvse. The eggs in solutions 3 and 4 were unde-
veloped and dead.

Twenty-second series. — It was evident that the addition
of 2 c.c. -^ n HC1 to 100 c.c. sea-water was able to cause
the development of the unfertilized eggs of -Chsetopterus,
especially if the eggs remained permanently in this solution.

1 intended to see how long the eggs must remain in such a
solution in order to reach the trochophore stage. Eggs
were put into such a solution and taken out in intervals of
ten, thirty, sixty, ninety, and one hundred and twenty min-
utes, respectively. One portion remained there permanently.
A large number of swimming larvae developed only in the
latter portion; in the former there were none. The control
eggs remained absolutely undeveloped.

Although these experiments are not yet finished, they
seem to indicate tthat in a solution of 100 c.c. sea-water -f-

2 c.c. ^n HC1 the unfertilized eggs of Chsetopterus can
reach the trochophore stage.

V. MORPHOLOGICAL OBSERVATIONS ON THE DEVELOPMENT
OF THE UNFERTILIZED EGGS OF CH^TOPTERUS

I have thus far confined myself to the statement that
certain solutions are capable of causing the unfertilized eggs
of Chsetopterus to reach the trochophore stage and swim
about. Nothing has been said as yet concerning the mode
of development of these parthenogenetic eggs. I have
watched their development very carefully and have made a
number of camera drawings. This part of the work is
essential for experiments on parthenogenesis. If one wishes
to be absolutely certain in regard to the parthenogenetic
character of the development, a close continuous observation

668 STUDIES IN GENERAL PHYSIOLOGY

and study of the eggs during the first seven to nine hours
is necessary. During this time the parthenogenetic eggs
throw out their polar bodies, segment, and become trocho-
phores, while the control eggs or the eggs treated with
ineffective solutions remain quite spherical and unchanged.

The egg of Chsetopterus is very
dark and opaque, and it is for this
reason much more difficult to deter-
mine the number of cleavage cells in
it than in the egg of most Echino-
derms. The fertilized egg of Cha3-
*~~/' N topterus develops very quickly. At
I la favorable temperature the cilia de-

^^/^-^ velop five hours after fertilization,
and the larvae begin to swim. The
^—^_ development of the unfertilized eggs
f ^\ differs in most cases from that of

( i the fertilized eggs. It is a little

V-**^ — / slower, and the nature of the seg-
mentation and the distinctness of the

single cleavage spheres vary considerably with the nature
of the ions that are added to the sea-water, or the agency
employed to bring about artificial parthenogenesis. If K
salts are used, one does not, as a rule, notice much more of
the beginning development, except that the eggs become
irregular in their outline and amoeboid. In the experiments
with Ca salts and acids, the cleavage spheres were much
more distinct and regular. Fig. 157 gives a good average
picture of the amoeboid character of the K eggs. In the
experiment in which these eggs were drawn the unfertilized
eggs of a Chsetopterus were put into a mixture of 98 c.c.
sea-water +.2 c.c. 2-Jw KC1 at 9:43. They remained in this
solution forty minutes, and were then put back into normal
sea- water. Three hours later, at 1:40, the drawing (Fig.

AKTIFICIAL PARTHENOGENESIS IN ANNELIDS 669

157) was made. It is impossible to recognize any distinct
cleavage spheres in these eggs. All that can be said is that
they have lost their spherical outline and are amoeboid. I
have never seen anything like this in fertilized eggs, or in
the unfertilized control eggs that are left in normal sea-water.
If the latter segment at all, they do not begin to do so until
after seven to nine hours or later, and they form more dis-
tinct cleavage cells.

The appearance of the eggs and the form of segmenta-
tion are thus distinctly a function of the constitution of the
sea-water. Inasmuch as the K eggs give rise to trocho-
phores which may look as normal as those developing from a
fertilized egg, it is evident that the appearance of the cleav-
age cells is of very little importance in the formation of the
embryo.

The difference between the development of unfertilized
K eggs and fertilized eggs can be seen from Fig. 158. In
the experiments in which these drawings were made the eggs
of one female were divided into two lots. The one was
fertilized at 11:45 by the addition of sperm; the other was
put at the same time for fifty-five minutes into a mixture of
98 c.c. sea-water -f- 2 c.c. 2^n KC1. In about fifteen to
twenty minutes after the eggs were put into this mixture
they threw out their polar bodies; sometimes one, sometimes
two were visible. This harmonizes with Mead's observations.
In the unfertilized control eggs that had remained in normal
sea-water nothing of this kind was noted.

Fifty-five minutes after the eggs had been put into the
KC1 mixture they were put back into normal (sterilized) sea-
water. In from ten to thirty minutes they began to lose their
spherical shape, and in some eggs little processes or knobs
appeared and remained or were withdrawn. The eggs
resembled amoebae in their behavior. In Fig. 158, on the
left side, the development of the fertilized lot is represented;

670 STUDIES IN GENERAL PHYSIOLOGY

Fertilized.

12.45

Unfertilized.

2255

120

1.30

210

3.40

3JS

4.30

7.40

FIG. 158

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 671

on the right side the development of the unfertilized eggs.
At about the same time a drawing of the fertilized and
the unfertilized K eggs was made. At 12:45 some of the
fertilized eggs were found in the four- to eight-cell stage.
The unfertilized eggs were only amoeboid at that time.
Some of them (see Fig. 158) at 12:55 showed an incision, as
if they were about to divide. At 1 : 20 some of the fertilized
eggs had reached the sixteen-cell stage, and at -1 : 30 only a few
eggs were found among the K eggs that seemed to be seg-
mented. At 2 : 10 the fertilized eggs were in an advanced
stage of cell-division, while the K eggs were not distinctly
segmented. At 3:40 the fertilized eggs had reached the
trochophore stage, with a clear edge and a dark center. At
that time the most differentiated eggs of the parthenogenetic
lot were in the condition that is represented at 3:15 in Fig.
158. At 4:30 we find these eggs still in the same condition,
and not until 7:40 did the parthenogenetic eggs reach the
beginning of the trochophore stage — clear edge and dark
center (Fig. 158). The fertilized eggs had formed their
cilia, and at about 5 o'clock were swimming around, while
the K eggs did not begin to swim until 8 or 9 o'clock. The
unfertilized control eggs which had remained in normal sea-
water during this time were at 8 o'clock still absolutely
spherical, and had given no signs of development of change.
Although the drawings in Fig. 158 give an idea of the
development of the parthenogenetic eggs, this idea has to be
supplemented by the statement that not all the eggs behaved
like those drawn. The majority of parthenogenetic eggs
never showed any higher degree of differentiation during
their development than those drawn in Fig. 157 ; many eggs
even remained spherical. The number of trochophores was
always considerably larger than the number of eggs that
became amoeboid. The majority of parthenogenetic trocho-
phores are perfectly spherical. I have often wondered

672

STUDIES IN GENERAL PHYSIOLOGY

whether it was possible for the unfertilized K eggs to reach
the trochophore stage without any visible external signs of
cleavage.1 I shall have to postpone a definite answer to
this question until next year.

Another point worth mentioning is the fact that phenomena
of cleavage seem to be reversible in this form, inasmuch as

an egg divides

Ointo two spheres
which very soon
fuse again. Such

8.04 8.WJ 805 8.05i changes> which

occur very sud-
denly, may be
occasionally ob-
served in unfertil-
ized Chsetopterus
eggs. Fig. 159
shows the succes-
sive stages which
were observed in
one egg within
four minutes. I
had watched the^ e

lively changes for several minutes before I decided to draw
them. The egg had been for an hour in a mixture of 95 c.c.
sea-water -f- 5 c.c. 2^n NaCl, and had been back in sea-water for
eight hours. When I began to draw the egg, it had the appear-
ance of being in the two-cell stage (Fig. 159, 8:04). Ten
seconds later it changed suddenly into a three-cell stage, the
upper sphere breaking into two cells (8 :04J). A few seconds
after this the lower sphere began to flow into the right upper
sphere (8:05), and at 8:05^ it had disappeared completely.
The egg was again in the two-cell stage (8:05J). Then the

i Professor F. Lillie in the following year confirmed this suggestion. [1903]

8.06

8.06*

808

FIG. 159

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 673

two spheres fused, and a small sphere or droplet appeared
above (8:05J). This disappeared almost immediately, and a
new little droplet broke loose at the right lower side of the
egg (8:06). It disappeared in a few seconds, and the egg
once more divided, but with an altogether different position
of the cleavage plane (8:06|, 8:07J). In a few seconds
the two spheres fused into one cell, and
a number of small droplets appeared
below (8:08). Of course it is impos-
sible to tell whether or not these single
spheres or droplets contained nuclei.

FIG. 160

These phenomena are of importance for

the mechanics of development, inasmuch as they show that the
bulk of the egg is liquid, and that in the case of Chsetopterus
its viscosity is very small, and less than in the case of the
sea-urchin's egg. It is hard to understand what kind of
structure could be preformed in a liquid mass of such low
degree of viscosity beyond the differentiation into nuclear
and protoplasmic material and possibly centrosomes.

The appearance of the trochophores originating from un-
fertilized eggs is exactly like that of those arising from fer-
tilized eggs, if one compares equal stages of development.
Fig. 158 gives no good idea of the trochophore, inasmuch
as the latter is at first spherical. Fig. 160 shows two
parthenogenetic trochophores, drawn by the camera with
the exception of the cilia, which are more or less diagram-
matic. The eggs from which these trochophores originated
had been treated with KC1. It is hardly necessary to men-
tion that the appearance of the trochophores developing
from parthenogenetic eggs depends greatly upon the treat-
ment the egg had received. I mentioned this point in
connection with the artificial parthenogenesis of sea-urchins.

A point which must be discussed is the duration of
life of the parthenogenetic trochophores. All the Cha3-

674 STUDIES IN GENEKAL PHYSIOLOGY

topterus larvae, those that developed from fertilized eggs as
well as those that developed from unfertilized eggs, died
after two days. As the fertilized eggs developed faster
than the unfertilized eggs, the trochophores that had devel-
oped from the former eggs were in a more advanced stage
at the time of death than the parthenogenetic trochophores.
But to judge from the energy of their motion, the vitality
of the parthenogenetic trochophores equaled that of the
trochophores emanating from fertilized eggs.1 The cause of
death was apparently the development of micro-organisms
in the poorly aerated culture dishes. The parthenogenetic
larvae of Arbacia lived, under similarly unfavorable con-
ditions, as long as ten days.

VI. ON THE EFFECT OF VARIOUS IONS ON THE ARTIFICIAL
PRODUCTION OF PARTHENOGENETIC GIANT AND DWARF
EMBRYOS IN ARBACIA AND CH^TOPTERUS

In a former paper on the artificial parthenogenesis of
sea-urchins I have mentioned the fact that as a rule more
than one embryo originates from one egg.2 It was not un-
usual to see 3, 4, or even 6 blastulse arise from one egg. Of
course each of these embryos was smaller than the normal
embryo of Arbacia in which the whole mass is utilized for
one embryo. In my first experiments I had caused the
parthenogenetic development of the eggs of Arbacia by
raising the osmotic pressure of the sea-water through the
addition of Mg013. I have since found that it depends
upon the nature of the substance which is added to the sea-
water whether the parthenogenetic larvae are dwarfs or of
normal size. If th'e unfertilized eggs of Arbacia are put

1 In the following year I found that the vitality of these parthenogenetic
larvae is considerably lower than that of the larvee which came from fertilized
eggs. [1903]

2 Part II, p. 576.

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 675

into sea-water whose osmotic pressure has been raised by the
addition of KC1 (e. g., 88 c.c. sea-water + 12 c.c. 2±n KC1),
and if after two hours they are put back into normal sea-
water, they will develop into swimming Iarva3. In this case,
as a rule, only one embryo develops from an egg, and dwarf
larvae are an exception. If, however, instead of KC1 the
corresponding quantity of NaCl or MgCl2 is added to the
sea- water, as a rule more than one embryo originates from
one egg, and larvaB of normal size are rare. I have not
made many experiments with CaCl3, but it seems to act
more like KC1 than like NaCl. In the experiments in which
the osmotic pressure of the sea-water was raised by cane-
sugar, dwarf blastulge were also observed.

I have already mentioned in an earlier paper that the
lack of a membrane favors the origin of more than one
embryo from the unfertilized egg. The fertilized egg has a
membrane which keeps the cleavage cells together. But if
the membrane be destroyed, the egg may give rise to more
than one embryo. In a small number of unfertilized eggs
the treatment with KC1 gives rise to a very thin film, which
may act as a membrane and prevent the cleavage cells from
becoming separated. But such a fine film is lacking in the
majority of eggs treated with KC1 (or CaCl2) in the right
proportions to produce parthenogenesis. And yet we do not
notice the falling apart of cleavage cells which in the case of
the NaCl eggs orMgC!2 eggs leads to the formation of more
than one embryo from an unfertilized sea-urchin's eggs.
The observation of the process of cleavage shows that the
treatment of the eggs with KC1 increases their power of
adhesion. The various cleavage cells of a K egg stick to-
gether, while after a treatment with NaCl the cleavage cells
adhere much less to one another and fall apart. The same
tendency is produced by the addition of MgClg to sea-water.
It is quite possible that the relative amount of the various

676 STUDIES IN GENERAL PHYSIOLOGY

ions influences the degree of agglutination in the cleavage
cells. Herbst has observed that in sea-water without Ca the
cleavage cells of fertilized eggs show a tendency to fall
apart.1

It was to be expected that if KC1 makes the cells of the
same egg stick together, it might also cause several eggs to
agglutinate. We know, from the experiments of Driesch2
and Morgan 3 on the eggs of sea-urchins and of Zur Strassen*
on the eggs of Ascaris, that if two eggs stick together they
may give rise to a single embryo of larger dimensions. I
have never observed giant embryos in the parthenogenetic
eggs of sea-urchins. But I have seen them in almost every
experiment in which the Chsetopterus eggs had been treated
with potassium. In such cases often two or more eggs would
stick together, and the result was either two or more trocho-
phores grown together or a single giant embryo of twice or
three times the mass of a normal trochophore. Of course
there were all kinds of transitions between the two extremes.
The formation of one giant embryo through the fusion of
two or more eggs is the more remarkable as the ChaBtopterus
eggs possess a membrane even in the unfertilized condition.
This membrane is evidently liquefied at the point of contact
of two eggs. This agglutination caused by K is not only
noticeable in unfertilized but also in fertilized eggs of Cha3-

oo

topterus. Fig. 161 shows a number of trochophores which
originated from agglutinating fertilized eggs of ChaBtop-
terus. All these and many other specimens of this kind
were found in a few drops of the culture taken out with a
pipette. I have tried to make camera drawings of the
various types that occurred. The embryos were eight hours
old, and began to move. No. 1 (Fig. 161) is a trochophore

1 HERBST, Archiv fiir Entwickelungsmechanik, Vol. IX (1900), p. 424.

2 DRIESCH, ibid., Vol. X (1900), p. 411.

3 MORGAN, ibid., Vol. II (1895), p. 65.

STRASSEN, ibid., Vol. VII (1898), p. 642.

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 677

developed from one egg ; No. 2 shows two trochophores which
are grown together but are otherwise independent. In No. 3
we notice the beginning of a common organization, inasmuch
as the clear peripheral areas (on the right side) are fused
together. In Nos. 4, 5, and 6 the clear areas are almost com-

FIG. 161

pletely fused together, and only the dark centers remain
separated. In No. 7 both eggs are fused completely and
form one giant embryo with one set of organs. Cases like
this are very frequent in the material treated with KCL
Nos. 8 and 9 are examples of the fusion of more than two
eggs. I have seen four eggs form one giant embryo with one
common dark center and one common clear area. Such mon-
sters swam, but usually died sooner than the single embryos.

678 STUDIES IN GENERAL PHYSIOLOGY

The fact that the fusion of two eggs into one giant embryo
occurs so much more readily in Chsetopterus than in Arbacia
may be due to the difference in the viscosity of the two eggs.

The formation of one giant embryo from two eggs in
Chsetopterus is so very interesting for the reason that the
Chsetopterus egg possesses a characteristic cell-lineage. We
must conclude from this that the cell-lineage is either a sec-
ondary element in the formation of the embryo or that the
earlier processes of differentiation in the Chsetopterus egg
are partly or wholly reversible (see section x).

I have made very few experiments with CaCl2, but in
these giant embryos were formed. Eggs that had been in a
solution of 90 c.c. sea- water -f~ 10 c.c. 5n CaCl2 for one hour
gave rise to a number of giant embryos. A sure way to
produce giant embryos in Chsetopterus is to put the unfertil-
ized eggs for about one hour into a mixture of 97 c.c. sea-
water +3 c.c. 2| n KC1.

I have occasionally, but very rarely, found that the fertil-
ized eggs of Chsetopterus show agglutination in normal sea-
water. The same phenomenon seems to occur in the eggs of
Ascaris, according to Zur Strassen.1

Dwarf embryos are rarely found in Chsetopterus. I have
found them in the experiments with HC1. Perhaps the
existence of a membrane prevents the unfertilized eggs of
Chsetopterus from forming dwarf embryos as easily as the
unfertilized eggs of the sea-urchins.

VII. ON DIFFERENCES BETWEEN THE ARTIFICIAL PARTHENO-
GENESIS OF ECHINODERMS AND CH^TOPTERUS AND THE
POSSIBILITY OF A HYBRIDIZATION BETWEEN THE TWO

It is impossible to hybridize Arbacia and Chsetopterus in
normal sea-water. I have tried a number of experiments
with negative results, as was to be expected. The negative

i ZUK STKASSEN, loc. cit.

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 679

result may be due to the impossibility of the spermatozoon
of the one species entering the egg of the second species, or
to the fact that the spermatozoon of Chaetopterus brings
about the development of the Chsetopterus egg by substances
which are ineffective in the Arbacia egg, and vice versa, or
the spermatozoon of the one species is poisonous for the egg
of the other species, or vice versa.1 The second possibility
is of interest to us on account of the fact that we can bring
about the parthenogenetic development of the Chsetopterus
eggs by means which have no effect upon the Arbacia egg.

When we intend to produce artificial parthenogenesis in
the eggs of Echinoderms, it is only necessary to put them for
from one and one-half to two hours in sea-water, the osmotic
pressure of which has been raised about 37^ to 75 per cent. ;
that is, into sea-water to which has been added 12^ to 25 per
cent, of its volume of a 2^n Nad solution or of a solution
isosmotic with the latter. We have not yet determined the
osmotic pressure of the sea-water at Woods Hole, and on
indirect data assume that it is about isosmotic with a f n
NaCl solution. The optimal increase of osmotic pressure
varies for different species and even for different females of
the same species. It may be that the temperature of the
water and the degree of maturity of the eggs play a role.
In making experiments of this kind, it is necessary to use
always a series of solutions of different osmotic pressure and
to take the eggs out at various intervals, from one-half to two
hours or more, until the optimum concentration and time
have been ascertained.

An increase in the osmotic pressure of the sea- water is
also able to cause artificial parthenogenesis in Chsetopterus.
The chief difference between the Chsetopterus and Arbacia
eggs is that at the same temperature the Chsetopterus eggs

i Certain constituents of the blood (globulins, enzymes?) frequently destroy the
blood corpuscles of other species that are not closely related.

680 STUDIES IN GENEKAL PHYSIOLOGY

do not need to stay so long in the more concentrated solution
as the eggs of Arbacia.

Although in this regard the difference between Chsetop-
terus and Arbacia is slight, a very striking difference exists
in regard to the specific effects of K ions upon the develop-
ment. While a pure KC1 solution of lower osmotic pressure
than sea- water, or sea-water with a slight increase of K, e.g.,
a mixture of 98 c.c. sea-water -|- 2 c.c. 2^n KC1, causes the
parthenogenetic development of the eggs of Chsetopte'rus that
have been exposed to such a solution only a few minutes,
such solutions are without any effect upon the unfertilized
eggs of sea-urchins (Arbacia). I left the unfertilized eggs
of Arbacia repeatedly in a mixture of 98 c.c. sea- water -f- 2
c.c. 2^n KC1 or 97 c.c. sea- water + 3 c.c. 2^n KC1 for from
three minutes to twenty-four hours without any develop-
ment following, with the exception of a few eggs that reached
the two-cell stage after about twenty hours. But this hap-
pens just as well in normal sea-water.

As far as the Arbacia eggs are concerned, I can only state
that if we increase the osmotic pressure of the sea- water by
adding KC1, a slightly smaller increase in the osmotic pres-
sure is required to bring about the parthenogenetic develop-
ment than if we add NaCl. I found regularly that while
90 c.c. sea- water -j- 10 c.c. 2^n KC1 sufficed to cause a great
many eggs to reach the blastula stage, a mixture of 90 c.c.
sea- water -f 10 c.c. 2^n NaCl was practically ineffective. I
had to take 87 \ c.c. sea-water -j- 12^ c.c. 2^n NaCl. It is,
however, possible, that this difference is only apparent. As
the sea- water consists chiefly of NaCl, the addition of 10 c.c.
of a 2^n NaCl to 90 c.c. sea-water will increase the osmotic
pressure of the sea-water less than the addition of 10 c.c. of
a 2Jn KC1 solution, as the degree of dissociation is less if
the concentration is higher. Further experiments with pure
NaCl and KC1 solutions will have to decide whether the dif-

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 681

fere nee in the degree of dissociation is responsible for the
result. A second typical difference between the Arbacia
egg and the Chsetopterus egg consists in the fact that the
latter can be caused to develop by a small addition of HC1
to sea- water. Any other inorganic acid would probably act
in the same way, as the addition of a small amount of Cl ions
has no such effect. This small addition of acid diminishes
or neutralizes the alkalinity of the sea-water, but I have
failed to test whether the latter is rendered acid.

The same treatment does not cause the Arbacia eggs to
develop beyond the two- or four-cell stage, even if they are
left in the solution for twenty-four hours. I have made a
number of new experiments this summer, but I have only
been able to confirm the experiments mentioned in a former
paper.1

I have pointed out that the experiments on artificial par-
thenogenesis force us to assume that the influence of the
spermatozoon upon the development and the transmission of
the qualities of the male depend upon different constituents
of the spermatozoon. On the basis of this assumption the
possibility of a successful hybridization between animals as
far apart as Worms and Echinoderms might be considered.
If we could cause the egg of Chsetopterus to develop by
treating it with KC1 and at the same time force the sperma-
tozoon of an Arbacia (or a similarly distant animal) to enter
into the egg, we might carry Echinoderm qualities into an
Annelid egg.2 But in all my attempts at thus crossing the
female Chsetopterus with the male Arbacia perfect trocho-
phores without Echinoderm characteristics resulted. Al-
though the problem may not be capable of solution in these
two forms, I think that the experiments on artificial par-

1 Part II, p. 576.

2 Provided the spermatozoon of the Echinoderm contains no poison for the
Annelid egg.

682 STUDIES IN GENERAL PHYSIOLOGY

thenogenesis will ultimately make hybridizations possible
which otherwise would be impossible. I intend to continue
these experiments.

VIII. PRELIMINARY EXPERIMENTS ON PHASCOLOSOMA, FUNDU-
LUS, GONIONEMUS, AND PODARKE

I will report briefly on experiments which I began but
was not able to finish, partly from lack of material and partly
from lack of time. My experiments on Phascolosoma were
carried further than the rest. I began with putting the
unfertilized eggs of this form in mixtures of 90 c.c. sea- water
-f- 10 c.c. 2Jn KC1 and leaving them in this solution from
thirty to one hundred and fifty minutes. I never saw an
egg reach the two-cell stage. Then stronger solutions were
tried, and now some of the eggs began to segment. When
the eggs were put into a mixture of about 30 c.c. 2^n KC1
-f 70 c.c. sea- water for about thirty minutes, they reached
a thirty- to sixty-cell stage. The appearance of the eggs
was so good that possibly in a continuation of these experi-
ments parthenogenetic Iarva3 will be produced. In these
experiments I received valuable advice from Dr. Gerould of
Dartmouth College, who is thoroughly familiar with the
biology and embryology of this form.

In Fundulus, a teleost fish, I succeeded in causing the
unfertilized eggs to reach the two-cell stage, but lack of
material prevented my carrying the experiments further.

In my experiments on Gonionemus, a Medusa, I was assisted
by Dr.Murbach, who was kind enough to select the females for
me. Dr. Murbach had observed that by putting these animals
into the dark they can at any time be caused to lay eggs.

My attempts (four experiments) to cause artificial par-
thenogenesis in these eggs have failed. All I was able to
accomplish was to force the eggs to become amoeboid and
creep about, but no segmentation occurred.

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 683

In Podarke, an Annelid, I succeeded in producing the
first segmentation in unfertilized eggs. I interrupted these
experiments to go on with experiments on Chsetopterus which
were much more promising.

IX. NATURAL AND ARTIFICIAL PARTHENOGENESIS

In a definite although very small number of animals each
egg possesses the quality to develop parthenogenetically.
Instances of this are to be found in the bees, social wasps,
Bombyx, Psyche, Daphnia, plant lice and others. In all
these animals the egg can be fertilized also by a spermato-
zoon. How does it happen that in these forms, although
fertilization may occur, the egg is, under certain conditions
at least, able to develop parthenogenetically? Our experi-
ments show, that if the constitution of the sea-water were
only slightly different, that is, if it contained a little more
K, Chsetopterus would have to be added to the list of nor-
mally parthenogenetic animals. What I stated in my pre-
liminary report is certainly true for Chsetopterus, namely,
that it is the constitution of the sea-water which prevents
many or certain forms from being "naturally" parthenoge-
netic. By reversing this statement we may say that in the
naturally parthenogenetic animals it may be due to the con-
stitution of the blood (or the sea-water?) that the egg can
develop without fertilization.

The bridge between the phenomena of natural and artifi-
cial parthenogenesis is formed by those animals in which
physical factors decide whether or not their eggs develop
parthenogenetically. In plant lice parthenogenesis is the
rule only as long as the temperature is high or the plant has
plenty of water. If we lower the temperature or let the
plant dry out, sexual reproduction occurs. The drying out
of the plant causes the tissues of the lice to lose water. The
factor, loss of water, makes the artificial parthenogenesis of

684 STUDIES IN GENERAL PHYSIOLOGY

Echinoderms and Chsetopterus possible. In plant lice the
effect is of the same kind, only in the opposite direction.

I have read somewhere the statement that Artemia salina
is parthenogenetic, while Branchipus is not. Branchipus is
a fresh-water Crustacean which, if raised in concentrated
salt solutions (salt lakes), becomes smaller and undergoes
some other changes. In that case it is called Artemia. If
Artemia is parthenogenetic while Branchipus is not, it would
mean that the unfertilized eggs of the Branchipus cannot
develop in fresh water, while they are able to develop in
solutions of much higher osmotic pressure. This would be
identical with our observation on the artificial parthenogenesis
of Echinoderms and Chsetopterus.

As I have mentioned in a former paper, O. Hertwig makes
the statement that the unfertilized eggs of a number of marine
animals which deposit their eggs in sea-water begin to de-
velop after a number of hours, but do not develop beyond the
first cleavage stages. Arbacia eggs reach the two-cell stage
in about twenty hours ; the egg of Chsetopterus may develop
as far as twelve or sixteen cells. According to Hertwig, not
only the eggs of Annelids and Echinoderms, but also those
of certain Crustaceans show this peculiarity. I have men-
tioned in a former paper the observation made by Janosik
that in the ovary of mammals occasionally eggs are found in
the process of cell-division. We shall make use of these
facts in the next section.

I finally wish to say a few words concerning experiments
published by Mr. Viguier of Africa, who maintains that the
eggs of Arbacia, Toxopneustes, and other sea-urchins are
naturally parthenogenetic.1 It would contradict neither my
experiments nor my views if his statement were correct, as
in all my papers I have assumed that these and many other
(if not all) eggs have a tendency to develop parthenogeneti-

i VIGUIER, Comptes rendus de I ' Academie des Sciences, Paris, July 2, 1900.

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 685

cally, and that it is only due to the constitution of the sea-
water (or blood?) if they do not do so under natural con-
ditions.1 It might be that the constitution of the sea-water
at Algiers differs from that of the rest of the world, and
allows the eggs of the sea-urchin to develop parthenogenetic-
ally. The experiments of Mr. Viguier are, however, not
of such a character as to make this probable. They are few
in number, and he seems to have omitted no possibility
which could further the contamination of his eggs by sper-
matozoa. He always handled males and females together,
and opened males and females in the same experiment. No
mention is made of a sterilization of his hands or instruments.
Whenever males and females are in the same dish there is
danger that the water may be full of spermatozoa, especially if
the material is fresh. The sperm sticks to the surface of the
females and it is absolutely impossible to avoid fertilization
of the eggs. To be sure, Viguier mentions a precaution he
took, but this precaution shows that he is not familiar with
the methods of sterilization or disinfection. He washed the
females off in filtered sea-water. As everybody knows, the
spermatozoa go through filter paper, and, in addition, sea-
water does not remove the spermatozoa from the surface of
the female, for the latter stick to solid bodies, as Dewitz has
proved. In order to avoid this source of infection I washed
the surface of the female several minutes in distilled water, or
under a powerful stream of fresh water which kills the sper-
matozoa. I have in my former papers given a description of
the precautions necessary in experiments on parthenogene-
sis. These were by no means exaggerated if one wished to
guard absolutely against contamination. I did not even
succeed in excluding contamination by spermatozoa in my first
Chaetopterus experiment (see p. 649), although my precautions
were vastly superior to those taken by Viguier.

i Part II, p. 539.

686 STUDIES IN GENERAL PHYSIOLOGY

Another surprising fact in Viguier's paper is that he does
not mention whether or not his unfertilized eggs had a
membrane. In my researches on Arbacia I have considered
the lack or presence of a membrane the most important
criterion for deciding whether the development of the eggs
is due to the entrance of a spermatozoon or to the osmotic or
chemical treatment they have received. The fertilized eggs
form a thick membrane, while the unfertilized eggs generally
have no membrane (unless treated with certain salts in exces-
sive quantities and for a long time). The cleavage of the
parthenogenetic egg that has no membrane differs so radi-
cally from that of the fertilized egg within a membrane, that
it must arouse the interest or surprise of any morphologist.
These differences are most noticeable during the first hours
of the development. As soon as the egg approaches the
blastula stage the membrane very often begins to disintegrate.
I do not think that any experienced observer would have
dared to publish the statement that the unfertilized eggs of
Arbacia reach the pluteus stage, without having convinced
himself that the "unfertilized" eggs had no membranes.1

Mr. Viguier makes the statement that he tried to repeat
my experiments but was not able to confirm them. This
does not surprise me, as he had not read my papers, and as
he did not even know how my solutions had been prepared.
My experiments have been repeated and confirmed by the
following authors: Dr. C. Herbst (Naples), Professor E. B.
Wilson (Columbia University), Dr. Hans Winkler (Tubin-
gen), and Dr. S. Prowazek (Prague), and partly by Professor
A. Giard (Paris). In addition they were repeated with
success by all the members of the class in physiology and
embryolgy at Woods Hole last summer. As far as the state-
ment is concerned that the unfertilized eggs of Arbacia or

i Viguier's paper has been criticised by A. GIARD, Comptes rendus de la Soci6t6
de Biologie, Vol. LII (1900), p. 76L

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 687

Strongylocentrotus are able to develop into plutei in normal
sea-water, I can say that this is most certainly not the case
at Woods Hole, in California (according to my own very
numerous observations), in Beaufort, N. C., and at Naples
and other places on the Mediterranean, that have been
visited by competent experimenters.

X. THE BEARING OF ARTIFICIAL PARTHENOGENESIS ON THE
THEORY OF FERTILIZATION AND OF LIFE PHENOMENA IN
GENERAL

The general opinion concerning the r6le of the sper-
matozoon in the process of fertilization is that it acts as a
stimulus, and that as such it starts the development of the
egg. This statement is certainly wrong for those eggs in
which we have been able to produce artificial partheno-
genesis. For these eggs, like many others, begin to segment
without any spermatozoon, if they are left long enough in normal
sea-water. The only difference between these and the fertil-
ized eggs is that the former begin to segment much later and
their development stops in the early segmentation stages (two
to sixteen cells at the most). The latter may be due to the
fact that the egg dies before it has time to develop further.

If we consider the fact that the eggs show at least a be-
ginning of a segmentation under "normal" conditions, the
act of fertilization assumes a different aspect. The sper-
matozoon can no longer be considered the cause or the stimu-
lus for the process of development, but merely an agency
which accelerates a process that is able to start without it,
only much more slowly. Substances that accelerate chemical
or physical processes which would occur without them are
called catalyzers (Ostwald). According to this definition we
may assume that the spermatozoon carries a catalytic sub-
stance into the egg, which accelerates the process that would
start anyhow, but much more slowly.

688 STUDIES IN GENEKAL PHYSIOLOGY

Through these facts and conceptions the phenomena of
artificial parthenogenesis assume a different aspect. It
would be wrong to say that the K ions are the stimulus that
causes the developmental process. They merely act as
catalyzers, accelerating a process that would otherwise
proceed too slowly. The loss of water on the part of the egg
cell must have a similar effect, but possibly a less direct one.
It may be that the loss of water alters the chemical processes
in the egg in such a way as to give rise to the formation of
a substance which acts catalytically.

Whether or not the catalytic substances introduced by
the spermatozoon are identical with those employed in my
experiments, I cannot say. I consider it probable that in
the case of Chsetopterus the natural fertilization is not
brought about by K ions, inasmuch as the normal develop-
ment does not show the characteristics of a treatment of the
eggs with K.

I have made a series of experiments with various enzymes
to bring about the development of the unfertilized eggs of
Arbacia, thus far without any results. The only enzyme
that caused the egg to segment at all was papain. But
I cannot be certain whether this was not due to some acci-
dental constituent of the enzyme preparation used. The
other enzymes were absolutely without effect. If we wish to
find the active principle in the spermatozoon, we must make
experiments in the direction of those begun by Winkler.1
This author used extracts of the spermatozoon, and found
that such extracts caused the eggs of sea-urchins to reach
the two- or four-cell stage. As such a result can be brought
about by slight alterations in the osmotic pressure or con-
stitution of the sea-water, and as such alterations occurred
in Winkler's experiment, I am not yet certain that these

i WINKLER, Nachrichten der kdniglichen Gesellschaft der Wissenschaften, GOt-
tingen, 1900.

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 689

results were actually due to the substances extracted from
the spermatozoon. But his experiments are certainly in the
right direction.

The idea that the spermatozoon and the substances which
cause parthenogenesis act only catalytically, has a great
bearing upon the theory of life phenomena. It means that
if we accelerate the processes of cell-division in the mature
egg (by specific catalyzers) the egg can live; but if these
processes occur too slowly at the ordinary temperature (as is
the case in the unfertilized egg in normal sea-water), the
egg dies. The introduction of the catalytic substances which
accelerate the processes of development saves the life of the
egg. This may be made intelligible on the following
assumption. Two kinds of processes are going on in the
mature egg after it has left the ovary. The one leads to the
formation of substances which kill the egg; the other leads
to the formation of substances which allow growth and cell-
division, and are not poisonous. We may use as an illustra-
tion Pasteur's well-known experiments on the behavior of
yeast cells in the presence and absence of atmospheric
oxygen. In the presence of oxygen the yeast cells multiply
on a sugar solution, while the zyma&e effect is comparatively
small. In the absence of oxygen the multiplication of cells
is limited or may stop, while the zymase effect becomes more
prominent. The products of alcoholic fermentation are
comparatively harmless for the yeast cell, and for this reason
an increase in the fermentative activity of the cell does not
cause the death of the yeast. I imagine that matters are
similar in the mature egg cell after it has left the ovary,
with this difference, perhaps, that the substances formed (by
fermentation?) in the egg cell are more poisonous for the
egg than the alcohol and the other products of fermentation
are for the yeast. The process that causes the death of the
egg cell and the one that causes cell-division are at least

690 STUDIES IN GENEKAL PHYSIOLOGY

partly antagonistic. They are both inhibited by a low tem-
perature, so that in this case death does not occur, although
no cell-division is possible. If we succeed in finding a sub-
stance which accelerates the process of cell-division at the
normal temperature, this will at the same time lead to a
suppression or a reduction of the antagonistic process that
shortens life. In the case of the egg of Chsetopterus a trace
of K ions acts as such a catalytic substance; possibly a trace
of H ions ; and perhaps certain substances that are formed
when the egg loses a certain amount of water. For the
Echinoderm egg we know at present only the last factor. In
addition there are the catalytic substances carried or pro-
duced by the spermatozoon (ions? enzymes?). But there are
certainly other catalytic substances, as is proved by tumors
and galls, in which the variety of structures corresponds to
an almost equal variety of parasites.1

It is very important to realize that the introduction of
catalytic substances into the egg does not prolong its life
unless the egg has reached a critical point determined by
two sets of conditions. The one is the maturity of the egg,
the other the change of conditions connected with the egg
leaving the ovary. As long as the egg is immature it lives
without the introduction of these substances or the sperma-
tozoon, and this may be true for the mature egg as long as
it remains in the ovary. The fact that there is an age limit
for the development of carcinoma may be a similar phenomenon.
The catalytic substances which are given off by the cancer para-
site may not be able to bring about cell-division in the epithelial
cells unless the latter have reached a critical point, which is
at least partly determined by the age of the individual.

i We do not need to assume a specific parasite for each kind of tumor. Tera-
tomas may be explained on the basis of the parthenogenetic tendency of the
mammalian egg in connection with some chemical change that furnishes the
catalytic substance. But it is not impossible that even in benign tumors, such as a
teratoma, the catalytic substance may be due to parasitic organisms.

ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 691

We generally consider development as a process which
can only occur in one direction, or, in other words, is irre-
versible. But this is certainly not generally the case. I
showed in a recent paper that the morphogenetic processes
in Hydroids are reversible. If the polyp of a Campanularia
is brought in contact with a solid body, it is transformed into
undifferentiated material and later into a stolon. If the
same organ is brought in contact with sea-water, it gives rise
to a polyp again.1 The same may be done with Margelis
and other Hydroids. In Antennularia a change in the
orientation of a branch with polyps will bring about the
transformation of this material into a stolon. Between the
two phases the material must pass through an undifferentiated
stage where it is neither polyp nor stolon. It will be the
task to determine how far in the animal kingdom the develop-
mental processes are found to be reversible. It is obvious
that in a form with a reversible development death will not
necessarily follow a certain stage of development (corre-
sponding to senility in man).

It is not impossible that "natural" death is comparable
to the situation which is present in the mature egg after it
leaves the ovary. Nature has shown us the way by which
at this critical point death can be avoided in the case of the

i Part II, p. 627.

XXXIV

ON AN APPARENTLY NEW FORM OF ABNORMAL IRRI-
TABILITY (CONTACT- IRRITABILITY?) PRODUCED
BY SOLUTIONS OF SALTS (PREFERABLY SODIUM
SALTS) WHOSE ANIONS ARE LIABLE TO FORM
INSOLUBLE CALCIUM COMPOUNDS1

I. INTRODUCTION

A SERIES of papers published from my laboratory has
furnished the proof that the rhythmical contractions of
striped muscles, the swimming bell of jelly-fish, the heart
and the lymph hearts depend upon the presence of Na ions
in the surrounding solution. Calcium ions have a tendency
to diminish or inhibit the contractions altogether, although
a small number of them must exist in the tissues in order to
preserve contractility.2 This point having been settled, I
next tried whether the sodium ions bring about these effects
directly or indirectly. I have not finished these researches
so far as the rhythmical contractions of the muscle are con-
cerned, but in pursuing this problem I have found a number
of facts which show that certain salts can bring about effects
indirectly by giving the muscle or nerve properties which
they do not possess normally and which to my knowledge
have not yet been described. If we put a fresh muscle
(gastrocnemius) of a frog for a short time (e. </., one to three
minutes) into a solution of a sodium salt whose anion is liable
to form insoluble calcium compounds (e. g., NaFl, Na2CO3,
Na2HPO4, sodium pxalate, sodium citrate, etc.), the muscle
will as a rule not show any reaction except perhaps a slight

1 American Journal of Physiology, Vol. V (1901), p. 362.

2 It is possible that certain other ions may act as a substitute for the Ca ions for
this purpose.

692

ABNORMAL IRRITABILITY PRODUCED BY SALTS 693

shortening. But as soon as it is taken out of the solution
and comes in contact with air, it goes into tetanus or per-
forms a series of powerful contractions. The tetanus or the
contractions cease at once and relaxation of the muscle
occurs when the muscle is put back into the solution.

It was found that not only the change of contact from
the above-mentioned solutions to air but
also to a number of other media produce
these contractions. What the nature of
the stimulus in this case is I cannot say
definitely. Provisionally I will assume
that we are dealing with contact-irrita-
bility and I will call the above-mentioned
reaction of the muscle the contact-reac-
tion. It would seem as though the en-
trance of the anions of the above-men-
tioned solutions caused a change in the
superficial layer of the muscle or its
individual fibers, either by precipitating
calcium or by otherwise altering the con-
stitution of the protoplasm. This change
is intensified by the increase in Na ions
in the same layer. In this condition the muscle is sensitive
to the nature of the substance with which it comes in contact.

In these experiments one end of the gastrocnemius of a
frog is tied to a glass rod, Gr (Fig. 162), and the other end
is tied to the lever, L. A dish, D, containing the solution
is raised from below when we wish to submerge the muscle,
and is lowered when we wish to bring the muscle into con-
tact with air.

In order to demonstrate the contact-irritability I used a
solution of 1 gram-molecule of sodium fluoride or sodium
citrate, etc., in about 8 or 10 liters. If the fresh gastrocne-
mius of a frog be put into such a solution for about one

FIG. 162

694 STUDIES IN GENERAL PHYSIOLOGY

minute, the muscle will show a slight contraction when
taken out of the solution. If the process be repeated, a
stronger contraction will follow when the muscle is removed,
and after a series of submersions have occurred the muscle
will give one or a series of powerful contractions every time
it is taken out of the solution and brought into contact with
air. After a certain time, which may be an hour or more,
and which varies according to the solution, the reaction
becomes weaker and finally ceases.

If we use a stronger concentration than 1 gram-molecule
in 8 liters, we get more powerful contractions, but the irri-
tability of the muscle disappears sooner.

II. THE NATURE OF THE SOLUTIONS WHICH PRODUCE CONTACT-
IRRITABILITY IN MUSCLE

Solutions of cane-sugar or urea were unable to produce
the contact-reaction in muscle. I have tried these solutions
in all concentrations from 0 to normal or even 2n. A large
number of electrolytes were then tested. None of the salts
of Li, K, Ca, Mg, and NH4 gave rise to the contact-reaction.
This statement is based upon experiments with LiCl, Li2SO4,
Li2C03, KC1, K citrate, K oxalate, MgCl3, MgSO4, NH4C1,
(NH4)2CO3, and ammonium citrate. The degree of dilu-
tion used was as a rule 1 gram-molecule in about 8 or 10
liters. In some instances stronger solutions were tried, but
with the same negative result.1

In my experiments on rhythmical contractions I have
shown that the sodium ions have a specific role in the pro-
duction of these contractions. It seemed also possible that
they play such a role in the production of the contact-irrita-
bility. But I found that f or even stronger solutions of
NaCl, NaBr, Nal, NaNO3 did not bring about the contact-

i Zoethout showed later in my laboratory that the addition of a trace of potas-
sium citrate to the sodium-citrate solution facilitates the production of contact-
irritability. [1903]

ABNORMAL IRRITABILITY PRODUCED BY SALTS 695

irritability ; neither did sodium acetate nor other salts whose
anions form soluble calcium compounds.

But the sodium salts whose anions precipitate calcium
promptly produce these reactions. NaFl, Na 3 CO s , Na 2 HPO 4
sodium oxalate, sodium citrate,1 sodium tartrate give the
contact reaction in a dilution of 1 gram-molecule in 8 or 10
liters of water or even less. NaHCO3 gives the reaction but
requires a higher concentration, e. g., 1 gram-molecule in 4
to 5 liters of water. If we put the muscle into a solution of
Na3PO4, it goes at once into a powerful tetanus. This
tetanus may be partly or wholly due to the high concentra-
tion of HO ions in this solution. When a muscle goes into
tetanus in a solution, we cannot, as a rule, demonstrate the
contact-reaction. Thus I have never succeeded in producing
contact-reaction by a Na3PO4 solution. NaH2PO4 does
not cause contact -irritability, but this is in harmony with
our general result.

The HO and H ions deserve special attention. In my
experiments on rhythmical contractions I found that while
they are not able to produce rhythmical contractions directly,
they accelerate the beginning of these contractions in the
presence of Na ions. In addition to such a catalytic action
common to both the HO and H ions, the former have another
effect which they do not share with the H ions. The muscle
produces constantly H3CO3 and possibly other acids. These
acids will increase the solubility of Ca salts and increase
the number of Ca ions in the tissues. An addition of HO ions
will counteract this effect.

It is due to the presence of free HO ions that solutions
of Na valerianate and Na formate give rise to a slight degree
of contact -irritability in muscle, although calcium formate
and calcium valerianate are soluble. If we diminish the

iThe citrates require an alkaline reaction for the precipitation of calcium
This condition is fulfilled in the fresh normal muscle.

696 STUDIES IN GENERAL PHYSIOLOGY

alkalinity of a sodium formate and sodium valerianate solu-
tion by adding a small amount of free formic or valerianic
acid (without, however, rendering the solution entirely
neutral) they no longer produce the contact-irritability in
muscle. A small amount of alkali added to a NaCl solu-
tion may or may not produce a slight degree of contact-
irritability.

The solubility of CaSO4 is comparatively high, and we
therefore cannot expect Na2SO4 to be very effective for the
production of contact-irritability. In solutions of 1 gram-
molecule Na2SO4 in 10 liters or less, I sometimes got and
sometimes failed to get the contact-reaction. May it not be
possible that the amount of free Ca ions in the muscle of
a frog varies at different periods of the year, and may not
this fact account for the seasonal variation in the irritability
of these animals? But if a Na2SO4 solution fail to produce
contact-irritability in a muscle an addition of some HO ions
will produced the desired effect. As a rule 4 c.c. -f- LiHO
or any other hydrate to 100 c.c. of the Na2SO4 solution is
the optimum. We can produce the contact-reaction also
through the addition of a small amount of acid to the
Na2SO4 solution, e. g., 4 c.c. of ^ HNO3 (or any other in-
organic acid) to 100 c.c. of the Na2SO4 solution. The effects
are not so strong as if we add alkali.

The sulphates showed an exceptional behavior in still
another direction. With one exception only sodium salts
give rise to contact-irritability and this exception is a
sulphate, namely (NH4)2SO4. It would almost seem that
the sulphates have physiological effects aside from their
effect upon calcium.. This is in harmony with Miss Moore's
experiments, in which she found that sulphates are as cap-
able of antagonizing the poisonous effects of a pure NaCl
solution as calcium salts.1

1 MOOKE, American Journal of Physiology, Vol. V (1901), p. 87.

ABNORMAL IRRITABILITY PRODUCED BY SALTS 697

It should finally be mentioned that sodium butyrate,
sodium succinate and sodium asparaginate did not produce
the contact-reaction.

Having thus proved that sodium salts, whose anions
precipitate calcium give rise to contact-irritability, it was to
be expected that solutions of calcium salts would prevent or
antagonize the contact-reaction. I found that by adding a
small amount of CaCl3 to a Na-citrate solution .the latter solu-
tion no longer produced the contact-reaction. The addition
of 1 c.c. of a 5n CaCl2 solution to 100 c.c. of an effective
sodium-citrate solution was sufficient to cause a muscle to
lose its contact-irritability at once. Only after a prolonged
stay in pure sodium-citrate solution does the contact-irrita-
bility return.

While all the facts thus seem to harmonize with the view
that a decrease in the amount of Ca ions in the tissues (and
possibly an increase in the amount of Na ions) is the essen-
tial condition for the production of the contact-reaction, it is
yet possible that the sodium salts whose anions form insoluble
calcium compounds may have a specific effect upon other
constituents of the protoplasm, e. g., proteids.

III. ON THE NATURE OF THE APPARENT CONTACT -REACTION

The reaction which we have provisionally called the con-
tact-reaction appears when a muscle, after having been sub-
merged in a sodium-citrate or any of the other above-
mentioned effective solutions, is brought into contact with air.
In this change from solution to air a number of conditions
change and it is now our task to determine which is the
essential one.

As soon as the muscle is taken out of the solution and
brought into air, more O2 may diffuse into and more CO3 may
diffuse from the muscle. These two conditions have, how-
ever, nothing to do with the reaction. The experiments

698 STUDIES IN GENERAL PHYSIOLOGY

were repeated in an almost pure atmosphere of CO2
instead of air and the contact-reaction was as powerful
as in air.

A second change is the sudden evaporation of water from
the surface of the muscle upon its leaving the solution.
The following experiment might suggest that this evapora-
tion is the cause of the contact-reaction. If we pack a
muscle, that gives powerful contact contractions, tightly in
moist filter paper the reaction will not occur when the muscle
is taken out of the solution, but will occur when the filter
paper is removed. Nevertheless, evaporation has nothing to
do with the reaction. We get the contact-reaction quite as
well in a moist chamber as in dry air. Furthermore we get
the reaction if we bring the muscle directly from the sodium-
citrate or fluoride solution into oil, without exposing it to
air. We can make this experiment in the following way.
The lower half of the dish, D (Fig. 162), is filled with the
effective sodium-citrate solution, the upper half with oil
(I used sperm and olive oil). The muscle is first brought
into the sodium-citrate solution and then, by lowering the
support S, into the oil. Powerful contractions occur. Evap-
oration of water from the surface of the muscle is, there-
fore, not the cause of the contractions.

After this had been established it was to be expected that
changes in temperature were not responsible for the contact
reaction. Experiments in which the muscle was rapidly
cooled and heated yielded only negative results.

The next possible cause to be considered was electricity.
The fact that a change from the salt solution to a non-con-
ductor (air, oil) caused contractions suggested the possibility
that these contractions were in reality electrical break con-
tractions, the muscle itself acting as a battery. The only
fact which did not seem to accord with this explanation was
the lack of a make contraction when the muscle was put into

ABNOKMAL IRRITABILITY PRODUCED BY SALTS 699

the solution. A number of experiments excluded the assump-
tion that the contraction or tetanus of the muscle which
occurs when it leaves the sodium-citrate solution is due to a
break shock. I connected the two opposite ends of the
muscle by means of a thick copper wire. In this case the
muscle contracted just as powerfully as before when taken
out of the sodium-citrate solution, although no break shock
of any strength was possible. Another still -more decisive
fact was found. After the muscle had been treated for some
time with a sodium-citrate solution the break contraction
could be produced by dipping the muscle for a short time,
e. g., thirty seconds, into a ^ or ^ solution of cane-sugar.
As soon as the muscle was brought into contact with air,,
contractions occurred. The same was true for glycerin solu-
tions. Both the sugar and the glycerin solution are non-
conductors. The possibility of a mechanical stimulation as
the cause of the contact-reaction was next to be considered.
As long as the muscle is in the solution each of its elements
is under the hydrostatic pressure of the column of liquid
above it. If we expose the muscle to the air this pressure
ceases. This might suggest the idea that a decrease of the
hydrostatic pressure upon the muscle causes its contraction.
The dipping of the muscle into the solution causes a relaxa-
tion of the concentrated muscle, and the inference should be
drawn that an increase of the hydrostatic pressure causes
relaxation. The following experiments prove the erroneous-
ness of this view. The bottom of the dish was filled with
a liquid of much higher specific gravity than the sodium-
citrate solution, e. </., with chloroform, 2n cane-sugar solution,
or metallic mercury, and the sodium-citrate solution was put
carefully above the sugar solution or chloroform. The
muscle was then brought from the sodium-citrate solution
into the sugar solution by raising the dish D (Fig. 162). In
this case I noticed regularly one or more powerful contrac-

700 STUDIES IN GENERAL PHYSIOLOGY

tions, although the hydrostatic pressure on the surface of the
muscle was increased.

It thus seems to me that none of the known forms of
muscular irritability suffices to explain the phenomena
with which we are dealing. We have before us an appar-
ently new form of muscular irritability, probably contact-
irritability.

Contact- irritability is a very general form of irritability
among plants and lower animals. I need only to remind the
reader of the phenomena of stereotropism and of the fact
that by mere contact-effects a polyp of a campanularia can
be transformed into a stolon. But contact-irritability cer-
tainly exists among certain cells of vertebrates, for example,
the leucocytes. The nature of the body with which leuco-
cytes come into contact determines whether or not they give
off fibrin ferment and cause coagulation of the blood or other
liquids which contain fibrinogen. How the nature of the
contact can influence the leucocytes is still a mystery. One
might think of surface tension phenomena or the formation
of double electric layers at the surfaces in contact.

If the phenomena described in this paper were really con-
tact-phenomena, a further search should reveal that only a
change of contact from certain bodies to other bodies can
cause contractions of the muscle.

I have begun experiments in this direction, and have thus
far found the following facts:

Contractions occur when the muscle passes:

f Sodium-citrate solutions

Sodium-fluoride solutions
From •{ Sodium -oxalate solutions To
Sodium-carbonate solutions
etc. (see above)

Air

CO2

Oil

2n sugar solution

Glycerin

Chloroform

Toluol

ABNOKMAL IRRITABILITY PRODUCED BY SALTS 701

Relaxation of the contracted muscle will occur when the
muscle passes from any medium in the right column above
to any medium in the left column.

After the muscle has been treated for some time with any
of the efficient solutions (Na citrate, etc.) the contractions are
also produced when the muscle passes:

From f or | sugar solution to air
From g or | glycerin to air
From any salt solution to air

A very interesting and theoretically important fact is that
the muscle loses this particular form of irritability very soon
when it remains in contact with the air, oil, sugar solution,
glycerin, or salt solutions different from those that produce
this specific irritability. In LiCl or NaCl solutions the
contact-irritability is lost as fast as, if not faster than, in a
sugar or glycerin solution. We can re-establish the irrita-
bility, however, if we put the muscle back into the sodium-
citrate solution for some time. This fact, together with those
mentioned before, suggests the following as the most prob-
able explanation of the peculiar phenomena of contraction
with which we have been dealing : the solutions which pro-
duce the contact-irritability possess anions that are liable to
form insoluble calcium compounds. They are all with one
exception — (NHJ 2SO4 — Na salts. Whatever the effects
of these anions may be, the fact that in less than a minute
the contact effects are noticeable indicates that only the sur-
face layer of the muscle or, what is less probable, the surface
layer of each individual fiber, is altered. It is impossible for
the anions to migrate deeper into the muscle in so short a
time. In the surface layer of the muscle or the individual
fibers we have temporarily a diminution of Ca ions. We
have, then, a muscle, whose surface layer differs from that of
an ordinary excised muscle. If this layer is once established
the muscle contracts at any change from the media of the

702 STUDIES IN GENERAL PHYSIOLOGY

left column of the above list to those of the right column.
But it is obvious too that as soon as this change occurs the
surface layer gradually undergoes an alteration, for example,
in air, sugar solution, NaCl solution, etc. This change, in
which the contact-irritability is lost, occurs most rapidly in
a CaCl2 solution. This suggests the following possibility.
The loss of contact-irritability of the muscle in air or oil,
etc., is due to the migration of Ca ions from the interior of
the fiber or the muscle to the surface, thus re-establishing
approximately the original normal surface condition. If we
then put the muscle back for a short time into a sodium-
citrate or sodium-fluoride, etc., solution, a diminution of
Ca ions will again occur in the surface layers and the con-
tact-irritability will be re-established, As is to be expected
the time the muscle remains in the solution is as important
as the concentration of the solution. If we dip a muscle for
a few seconds only into a sodium-citrate solution (1 gram-
molecule in 10 liters) the contact-irritability cannot be pro-
duced, as there is not time for a large enough number of
anions to diffuse into the muscle.

Still another fact harmonizes with our assumption. If we
lift only a piece of the muscle out of the sodium-citrate
solution, not the whole muscle contracts, but only the in-
dividual fibers that come in contact with the air. Similarly
a more powerful contraction occurs when we lift the thick
femur end of the gastrocnemius out of the solution than if
we expose the thin tendon-Achilles end to the air.

Finally it should be mentioned that the latent period is
somewhat long in these experiments. I have not measured
it yet exactly; but it may be a considerable fraction of a
second, especially when the contact-irritability is about to
disappear. This somewhat long latent period would harmo-
nize well with the assumption of contact-phenomena.

Although I have spoken chiefly of the diminution of

ABNORMAL IRRITABILITY PRODUCED BY SALTS 703

Ca ions as the effect of the sodium-fluoride and similar
solutions, I wish to state that I consider it possible that
solution may have other effects which play a r6le in these
phenomena.

IV. THE EFFECTS OF SODIUM FLUORIDE AND CORRESPONDING
SOLUTIONS UPON THE NERVE

If we try the experiments described above on curarized
muscles we get little or no result. This would indicate that
the contact-reaction is not due to an effect of these solutions
upon the muscle but upon the nerve elements in the muscle.
There is a second possibility, namely that curare, although
it does not abolish the electrical irritability of muscle, may
yet alter its substance enough to prevent the effects of con-
tact stimuli, or prevent the formation of the hypothetical
surface layer.

It may be said with certainty that sodium-fluoride, sodium-
citrate, and the corresponding solutions act upon the nerve
in a way altogether different from that in which they act
npon muscle. If we put the nerve alone (without the muscle)
into one of these solutions which contains 1 gram-molecule
in about ten liters, as a rule nothing will happen during
the first five minutes. The removal of the nerve from the
solution will not call forth a contraction of the muscle. After
about five minutes the muscle will begin to twitch rhythmic-
ally, and very soon the muscle will shorten steadily until it
reaches a high degree of tetanic contraction. This twitching
continues as long as the nerve is in the solution. As soon
as the nerve is taken out of the solution and exposed to the
air the muscle relaxes more or less completely, and the
twitchings become less numerous. As soon as the nerve is
put back into the sodium- citrate solution the contraction in-
creases again and the twitchings become more powerful.
This may be repeated very often. It is obvious that the

704 STUDIES IN GENEEAL PHYSIOLOGY

nerve behaves in exactly the opposite way from the muscle.
The latter contracts when taken out of the solution and ex-
posed to the air, and relaxes when put back into the solution.
If the nerve alone (without the muscle) be put into the
solution, contractions of the muscles occur while the nerve is
in the solution, and partial or complete relaxation is observed
when the nerve is taken out.

These experiments on the nerve give one the impression
that the sodium-citrate solution and the solutions of the
other sodium salts whose anions precipitate calcium stimu-
late the nerve chemically. Albert Mathews has recently
found that weak solutions of sodium salts can cause con-
tractions of the muscle when the nerve alone is put into the
solution, while the salts of the other metals can only produce
contractions when their osmotic pressure is considerably
higher than that of the tissues. I have confined my experi-
ments chiefly to those sodium salts whose anions precipitate
calcium. But I think I can show definitely that these salts
are not the direct stimulus that calls forth the contractions
of the muscle, but play only an indirect r6le, inasmuch as
they make the nerve more sensitive for another kind of
stimulus, either a mechanical- or a contact-stimulus. When
the nerve alone has been put into a sodium-citrate solution
(of 1 gram-molecule in about 10 liters) and the muscle has
begun to contract powerfully, a gradual relaxation of the
muscle is observed when the nerve is taken out of the solu-
tion and allowed to hang in the air. But at any time the
contractions and the final tetanus of the muscle will begin
again when the nerve is brought into contact with any solid
or liquid body, no matter whether it is a conductor or a non-
conductor. As soon as the contact ceases and the nerve is
surrounded by air again on all sides the muscle gradually
relaxes. This can be repeated quite often with the same
result. Among; the substances whose contact causes con-

ABNORMAL IRRITABILITY PRODUCED BY SALTS 705

traction I may mention hard rubber, glass, filter paper, var-
nished and unvarnished wood, bone, muscle, all. kinds of
metals. Among the liquids tried were oil, glycerin, sugar
solutions and several salt solutions. It is thus obvious that
in a sodium-citrate solution two influences are united, first
the effects of the citrate ion which causes a modification or
an increase in the irritability of the nerve, and second, the
liquid character of the solution. The latter is the direct
cause for the contraction.

Another point is of interest in this connection. The
sodium-citrate or sodium-fluoride solution increases the elec-
trical irritability of the nerve so that it can easily be stimu-i
lated by its own current of demarkation. This increase
occurs regularly before the twitchings of the muscle begin.

In my experiments on artificial parthenogenesis in Chse-
topterus I found that there are two ways by which the
unfertilized egg can be caused to develop — first, by certain
ions (K, H), and second, by causing the egg to lose water.
It follows from the facts of dissociation that a loss of water
on the part of the egg must alter the proportion of ions in
the egg. It thus becomes possible that the artificial par-
thenogenesis produced by the loss of water is in reality an
ion effect. In regard to the twitchings caused by putting
the nerve into solution Mathews has shown that two cases-
must be distinguished — first, the effect of specific ions, and
second, the effect of loss of water. Any solution whose
osmotic pressure is high enough can cause contractions if
the nerve be put into it. Is it not possible that the loss of
water in the nerve acts in the same way as the citrate or
fluoride ions ? The limited solubility of CaSO4 would make
this possible. I tried whether a nerve after having been
put into a 2n sugar solution long enough to cause muscular
contractions would show the above-mentioned mechanical or
contact-irritability. This was indeed the case. If such a

706 STUDIES IN GENERAL PHYSIOLOGY

nerve is taken out of the sugar solution and brought into
contact with solid bodies, it gives rise to stronger contrac-
tions. But, as was to be expected, the nerve loses this irri-
tability again when put into £ NaCl or Na citrate solution.
In such a solution water will enter the muscle and restore
the original condition, and only later will the entrance of
citrate ions show its effect.

It now remains to be seen how far these facts can throw
light upon the heart-beat. The fact that a heart which has
ceased to beat in a solution often begins to beat again when
taken out of the solution reminds us of the contact-reaction
of muscle described above.

V. SUMMARY

1. Certain salt solutions (1 gram-molecule in 8 or 10
liters) bring about an apparently new form of irritability in
muscles, which may be called provisionally contact-irrita-
bility. A muscle that has been treated in this way will
contract powerfully when it passes from the salt solution to
air, CO2, oil, sugar solution, etc., or from glycerin solutions,
sugar solutions to air.

2. The salts whose solutions produce this form of irrita-
bility are (with one exception) sodium salts, whose anions
are liable to precipitate calcium,. namely:

Sodium fluoride Na2HPO4 Sodium citrate

Sodium carbonate Sodium oxalate Sodium tartrate

3. If the nerve alone (without the muscle) be put into
one of these salt solutions (1 gram-molecule in 8 or 10 liters),
the muscle begins to twitch in about five minutes and finally
goes into tetanus. If the nerve be taken out of the solu-
tions, the contractions cease. Although this seems to indi-
cate that the salts or their ions stimulate the nerve directly,
it can be shown that they only modify or increase the irrita-
bility of the nerve. For when the same nerve is brought

ABNORMAL IRRITABILITY PRODUCED BY SALTS 707

into contact with any solid or liquid body (conductor or non-
conductor) the contractions of the muscle will be resumed,
while they will gradually cease or diminish when the nerve
is again surrounded by air on all sides.

4. The fact that certain ions are capable of bringing
about forms of irritability in nerves and muscles which do
not exist normally may perhaps furnish the explanation of a
number of certain morbid phenomena (neuroses, hysteria) in
which the motor and sensory reactions of the patient are
modified.

XXXV

THE TOXIC AND THE ANTITOXIC EFFECTS OF IONS
AS A FUNCTION OF THEIK VALENCY AND POS-
SIBLY THEIR ELECTRICAL CHARGE1

I. INTRODUCTION

FIVE years ago I published a series of papers on the
physiological effects of the electric current which impressed
upon me the long-known fact that the galvanic current is the
most universal and effective stimulus for life-phenomena.
This fact suggested the idea that it should be possible to
influence life-phenomena just as universally and effectively
by the electrically charged molecules — the ions — as we can
influence them by the electric current.

My first aim was to find out whether or not it is possible
to alter the physiological properties of tissues by artificially
changing the proportion of ions contained in these tissues.
In this way originated the investigations on the effect of ions
upon the absorption of water by muscles,2 the effects of ions
upon the rhythmical contractions of muscles, and Medusae,3
the heart of the turtle,4 and the lymph hearts5 of the frog,
the role of ions in chemotropic phenomena6 and the influence
of ions upon embryonic development,7 and the development

1 American Journal of Physiology, Vol. VI (1902), p. 411. A preliminary report
of these experiments appeared in Pflilgers Archiv filr die gesammte Pysiologie,
Vol. LXXXVIII (1901), p. 68.

2 Part II, pp. 450, 501, and 510.

3 Part II, pp. 518, 559, and 692; Archiv filr die gesammte Physiologie, Vol. LXXX
(1900), p. 229.

4 D. J. LINGLE, American Journal of Physiology, Vol. IV (1900), p. 265.

5 A. MOORE, ibid., p. 386.

e W. E. GAEEEY, ibid., Vol. Ill (1900), p. 291.
1 Part II, pp. 559 and 576.

708

Toxic AND ANTITOXIC EFFECTS OF IONS 709

of unfertilized eggs (artificial parthenogenesis).1 Those
who have followed my work on artificial parthenogenesis
may have noticed that from the start I aimed at bringing
about artificial parthenogenesis through ions. It seemed to
me that I could not find any better test for my idea that the
electrically charged ions influence life-phenomena most
effectively than by causing unfertilized eggs to develop
by slightly altering the proportion of ions contained in
them. I believe that all these experiments proved what I
expected they would prove, namely, that by slightly chan-
ging the proportion of ions in a tissue we can alter its physi-
ological properties.

The next step taken consisted in proving that it was in-
deed the electrical character of the ion that determined its
specific efficiency. I succeeded in doing this three years
ago. It was known that a frog's muscle gives rise to twitch-
ings or rhythmical contractions when immersed in certain
solutions. I showed that such contractions occurred only in
solutions of electrolytes, and not in solutions of non-con-
ductors (distilled water, various sugars, glycerin, urea).2
Soon after I showed the same to be true also for the rhyth-
mical contractions of the Medusae.3 From observations made
in my laboratory, the same fact was shown to hold for the
turtle's heart by Mr. Lingle,4 and for the lymph hearts of
the frog by Miss Moore.5 I am confident that this fact
will be proved universally.

In the physiology of the heart one frequently encounters
the statement that calcium is the stimulus for the contraction
of the heart. I had found that a muscle is able to twitch

1 Part II, p. 646. Archiv filr die gesammte Physiologic, Vol. LXXXVII (1901),
p. 594.

2 Part II, p. 518.

3 Part II, pp. 553 and 559.

* LINGLE, American Journal of Physiology, Vol. IV (1900), p. 265.
& MOOBE, ibid., Vol. V (1900). p. 87.

710 STUDIES IN GENERAL PHYSIOLOGY

rhythmically when immersed in the solution of salts with a
monovalent kation — I obtained contractions in Na, Li, Rb,
and Cs salts — but that the addition of a small quantity
of a bivalent kation — Ca, Mg, Sr, Be, Mn, Co — inhibits
these rhythmical contractions.1 This seemed to be a direct
contradiction to the statement that calcium salts are the
"cause" of the heart-beat. The significance of the calcium
had to be looked for, then, in another direction. It was
soon found that the muscle, the apex of the heart, and a
Medusa contract rhythmically in a pure sodium-chloride so-
lution, but that they soon come to a standstill. If, however,
a trace of a soluble calcium salt is added to the sodium-
chloride solution, the contractions continue much longer. I
concluded from this that the pure sodium-chloride solu-
tion acts, in the long run, as a poison — that is to say, brings
about definite, but at present unknown, physical changes in
the protoplasm — but that a trace of a calcium salt anni-
hilates this toxic action. The amount of calcium neces-
sary for this antitoxic effect is, of course, much smaller than
the amount necessary to inhibit the rhythmical contractions.
Soon after I succeeded in demonstrating conclusively the
poisonous effect of a pure sodium-chloride solution, and the
annihilation of this effect by calcium.2 The eggs of a
marine fish (Fundulus) develop normally in sea-water, but
they can develop just as well, as I had previously found, in
distilled water. The addition of ions from the outside is
consequently not necessary to the development of this animal.
I found, now, that if the freshly fertilized eggs of this fish
are put into a pure sodium-chloride solution having a con-
centration equal to, the concentration of the sodium chloride
in the sea- water (about f-tn), not a single egg can develop
into an embryo. If, however, a trace of a calcium salt is

1 Part II, p. 518.

2 Part II, p. 559; Archiv fur die gesammte Physiologic, Vol. LXXX (1900), p. 229

Toxic AND ANTITOXIC EFFECTS OF IONS 711

added to a sodium-chloride solution, as many eggs develop,
and in just as normal a manner, as in ordinary sea-water.
The calcium ions in this case undoubtedly serve the purpose
of annihilating the poisonous effect of a pure sodium-
chloride solution.

In the meantime I had become familiar with the bril-
liant experiments of Hardy upon the influence of ions
and the galvanic current upon colloidal solutions.1 They
indicated to me that the next step I had to take was to
see whether or not the valency and the sign of the elec-
trical charge of an ion determine its physiological effects.
I suspected that the antitoxic effect of the calcium ion in
the above-mentioned experiment was due to its electrical
charge and decided to investigate in a more systematic
way whether or not the sign and quantity of the electrical
charge influence life-phenomena. My experiments carried
on at Woods Hole this summer showed conclusively that
this is the case for the antitoxic effects of ions, and prob-
ably for the production of rhythmical contractions through
ions. It seems at least possible that it is true also for
artificial parthenogenesis.2

II. THE ANTITOXIC EFFECT OF IONS AS A FUNCTION OF THEIB
ELECTRICAL CHARGES AND VALENCY

1. The development of an embryo from the freshly fer-
tilized egg of the before -mentioned fish, Fundulus, served as
a test for the toxic and antitoxic effects of ions. I chose
this particular animal for two reasons. First, the process of
development in this form is to an astonishing degree inde-
pendent of the osmotic pressure of the surrounding solution.
The egg will develop not only in sea-water, the osmotic

i HARDY, Proceedings of the Royal Society, Vol. LXVI (1900), p. 110.

2 1 have not altered this introduction, although I now think it probable that the
ions act chemically in all these cases. [1903]

712 STUDIES IN GENEKAL PHYSIOLOGY

pressure of which is about equal to that of a ^m sodium-
chloride solution,1 but also in distilled water, or in sea-water
the concentration of which has been doubled (by the addi-
tion of NaCl). In the following experiments, therefore, we
need not at all consider the osmotic pressure of the sur-
rounding solution. Secondly, since enormous numbers
of the eggs can be obtained, it is an easy matter to per-
form the experiments upon hundreds or thousands of eggs
at once.

The eggs were artificially fertilized in the laboratory by
the addition of sperm, and then immediately distributed into
the various solutions. The embryo forms in from about
twenty-six to forty-eight hours — varying with the tempera-
ture— and twenty-four hours later the heart begins to beat,
and the circulation is established. Usually about two hun-
dred eggs were put into a solution, and after two or three
days the developed embryos were counted and the per-
centage of the eggs which had developed was determined.
The eggs were kept under observation as long as the
embryos remained alive. Usually when an embryo was
once formed, development went farther, and the circulation
was established.

2. First of all the toxic effects of a pure sodium-chloride
solution at various concentrations were tested. In a J NaCl
solution every egg produced an embryo which died, how-
ever, before, or immediately after, emerging from the egg.
(The embryo hatches from between twelve to twenty days
after fertilization.) On the other hand, in a f m NaCl solu-
tion only a few of the eggs gave rise to embryos — about 1 to
5 per cent. In a f m NaCl solution an embryo forms but
rarely, and in a -|ra NaCl solution the formation of embryos
is rendered impossible. The egg goes through the first

i m represents that degree of dilution of a solution which contains one gram-
molecule of the substance in one liter of the solution.

Toxic AND ANTITOXIC EFFECTS OF IONS 713

stages of segmentation, but dies when it reaches the thirty-
two- or sixty -four cell stage. The concentration of a -| m
sodium-chloride solution is indeed so high above the point
of the fatal concentration of sodium chloride that a slight
decrease in the degree of dissociation of the NaCl solution
brought about through the addition of a small amount of
another salt having a common ion, could be entirely dis-
regarded. In the following experiments, howeyer, salts with
different ions were combined, wherever this was possible.

If to a pure sodium-chloride solution a trace of a
calcium salt is added, as many eggs develop as in ordi-
nary sea-water, as shown by Table I.

TABLE I

Solution

Percentage of
Eggs Yielding
Embryos

1

100 c.c.
100
100
100
100
100

fmNaCl

" " +ic.c.em*CaS04

tt tt 1 1 tt tt

t< tt _|_2 " "

tt tt 1 A tt tt

«. « _|_8 " "

0
3
3
20
75
70

2 . ..

3

4

5

6

This series of experiments does not show whether it is
the Ca or the SO4 ion that has the antitoxic effect. To
determine this point the same series of experiments was
twice repeated with certain modifications. In the first of
these Ca(NO3)2 was added to the f NaCl solution instead of
CaSO4. The result was practically that given in Table I.
In the second Na2SO4 was added to the sodium -chloride
solution. The addition of Na2SO4 did not inhibit the toxic
action of the sodium chloride, and the eggs developed no better
than in the pure sodium-chloride solution. We shall return
to this point later. However, in order to eliminate entirely
the effect of the anions in the antitoxic effects produced, a

714

STUDIES IN GENERAL PHYSIOLOGY

series of experiments were instituted in which the toxic and
antitoxic salt both had the same anion.

TABLE II

Solution

Percentage
of Eggs Yielding
Embryos

1..

100 c.c.

| m NaNO3

8

2

LOO

" " 4- £ c.c.

£VCa(N(X)2

1

3

100

" " +1

u u

10

4

100

" tt o

« u

15

5

100

" " 1 A

(« 44

15

6

100

tt a 1 g

t( l(

70

It is undoubtedly true, therefore, that the addition of
even a small amount of Ca ions diminishes the toxic
action of a pure sodium-chloride solution. It can further
be shown that the concentration of the Ca ions necessary
to abolish the poisonous effects of a sodium-chloride solu-
tion increases as the concentration of the latter increases
(see Table III).

Tables II and III show clearly that the amount of calcium
necessary to annihilate the poisonous effect of a solution of
a sodium salt increases with the concentration of the sodium
salt in the solution.

The embryos formed in these solutions, rendered harmless
through the addition of calcium, developed a normal circula-
tion and lived several weeks. As a rule, however, they did
not hatch. It was further found that the addition of 5 c.c.
of a -g1 CaSO4 solution could annihilate absolutely the toxic
effect of a f m, ^m, or -f- m NaCl solution. These experiments
leave no room for doubt that the presence of a trace of Ca
ions is capable of rendering inert the poisonous effects of a
pure sodium-chloride solution.

3. It was next shown that Sr, Ba, and Mg ions are
also capable of annihilating the poisonous effects of a

Toxic AND ANTITOXIC EFFECTS OF IONS 715

pure NaCl solution in a way similar to that of Ca ions
(see Table IV).

TABLE III

Solution

Percentage
of Eggs Yielding
Embryos

1

100 C.C.
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100

|m

u
tt

11

u

u
M

u

tl
11

f

M

u

NaNO3

u _

11 _
11
11

11 _
11 _

(I _
11 _
tl
11 _
u
H
n _
tl _

-fcc.c.ftCaCNO,),

_1 « u

_ 2 *< «

-8 " "

_ i « «
-2 " "
-4 " "

-8

-It

_J « «

-2
_4

-8 " "

5
48
40
63
66
70
1
8
9
45
42
70
0
0
2
7
10
50

2

3

4

5

6

7

8

9

10

11
12... .

13

14

15

16

17

18

TABLE IV

Solution

Percentage
of Eggs Yielding
Embryos

1. .

100 c.c. f m NaCl
100 " " -
100 " "
100 " " H
100 " " -
100 " " -

-f c.c.m BaCls

-2 l> "
-2 " MgCl2
-2 c.c. ^m SrCl2

-1| " | " Ca(NO3)2

0
75

90
75
90
80

2

3

4
5

6

That the threshold for the antitoxic effects of Ba, Mg,
Sr has the same magnitude as that of Ca may be indicated
by a single experiment with Ba (Table V).

It can be seen that the threshold of the antitoxic effect
of barium is almost identical with that for Ca under similar
conditions.

716

STUDIES IN GENERAL PHYSIOLOGY

TABLE v

Solution

Percentage
of Eggs Yielding
Embryos

1

100 c c f m NaCl

o

2

100 " " +^c.c

ft BaCl2

8

3

100 « « -|-1

u «

4

4

100 " " +2

11 it

27

5

100 " *' +4

u u

76

6

100 " " +8

a (i

75

Since all these ions are related chemically, the objection
was possible that we were dealing here, not with the effects
of the valence or the electrical charge of the ions, but with
a specific chemical effect. It was, therefore, necessary to
show that the same effect can be produced by bivalent kations
which lie outside of the calcium group. My first experi-
ments failed me, since I at first employed too large amounts
of the antitoxic salts. I discovered only gradually that the
poisonous effects of a sodium-chloride solution may be
annihilated by a bivalent kation in quantities much smaller
than are given in Table IV, which presents the results of one
of my first experiments. My experiments now succeeded.

4. A large number of experiments were performed with
ZnSO4 as the antitoxic substance for NaCl. The Nad
solution used was somewhat more concentrated than that
usually employed, namely \\m instead of j^m.

TABLE VI

Solution

Percentage of
Eggs Yielding
Embryos

1

100 cc

i^mNaCl

o

2

100

' " _|_ ^ c c 1f^- ZnSO4

3

100

*.«'_!_! « u

2

4

100

« « _j_ 2 " "

22

5

100

' « _4_ 4 » «

50

6

100

i 11 1 Q U ((

75

Toxic AND ANTITOXIC EFFECTS OF IONS 717

To supplement these results the following table dealing
with the effects of a more concentrated ZnSO4 solution and
a more dilute Nad solution than that of the previous table
may be given:

TABLE VII

Solution

Percentage of
Egss Yielding
Embryos

1..

2

lOOc.c. fraNaCl
100 •< " + J c.c. 3wa ZnSO4

5

90

3

100 " " -}- 1 " "

80

4

100 " "+2 " "

86

5

100 " " + 4 " "

88

The remaining experiments showed a similar agreement
in the results obtained. It is worthy of note that these
embryos remained alive over a week, possessed an entirely
normal circulation, and moved in the egg.

The experiments with freshly prepared FeSO4 yielded as
striking results as the above. Only in these experiments the
transformation of the bivalent into the trivalent Fe ion
introduces a disturbing element. We shall see later that the
ferric ion is apparently extremely poisonous. The addition
of ^ c.c. or 1 c.c. of a freshly prepared ™ FeSO4 solution
to 100 c.c. of f m NaCl solution annihilates the poisonous
effect of the pure sodium-chloride solution just as com-
pletely as the addition of the Zn ions in the previous ex-
periment.

Then I tried whether cobalt ions are capable of anni-
hilating the antitoxic effects of a pure sodium-chloride solu-
tion. The results were very clear indeed.

Since the amount of the bivalent kation capable of exhib-
iting its antitoxic properties was so extraordinarily small, I
risked the attempt to annihilate the poisonous effects of a
pure sodium -chloride solution through the addition of Pb,

718

STUDIES IN GENERAL PHYSIOLOGY

TABLE VIII

Solution

Percentage of
Eggs Yielding
Embryos

1

100 c.c.

| ra XaCl

0

2
3

100
100

" " + lc-c- iri CoCl8

« -f 2 " "

6

2

4

100

« _i_ 4. « «

2

5

100

u _j_ g « «

50

6 ....

100

" +2 ?

88

7

100

« _|_5 « «

62

Cu, and Hg ions. Had I not before demonstrated the anti-
toxic effects of so poisonous an ion as the zinc ion, such an
attempt would have appeared to me only ridiculous. With
copper acetate and mercuric chloride I obtained negative
results throughout, for these two ions are so poisonous indeed
that the small amount necessary to render inert the poison-
ous effects of a sodium -chloride solution are sufficient to
kill the egg or cause its coagulation. With lead ions, how-
ever, I had a distinct success. For the antitoxic salt lead
acetate was used, and for the toxic salt, sodium acetate. It
was proved that the latter was slightly more toxic than NaCl.

TABLE IX

Solution

Percentage of
Eggs Yielding

Embryos

IV.

100 c.c.

|m CH3CO8Na

1

2.. .

100

+ \ c.c. fi Pb acetate

8

3.. .

100

« <« _|_ ! » «

12

4.. .

100

«( u i 2 " "

23

5.. .

100

(( « I 4 « u

34

In another case 40 per cent, of the eggs formed embryos.
The objection was here again at hand that the decrease in
the degree of the dissociation of the sodium acetate had
played a r6le. Although lead chloride is only very slightly

Toxic AND ANTITOXIC EFFECTS OF IONS 719

soluble, I tried to see if the few lead ions that go into solu-
tion when lead acetate is added to sodium chloride would
still suffice to weaken the poisonous effects of a pure NaCl
solution. Such was indeed the case.

TABLE X

Solution

Percentage of
Eggs Yielding
Embryos

1

100 c.c. f m NaCl

3

2

100 " " + i c.c. ff Pb acetate

7

3

100 " " + 1 " "

17

In the remaining solutions the number of embryos could
not be determined, since the eggs had been rendered opaque
by the precipitation of the lead salts.

5. Experiments were now made to see if it were possible
to annihilate the toxic effects of a sodium-chloride solution
through the addition of salts having a trivalent ion. A1C13,
Cr 2 (SO 4)3 and FeCl3 were used. The experiments with
Fed 3 all yielded negative results. No concentration could
be found at which this salt exhibited antitoxic properties.
Perhaps the strongly acid character of this solution had
something to do with this result. The experiments with the
two other salts, however, yielded positive results.

TABLE XI

Solution

Percentage of
Eggs Yielding
Embryos

1..
2. . .

100 c.c.
100

fraNaCl

0
0

3

100

" 1 i « "

4

4

100

« _^_ i « «

25

5

100

« -]- 2 " "

39

6

100

« 1 A it U

25

720

STUDIES IN GENERAL PHYSIOLOGY

Two other series of experiments yielded the same results.
It is worthy of note that the amount of a trivalent kation
capable of exerting a certain antitoxic effect is considerably
less than the amount of a bivalent kation necessary for the
same purpose. At the same time one notices, however, that
the number of eggs forming embryos is, even at the best,
lower than when bivalent kations are employed. The reason
for this lies, as I believe, in the fact that the trivalent ion
causes readily a coagulation of the egg contents, as direct
observation shows. But this coagulation is not exclusively
a function of the valency of the ions, for Cu, Hg, and to a
slight extent Pb have the same influence upon the egg.
The influence of the Cr ion in bringing about coagulation
is much more marked than is the case with Al, and its anti-
toxic effects are correspondingly slight, but yet definite.

TABLE XII

Solution

Percentage of
Eggs Yielding
Embryos

1..

2 ....

lOOc.c. fmNaCl
100 " "4-4- c.c ^ Cr (SCM

0

3

3

100 " "4-1- " "

8

4

100 " " -j- 1 " "

8

5

100 " "4-2 " "

10

6

100 " "4-4

6

6. Since traces of trivalent kations and small amounts of
bivalent kations suffice thus to annihilate the poisonous
effects of a sodium-chloride solution, experiments were made
to ascertain if the same could also be brought about by
monovalent kations.. The experiments have thus far led to
no positive results. I tried to see if the poisonous effects of
a pure sodium-chloride solution could be done away with by
the addition of potassium salts (KC1 and K2SO4). Small
amounts of potassium salts were entirely without effect.

Toxic AND ANTITOXIC EFFECTS OF IONS 721

The addition of ^ to 2 c.c. of m KC1 or K2SO4 occasionally
yielded results, in that 1-5 per cent, of the eggs formed
embryos. Lithium salts showed themselves to be even less
active. I occasionally obtained a slight antitoxic action by
the addition of large amounts of NH4 salts. Whether
hydrogen ions can yield better results must be determined
through further experiments.

7. Not only can the poisonous effects of a pure sodium-
chloride solution be annihilated through the addition of
small amounts of bivalent or trivalent kations, but it seems
as though the same holds for all salts which, like NaCl, have a
univalent kation and anion. No embryos develop in a f m LiCl
solution. By the addition of small amounts of Ca(NO3)2,
BaCl2, SrCl2, or MgCl2, 50-60 per cent, of the eggs were
caused to form embryos, which developed normally. Other
kations of a higher valency were not tested. I obtained
entirely similar results in regard to KC1. In a -f in or even
a ^ra KC1 solution an egg may occasionally develop. When
a small quantity of MgCl2, Ca(NO3)2, SrCl2, BaCl2 or
FeSO4 was added, the poisonous effects of the pure KC1
solution were annihilated. Of salts having other bivalent
kations, only ZnSO4 (a single experiment) was used. An
effect was obtained in this case also, but it was less striking
than in the case of the other bivalent kations.

NH4C1 seems to be the least toxic of all the salts men-
tioned thus far. Even in a ^m NH4C1 solution an embryo
could form occasionally. This immunity of the Fundulus
egg against NH4C1 is perhaps related to its great immunity
against urea. I cannot get rid of the suspicion that a per-
centage of the NH4 ions is perhaps done away with in the
metabolism of the egg. I obtained striking antitoxic effects
with small amounts of SrCl2 and, although less definite,
of FeSO4. Ca(NO3)2 increased the number of embryos
formed, though not as greatly as the other salts with a

7'22 STUDIES ^N GENERAL PHYSIOLOGY

bivalent kation, but the life of the embryos was very con-
siderably prolonged.

The shortness of the spawning season limited the number
of my experiments, so that I decided to bring my experi-
ments upon the annihilation of the poisonous effects of a
pure sodium-chloride solution to a close, and to carry the
remaining experiments only far enough to decide if we are
dealing here, in the main, with the same condition of affairs.
That, I believe, is undoubtedly the case, so that I feel my-
self justified in making the following statement : The salts
of monovalent kations (Na, Li, K, NH^) with monovalent
anions (Cl, NOS, CH3COO) exert a toxic effect at certain
concentrations. This toxic effect can be annihilated through
the addition of a small amount of a salt having a bivalent
kation. For NaCl, proof has been brought forward that
trivalent kations exhibit even a much more energetic anti-
toxic effect than bivalent kations. Further experiments
are yet to be made, to decide if the poisonous effects of the
other salts (LiCl, KC1, NH4C1) can also be done away with
through the addition of such small amounts of trivalent ka-
tions as suffice for NaCl.

8. While the preceding experiments show an undoubted
influence of the valency of the ions upon their antitoxic
effects, it was now necessary to prove that the sign of the
electrical charge was the second determining variable. I
instituted a large number of experiments in which I
attempted to annihilate the poisonous effects of a -|m NaCl
and a ^m KC1 solution by the addition of salts having a
univalent or bi- or trivalent anion. The antitoxic effects of
the following salts are investigated; KOH, NaBr, Nal,
NaHCO3, Na2CO3, NaSO4, Na2HPO4, sodium citrate,
K2SO4. Extensive quantitative experiments were made
with Na2SO4, K2SO4, NaHCO3 and Na2HPO4. The
results were negative throughout. In the best cases 1 per

Toxic AND ANTITOXIC EFFECTS OF IONS 723

cent, of the eggs formed embryos. It followed from these
experiments that the toxic effects of salts with a monov-
alent kation and a monovalent anion can be annihilated
only by bi- or trivalent kations, but not by mono-, bi-, or triv-
alent anions. If we correlate this fact with that previously
found, that spontaneous, rhythmical contractions of muscles,
Medusae, and hearts are possible only in solutions of electro-
lytes, then the idea can certainly not be repudiated that the
antitoxic effect of salts in the above-mentioned experiments
may be a function of the magnitude and the sign of the
electrical charges of the ions.

9. If the toxicity of a pure CaCl2, MgCl3, BaCl2 or
SrCl2 solution is compared with the toxicity of a solution of
a chloride of a monovalent kation, then it is found that the
former are the more poisonous. In a f Ca(NO3)2 solu-
tion no embryo develops. This same toxic concentration is
reached in a MgClg solution at the dilution of ™. Can
the toxic effects of these solutions also be overcome? One
can indeed easily overcome the poisonous effects of a ™
Ca(NO3)2 solution by adding large amounts of a KC1 or
NH4C1 solution. NaCl and LiCl solutions are almost with-
out effect.

TABLE XIII

Solution

Percentage of
Eggs Yielding
Embryos

1

100 c.c.
100
100
100
100
100

f Ca(N03

U It

11 it

« u
<t Cl

2 4-^ c.c. 2£mKCl

+1 ;; ;;

+ 4 « «
_j_8 u «

0
15
34
40
55
67

a.

3.

4. . .

5

6

As one can see, the number of embryos formed shows a
definite increase with an increase in the concentration of the
KC1. I tried still stronger solutions of KC1 in further

724

STUDIES IN GENERAL PHYSIOLOGY

experiments, and found that in a mixture of 100 c.c. ^ Ca
(NO3)2 +20 c.c. 2| ra KC1 a still larger number of eggs
formed embryos than in the preceding experiments. On the
other hand, it could be shown that the addition of small
amounts of KC1 was without effect.

TABLE XIV

Solution

Percentage of
Eggs Yielding
Embryos

1

100 c.c.
100
100
100
100
100
100

y Ca(NO,

(1

((

u
u

«

)•

+ frc.c.ftKCl

+ 1 " "
+ 2 •• «'
+ 4 " "
+ 8 •« «
+2 c.c. 2£ m KC1

0
0

0
0
0
2
12

2

3

4

5

6

7

The size of the antitoxic dose of KC1 in Ca(NO3)2 poi-
soning is, in fact, extraordinarily larger than the antitoxic
dose of Ca(NO3)2 in the case of KC1 poisoning. Similar
relations exist for the antitoxic effect of NH4C1 upon CaCl2
poisoning.

TABLE XV

Solution

Percentage of
Eggs Yielding
Embryos

1

100 c.c. f Ca(NO3)a
100
100
100

100 " " H
100

-1 c.c. 2^m NH4C1
-2 *' "
_ 4

-8
-16

0
9
8
16
21
16

2

3

4

5

6

The antitoxic effects of NH4C1 are not as great as those
of KC1. Similar experiments with NaCl and LiCl as anti-
toxic substances were without positive result.

Similar experiments were then performed with MgCl2.

Toxic AND ANTITOXIC EFFECTS OF IONS 725

By the addition of MgSO4 the toxic effects of MgCl2 could
not be done away with. But through the addition of large
amounts of KC1, NH4C1, or small amounts of SrCl2 this was
possible, as also — to a slight extent — through the addition
of Ca(NO3)2. The dilution at which a MgCl2 solution
hinders the development of an embryo is ^ra MgCl2.
Table XVI shows a series of antitoxic experiments. NaCl
and LiCl were just as unable to annihilate the toxic effects
of the MgCl2 solution as they were unable to annihilate the
poisonous effects of a Ca(NO3)2 solution. When less than
-| c.c. of a ^ SrCl2 solution was added, not a single egg
could develop.

10. If, in these experiments, only the kations have an
antitoxic effect, and this the greater, the greater their elec-
trical charge ; and if in these antitoxic effects we are dealing
only with electrical effects, then it is to be logically expected
that the toxic effects which are inhibited in these cases are
also electrical effects, and indeed the effects of the negative
electrons. If the antitoxic ions are the strongly charged
positive ions, then the toxic ions in the sodium-chloride solu-
tion must be the Cl ions. But in a pure sodium-chloride
solution we have just as many kations as anions, and in con-
sequence just as many positive as negative electrical units.
It is therefore not at once intelligible why the negative
charges of the chlorine ions should be able to call forth
poisonous effects in a sodium-chloride solution. If it is
necessary for us to accept the fact that we are here dealing
with electrical effects, then we are forced further to conclude
that, for some reason or other, the negative charges of the
chlorine ions attain a greater activity than the positive
charges of the sodium ions. Nernst has pointed out the fact
that the metallic ions tend to bind their electrical charges
more strongly than the anions, and he brings this into con-
nection with the fact that we are acquainted with kathode

726

STUDIES IN GENEEAL PHYSIOLOGY

TABLE XVI

Solution

Percentage of
Eggs Yielding
Embryos

1.

100 c.c. Te
100 «
100
100 -
100
100
100
100
100
100
100

, m MgCl8

" H

« _

" -

" -

u _
« _
« _
a _

11 H
« r

- ic.c:2^

h 2
- 4
- 8
-16

-1,CA

- 4
- 8
-16

m NH4C1

M
<c

m SrCl2
«

< (i

* "

0
16
22

34
9
3
25
22
9
0
0

2

3

4

5 :...

6

7

8 . ..

9

10

11

rays, but not with anode rays. Another possibility may be
thought of. The egg — and all protoplasm — is a system
with various phases; we have solid parts (membranes), and
liquid parts which are either rich or poor in colloids. It is
conceivable that the coefficient of distribution for the posi-
tive and negative ions is unequal in the various phases, and
that this fact leads to the toxic effects of the negative ions
which can be annihilated by the addition of a small number
of positive ions holding a double or triple charge.

I was long inclined to look upon the sodium ions as the
toxic ions in a pure sodium-chloride solution, and I have
upheld this view in my preliminary communication concern-
ing these experiments. What led me to this conclusion was
the following experiment : I tested the relative toxicity of H
and OH ions for the eggs of Fundulus. As was to be
expected, it came to light that the hydrogen and hydroxyl
ions differ in their toxicity. In a -%%-$ KOH solution the
eggs developed and, formed embryos, while a T^¥ HC1 solu-
tion killed the eggs almost immediately. The hydrogen
ions are therefore at least as much as five times as poisonous
as the hydroxyl ions. But I do not believe that we are
forced to conclude from this that the poisonous effects of a

Toxic AND ANTITOXIC EFFECTS OF IONS 727

sodium-chloride solution necessarily originate from the posi-
tively charged ions. Besides the electrical charge other fac-
tors may have to be considered in the toxicity of ions for the
determination of which physical chemistry and physics must
first furnish us the data. In this category belongs, for
example, the fact that the toxic effects of the sodium salts
of the halogens upon fish eggs (perhaps upon protoplasm in
general) increase in the following order: Nad, NaBr, Nal,
NaF. In addition we find that the bivalent anions are in
general more poisonous than the monovalent, and the triv-
alent more poisonous than either. The same also holds true
to a certain extent for the poisonous effects of kations.

In order to make the bulk of this, paper no greater than
it is already, I shall discuss my experiments on the toxic
effects of ions no further at this point.

XXXVI

MATURATION, NATURAL DEATH, AND THE PROLON-
GATION OF THE LIFE OF UNFERTILIZED STAR-
FISH EGGS (ASTERIAS FORBESII) AND THEIR
SIGNIFICANCE FOR THE THEORY OF FERTILIZA-
TION1

I. INTRODUCTION

I HAVE pointed out in my earlier publications that fertili-
zation of the egg serves to prolong the life of the egg.2

The mature unfertilized egg dies in a comparatively short
time. Because of this fact the egg becomes of importance
as an object of experiment, to study the question of natural
death and the prolongation of life. For by no means has it
been decided that there is a "natural" death. We only
know that with an increase in age a critical period is reached
in which every living organism dies under the influence of
conditions which do not affect a younger organism. It may,
therefore, be of interest that we are able to show, as I believe,
that a critical period exists in the life of many eggs in which
they die a "natural" death, and that the life of the eggs can,
during this period, be saved or lengthened only through
various external conditions.

The egg of the starfish (Asterias Forbesii) serves as a
very favorable object of experiment in the study of this
question. When removed from the ovary this egg is gen-
erally "immature," but as soon as it comes in contact with
sea- water it begins to "maturate."

Morphologically,' the immature state is characterized by

1 Biological Bulletin, Vol. Ill (1902), p. 295.

2 Part II, p. 689; LOEB AND LEWIS, American Journal of Physiology, Vol. VI
(1902), p. 305.

728

NATURAL DEATH AND FERTILIZATION 729

a very large plainly visible nucleus.1 The process of matura-
tion consists morphologically in this, that the nucleus
becomes invisible and the polar bodies are thrown out.

This process is completed within one or two hours after
the eggs are removed from the ovaries and placed in sea-
water. Only when maturation is complete is it possible to
cause the egg to develop through the addition of sperm or
through the physical and chemical agencies that have been
described by me, Delage, Mathews, and Greeley.

II. THE NATURAL DEATH OF THE MATURE UNFERTILIZED
STARFISH EGGS

The living eggs of Asterias are light yellow in color and
homogeneous. They retain this appearance during the pro-
cess of maturation as long as they are alive. They retain
this appearance also when they are made to develop through
the entrance of a spermatozoon or through the proper
chemical or physical means.

If, however, the mature eggs are not fertilized or do not
develop, they die in the course of four to twelve hours, and
this process of dying is accompanied by a characteristic
change in the color of the egg. The egg becomes at first
opaque, then almost black, and the homogeneous structure of
the protoplasm becomes granular. If such a culture of
unfertilized eggs is examined under the microscope after
twenty-four hours, two kinds of eggs are found, first, the
just-described dark, dead eggs which are mature, and
secondly, living, normally colored, but immature, eggs. For
usually not all the eggs that are removed from the ovaries of
a starfish mature at once ; many mature very late, others not
at all. It is readily seen that the immature eggs remain

i The recent beautiful experiments of Delage have shown that, besides these
visible changes in the nucleus, chemical, but morphologically invisible, changes also
occur in the protoplasm. DELAGE, "Eludes experimentales sur la maturation
cytoplasmique et sur la parth6nogenese artificielle chez les E\;hinodermes," Arch, de
zoologie experiment., Vol. IX (1901).

730 STUDIES IN GENERAL PHYSIOLOGY

alive for several days until they finally become the prey of
bacteria; while the mature eggs become opaque and die in
from four to twelve hours after maturation has been com-
pleted.

Is the death of the mature but undeveloped egg brought
about through internal conditions, or through the bacteria
contained in the sea-water?

A trustworthy way of determining this consists in making
sterile culture of the eggs in sea-water. This is a relatively
simple procedure in the case of starfish. Eight flasks were
sterilized, filled with sterilized sea-water, and again heated
for twenty minutes on three successive days to 100° C. A
female starfish was thoroughly washed externally, an arm
was opened, and one of the ovaries removed with sterilized
forceps and placed in sterilized sea-water. From the thick
stream of eggs which at once flowed out of the ovary, a few
drops were quickly introduced with a sterilized pipette into
each of the sterilized flasks. A second series of eight flasks
contained normal, unsterilized sea- water, and a few drops of the
same eggs were introduced into these flasks also. A third series
of flasks were filled with sea-water, to each of which were
added 2 c.c. of a putrid, foul-smelling culture of old starfish
eggs in order to bring about a rapid development of bacteria
from the beginning. Each of these flasks also contained
eggs from the same culture as those in the sterilized flasks.

That perfect sterilization had been attained in the first
eight flasks was proved by the fact that all the flasks remained
absolutely clear and cloudless during the course of the experi-
ment, and that three of the flasks which had not been opened
are even today (after six weeks) absolutely clear, and every
egg can be individually recognized. The flasks containing
the unsterilized sea-water became cloudy within as short a
time as twenty -four hours, and after two days the eggs had
become the prey of bacteria and no individual egg could be

NATURAL DEATH AND FERTILIZATION 731

recognized. The sterilized flasks which were opened were
at all times free from foul odor, while the unsterilized flasks
gave off a penetrating stench, often after one, invariably
after two, days. The microscopic examination of the sea-
water for bacteria was always negative in the sterilized flasks,
always positive in the other flasks. In those flasks to which
2 c.c. of the putrid culture of starfish eggs had been added,
bacteria and infusoria were exceedingly numerous from the
beginning.

Six hours after the beginning of the experiment one flask
of each of the three series was opened, and the eggs
examined microscopically. The picture was the same in all
three flasks. Nearly all the eggs were mature, and a small
number of them were opaque or black. But what is of the
greatest importance to us is the fact that the percentage of
opaque dead eggs was just as great in the sterile culture (if
not greater) than in the unsterilized or the infected sea-
water.

Twelve hours later, that is to say eighteen hours after the
beginning of the experiment, one of the flasks of each of the
three cultures was again opened. At this time nearly all the
eggs of the sterile culture were opaque or black, and a few
were already granular. In the two other cultures an equal
percentage of the eggs were opaque. The eggs, therefore,
die just as rapidly in the sterilized flasks which are abso-
lutely free from bacteria as in the flasks containing bacteria.
Death follows from internal causes, and so rapidly that the
few bacteria in the sea-water are scarcely able to accelerate
the death of the eggs. The eggs have already died from
internal causes before the bacteria can attack them in suffi-
cient numbers to threaten their existence.

The flasks which were opened later served only to corrob-
orate what has been said. The experiment was repeated
with the same result. Each of the flasks that were opened

732 STUDIES IN GENERAL PHYSIOLOGY

during the first few days also contained a small number of
living transparent eggs. The latter were, without exception,
immature. The experiment, therefore, shows that the mature
eggs of starfish die in the course of a few hours, and that
the cause of this death cannot be sought in the bacteria of
the sea-water; and further, that under exactly the same con-
ditions the immature eggs remain alive.

III. THE CHEMICAL CONDITIONS NECESSARY FOR MATURA-
TION IN STARFISH EGGS

Since the eggs of Asterias are usually immature in the
ovary, but, in part, at least, maturate in the course of one or
two hours when introduced into sea-water, the suspicion was
aroused that some of the substances contained in the sea-
water brought about the maturation. In order to determine
which substance this might be, a series of solutions were
prepared having approximately the osmotic pressure of the
sea-water. The result was so simple that it is not necessary
to describe all the experiments here. For it was found that
when the eggs are introduced into solutions which contain
free hydroxyl ions, maturation soon follows, but that this
does not occur in solutions containing no hydroxyl ions.
So, for example, the eggs retain their nucleus in a -|-m NaCl
solution, or in NaCl solutions to which some potassium or
calcium has been added. If, however, 0.5 to 2 c.c. ^
NaOH is added to each 100 c.c. of such solutions, matura-
tion soon follows: that is to say, the nucleus becomes in-
visible. Since sea-water contains free hydroxyl ions the
conclusion is justified that these are one of the causes for
the maturation of the starfish egg. It was possible to prove
this assumption through further experiments. If a small
amount of acid is added to sea-water, the free OH ions dis-
appear, and the water becomes acid in reaction (through the
addition of 1.5 c.c. or more ^ HC1 to 100 c.c. sea-water).

NATURAL DEATH AND FERTILIZATION 733

Immature eggs were introduced directly into sea-water to
which 1, 2, 3 and 4 c.c. of a ^ HNO3 solution had been
added to each 100 c.c. of sea- water. While, as is usual, a
large percentage of eggs soon maturated in the normal sea-
water, maturation did not occur at all in the vast majority or
in all the eggs contained in the sea- water to which 2 or more
c.c. acid had been added. The addition of even 1 c.c. of
acid diminishes the number of eggs that maturate. But it is
not even necessary to keep the eggs permanently in neutral
or acid sea-water in order to inhibit maturation. If 4 or 5 c.c.
of a ^ HNO3 solution are added to 100 c.c. sea- water, and
immature eggs are introduced into such a solution for only
about fifteen minutes, relatively few eggs maturate when
they are returned to normal sea-water. Such acidified sea-
water does not kill the starfish eggs.

We shall see later that the procedure described here
which, when used upon immature eggs, prevents maturation,
brings about artificial parthenogenesis when used on mature

I have, moreover, been able to convince myself of the
fact that the eggs which are introduced into acidified sea-
water in an immature state, can be fertilized by sperm if
they finally maturate. It is possibly in harmony with what
has just been said that the addition of NaHCO3, or larger
amounts of sodium citrate to the sea-water accelerates the
process of maturation. Free hydroxyl ions are present in the
solutions of both substances, and it is possible that their
addition to the sea-water increases the concentration of the
free hydroxyl ions in the sea-water.

But the hydroxyl ions are certainly not the only sub-
stances in the sea-water which favor or cause the maturation
of the starfish egg. I soon found that when different speci-
mens of eggs are taken from the same culture, and the per-

i LOEB, FISCHER, AND NEILSON, Pfl&gers Archiv, Vol. LXXXVII (1901), p. 594.

734 STUDIES IN GENEKAL PHYSIOLOGY

centage of mature eggs is determined, this percentage is
subject to the greatest variations. The cause of these
variations was soon discovered. For it was found where
the eggs lie together in a heap maturation occurs slowly,
but where they lie in a thin layer, maturation occurs quickly.
This fact suggested the importance of oxygen for matura-
tion. Where the eggs lie in a heap the appropriation of the
oxygen by the superficial layers of eggs prevents the diffu-
sion of the oxygen to those lying deeper.

Experiments were now made in which the oxygen of a small
flask containing a small amount of sea-water was replaced
by hydrogen. When, in such experiments, all the oxygen
was entirely removed maturation did not occur in any, or at
least the majority of the eggs, in spite of the presence of
the hydroxyl ions in the sea- water. There are, therefore, at
least two substances in sea-water which cause or accelerate
maturation, oxygen and hydroxyl ions. Possibly other con-
stituents of the sea-water are also concerned in the process,
but NaCl, Ca, and K have apparently no beneficial effect upon
maturation.1

It seems, therefore, that the absence of oxygen and
hydroxyl ions in the ovaries belongs to the conditions which
inhibit maturation of the eggs in the ovary.

IV. THE PROLONGATION OF THE LIFE OF THE UNFERTILIZED
STARFISH EGG BY THE PREVENTION OF MATURATION

We have shown above that the mature eggs of a culture
of unfertilized starfish eggs die within a short time (which
decreases with an increase in temperature), while the imma-
ture eggs remain alive a relatively long time. It was
necessary now to show that when the maturation of a culture
of unfertilized egg of Asterias is prevented artificially, the

1 Professor Whitman informs me that the maturation of the eggs of Clepsine
does not begin until after they are laid. Possibly the oxygen contained in the water
is in this case also a necessary condition for maturation.

NATURAL DEATH AND FERTILIZATION 735

eggs live longer. We begin with the experiment which is
technically most simple. The eggs streaming from the
ovary are divided into two portions. One portion of eggs
is carefully distributed without mechanical agitation, by
carefully tipping the vessel, in a thin layer over the bottom
of the vessel. The vessel must be low and the layer of sea-
water covering the eggs not too deep, so that the diffusion
of oxygen to the eggs can occur with ease. A 'second portion
is introduced with just as great care into a small-calibered
glass tube sealed at one end. This glass tube is half filled
with eggs so that one is certain that the lower layers of the
eggs in the pipette receive little or no oxygen. It is self-
evident that the eggs must be introduced into the tube
immediately after being laid. When, after twenty-four
hours, the eggs which are distributed over the bottom of the
glass dish and which receive a large amount of oxygen are
compared with those at the bottom of the glass tube, a
striking difference is found between them. The eggs richly
supplied with oxygen contain a much larger percentage of
mature dead and black eggs than those kept in the lack of
oxygen. In the latter the living immature eggs are in the
majority, and a part of these maturate when spread out in a
thin layer over the bottom of a vessel. These experiments
are also well adapted to show that the rapid death of the
mature unfertilized sea-urchin eggs is determined through
internal conditions and not by the bacteria contained in the
sea-water. I will cite an example.

One portion of a lot of eggs was spread out in a thin
layer over the bottom of a dish ; another was heaped in a
mass in the same dish. The sea-water was the same in both
cases. The first portion of eggs matured in a few hours and
were, in less than twelve hours, opaque and dead, while the
water was still absolutely clear and without odor of putrefac-
tion. After twenty-four hours the water became putrid and

736 STUDIES IN GENERAL PHYSIOLOGY

contained many bacteria. Even after three days, when the
water was exceedingly foul and cloudy, a portion of the eggs
which had lain in a heap, that is to say, without oxygen,
were immature and living. They were introduced into fresh
water and spread out into a thin layer. They maturated and
developed into swimming larvae upon the addition of sperm.
It is self-evident of course that even immature eggs finally
become the prey of bacteria, and so go to pieces in the sea-
water.

The same experiment can be made in a somewhat more
complicated way with pure oxygen and hydrogen. The
freshly laid eggs of a starfish were distributed into two
series of eight flasks. The one series of flasks was connected
with a hydrogen generator; the other with a tank contain-
ing pure oxygen. Before the beginning of the experiment
all the air in one of the series of flasks was driven out by
the current of hydrogen. During the course of the experi-
ment a vigorous current of hydrogen was maintained. Both
series of flasks contained freshly laid immature eggs of
Asterias. The experiment lasted three days, and from time
to time a flask was removed and its contents examined. The
eggs which had been exposed to the current of oxygen
maturated just as rapidly and as numerously as those in ordi-
nary sea-water, and the mature eggs soon died. In the cur-
rent of hydrogen maturation did not occur in the majority of
the eggs, and these remained alive. In the hydrogen
cultures a rapid development of bacteria occurred, while in
the oxygen cultures this occurred to a small degree.1

Treatment with acids which, as we have shown above,
prevents the maturation of the eggs (without killing them)
also prevents their death and disintegration.

Eggs which, without having been in contact with pure

1 Care must be taken in these experiments that the air is thoroughly removed
from the sea-water in the hydrogen flasks before the eggs are introduced into them.
Of course the hydrogen apparatus must also be free from air.

NATURAL DEATH AND FERTILIZATION 737

sea-water are introduced for ten or fifteen minutes into 100
c.c. sea-water plus 4 c.c. ^ HC1 maturate very slowly or
not at all when they are returned to normal sea- water. They
also retain, as long as they are immature, the transparent,
normal appearance of living eggs until they become the
prey of bacteria.

It seems to follow from these experiments that the same
processes which underlie the maturation of starfish eggs
also lead to their death (if they are not inhibited through
circumstances which we designate by the term fertilization).
I tried to see, now, whether it was also possible to maintain
the life of the mature egg through lack of oxygen. I
indeed obtained in a few cases positive results in this direc-
tion. The eggs of a starfish were spread in a thin layer
over the bottom of a dish. After three hours 75 per
cent, of the eggs were maturated. A portion of the mature
eggs was carefully introduced into the glass tube described
above, in which the deeper layers suffered from lack of
oxygen. A second portion was introduced into a small flask
through which a steady stream of pure oxygen was passed.
On the following morning, that is to say, fifteen hours after
the eggs were brought into the atmosphere of pure oxygenr
the various portions of the eggs were examined. The eggs
introduced into the current of oxygen showed in one vessel
98 per cent, mature and dark, dead eggs 'and 2 per cent,
immature living eggs. The eggs which had remained in
normal sea- water contained, as before, about 75 per cent,
mature eggs, all of which, however, were black and dead,,
with the exception of a few eggs which had begun to divide,1
and were living.

The immature eggs were also still living. Upon the other
hand, the eggs which had been left in the glass tube in

iThis cleavage was possibly brought about through mechanical agitation; I
had repeatedly shaken the dish to facilitate the introduction of oxygen into the
sea-water.

738 STUDIES IN GENERAL PHYSIOLOGY

absolute or relatively high lack of oxygen, were nearly all
living! This observation seems to show that the same
processes which lead to the maturation of the egg bring
about its death if they are not inhibited at the right time.
In this way the process of fertilization becomes a life-saving
or life -prolonging act.

V. DO THESE FACTS HOLD FOR OTHER FORMS?

The question of the relation between maturation and
natural death can be studied most beautifully in the starfish
egg because it is possible to obtain it in an immature condi-
tion, and because maturation follows very rapidly. With
sea-urchin eggs conditions are much less favorable since the
egg maturates within the ovary, and since it is difficult to
obtain immature eggs during the spawning season. I have,
therefore, been unable to discover which chemical factors
determine the maturation of the sea-urchin egg, and to decide
whether the same circumstances cause the death of the sea-
urchin egg that bring about the death of the starfish egg;
and whether the life of the sea-urchin egg can be prolonged
through a prevention of these circumstances. In an indi-
rect way Lewis and I attempted to answer this question last
year when we assumed that the destructive processes which
bring about the death of the unfertilized egg are enzymatic
(autoly tic ?) processes which can be inhibited through poisons
such as KCN.1

We did in fact succeed in showing that the addition of a
small amount of KCN to the unfertilized sea-urchin eggs mark-
edly lengthens their life. Even after seven days such eggs
can be fertilized as soon as they are returned to normal sea-
water. We also pointed out that, because of the well-known
bactericidal properties of potassium cyanide, the experiments
on sea-urchin eggs were not in themselves decisive, and so

1 LOEB AND LEWIS, American Journal of Physiology, Vol. VI (1902), p. 305.

NATUKAL DEATH AND FERTILIZATION 739

began experiments on starfish eggs1 which, however, we were
not able to complete at that time. In dealing with eggs
which are as long lived as sea-urchin eggs a great develop-
ment of bacteria in normal sea-water cannot be prevented,
since a few of the eggs always die and so serve as an excel-
lent culture medium for the further development of bacteria.
It need, therefore, surprise no one that the unfertilized eggs
of sea-urchins, as I was able to show this year,- live in sterile
sea-water for five days, or possibly longer, while they die
much earlier in ordinary sea-water (about two days). The
very fact that the eggs of sea-urchins are found mature in the
ovary indicates that they are able to live a considerable time
after maturation and that they differ in this respect from
the starfish egg.

It is, however, a fact that in the same sea-water the fer-
tilized and developing sea-urchin eggs live longer than the
unfertilized eggs.

It almost seems as if in certain of the higher animals
there are eggs which develop only when they are fertilized
immediately after leaving the ovary. Under the direction of
Professor C. O. Whitman, Harper has shown that the eggs
of pigeons are fertilized the moment they leave the ovary.
The sperm lives in a gelatinous mass upon the surface of the
ovaries,2 so that provision is made for the necessary contact
between sperm and egg. This also does away with the diffi-
culty which many have found in explaining how the sper-
matozoon finds its way to the egg in animals in which fer-
tilization occurs within the body. Definite directive forces
are clearly not necessary, since a portion of the spermatozoa
must reach the ovary, through their ciliary motion, by way
of the uterus and Fallopian tubes. Experiments similar to

1 Ibid.

2 Spermatozoa are in general much longer lived than mature eggs, even though
great differences exist in this regard in different animals. In the spermatic vesicles
of the queen bee spermatozoa are believed to remain alive more than a year after
copulation.

740 STUDIES IN GENEEAL PHYSIOLOGY

those made by Harper upon pigeons must yet be made upon
mammals. Yet there seems to be no doubt that the mam-
malian egg of many species is also fertilized before it reaches
the uterus. Cases of extra-uterine pregnancy also point to
the possibility that fertilization may occur at the surface of
the ovary.

VI. THE PEOLONGATION OF LIFE AND THE THEORY OF
FERTILIZATION

Our experiments seem to have proved that the mature
unfertilized starfish egg dies within a few hours through
internal changes, but that the process of fertilization saves
the life of the egg. This is true, not only of the fertiliza-
tion of the starfish egg by spermatozoa, but also for the
chemical fertilization through hydrogen ions. Mr. Neilson
succeeded this year in keeping the parthenogenetic larvae of
starfish alive much longer than has thus far been the case
(over thirty days), and Dr. Fischer was able to accomplish
the same for the larvae produced osmotically from unfertil-
ized sea-urchin eggs. It is therefore possible that the chemi-
cal or osmotic fertilization of these eggs can give rise to aa
long-lived larvae as the fertilization of the egg through
sperm.

But how does the spermatozoon, or the physical and
chemical means substituted for it, save the life of the egg,
and why does the mature egg die when it is not fertilized
by sperm or artificial means ? I believe that the answer
lies in this, that the fertilizing agencies accelerate metabolic
processes in the egg which, before fertilization, went on only
slowly. After fertilization by sperm or by the chemical or
physical means substituted for it, the egg divides and grows
which it did not do before fertilization occurred. Growth
is inconceivable without a preponderance of synthetical over
hydrolytical processes. I believe it possible that the deter-

NATURAL DEATH AND FERTILIZATION 741

mining factor in the chemical forces set in motion within the
egg through fertilization consists in this that the synthetical
processes in the egg are accelerated. If these processes are
not inaugurated or accelerated the egg dies. The wasting
of the body in old age also indicates a decrease in syntheti-
cal processes. Whether the second critical period occurring
in old age is similar to the critical period of the egg cannot
yet be determined. Yet it is not impossible -that the ques-
tion of the prolongation of life at this period should pass
over into the question of the possibility of accelerating
synthetical processes.

We, therefore, come to the conclusion that fertilization
accelerates a series of chemical changes (syntheses?) in the
eggs which do not occur sufficiently rapidly without spermatic,
chemical, or osmotic fertilization in the eggs of the majority
of animals. But why does the mature egg die when these
processes are not accelerated, and why does it remain alive
before it maturates ? The egg must often exist for years in
the immature condition in the ovary. In answer I can
only suggest that the processes underlying maturation are
at least in some form of a destructive nature (one might
think of autolytic processes) which the egg cannot withstand
for an indefinite length of time without dying. In many
eggs the velocity of these destructive (autolytic?) processes
may be greater than in others and this may determine the
differences in the velocity with which the mature, unfer-
tilized egg dies. It is in harmony with this view that
when maturation is prevented, or the mature egg is put un-
der conditions which inhibit the process of maturation or the
chemical processes underlying it, the life of the egg is
lengthened. Lack of oxygen or the addition of an acid
works in this way in the case of starfish eggs ; a slight addi-
tion of potassium cyanide, in the case of starfish and sea-
urchin eggs. But since all of these substances injure the

742 STUDIES IN GENERAL PHYSIOLOGY

eggs indirectly and do not entirely do away with the
destructive (autolytic?) processes occurring within the egg,
life is not prolonged to the same extent by these means as
by fertilization in which case life is prolonged not only
through an inhibition of the destructive but also through
an acceleration of the synthetical processes.

That the chemical processes which underlie maturation are
not identical with those which bring about fertilization
seems to be supported by the observation made above, that
the same means — the treatment with acid — which causes
the mature egg to develop and live beyond the bipinnarian
stage, inhibits the maturation of the immature egg. When
the mature unfertilized eggs of a starfish are introduced
for fifteen to sixty minutes into a mixture of 100 c.c. sea-
water plus 3 c.c. ^ HC1 90 per cent, of the eggs can,
under favorable conditions, develop into larvaB. If, how-
ever, the eggs are introduced into such a solution for the
same length of time before maturation, the maturation of
the eggs is prevented either permanently or for a long time.1
The difference is still more striking when the eggs are kept
for a shorter time in a mixture of 100 c.c. sea- water and 5 c.c.
^ HC1. This shows that acid affects the process of devel-
opment and the process of maturation in opposite or at least
different ways.

We must now raise the question, How does the behavior
of naturally parthenogenetic eggs, such as the eggs of bees,
harmonize with these ideas?

In naturally parthenogenetic eggs it seems as if the
processes which underlie maturation pass over into those
underlying development. But it is possible that this is only
apparently the case, and that in reality it so happens that in
the processes underlying maturation a metabolic product is

1 The eggs which finally maturate in spite of the previous treatment with acid
often begin to cleave when maturation is complete and develop into larvae, while the
control eggs kept in normal sea-water do not develop.

NATURAL DEATH AND FERTILIZATION 743

formed in the parthenogenetic animals which favors the
processes of development. We know that an exceedingly
small amount of hydrogen ions suffices to bring about devel-
opment in unfertilized starfish eggs; that an exceedingly
small amount of calcium causes the unfertilized eggs of
Amphitrite to develop; and that a trace of potassium ions
brings about the development of unfertilized ChaBtopterus
eggs.1 It is entirely possible that the specific ions or other
substances necessary to start the development of the eggs of
the bee are formed within the egg itself through the chemi-
cal changes taking place during or after maturation, and
that without the formation of these substances develop-
ment is impossible. In the case of sea-urchins and starfish
eggs one might also believe that the processes of maturation
and the processes of development pass over into each other.
For it has often been observed that the unfertilized eggs of
these forms after having resided in "normal" sea- water for
about twenty-four hours begin to cleave shortly before death.
This cleavage, however, never goes beyond the two- or four-
celled stage. This might be explained by the fact that the
eggs begin to die at this time. After I had found this year
that the eggs of sea-urchins can still be fertilized after a
residence of five days in sterilized sea- water (at summer tem-
perature), I decided to study this question of spontaneous
cleavage somewhat more closely. If it were true that indi-
vidual sea-urchin eggs begin to cleave in ordinary sea-water
after about twenty hours, and cease to develop any further
only because they soon die, it would be expected that many or
all should cleave when kept alive four or five days, and that
a number of them should reach a fairly advanced stage of
development. A lot of sea-urchin eggs were distributed
into a series of flasks containing sterile sea-water. One of
the flasks was opened every morning and a careful search
was made for developed eggs.

, FISCHER, AND NEILSON, loc. cit,

744 STUDIES IN G-ENEKAL PHYSIOLOGY

In the course of five days I never found a single divided
egg, either in the two-celled stage or in later stages of devel-
opment. It is possible that during the last days of the
experiment a few eggs divided, and that the cleavage cells
fell apart. Lewis and I found last year that when eggs
are fertilized forty-eight or more hours after their removal
from the ovaries they form no membrane and the cleavage
cells fall apart. I have corroborated this fact this year.
Usually more than one embryo develops from such an egg,
because the cells drop apart. I kept this fact in mind and
will not deny that a few small eggs were present, which per-
haps represented only half the mass of an ordinary egg.
But nearly all the eggs were of normal size, and since small
eggs are occasionally found even under normal conditions,
the experiment shows that in sea-urchin eggs also the
processes of maturation are not continuous with those of
cleavage, and that entirely different conditions which we can
bring about through the abstraction of water or the entrance
of a spermatozoon are necessary that division may occur.
It cannot be urged that the sterilized water perhaps pre-
vented the cleavage. When at the conclusion of the experi-
ment these same eggs were fertilized in sterilized water by
adding a drop of sperm, they developed to the pluteus stage
in sterile sea-water. I, therefore, consider it possible that
where authors describe a cleavage of the unfertilized sea-
urchin eggs in "normal" sea-water, the sea- water or the
egg had in reality suffered some change which had escaped
the notice of the observers. One might think of evaporation
and increase in the osmotic pressure of the sea-water. A
very slight increase in the osmotic pressure of the sea-water
is sufficient to cause the sea-urchin egg to divide into two
cells in the course of twenty hours. One might also think
of a change in the sea-water brought about by the putre-
faction of the dead eggs. Finally it is possible that a sub-

NATURAL DEATH AND FERTILIZATION 745

stance is perhaps formed (for example, an acid) in the dying
eggs which brings about a single cleavage.

The relations which exist between maturation and natural
death upon the one hand, and fertilization and prolongation
of life upon the other, lead us to the conclusion that a "fer-
tilization" must perhaps come to pass in every egg, even in
those naturally parthenogenetic. Only, according to our
idea, the act of fertilization is not identical with the mor-
phological process which is designated fertilization. It is
rather a chemical or a physico-chemical act which accelerates
certain (synthetical?) metabolic changes in the egg, which
occur in the egg under ordinary conditions also, only much
too slowly, The difference between naturally partheno-
genetic eggs and the eggs which must be fertilized before
they can develop consists perhaps in this, that to the latter
the catalytically working substance or complexus of condi-
tions must be added from the outside in order to accelerate
the synthetical ( ?) processes, while in the naturally partheno-
genetic eggs these substances are formed within the eggs
(possibly in conjunction with the processes of maturation).

The connection between the prolongation of life and fer-
tilization clearly points out that every purely morphological
theory of fertilization is incomplete and that a correct theory
of this process must have a physico-chemical basis. The
means of reaching this basis I see in further attempts at
causing development of unfertilized eggs through unequiv-
ocal physical and chemical means.

VII. CONCLUSIONS

1. Our observations and experiments seem to show that
in the same sea-water and under otherwise identical con-
ditions, mature but unfertilized starfish eggs soon die, while
immature as well as mature but fertilized eggs live longer.

2. It seems certain that the rapid death of the mature

746 STUDIES IN GENERAL PHYSIOLOGY

unfertilized starfish eggs is determined by internal condi-
tions connected with maturation and not by the bacteria
contained in the sea-water. The proofs for this are: First,
mature eggs die just as rapidly in sterilized sea-water free
from bacteria as in unsterilized water, and secondly, when
maturation is prevented artificially the eggs may continue to
live in water containing many bacteria.

3. We have shown that oxygen and free hydroxyl ions
accelerate the maturation of starfish eggs; that lack of
oxygen and a neutral or faintly acid reaction of the sea-
water inhibit or prevent maturation. The fact that the eggs
which remain immature in the ovaries of the starfish
maturate when brought into sea- water seems to find its
explanation in part at least through this.

4. When the maturation of starfish eggs is prevented
artificially through lack of oxygen, or the addition of an
acid to the sea-water, they remain alive much longer than
when they maturate. The eggs in which maturation has
already begun, or has just been completed, seem also to be
saved from rapid death by these means.

5. It seems to follow from these facts that the same
chemical processes do not necessarily underlie the process
of maturation and the process of fertilization. Fertiliza-
tion by spermatozoa, chemical or physical agencies, lengthens
the life of the egg, while the changes following the matura-
tion of the egg lead, sooner or later, to death (through
autolysis?). It is in harmony with what has been said that
the same treatment with acid which brings about artificial
parthenogenesis in mature starfish eggs inhibits the process
of maturation when used upon immature starfish eggs.

6. These facts corroborate a suggestion which I have
made before, that the fertilizing action of the spermatozoon
consists in this, that it carries into the eggs substances which
accelerate the course of certain (synthetical?) processes in

NATURAL DEATH AND FERTILIZATION 747

the egg. Such an acceleration might, for example, be
brought about through certain ions (for example, the
hydrogen ions of nucleic acid), yet the possibility that
such catalytic effects might also be brought about through
enzymes or other substances is of course not shut out. Yet
this fact must be considered: that we have been able to
produce artificially normal embryos capable of development
through ions, while the careful experiments of Cries con-
ducted in my laboratory, in which he attempted to find the
same to hold for enzymes, have thus far failed.

In conclusion I wish to thank my assistant, Mr. Neilson,
for the assistance which he rendered me in these experi-
ments.

XXXVII

ON THE PRODUCTION AND SUPPRESSION OF MUSCU-
LAR TWITCHINGS AND HYPERSENSITIVENESS OF
THE SKIN BY ELECTROLYTES1

IT has been shown in former publications that a slight
variation in the proportion and character of the electrolytes
in a tissue is capable of imparting to that tissue properties
which it does not possess ordinarily, and it has been sug-
gested that this fact might help us in recognizing the nature
of a number of nervous and muscular diseases, and also
possibly furnish a means of curing or mitigating them.
This paper contains some further contributions to the same
subject. It deals with the determination of electrolytes which
are liable to produce and inhibit hyperactivity of muscles and
hypersensitiveness of the nerves of the skin; and tries to
answer the question whether or not the stimulating and
inhibiting effects of ions are a function of their valency and
electrical charge.

I. THE PRODUCTION AND SUPPRESSION OF MUSCULAR TWITCH-
INGS BY ELECTROLYTES

1. Our muscles do not normally contract or twitch rhyth-
mically, but they do so in certain diseases. The main
electrolyte in our blood is sodium chloride. When we put a
muscle into a pure sodium-chloride solution of the right
osmotic pressure (i. e., isotonic with the muscle), the muscle
soon begins to twitch rhythmically, and these twitchings may
last for several days, or about as long as the muscle lives.
But when we add a very small, though definite, amount of a

1 University of Chicago Decennial Publications (1902), Vol. X, p. 3.

2 Part II, pp. 544, 559, and 692 ; PjOlgers Archiv, Vol. LXXXVIII (1901), p. 68.

748

MUSCULAR TWITCHINGS 749

soluble calcium salt, the twitchings will not occur, though
the muscle lives longer in such a solution than in a pure
sodium-chloride solution. I concluded from this that we
owe it to the calcium ions in the blood that our muscles do
not twitch or beat rhythmically like our heart.1

To test this idea further, Mr. W. E. Garrey and I under-
took, in 1889, a series of experiments, not yet published, on
the behavior of muscles in solutions of sodium salts whose
anions precipitate calcium. The muscle itself contains cal-
cium salts, and we considered it likely that these calcium
salts might help in preventing contractions. We therefore
thought that by putting the muscle into solutions of sodium
salts, which, by entering the muscle, precipitate the calcium
contained in it, we might produce still more powerful rhyth-
mical contractions than in a pure sodium-chloride solution.
This was found to be true. In solutions of sodium-fluoride,
-oxalate, -carbonate, -phosphate, etc., of the proper concen-
tration (1 gram-molecule in 8 liters of the solution), we
obtained similar, but more powerful, rhythmical contractions
than in sodium-chloride solutions of the same osmotic pres-
sure. Another series of observations confirms the idea that
it is due to the calcium salts in our body that our muscles do
not show any rhythmical contractions or twitchings. When
we inject into the body of an animal any salts that are liable
to precipitate calcium, we notice almost immediately twitch-
ings of all the muscles.2 It seems, therefore, rational that in
the pathology of muscular twitchings the concentration of
the calcium ions in the blood should be taken into considera-
tion. It is quite possible that abnormal conditions may arise
in the body which lead to an increase of such acids in the
circulation as diminish the amount of calcium ions in the

1 Part II, p. 518. See also S. RINGEB, Journal of Physiology, Vol. VII (1886) , p. 291
In this paper Ringer also mentions briefly the fact that Ba differs in its action from
Ca and Sr.

2 FBIEDENTHAL, Engelmanri's Archiv, 1901, p. 145.

750 STUDIES IN GENERAL PHYSIOLOGY

body, e. (/., oxalic acid, or others. The necessary outcome
would be muscular twitchings. In that case the administra-
tion of calcium salts might cure the disease.

2. In a recent paper I have shown that the antitoxic
effects calcium produces when added to a pure NaCl solution
are a function of its valency and the sign of its charge, inas-
much as similar effects can be produced by other bivalent or
trivalent kations (e. g., Mg, Sr, Ba, Zn, Fe, Co, Pb, Al, Or),
but not by bivalent or trivalent anions.1 The question arises
whether or not the inhibiting effects of Ca ions in the case
of rhythmical contractions of muscles are also a function of
the valency and electrical charge of the Ca ion. My earlier
experiments were not opposed to such a conclusion. I had
found that in ™ solutions2 of LiCl, NaCl, RbCl, and CsCl
rhythmical contractions occur, while small amounts of the
chlorides of Ca, Mg, Sr inhibit these contractions. I have
since continued these experiments, with the following results :
When we put a muscle (the gastrocnemius of the frog was
used in these experiments) into a ^ sodium-acetate solu-
tion, the twitchings of the muscle begin at once. The addi-
tion of from 3 to 4 c.c. of a m CaCl2 solution to 100 c.c. of a
^ sodium -acetate solution absolutely suppresses all twitch-
ings. But even half the amount suffices for practical pur-
poses, inasmuch as in this case only a few beats occur at the
beginning. MgCl2 and SrCl2 act like CaCl2. But BaCl2
acts altogether differently. An addition of 5 c.c. of a m
solution of Bad 2 to 10 c.c. ^ sodium-acetate solution not
only does not stop the rhythmical contractions, but makes
them more powerful. Instead of the rapid and rather weak
fibrillary twitchings which occur in a ^ sodium-acetate
solution, more tetanic and energetic contractions occur when
BaCl3 is added. I then tried whether the muscle is able to

1 Part II, p. 708.

2 By a ™ solution is meant a solution which contains 1 gram-molecule in 8 liters.

MUSCULAR TWITCHINGS 751

beat in a pure BaCl3 solution. It goes without saying that
in pure solutions of MgCl2, CaCl2, and SrCl2 a muscle does
not show any rhythmical contraction. In a ^ BaCl2 solu-
tion, however, the muscle beat for forty minutes; in a -f1
solution, for one and a half hours; in a f^ BaCl2 solution,
for over an hour; and in a ^ BaCl2 solution, for about
half an hour. The beats showed the same tetanic form char-
acteristic of the presence of barium. The fact that the beats
stop sooner in a f BaCl2 solution than in a fj. solution
is due to the poisonous effects of barium. The fact that the
beats stop also very soon in a -jfe BaCl2 solution is due to
the enormous absorption of water which occurs in such weak
solutions.1

Similar facts were found for Ba(NO3)2 and Ba (HO)S.
The most striking fact is that the stimulating power of Ba
salts is greater than that of the corresponding Na salts.
In a ^ Bad 2 solution the muscle may begin to beat in a
few minutes and may continue to do so for half an hour. I
have even occasionally noticed rhythmical contractions in a
y^g BaCl2 solution. But I have never noticed any rhyth-
mical contractions of muscle in a ^ NaCl solution, and as
a rule even in a ^ NaCl solution the twitchings begin only
after a long latent period and last but a short time.

Barium is, however, not the only bivalent kation whose
chloride possesses a higher stimulating power than that of
the chloride of univalent kations. It was found that the
chlorides of the heavy metals are also capable of producing
rhythmical twitchings or beats of muscles in much lower
concentrations than those found effective in the case of NaCl
or LiCl. In T^ solutions of ZnCl3 strong beats occurred,
which, however, did not last long on account of the rapid
imbibition of the muscle with water, as well as on account

iThe beats in BaCl2 solution often do not begin at once, but after a latent period
of from two to fifteen minutes.

752 STUDIES IN GENEKAL PHYSIOLOGY

of the poisonous effects of Zn. In stronger solutions than
^ no beats occurred. The same was true for ZnSO4.
Solutions of CdCl2 and Pb(No3)2 also gave rise to a few
contractions in the concentration of about ^¥ to T^.

The fact that the more concentrated solutions of the salts
of heavy metals did not act is probably due to their poison-
ous effect. It is, therefore, evident that there are a number
of chlorides with bivalent kations which are able to produce
rhythmical contractions at a lower concentration than NaCl.
It would, therefore, be wrong to ascribe the inhibiting effect
of Ca salts upon rhythmical contractions to the double
valency and the positive charge of the Ca ions.

3. Does the effectiveness of salts for the production of
rhythmical muscular contractions increase with the valency
of the anion? This is decidedly not the case, as the fol-
lowing table shows. In this table are given the minimal
concentrations of the solutions of various sodium salts in
which rhythmical contractions occur:

Salt Minimal Effective

Concentration

NaCl {|

NaBr ft

NaJ

Na acetate

Univalent anions -<

^Na formiate ^

f Na2 succinate ft

Bivalent anions -i ^HPO Sm

[Na2 oxalate ^

Trivalent anions \ XT

It is obvious that the power of favoring rhythmical con-
tractions in muscles is not an unequivocal function of the
valency of the anion. It is likewise obvious that the sodium
salts whose anions precipitate calcium powerfully, like sodium
fluorides, sodium oxalate, and sodium phosphate, are among

MUSCULAR TWITCHINGS 753

the most favorable salts to produce rhythmical twitchings.
Sodium citrate does not precipitate calcium in the tissues, but
prevents its precipitating other compounds, and therefore
practically makes calcium inactive. 1 But that the precipitation
and inactivation of calcium is perhaps not the only factor
involved is shown by the efficiency of sodium formiate. It
may be, however, that sodium formiate undergoes further
changes in the tissues, and that one of the products formed
acts upon calcium.

All these facts suggest that it might be worth while to
test the idea whether or not the pathological cases of mus-
cular hyperactivity and twitching are due to a lack of cal-
cium in the muscles (or blood), and whether the evil can be
mitigated by giving calcium salts to such patients. Experi-
ments must be made in animals to find out whether or not
such a treatment would do any harm before any therapeutical
experiments on patients should be attempted. It is our in-
tention to take up these experiments in the laboratory.

II. THE DIFFERENT EFFECTS OF CALCIUM IN THE CASE OF
MYOGENIC AND NEUROGENIC RHYTHMICAL CONTRACTIONS

1. While it seems easy to suppress, by the addition of
Ca, Sr, and Mg, rhythmical twitchings which originate in
the muscle itself, the question arises whether the same
means allow us to suppress, with equal ease, muscular twitch-
ings which originate through the central nervous system.
The simplest organism in which this can be tested is probably
the jelly-fish. These animals contract rhythmically. Their
central nervous system is contained in the margin of the
swimming-bell, while the center of the animal is said to con-
tain no nerve-elements except scattered neurons. By a cut

l SABBATANI has shown that although sodium citrate does not precipitate cal-
cium, it renders it inactive. In the presence of a sufficient quantity of sodium
citrate, calcium loses, e. g., its coagulating effect: Archives Italiennes de Biologic,
Vol. XXXVI (1901), p. 397.

754 STUDIES IN GENERAL PHYSIOLOGY

parallel to the edge we can divide the animal into a marginal
part, which contains the central nervous system, and a cen-
tral part, without a central nervous system.

When this operation is performed, the margin will go on
beating in sea-water, while the center will not beat. Ro-
manes, who was (as I believe) the first to make this experi-
ment, drew the conclusion that the central nervous system
was the originator of the automatic contractions of this
animal. From previous experiments of Aubert,1 Howell,2
and Greene3 on the heart, and my own experiments on the
muscles, I concluded that the center of a jelly-fish (Gonio-
nemus) did not beat in sea- water on account of the presence
of certain ions in sea- water, especially calcium, and I showed
that the center of a Medusa will beat rhythmically in pure
NaCl or NaBr solution.* The center of a Medusa whose
margin is cut off seems then to behave, to a certain extent,
like the striped muscle. It was of some importance to
find out how far this analogy goes. The following six solu-
tions were prepared:

1. 100 c.c. £ NaCl

2. 100 c.c. f NaCl + Jc.c. -ft Ca(No3)2

3. 100 c.c. f NaCl+ 1 c.c. -ft Ca(No3)2

4. 100 c.c. f NaCl + 2 c.c. -ft Ca(No3)2

5. 100 c.c. £ NaCl + 4 c.c. -ft Ca(No3)2

6. 100 c.c. f NaCl + 8 c.c. -ft Ca(No3)2

In solution 1 the center of a Medusa begins at once to con-
tract very rapidly. The velocity of contractions steadily in-
creases and very soon it becomes impossible to count the
contractions. Occasionally the same happens in solution 2.
But in the solutions 3 to 6 the center at first remains per-
fectly quiet. After a latent period of about ten minutes,
often, but not always, contractions begin in solutions 3 to 5,

1 AUBERT, Pflilgers Archiv, Vol. XXIV (1881), p. 361.

2 HOWELL, American Journal of Physiology, Vol. II (1898), p. 47.

3 GREENE, ibid., p. 82. * part n, p. 559.

MUSCULAE TWITCHINGS 755

or even in 6, which last sometimes as long as fifteen minutes.
These contractions are not as rapid as those observed in a
pure NaCl solution, and resemble more the normal contrac-
tions of a Medusa in sea-water. A series of experiments
was undertaken to find out the minimal amount of Ca re-
quired to prevent completely all contractions in a pure NaCl
solution. In a mixture of 100 c.c. of a f NaCl solution
-f- 3 c.c. of a \m Ca(NO3 ) 2 solution no contractions occurred.
A series of experiments with a slightly greater amount of
CaCl2 were made with the same result.

The same inhibitory effect can be produced if, instead
of Ca, Sr or Mg is used. But Ba behaves altogether differ-
ently. The following solutions were tested:

100 c.c. f NaNo3

100 c.c. f NaNo3 + 1 c.c. m BaCl2
100 c.c. » NaNo3 + 2 c.c. m BaCl2
100 c.c. f NaNo3 + 4 c.c. m BaCl2
100 c.c. f NaNo3 + 8 c.c. m BaCl2
100 c.c. f NaNo3 -f 16 c.c. m BaCl2

When the center of a Medusa was thrown into any of
these solutions, the rhythmical contractions began at once.
The center behaved as if the Ba ion had not been present,
with this difference, however, that the solutions with a
larger amount of barium were more poisonous than a pure
NaCl solution. Ba has, therefore, little or no inhibitory
effect upon the center of a Medusa.1

The analogy between the effect of ions upon muscle and
the center of a Medusa goes still farther. I pointed out
that possibly the Ca ions in the sea-water and the tissues of
the Medusa prevent the isolated center from beating in sea-

i Since this was written I have received, through the kindness of Professor
Sabbatani in Cagliari, a paper published by his assistant, Dr. Regoli, in which the
latter shows that, while Ca and Sr diminish the irritability of the cerebral
cortex, Ba has the opposite effect ; EEGOLI, "Azione dei metalli alcalino-terrosi sulla
eccitabilita elettrica della corteccia cerebrale," Bollentino d. Societate tra i Cultori
delle Scienze etc. in Cagliari, Torino, 1901.

756 STUDIES IN GENERAL PHYSIOLOGY

water in the same way as the presence of Ca in the blood
seems to prevent our muscles from beating. In order to
test this idea, I added to the sea-water various salts which
precipitate Ca, e. g., NaF and Na3HPO4. I found that
when a little more of these salts had been added than re-
quired to precipitate all the Ca in the sea-water, the center
behaved indeed in the same way as if it had been put into a
pure NaCl solution. When a little less Na3HPO4 was
added, the beats began after a latent period, which varied
according to the amount of Na3HPO4 added. Kapid con-
tractions began at once when 32 c.c. of a ^ Na3HPO4
solution was added to 68 c.c of sea-water. The same result
was obtained when 16 c.c. of a normal NaF solution was
added to 100 c.c. of sea- water.

The addition of about 13 c.c. of m sodium-citrate solution
to 100 c.c. of sea- water also brought about immediate con-
traction of the isolated center. This salt does not bring
about a precipitation of Ca in the sea-water or the tissues,
but excludes the action of Ca ions in another way.

I did not succeed in bringing about such results with
the addition of Na3SO4 to sea-water. Even the addition of
32 c.c of m Na3SO4 to 100 c.c. of sea-water did not give
rise to contractions, although the irritability of the center
was increased. Experiments with the addition of NaHCO 3
remained also negative. But as only a few experiments
were made with Na3SO4 and NaCHO3, it is possible that a
continuation of the work might lead to positive results.

It is, therefore, obvious that the centers can be caused to
beat through a diminution of the amount of Ca they contain,
and it may be further argued that the presence of Ca in the
sea- water is the cause, or at least one of the causes, that pre-
vent the centers from beating in sea-water.

It should, however, be added that, while a certain diminu-
tion of Ca in the center is necessary for the development of

MUSCULAR TWITCHINGS 757

rhythmical contractions, the diminution has its limit. It
appears that, if too much Ca is removed from the tissues,
the beats will also cease. This is demonstrated by the fol-
lowing facts: When we put the center of a Medusa into
sea-water to which enough sodium citrate has been added,
beats begin at once, last for a certain time, and then cease.
If at this time the centers are put back into sea- water with
less or no sodium citrate, beats will begin again. The ex-
planation of this phenomenon seems to be as follows: The
normal center of a Medusa contains too much Ca for sponta-
neous rhythmical contractions. If we put a center into sea-
water to which a large amount of NaF, Na3HPO4, or sodium
citrate has been added, so much of the salt will diffuse at
once into the organism that at least in the superficial cells
enough Ca will be eliminated from the field of action to
allow the spontaneous contractions to begin. Subsequently
the same will happen in the deeper cells. The process of
elimination of calcium in the cell proceeds, and very soon
a period comes when the loss of Ca in all the cells will be too
great for the contractions to go on. If, as soon as this
occurs, the center is thrown into normal sea-water, or sea-
water with only a little sodium citrate or phosphate, citrate
and phosphate anions will diffuse back from the tissues into
the sea- water, or Ca ions will diffuse into the cells, or both
phenomena will occur, and beats will again begin. The same
reasoning applies probably to the rhythmical contractions of
muscles and the apex of the heart.

2. When we put the margin containing the central
nervous system into a pure NaCl solution, it behaves very
much like the center, e. g., it begins to beat very rapidly,
and the rapidity of the beats increases, at first steadily, until
the poisonous effects of the pure NaCl solutions make them-
selves felt. But even the addition of large quantities of Ca
does not inhibit these contractions. For instance, when we

758 STUDIES IN GENERAL PHYSIOLOGY

add from 2 to 5 c.c. of a f m solution of Ca(NO3)2 to 100 c.c.
of ^ NaCl solution, the margin at once begins its rapid
beats. The only effect the addition of calcium has is to make
the rate of the beats a little slower than without calcium. I
thought at first that the stimulus of the wound caused by
the cutting off of the margin might be responsible for these
contractions in the presence of calcium. But this is not the
case, for if we put a whole Gonionemus intact into any of
these solutions, it behaves like the isolated margin. The only
possible inference is that the margin is much more immune
toward the inhibiting effects of calcium than the center, a
fact which I have pointed out already in a former paper.1
In a pure CaCl2 solution the margin will not beat.

Inasmuch as the essential difference between center and
margin which accounts for this difference in the effect of cal-
cium is the presence of the central nervous system in the mar-
gin, it may follow from these observations that for the supres-
sion of twitchings of a nervous origin larger doses of calcium
might be required than for the suppression of twitchings of
muscular origin. Preliminary experiments on the motor
nerves of frogs seem to harmonize with this idea. This sug-
gests the possibility that, while a calcium treatment might
be advisable for the cure of myogenic twitchings, for the
suppression of neurogenic twitchings so much calcium might
be required as to exclude the use of this remedy. This, too,
is a point which further experiments on animals must decide
before the matter may be tried in patients.

III. THE PRODUCTION OF HYPERSENSITIVENESS OF THE SKIN
BY ELECTROLYTES

1. In a former paper I have shown that, aside from the
rhythmical twitchings, the salts whose anions precipitate or
inactivate calcium also make muscles and motor nerves sensi-

1 Part II, p. 692.

MUSCULAR TWITCHINGS 759

tive to stimuli which normally would not affect these organs.
For example, when we put a fresh muscle for one or more
minutes into a ™ solution of sodium citrate, a peculiar
form of irritability arises (contact-irritability).1 Whenever
the muscle is taken out of the solution it goes into powerful
tetanic contractions, which cease at once and give way to
relaxation of the muscle as soon as the latter is put back into
the solution. When this hypersensitive condition is once
established, the contractions can be produced whenever the
muscle is changed from any aqueous solution to any other
non-aqueous medium, while the contractions cease when the
muscle is put back into an aqueous medium, no matter whether
the latter be a solution of an electrolyte or a non-conductor.
It is rather striking that these phenomena do not occur when
the above-mentioned solutions call forth at once the rhyth-
mical contractions mentioned in the previous part of this
paper. It almost looks as if there existed two allotropic
states of the muscle substance, the one giving rise to rhyth-
mical twitchings, the other to the peculiar tetanic contrac-
tions (contact-reactions) just referred to.2 Ultimately, how-
ever, in all cases, rhythmical twitchings are produced.

As far as motor nerves are concerned, I have shown in
the same paper that the same salts which produce this con-
tact-reaction produce hypersensitiveness of the nerve and
ultimately rhythmical contractions of the muscle when acting
upon the nerve alone.

It might be mentioned here in parenthesis that these facts
may throw some light upon the action of cathartics. All the
salts which give rise to the above-mentioned contact-reaction
or hypersensitiveness act as cathartics when introduced into
the intestine. The common explanation of their action is

1 Part II, p. 692,

2 This difference is emphasized by the fact, found by my pupil, Dr. Zoethout
that an addition of potassium favors the contact-reaction. As far as rhythmical con-
tractions are concerned, K has an inhibiting effect.

760 STUDIES IN GENERAL PHYSIOLOGY

the one which, I believe, was first suggested by Schmiede-
berg, namely, that these salts prevent the absorption of
liquids from the intestine, and that this retention of liquids
causes the cathartic effect. I will not deny the effect of these
salts upon the phenomena of absorption of water from the
intestine,1 but it is obvious from our experiments that the
same salts must increase the irritability of the nerves and
muscles of the intestine, and that this must facilitate the
production of peristatic motions, possibly through the mechan-
ical or contact-stimuli of the fseces upon the nerve-endings
or the muscular wall of the intestine.

2. These experiments suggested the idea whether or not
electrolytes are capable of producing also a hypersensitive-
ness of the skin and conditions that may be comparable to
the conditions of hypersesthesia or hyperalgesia. It is well
known that when we suspend a pithed frog vertically so that
its legs hang down, the latter will be lifted at once when
they are dipped into an acid or alkali of a certain concentra-
tion, while no such reaction occurs when they are dipped
into water. The reaction of the animal to acid may be so
violent as to suggest to a layman the idea that it is suffering
intense pain. I wondered whether by an alteration of the
nature and proportion of ions in the skin the sensitiveness
could be increased or varied in such a way as to make the
skin as sensitive to the contact with pure water as it natur-
ally is to strong acid. The experiments resulted in my find-
ing certain solutions of electrolytes which did not seem to
affect the animal directly, but yet made it extremely sensi-
tive toward contact with water. The best solutions for this
purpose are, as far a§ my present experiments go, A1C13, and
sodium-citrate solutions. The way of proceeding is as fol-
lows: A number of solutions, say A1C13, are prepared,
namely, f|, "^ », «, and possibly f. Then the weakest

1 CUSHNY AND WALLACE, American Journal of Physiology, Vol. I

MUSCULAR TWITCHINGS 761

of the solutions is first brought in contact with the feet
of the frog. If the feet are not withdrawn, the next
stronger solution is used, and, if no reaction occurs, the next
stronger. If one thus succeeds in keeping the feet of the
animal for one minute or more in the A1C13 solution, subse-
quent contact of the feet with common tap-water or distilled
water makes the animal act as if the water caused the most
excruciating pain. The feet are violently withdrawn, rubbed
against each other in a way that one notices otherwise only
when the feet are dipped into strong acids. If the A1C13
solution chosen is too strong, the animal will not leave his
feet in the solution, but will try to withdraw them. But in
that case its attempts at withdrawing its feet from the solu-
tion are never as violent as the subsequent attempts at with-
drawing the feet when brought into contact with common
water. The stronger the solution of A1C13 is in which the
feet had been kept, and the longer they had been kept in
the solution, the stronger their sensitiveness toward water
will become.

Sodium citrate acts very similarly to aluminium chloride.
As the latter is slightly acid and sodium citrate slightly
alkaline, the possibility was suggested that the H and HO
ions are responsible for the hypersensitiveness. While it is
possible to produce occasionally a slight hypersensitiveness
toward common water by a pure solution of NaOH or HC1,
the results are very unreliable. It is practically the same if
one tries to use NaCl solutions to which slight and varying
quantities of HC1 or NaOH have been added. Better results
can be obtained with solutions of oxalates, sulphates, car-
bonates, and phosphates. The sodium salts are preferable
to the potassium salts, for the animal withdraws its feet
much more rapidly from the solution of a potassium salt
than from the solution of the corresponding sodium salt.
This makes it difficult in the case of potassium salts to

762 STUDIES IN GENEKAL PHYSIOLOGY

saturate the foot with the sufficient number of ions to induce
the hypersensitiveness.

It goes without saying that the hypersensitiveness which
can be produced by A1C13 and sodium citrate does not make
itself felt toward water alone, but to salt solutions also.
One can find a minimal concentration for each solution of
an electrolyte at which a pithed frog almost instantly with-
draws its feet when they come in contact with the solution.
This minimal concentration is considerably lowered after a
treatment of the foot with an A1C13 or sodium-citrate solution.

The production of hypersensitiveness is only one side of
the problem. The mitigation of the hypersensitiveness is
the other side. The violent reactions of a frog when its
feet are dipped in tap- water after a treatment with A1C13
can be stopped instantly when the feet are put into a normal
solution of cane-sugar. When weaker solutions of cane-
sugar are used the feet are withdrawn, and the attempts at
withdrawing become more noticeable and violent the weaker
the sugar solution is. Very concentrated solutions of urea,
e. g., 2n solutions, act similarly, but not so powerfully as
cane-sugar. Glycerin solutions gave no such results ;
neither have I been able to find as yet any solution of an
electrolyte which acted this way. The fact that only very
concentrated solutions of cane-sugar or urea inhibited the
hypersensitiveness gave rise to the idea that the diffusion of
water out of the foot might be the inhibiting factor, and
that a stream of diffusion in the opposite direction, namely,
from the outside into the skin, might give rise to a with-
drawal of the foot. The latter idea could be tested. When
the feet of a pithed .frog are dipped into a normal solution
of cane-sugar, they are not withdrawn, no matter how long
they remain in the solution. But if subsequently (after
several minutes) the feet are put into pure water, after a
few (five to ten) seconds the feet are energetically withdrawn.

MUSCULAR TWITCHINGS 763

In this case, obviously, water diffuses into the skin, which
previously had lost water.

There may be electrolytes which act similarly to cane-
sugar, but I have not yet found them. Every solution of
an electrolyte causes, above a certain concentration, an
immediate withdrawal of the feet, and this withdrawal is
the more energetic the more concentrated the solution.
This differs from the behavior of sugar and urea, which
above a certain concentration have the opposite effect.

The lowest concentration at which the solutions of various
electrolytes will cause a pithed frog to withdraw its feet
instantly or in from five to ten seconds, is about as follows :

HC1, 5f ff or less

NaOH, ft or less CaCl2 f

. XT ' o ™ >• % or a little less

AgNO3, ^ or less

FeCl3, ^ or less

HgCl2 to

AlCla, _ f

g III/ IU 2

It almost looks as if the coagulating effect of the kations
upon proteids was of some importance. The powerful effects
of Ag, Cd, and Hg interfere somewhat with the conclusion
that we are dealing with a pure valency effect, which otherwise
seems to make itself felt. If, instead of the chlorides, the nitrates
or sulphates of the same metals are chosen, the order of effi-
ciency seems to remain practically the same, as far as can be
judged from an as yet incomplete series of experiments.

As far as the anions are concerned, the order of efficiency
is for the sodium salts about as follows:

Na2 oxalate f NaHCO3

Na3 citrate, % Na formiate m

Na2SO4, ? Na2 succinate f 1 ° s

NaHP04,f NaCl

NaF, f to f

764 STUDIES IN GENEKAL PHYSIOLOGY

In this case, as in the case of rhythmical contractions,
the oxalates and citrates are the most powerful anions of
this series. It is clear that, in the determination of the
lowest concentration of a salt which is still able to cause the
immediate withdrawal of the foot, one must remember that
a number of solutions (e. g., AgNO3, A1C13, FeCl3 , HC1,
NaOH, Na3 citrate, etc.) have an after-effect which makes
itself felt in an increase of irritability. Other solutions
(e. g., those of calcium salts) may possibly have the opposite
effect, namely, to raise the threshold of stimulation for sub-
sequent tests.

It was of some interest to ascertain whether the results in
these experiments were produced through an action of the
electrolytes upon the nerve-endings, or upon the nerves
themselves. In experiments upon frogs whose skin had been
removed from the feet, the results described in this paper
could not be produced. The experiment of putting the nerves
themselves into the above-mentioned solutions remained prac-
tically without effect. It is possible that with solutions of
much greater concentration results may be obtained. It is,
therefore, certain that the results observed in our experiments
are due to an action of the electrolytes upon the nerve-endings
in the skin, and not to an action upon the sensory nerves.1

IV. CONCLUSIONS

The experiments mentioned in this paper were undertaken
with two aims in view, a practical and a theoretical one. A.B
far as the former is concerned, it follows from our investiga-
tions that abnormal muscular twitchings and contractions
may be brought about in an organism by a reduction in the
proportion of calcium (or magnesium) in the muscles or the
blood, or an increase in the proportion of Na and other

iThe chemical irritability of muscles is, as far as electrolytes are concerned,
also greater than that of motor nerves. The reverse is true for electrical stimula-
tion.

MUSCULAR TWITCHINGS 765

kations. In view of the fact that thus far no explanation
has been found for pathological phenomena of this kind, it
becomes of some importance to see whether or not in certain
of these diseases the relative amount of calcium ions in the
blood is diminished. If this should be the case, the adminis-
tration of calcium would be the cure for these diseases, which
thus far have been beyond medical control. It is also
apparent from our experiments that for the 'suppression of
neurogenic twitchings or contractions more calcium may
possibly be required than for the suppression of myogenic
twitchings. There has thus far been no clue as to the origin
of hypersensitiveness or hyperalgesia of the skin. Our ex-
periments show that slight variations in the proportion of
certain ions in the skin can cause an enormous hypersensi-
tiveness.

As far as the theoretical side of the paper is concerned,
it was our aim to test the idea whether or not the "stimulat-
ing" and inhibiting effects of ions are an unequivocal func-
tion of their electrical charge or valency. Over a year ago
I tested the same idea without being able to obtain positive
results, and nothing was said about the subject in the paper
in which the results were published.1 The test was con-
tinued in the above-mentioned experiments, with results
which, in my opinion, are equally questionable, if not alto-
gether negative.

1 Part II, p. 692.

XXXVIII

ON THE METHODS AND SOURCES OF ERROR IN THE
EXPERIMENTS ON ARTIFICIAL PARTHENOGENESIS1

1. BECAUSE of various papers by European authors who
have encountered difficulties in repeating or continuing my
experiments on artificial parthenogenesis I wish to make a
few remarks on the methods and the sources of error in
these experiments. I do not need to dwell upon the impor-
tance of sterilizing the sea- water, the instruments, the hands,
and the animals themselves; it is self-evident. I wish
in this connection to mention only the greatest sources of
error, namely, the tendency of males, especially ripe sea-
urchins, to fill the sea-water in the pail in which they are
brought into the laboratory with sperm. It is therefore
advisable to keep the females isolated for twenty-four hours
or if possible even longer in sea-water free from sperm
before using them in the experiments on artificial partheno-
genesis. If one has taken the necessary precautions against
infection with sperm, the next step is to bring the unfertil-
ized eggs to development. In the eggs of sea-urchins the
only effective method which is known thus far by which
they can be made to develop parthenogenetically consists in
keeping the eggs for about one and one-half hours in sea-
water the osmotic pressure of which has been increased a
definite amount. In general it is immaterial how this
increase in osmotic pressure is brought about, whether
through evaporation of the sea- water or through the addition
of salt or sugar or urea to the sea-water. If we wish to
obtain many and, as nearly as possible, normal Iarva3, the
choice of methods is somewhat more limited. I find after

1 Archiv fiir Entwickelungsmechanik der Organismen, Vol. XIII (1902), p. 481.

766

EXPERIMENTS ON ARTIFICIAL PARTHENOGENESIS 767

all my experience that the addition of potassium or sodium
salts, especially potassium chloride and sodium chloride, is
perhaps the best. The degree of increase in concentration
is of great importance. If the correct concentration is not
struck, failure will result, and it is remarkable how greatly
the necessary concentration varies in different series of
experiments. Whether the variations are exclusively of an
individual character and correspond to the different states of
maturation of the eggs I will not endeavor to say. Possibly
temperature also has some effect. In order to meet all these
possibilities I always work with a series of solutions. In
this way I am certain to obtain good results in at least one
of the solutions. I use as a stock solution a 2^ normal
NaCl or KC1 solution; that is, a solution which contains
about 186 g. of KC1 in the liter of solution. In my experi-
ments the solutions were accurately titrated, but this is
superfluous for most purposes. I take six dishes, each con-
taining 100 c.c. sea-water, and add to these six dishes the
series of 8, 10, 12, 14, 16, 18 c.c. of the above 2^ normal
NaCl solution. The unfertilized eggs of the sea-urchin are
then distributed into these six dishes (and in addition into a
control dish containing pure sea- water).

Differences also exist regarding the time during which
the eggs must remain in these solutions. It is therefore
necessary to remove, not all the eggs at once, but at various
intervals after about one-half, one, one and one-half, and two
hours. In this way one will certainly strike the optimal
concentration and time of experiment. Potassium chloride
has the advantage that it leads usually to the formation of a
single embryo from each egg, while when sodium chloride
is used more than one embryo is usually formed from an egg.
The formation of the skeleton, however, probably occurs
somewhat better when sodium salts are employed than when
potassium salts are used. Mr. Hunter obtained very satis-

768 STUDIES IN GENERAL PHYSIOLOGY

factory parthenogenetic plutei by using sea-water the con-
centration of which had been increased 30 per cent, to 40
per cent, by evaporation. When the eggs were introduced
for one to two hours into this concentrated sea- water they
developed beautifully when returned to normal sea-water.

A second important circumstance which perhaps plays a
role in these experiments is the temperature. The experi-
ments at Woods Hole (as well as Wilson's experiments in
Beaufort) were all made at summer temperature when the
temperature of the water was 20° C. or higher. In Califor-
nia the temperature varied considerably in my experiments.
It was often pretty low and I was occasionally unsuccessful
in bringing about artificial parthenogenesis. I attributed this
at that time to the immaturity of the eggs. Possibly this was
right, and possibly this explains the negative results of most
of the European investigators who worked in winter. Since
then, however, I have thought that perhaps the temperature
affects the results of the experiments in such a way that
below a certain temperature artificial parthenogenesis does
not occur, -or at least only with difficulty. This idea is
strengthened by a letter from Mr. Doncaster who has
worked in Naples, and who informs me that he at first
obtained only negative results, that he then suspected, how-
ever, that the temperature of the water in Naples was too
low, and so made experiments in water of the temperature
of about 20° C. In the latter case he obtained positive
results.

2. What has been said thus far refers only to experiments
on sea-urchin eggs, especially Arbacia. Especial care is
necessary when working with starfish eggs. A. Mathews
has observed that the unfertilized eggs of starfish (Asterias)
after maturation in sea-water can be made to develop by
shaking, and that a time exists at which the agitation con-
nected with transferring the eggs from one dish to another

EXPERIMENTS ON ARTIFICIAL PARTHENOGENESIS 769

is sufficient to obtain larvae.1 Eggs as sensitive as this must
be carefully handled in two directions if one does not wish
to obtain deceptive results. First it is necessary to transfer
the eggs from one dish to another in such a way that every
mechanical agitation is done away with. This is best done
by using pipettes with a wide opening for sucking up and
transferring the eggs. The latter manipulations must then
be made with the greatest care. The second precaution
consists in this, that whenever the experimental eggs are
transferred from one solution to another or into sea-water the
same mechanical manipulation must be repeated in exactly
the same way with the control eggs. In this way it can
be determined whether the parthenogenetic development in
individual cases is attributable to mechanical agitation, or to
other agents which one employs. With these precautions we
have made a series of experiments this summer on Asterias
eggs and have found up to the present time that, independ-
ently of mechanical agitation, only two methods lead to
artificial parthenogenesis in starfish eggs. First, the intro-
duction of the eggs for from three to twenty minutes into
sea- water to which 3 to 5 c.c. of a y1^ normal HC1 or some
other inorganic acid has been added to each 100 c.c. of sea-
water. The second method which was discovered by my
pupil, Mr. A. W. Greeley, consists in keeping the eggs, after
lying for a certain time in sea-water, on ice for a number of
hours. Other methods all gave negative results, especially
heating the eggs which Mr. Greeley also tried. Neither did
we succeed in obtaining clear results through the abstraction
of water from the egg, so that I suspect that in my earlier
experiments perhaps, in which I found starfish eggs to
develop through an increase in the concentration of the sea-
water, mechanical agitation really caused the development.

1 1 have since found that the eggs of the starfish can develop without any notice-
able external cause. [1903]

770 STUDIES IN GENEKAL PHYSIOLOGY

I believe also that Delage has in part been led into error by
this circumstance when he asserts that about every physical
and chemical factor brings about artificial parthenogenesis.
I do not believe that such an assertion could be made on the
basis of experiments on sea-urchin eggs. In sea-urchin
eggs agitation does not act this way and this source of error
which is so inconvenient in working with starfish eggs does
not exist here. Nevertheless, I made it a rule from the
first to expose the control eggs to the same mechanical agita-
tion in the experiments with sea-urchin eggs as the experi-
mental eggs themselves.

3. The precautions necessary for the experiments on star-
fish eggs must also be used in the experiments on the eggs
of Annelids, Chsetopterus, and Amphitrite. In both these
forms it has been possible this summer to bring about arti-
ficial parthenogenesis through shaking and mechanical agi-
tation of the eggs. In Chsetopterus, however, this result is
less certain than in Amphitrite. If the unfertilized eggs of
Annelids are allowed to remain in ordinary sea-water without
jarring the vessel, the eggs do not develop into larvae any
more than do starfish eggs. We cannot speak of a "natural"
parthenogenesis of these forms. If, however, they are
allowed to remain for thirty minutes in the sea-water the
unfertilized eggs of Amphitrite can»be made to develop into
larvae by squirting them from one vessel into another by
means of a pipette. This does not succeed with every culture,
but still very frequently.1 It is possible, however, to cause
the unfertilized eggs of Amphitrite to develop every time
without agitation, when they are introduced into sea- water to
which a small but definite amount of a soluble calcium salt
has been added. It is not necessary to return the Amphi-
trite eggs from such a solution to sea-water. They develop

1 1 suspect that the skaking affects the development of the egg only in an indirect
way. [1903J

EXPERIMENTS ON ARTIFICIAL PARTHENOGENESIS 771

in such a solution to swimming larvae. Just as hydrogen
ions bring about the development of larvae from starfish eggs
calcium ions bring about the development of Amphitrite
eggs. The addition of 2 to 5 c.c. of a normal calcium-nitrate
or calcium-chloride solution to 100 c.c. of sea-water is suffi-
cient for this purpose.

I repeated and confirmed this year my earlier experiments
on the specific effects of potassium ions on the development of
unfertilized Chaetopterus eggs. These eggs develop when
a small but definite amount of any soluble potassium salt
(KC1, KNO3, K2SO4) is added (about 1 to 2 c.c. of a 2^
normal solution of one of these salts to 100 c.c.) to sea-water.
It is not necessary to remove the eggs from this solution and
to return them to normal sea-water. It may perhaps be well
to emphasize especially that calcium and potassium ions have
no specific effect upon starfish eggs, that potassium ions are
in the same way unable to cause the development of Amphi-
trite eggs, and that calcium ions are ineffective in the case of
Chaetopterus eggs.

4. The following facts are also indirectly of importance
for the methods of the experiments on artificial partheno-
genesis. All the ions which bring about parthenogenesis in
starfish, Amphitrite, and Chaetopterus, also bring about at the
same time agglutination of these eggs and the formation of
giant embryos. The problem at which Driesch once worked,
and which in sea-urchins is beset with great difficulties,
namely, to bring about the coalescence of the contents of
several eggs, succeeds beautifully in these experiments, and
to a large extent especially in the eggs of starfish. I need
scarcely emphasize the fact that it is of great theoretical
importance that the ions which bring about artificial parthen-
ogenesis also in a definite sense and at the same time alter
the physical state of the egg. I have not yet succeeded in
finding a specific ion which brings about the development of

772 STUDIES IN GENERAL PHYSIOLOGY

the unfertilized eggs of sea-urchins. It would be of interest
to determine whether such an ion when it is found also brings
about an agglutination of the sea-urchin eggs.

In other forms, Nereis, Podarke, and Phascolosoma, the
experiments have been carried far enough so that we can say
that artificial parthenogenesis (swimming larvae) is possible in
these. The experiments, however, have not yet been worked
out sufficiently in order to allow them to be published.

We can say with certainty of the methods given here that
they lead to successful results in the American forms on the
Atlantic ocean. In the attempt to discover new methods it
may perhaps be well to keep the following (theoretical) con-
siderations in view, which I have discussed in greater detail
in various earlier papers. The artificial methods for obtain-
ing parthenogenesis must be able, first of all, to favor the
liquefaction or other destruction of the nuclear membrane.
Secondly, they must also alter in a definite way the physical
properties of the protoplasm (viscosity, etc.). It seems that
in the eggs in which artificial parthenogenesis has succeeded
thus far (and possibly in many, if not all, other eggs) chemical
changes take place under natural circumstances in the unfer-
tilized egg, which endeavor to alter the egg in the two direc-
tions mentioned above; that these, however, under ordinary
conditions occur so slowly that the egg dies before it under-
goes actual cell-division. Those circumstances which are
able to accelerate these natural processes will also bring about
the development of the unfertilized egg.

INDICES

AUTHORS' INDEX

Allman, 116, 117, 118, 128, 129.
Andrews, E. A., 289.
Araki, 372.
Arrhenius, 450.
d'Arsonval, 496.
Aubert, 187, 371, 404, 754.

Baer, 345.

Bardeen, 363.

B6clard, 426.

Bemmelen, van, 466.

Bert, Paul, 9, 378.

Berthold, 506.

Bickford, E., 336.

Bickford, Miss, 629.

Biedermann, 420, 518.

Birukoff , 449.

Bonnet, Charles, 118, 119, 228, 250, 627.

Boveri, 325, 331.

Breuer, 186, 189.

Budgett, 507, 636.

Bumpus, 282, 597.

Bunge, 310, 370.

Bunsen, 236.

Castell, 404.
Chauveau, 496.
Chun, 290.
Glaus, 356.
Conklin, Dr., 576.
Contarini, N., 167, 171.
Cooke, Miss, 450, 469.
Cremer, 499.
Cunningham, J. T., 190.
Cushny, 511, 760.

Dalyell, 116, 117, 127, 128, 129, 136, 175, 250,

627.

Danilewsky, 482, 491.
Darwin, 77, 81, 286, 358.
Delage, 187, 729, 770.
Demoor, 384.
De Vries, 231.
Dewitz, J., 23, 111, 580.
Diquemare, 167.
Dohrn, A., 338.
Doncaster, 768.
Driesch, 589, 629, 676, 771.
Driesch, Hans, 105, 143, 306, 307, 323, 332.
Dubrochet, 378.

Duclaux, 635.
Duhamel, 119.
Dutrochet, 426.

Edwards, 426.
Engelmann, 14, 15, 73, 77.

Faraday, 489, 496.
Farkas, 535,
Fernet, 236.
Fischer, 597, 733, 740.
Forchhammer, 225, 242.
Fraenckel, 535.
Friedenthal, 5a5, 749.
Friedlander, 438.
Friedlander, B., 221, 357 ff.

Galileo, 81.

Garrey, W. E., 708, 749.

Gemmill, 597.

Geppert, 479.

Giard, A., 686.

Gleichen-Russwurm, 88.

Goltz, 189, 368, 437, 438.

Graber, 12, 13, 16, £58.

Greely, 729.

Greeley, A.W., 769.

Greene, 530, 535, 754.

Groom and Loeb, 89, 180, 272, 290.

Gruber, 508.

Hardy, 711.

Hammarsten, 623.

Harper, 739.

Hegel, 38.

Heider, A. von, 201, 2:>0.

Hem pel, 310.

Herbst, 205, 307, 575, 589, 590, 595, 627,

Hermann, 370.

Hertwig, 255, 325, 580, 595.

Hertwig, O., 684.

Hertwig, R., 581.

Hilger, 229.

Hoeber, 535.

Hoek, 340.

Hoff, Van 't, 450, 496, 499.

Hoffmeister, 77, 78, 358, 513.

Hogies, 88.

Hoppe-Seyler, 225, 244, 245, 371, 644.

Howell, 513, 519, 533, 754.

Huxley, 286.

775

776

STUDIES IN GENERAL PHYSIOLOGY

Jacobsen, 241.
Jan6sik, 543.

Kahlenberg, 451, 452, 474.
Kohlrausch, 456.
Ktihne, 372, 440, 448.
Kulagin, 580.

Lewis, 728, 738.
Lingle, D. J., 708.
Locke, 526, 533, 549.
Lubbock, 10, 11, 55.
Ludwig, 238.
Lyon, 87.

Mach, Ernst, 69, 81, 87, 88, 187, 189, 202, 499.

Marshall, W., 117, 283.

Massart, 283.

Mathews, 579, 729, 768.

Maxwell, 447.

Mead, 540, 591, 656.

Metschnikoff, 637.

Miescher, 637.

Mingazzini, P., 217, 222.

Moore, A., 708.

Moore, Miss, 696.

Morgan, 577, 591, 607, 618, 644, 676.

Miiller, Johannes, 9.

Mtiller, Wilhelm, 43.

Neilson, 733, 740.
Noll, 202, 203.

Norman, 540, 577, 578, 607, 618, 644.
Norman, W. W., 399.

Nussbaum, 117, 205, 321, 322, 323, 336, 341,
508, 580.

Ostwald, 450, 456, 459, 476, 490.
Ostwald, Wolfgang, 292.

Pasteur, 636, 689.
Pauli, Dr. W., 548, 622.
Pemsel, 547.

Pfluger, 202, 323, 371, 404.
Plateau, 110.

Pleasanton, General, 426.
Preyer, 184, 185.
Prowazek, Dr. S., 686.

R&dl, 69, 87.
Ranke, 450.
Reaumur, 7, 37, 38, 41, 56. . ""'

Regoli, 755.

Ringer, 513, 519, 533, 549, 749.

Romanes, 37, 80, 81, 541, 560.

Roth, 226.

Roux, 448.

Sabbatani, 753, 755.

Sachs, 2, 4, 5, 6, 7, 14, 42, 73, 80, 89, 96, 97,

102, 176, 179, 207, 211, 212, 215, 249, 342, 343,

o44.

Saussure, 378.
Schrader, 88, 186, 347.
Schmankewitsch, 237, 238, 239, 240.
Schwann, 378.
Semper, 56, 229, 230, 248.
Sewall, 186.

Spallanzani, 370, 378, 627.
Spencer, Herbert, 229.
Spiro, 546, 547.
Spitzer, 506.
Stahl, 6, 14, 73, 75.
Steiner, 187, 345, 346.
Stieglitz, 546.
Strasburger, 5, 73, 283.
Strassen, zur, 676, 678.

Tichomirof, 580.
Tiedemann, 404.
Tornier, Gustav, 436.
Torrey, 167.
Traube, 506.

Trembley, 7, 8, 73, 116, 118.
True, 451, 452, 534.

Verworn, 506, 507, 508.
Vignier, 684.
Voit, 499.

Wallace, 511, 760.
Weismann, 319.
Wilson, E. B., 686, 768.
Winkler, Hans, 686, 688.
Wheeler, 287, 288.
Whitman, C, O., 734, 739.
Wortmann, 6, 212, 231.
Wtlllner, 238.

Young, Emil, 427.

Zoethout, 694, 759.
Zuntz, 236, 237, 309.

SUBJECT INDEX

ABSORPTTON OF WATER: relation of, to
regeneration, 223 ff . ; a basis for judg-
ing effects of ions, 453; effect of H.
and OH. ions on, 464 ff. ; by muscles
and soaps, 510 ff.

ACIDS: physiological effects of, 453 ff.,
501 ff.

ACTINIA CARA: heteromorphosis in, 166.

ACTINIA DIAPHENA : heteromorphosis in,
171.

ACTINIA EQUINA: heteromorphosis in,
166 ff . ; stereotropism in, 170.

ACTINIA MESEMBRYANTHEMUM: geotro-
pism in, 183 ; basal end preferred in new
growth, 201.

ACTINOSPH^ERIUM : exception to Pflii-
ger's law, 440; liquefaction of proto-
plasm of, 448.

ADAMSIA RONDELETTI : heteromorphosis
in, 166.

ADVENTITIOUS ROOTS : stereotropism of,
136.

AGLAOPHENIA PLUMA: heteromorphosis
in, 115, 130; morphology of, 130; deter-
minants of heteromorphosis in, 132;
stereotropism in, 135; longitudinal
growth of, 136 ; larvee of, 137 ; geotro-
pism in, 176.

ALG.E : influence of direction of rays of
light on, 5, 89; heliotropism of swarm-
spores of, 283.

AMBLYSTOMA : influence of central nerv-
ous system on development of, 436 ff . ;
galvanotropism in, 440 ft".

AMMONIUM CHLORIDE: effects on regen-
eration and growth, 246.

AMMOTHEA : segmentation in, 340.

AMOEBA: spontaneity in, 321.

AMPHIPYRA : heliotropism in, 21 ; stereot-
ropism in, 21 ; geotropism in, 44.

AMPHITRITE: artificial parthenogenesis
in, 770.

ANEMONIA SULCATA: heteromorphosis
in, 166.

ANISOTROPY: influence of intense light
on, 62.

ANTENNULARIA : heieromorphosis in,
628 ff.

ANTENNULARIA ANTENNINA: geotropism
in, 177 ; growth in, dependent on geot-
ropism, 191-204; variation in, 204; irri-
tability and growth in, 213; hetero-
morphosis in, 249.

ANTS : influence of less refrangible rays
of light on wingless form, 11 ; heliot-
ropism of winged form, 19,113; rela-
tion between heliotropism and sexu-
ality of, 52, 113; factors determining
nuptial flight of, 53.

ARBACIA (SEA-URCHIN) : effect of change
of concentration of sea-water on cleav-
age of eggs of, 253 ff. ; twin production
in, 303 ff. ; limit of divisibility of em-
bryo of t 323 ff. ; lack of oxygen and seg-
mentation of eggs of, 400 ff . ; effect of
ions on unfertilized eggs of, 576 ff. ; on
fertilized eggs of, 581 ff. ; artificial par-
thenogenesis in, 624 ff.

ARTEMIA MULHAUSENII: artificial con-
version into Branchipus, 237 ff .

ARTEMIA SAUNA: conversion into Miil-
hausenii, 237 ff.

ARTISTIC IMPULSE: anthropomorphic
conception of, 165.

ASCARIS : agglutination of eggs of, 678.

ASSOCIATIVE MEMORY : definition of, 365.

ASTERIAS (STAR-FISH): geotropism in
183 ff., 291 ; artificial parthenogenesis
by shaking eggs of, 768.

ASTERIAS FORBESII: artificial partheno-
genesis in, 644 ; prolongation of life of
unfertilized eggs of, 728 ff.

ASTERINA GIBBOSA : geotropism in, 183.

ASTERINA TENUISPINA: heliotropism in
183.

AUDITORY NERVES : relation of, to orien-
tation, 188.

AURELIA AURITA : relation of concentra-
tion of water to contractions of, 561.

BACTERIA : heliotropism of, 15 ; orienta-
tion influenced by assimilation, 15.

BALANUS PERFORATUS : negative stereot-
ropism in, 111; change of sense of
heliotropism in, 113, 272, 417: depth-
migrations of, 290.

BARANA CASTELLI : regeneration of, 338.

BASES : physiological effects of, 461.

BATHOMETRIC DISTRIBUTION : of pelagic
animals, 178: physiological conditions
determining, 289 ff.

BEETLES : heliotropism of, 56, 70, 85.

BLOOD-VESSELS : development of, 297 ff .

BOMBYX: parthenogenesis in, 580.

BOMBYX LANESTRIS : heliotropism in, 38.

BOMBYX NEUSTRA : geotropism in, 85.

BRAIN : regeneration of, 251 ; physiology
of, in worms, 345 ff.

BRANCHIOMMA: explanation of eye on
gill of, 97.

BRANCHIPUS : Schmankewitsch's experi-
ment on, 237.

BRYOPSIS: organization controlled by
external forces, 202.

BURSARIA : heliotropism in, 15.

BUTTERFLIES: sleep of, 37; heliotropism
in, 20, 37.

777

778

STUDIES IN GENERAL PHYSIOLOGY

CAMPANULARIA : heteromorphosis in, 629.

CATALYTIC SUBSTANCES: importance in
oxidation, 505.

CATERPILLARS: heliotropism in, 20, 42,
74.

CELL-DIVISION : mechanics of, 389.

CEREACTIS AURANTIACA : heteromorpho-
sis in, 166.

CEREBRATULUS MARGINATUS : brain
physiology of, 356 ff.

CERIANTHUS MEMBRANACEUS : secretion
of, due to friction, 99; lack of hetero-
morphosis in, 116; formation of ten-
tactes of, 145; heteromorphosis in, 150;
relation between form and irritability
of, 152; life phenomena of, 159; impor-
tance of turgor in, 162 if ; external con-
ditions in formation of tubes of, 165.

CH^ETOPTERUS : artificial parthenogene-
sis in, 540, 579, 646, 674, 770; specific
effects of K ions on, 656.

CHEMICAL STIMULI: orientation, of
Musca toward, 66.

CHLOROPHYLL-BEARING PLANTS: influ-
ence of light on movements of proto-
plasm of, 6.

CHROMATOPHORES : distribution in Fun-
dulus, 300.

CIONA INTESTINALIS : organization of,
215 ; regeneration of nervous system of,
217; threshold of stimulation of, 219.

CLADOCORA: geotropism in, 201; hetero-
morphosis in, 250.

CLEAVAGE: general remarks, 253 ff . ;
lack of oxygen and, 373 ff; carbon di-
oxide and, 393 ff. ; pure oxygen and, 394.

CLEPSINE: oxygen necessary for matu-
ration of eggs of, 734.

COCCINELLA: geotropism in, 85, 179.

COCKROACH : geotropism in, 86, 182.

COLOR PREFERENCE: anthropomorphic
idea of, 16.

COMPENSATORY MOVEMENTS : influence
of inner ear on, 186.

CONTACT-STIMULI: effect on orientation,
193; effect on organization of, 214.

COPEPODS: heliotropism of, 282; change
of sense of heliotropism, 283 ff., 417.

CRYPTOPS: stereotropism in, 110.

CTENOLABRUS : influence of lack of oxy-
gen on, 378 ff. ; influence of carbon di-
oxide on, 393 ff.

CUCUMARIA CUCUMIS: geotropism in,
180 ff.

CUMA RATHKII : heliotropism id, 73 ff .

CYCLAS : effect of hydrogen on heart-beat
of, 417.

DAPHNIA PULEX: Bert's anthropomor-
phic idea of, 9; Lubbock's idea of
"preference" in, 10.

DEPTH DISTRIBUTION AND MIGRATION :
conditions determining, 289 ff.

DEVELOPMENT OF ORGANS: influence of

light on, 425 ff.
DIVISIBILITY OF MATTER : orientation of

particles, 117; limits of, 321 ff.
DROSERA : reactions of, 286.

EARTHWORMS: heliotropism in, 73, 77;
brain physiology of, 357 ff.

ECHINUS: segmentation not influenced
by light, 427.

ELECTRICAL WAVES: physiological ef-
fects of, 482 ff .

EMBRYONIC TISSUE: effects of ions on,
565 ff.

EPHEMERHXE: geotropism in, 44.

EUDENDRJUM RACEMOSUM : heliotropism
in, 106; heteromorphosis in, 140, 172;
influence of light on growth of, 428.

EUGLENA : influence of direction of rays
on, 14; influence of refrangibility of
rays on, 14; sensitive spot of, 77.

EYES : relation between irritability and,
56.

FERTILIZATION : nature of, 539, 620, 638,
646 ; theory of, 683, 740.

FLY (LARV.E) : heliotropism of, 20, 68,
158.

FORFICULA AURICULARIA : heliotropism
of, 22; stereotropism of, 110, 158.

FREEZING : effects of, 225.

FRICTION : a cause of movement, 107, 110.

FUNDULUS : development of embryo of,
295 ff. ; sensitiveness to lack of oxygen,
309, 397; effects of lack of oxygen on
cardiac activity of embryos, 404; in-
fluence of light on embryos of, 434;
effects of ions on, 550 ff . ; partheno-
genesis in, 682.

FUSION : of cleavage cells through lack
of oxygen, 383.

GALVANOTROPISM : of Amblystoma, 440
ff. ; theory of, 440 ff. ; of Crustaceans,
447.

GAMMARUS LOCUSTA: heliotropism in, 73.

GASTROSTYLA: divisibility of, 321.

GEOMETRA PINIARIA : heliotropism in, 40.

GEOTROPISM: in Porthesiachrysorrhoea,
33; in Lepidoptera, 43; in Amphipyra,
44; in Bombyx, 85; theory of, 102; in
Hydroids, 174 ff. ; in free-swimming
animals, 178; in Asterina, 183; depend-
ence on inner ear, 185; influence of, on
axis of eyes, 186; in Antennularia au-
tennina, 251; relation of, to heliotro-
pism, 285; in Loligo, 292; in star-fish,
292.

GONIONEMUS : effects of ions on 553, 559 ;
parthenogenesis in, 682, 754.

GONOTHYREA LOVENii : abnormality of
growth in, 144; heteromorphosis in,
173.

GRAVITATION : relation to light, 95, 105 ;
effects of, on Cerianthus, 107, 109, 154
ff. ; effect of, on orientation of body,

SUBJECT INDEX

779

186 ; effect on position of eyes, 186 ; rela-
tion to heteromorphosis in Antennula-
ria, 191 ff.

GREEN SLIPPER ANIMALCULE: heliot-
ropism of, 15.

GROWTH: phenomena of importance in,
175 ; dependence of, on geotrqpism, 191 ;
relation between concentration of sea-
water and, 228 ff. ; relation of, to quan-
tity of water, 247; and regeneration,
252.

BLEMATOCOCCUS : heliotropism in, 114,
283.

HEART: action of potassium salts on,
296.

HELIOTROPISM: identity of, in animals
and plants, 1 ft'., 89; influence of direc-
tion of rays on, 2, 4, 5, 16, 53, 91, 108, 265;
in plants, 4; in Hydra, 8; in Euglena,
14; in Infusoria, 14; in bacteria, 15;
in Bursaria, 15; methods of experi-
mentation, 17; negative form of, 17, 56;
positive form of, 17, 24 ; effect of col-
ored rays on, 18, 29 ff . ; in ants, 19, 52 ;
in caterpillars, 20, 42, 74; in fly larvae,
20; inversion of sense of, 20, 68, 265 ff.,
417 ff . ; in Amphipyra, 21 ; in Forficula
auricularia, 22; effect of temperature
on 37; in butterflies, 37; in Bombyx
lanestris, 38; in Moths, 38; in Sphinx
euphorbiae, 38; in Geometra piniaria,
40; periodic variations in, 40; in Papi-
lio machaon, 42; in plant lice, 45; in
beetles, 56, 70, 85; in Mesocarpus, 56,
113; character of protoplasm in, 57;
in Musca, 57; in Tenebrio molitor, 70;
in June bugs, 71; in Melqlontha vul-
garis, 71 ; in Cuma rathkii, 73 ff . ; in
earthworms, 73, 77; in Gammarus lo-
custa, 73; in leeches, 73; in planaria,
73; in snails, 73; distribution of, in
animal kingdom, 73; variations of, ac-
cording to pole of animal, 79, 83; not
dependent on central nervous system,
84, 221; in sessile animals, 89; in Spiro-
graphis Spallanzanii, 90-110; Sach's
theory of, 102; in hydroids, 103; in Ser-
tularia, 103,266; relation of , to instinct,
109; sexuality in relation to, 113; in
Balanus perforatus, 113, 272,417; lack
of, in Tubularia mesembryanthemum,
126; in Eudendria, 141; effect of, on
heteromorphosis, 174; in Asterina ten-
uispina, 183; in Ciona, 221; in Limulus
polyphemus, 267; theory of, 270; in
Polygordius, 273; intensty of, 273; effect
of concentration of sea-water on, 279,
282; in Temora longicornis, 282; in
Copepods. 283; difference in locomo-
tion under influence of, 284; liberation
of energy in, 286 ; in Loligo, 291 ; lack
of oxygen in, 417.

HEREDITY : theory of, 319.

HETEROMORPHOSIS : in Aglaophenia
pluma, 115, 130; definition, 120; in Tu-
bularia mesembryanthemum. 120 ff . ;
in Plumularia pinnata, 137 ; in Euden-
drium, 138, 140, 172 ; effects of aeration
on, 140, 144; in Sertularia, 142; in Go-
nothyrea lovenii, 144, 173; in Cerian-

thus membranaceus, 150; in Actinia
cara, 166; in Actinia equina, 166; in
Adamsia rondeletti. 166; in Anemonia
sulcata, 166; in Cereactis aurantiaca,
166; in Actinia diaphena, 171; law gov-
erning, 173; influence of geotropism on,
191 ff. ; in uninjured organs, 196; casu-
istic ideas of, 249 ; in Antennularia an-
tennina, 249; in Cladocora, 250: in
horse-shoe crab, 267 ; in Hirudinee. 341 ;
in Hirudo, 343; in Crustaceans, 627; in
Hydroids, 627 ff . ; in Antennularia, 628
ff. ; in Campanularia, 629.

HYBRIDIZATION : possibility of, between
Chsetopterus and Echinoderms, 678.

HYDRA : heliotropism of, 8, 73 ; theory of
polarity in, 117, 118, 216 ; regeneration
in, 149, 150, 173, 205; amount of sub-
stance necessary for regeneration of,
336.

HYDROIDS: heliotropism in, 103-5; stere-
otropism in, 111; heteromorphosis in,
115, 116, 627 ff. ; geotropism in, 177.

HYDROMEDUS^E : influence of ions on con-
traction of, 541 ; Eomanes's ideas of, 541.

HYDROSTATIC PRESSURE : importance of,
for growth of tentacles of Cerianthus,
175 ; relation to geotropism, 181 ff .

HYDROTROPISM : in Plasmodia, 179, 182.

HYPERSENSITIVENESS OF SKIN: produc-
tion and suppression of, by electro-
lytes, 748 ff.

INFUSORIA: heliotropism in, 14, 73; re-
generation in; 205: importance of nu-
cleus in, 321; influence of lack of oxy-
gen, 636.

INSTINCT: inherited, 61; movements
called instinctive due to physical laws,
107; physical laws in, 107-110; anthro-
pomorphic idea of, 165.

ION-PROTEIDS : importance in absorp-
tion of water by muscles, 510 ff . ; r6le
in life-phenomena, 544 ff . ; relation to
ciliary movements, 555 ff . ; in parthe-
nogenesis, 646 ff.

IONS: physiological effects of, 450 ff., 501
ff . ; in relation to absorption of water
by muscles, 464 ff. ; velocity of migra-
tion of. in relation to toxicity of, 474
ff. ; and rhythmical contractions of
muscles, 518, 559 ; effects of, on devel-
opment of muscle, 565 ff. ; effect of, on
undifferentiated embryonic tissue, 565
ff. ; in artificial parthenogenesis, 576
ff. ; effect of, on fertilized eggs of Ar-
bacia, 581 ff. ; effects of, in artificial
parthenogenesis of giant and dwarf
embryos of Arbacia and Cheetopterus,
674 ff , ; effect of, on nerves, 703 ff. ; toxic
and antitoxic effects of. 708 ff.

IRRITABILITY : a function of tempera-
ture, 36 ; sex-differences in, 56 ; relation
between structure of body and, 76;
distribution of, 80; and form, 152; and
organization, 213; dependence of, on
concentration of medium, 262.

ISOPODS: change of sense of heliotro-
pism of, 419.

780

STUDIES IN GENERAL PHYSIOLOGY

JUNE-BUG LARVAE : heliotropism of, 20, 71.

LACK OF OXYGEN: influence of, on fish
embryos, 309 ff . ; and Perca fluviatilis,
320; physiological effects of, 370 ff.;
and Copepods, 373; and Ctenolabrus
eggs, 374; influence of, on Fundulus
eggs, 375, 397 ; and segmentation of egg,
400 ff . ; and cardiac activity of fish em-
bryo-', 404; and Cyclas larvae, 417 ; and
heliotropism, 417; and pigment cells,
420.

LEECHES: heliotropism in, 73: oral end
more sensitive to light, 78; stereotro-
pism in, 79; lack of regeneration in, 341
ff.; brain physiology of, 361.

LEPIDOPTERA: heliotropism in, 7, 37, 40,
54, 56, 61, 74, 86; chemotropism in, 112.

LEUCOCYTES : migration of, due to
stereotropism, 111.

LIGHT : effect of direction of ray on
movements, 2, 4, 5, 16, 53. 91, 108, 265;
effective rays of, 3, 5 ; effect of change
in intensity of, 3,82,276; effect of, on
swarm-spores of algae, 5 ; effect of, on
Lepidoptera, 7 ; effect of, on Protozoa,
7; mechanical effects of, 7; effect of,
on water fleas, 8 ; effect of, on Daphnia,
9; influencing "instinctive" move-
ments, 107, 109; effect of, on develop-
ment of organs, 42r> ff. ; effect of, on
Fundulus embryos, 434 ff.

LIMULUS POLYPHEMUS : heliotropism in,
267. 288; locomotion dependent on
sense of heliotropism of, 284.

LOLIGO: heliotropism in, 291; geotro-
pism in, 292.

Loss OF WATER : effect of, on cleavage,
253: influence of, on embryos, 309, 314.

LUMBRICUS FCETIDUS: brain physiology
of, 359.

LYMN^EUS STAGNALIS : growth in, 229.

MACH^ERITES: blindness of, as related
to sex, 56.

MARGELIS : heteromorphosis in, 628 ff .

MATURATION: of unfertilized eggs of
star-fish, 728 ff .

MEDUSA : periodic migration of, 366; ion-
prpteids in, 544; locomotion of, 553;
effects of Ca ions on, 753.

MELOLONTHA VULGARIS: heliotropism
in, 71.

MESOCARPUS : influence of light on, 6.

METAMORPHOSIS: influence of central
nervous system on, 436.

MICELLA : conceptions of, 334.

MIGRATIONS OF ANIMALS: influence of
geotropism on, 180; physiological con-
ditions determining, 289 ff.

MOTHS: heliotropism in, 7, 38.

MULTIPLE EMBRYOS : development of, 303.

MUSCA VOMITORIA: helitropism in, 56,
113 ; stereotropism in, 64, 68 ; effect of
heat on orientation of, 65; relation of
orientation of, to chemical stimuli, 66:
movements due to sum of stimuli, 112.

MUSCLE: absorption of water by, 510 ff. ;
rhythmical contractions of, 518 ff. ;
contact-irritability of, 692 ff. ; produc-
tion and suppression of twitchings of,
by electrolytes, 748 ff.

: stereotropism in, 110.

NEMERTINES : brain physiology of, 356 ff.
NEREIS: brain physiology of , 358 ; artifi-

cial parthenogenesis in, 772i
NERVE: regeneration of, 217, 252; effects

of fluorides on, 703 ff.
NUCLEUS : effect of, on growth. 321 ff. ;

the organ of oxidation, 508.

OCELLA : formation of, in Ciona, 216.
(EDEMA: influence of osmotic pressure
.on, 471 ; increase in osmotic pressure of
muscle causing, 515.

ORAL PLATES: in Cerianthus, 159, 160.

ORBITOLITES : changes of enucleated
pieces of, 506, 507.

ORGANIZATION: dependent on orienta-
tion, 191, 192; relationship of, to
growth, 191, 251; internal causes of,
205; Sach's theory of, 207, 211, 212, 215;
Loeb's theory of, 208; relationship of,
to irritability, 213; in Ciona, 215; em-
bryonal, 319.

ORIENTATION: influence of direction of
rays on, 2, 4, 5, 16, 53, 91, 108, 265 ; influ-
ence of temperature, 3; influence of in-
_ ^Bnsif.y of light on, 3, 4, 32; influence of
physical laws on, 51 ; influence of form
of body on, 75; of plants, 109; com-
position of forces in, 112; forms of irri-
tability influencing, 174, 251 ; influence
of gravitation on, 176, 177, 178; governs
regeneration and heteromorphosis,
193.

ORTHOTROPISM : of organs, 2; in dorsi-
ventral animals, 97.

OSMOTIC PRESSURE: relation of, to
growth, 228, 240; in bursting of egg-
membrane, 306 ; relation of, to absorp-
tion by muscle, 466 ; in parthenogenesis,
640, 648 ff.

OTOLITHS : relation of, to geotropism,
187, 189.

OXYGEN (see Lack of Oxygen): role of ,
in heliotropism, 15 ; relation of, to re-
generation, 240, 252; Hoppe-Seyler's
theory of action of, 371 ; relation of, to
cleavage, 394 ; relation of, to growth,
400.

PANTOPODS : regeneration of, 338.
PAPILIO MACHAON : heliotropism in, 42.
PARTHENOGENESIS : artificial, 539 ff.,

638 ff.; in Arbacia, 576 ff., 624 ff . ; in

Annelids (Cheetopterus), 646 ff.; in

Fundulus, 682; in Gonionemus, 682;

in Podarke, 682; in Phascolosoma.

682, 772 ; in Crustaceans, 684 ; sources of

error in, 765 ff.
PERCA FLUVIATILIS: effect of lack of

oxygen on, 320.

SUBJECT INDEX

781

PHASCOLOSOMA : artificial parthenogen-
esis in, 682, 772.

PHOTOKINETICS : definition, 265; reac-
tions influencing, 286 ff .

PHOXICHILIDIUM MAXILLARA: regenera-
tion of, 338 ff .

PHYSIOLOGICAL PROBLEMS : discussion
of, 497 ff.

PHYSIOLOGIC UNIT : idea of, 322.

PIGMENT : relation of, to gravitation, 190 ;
in Tubularia, 210; influence of lack of
oxygen on, 420.

PLAGIOTROPIC ORGANS: definition, 2.

PLAN ARIA : heliotropism in, 73, 77 ; pho-
tokinesis in, 287 ; heteromorphosis in,
344 ; brain physiology of, 352 ff .

PLANORBIS: influence of light on seg-
mentation of, 427.

PLANT LICE : heliotropism in, 45, 49, 50,
112; geotropism in, 50; temperature
effects on, 51, 108 : flight movements of,
54; movements of, not voluntary, 109.

PLANULA : influence of light on, 433.

PLASMODIA : geotropism in, 179.

PLUMULARIA FINN ATA : heteromorpho-
sis in, 137, 172.

PODARKE: Parthenogenesis in, 682, 772.

POLARITY: heliotropism as a factor in,
76; Allmann's theory of, 117, 129; as
r -gards Tubularia mesembryanthe-
mum, 128; in Cerianthus, 206, 215.

POLYGORDIUS ; change of sense of heli9t-
ropism in, 273, 293r 417 ; locomotion in,
284.

POLYPS : formation of, 124.

PORTHESIA CHRYSORRHCEA : heliotropism
in, 19, 24-37, 112; stereotropism in, 21.

POTASSIUM: effects of, on 'regeneration,
242, 252 ; effects of, on Fundulus, 296, 297.

PRESSURE: effects of, on so-called in-
stinct, 110.

PROTOPLASM: influence of light on, 6;
movements of, 212 ; isotropy in, 334.

PROTOZOA : heliotropism in, 7.

PYCNOGONIDES: regeneration of, 338 ff.

QUANTITY OF WATER: influence on re-
generation and growth, 247, 252.

KAN A: influence of light on develop-
ment of, 427.

KEFLEXES: persistence of, in Ciona, 218;
in Planaria, 355 ff.

REGENERATION : Bonnet's theory of, 119;
definition of, 120; in Tubularia, 122; in
Ciona intestinalis, 217; influence of
concentration of sea-water on, 222;
necessity of oxygen for, 240; relation
of, to inorganic substances, 242, 245;
influence of quantity of water on, 247,
252; influence of magnesium on, 252;
quantitative limits of, 336; general
ideas on, 338; in Pantopods, 338; in
Chsetopods, 341; and nucleus, 505; of
organs in Hydroids, 627 ff.

RESEGMENTATION : of Ctenolabrus eggs

without oxygen, 378.
RESPIRATORY ORGAN : effect of light on,

97.
RHEOTROPISM: in Eudendria, 141; in

Plasmodia, 179, 182.
RHIZOPODS : regeneration in, 507.

SACCHAROMYCES CEREVISLE: a ferment
for glucoses, 498.

SCYLLIUM CANICULA: geotropism in, de-
pendent on inner ear, 187.

SEA-WATER: regeneration in, 222; con-
centration of, and influence on longi-
tudinal growth, 228; analysis of, 242;
influence of concentration of, on seg-
mentation, 257 ; effect of concentration
of, on heliotropism, 279-82.

SEGMENTATION : of nucleus without seg-
mentation of protoplasm, 258.

SERPULA: heliotropism in, 101, 106: pho-
tokinesis in, 289,

SERTULARIAHALECINA: heliotropism in.
136.

SERTULARIA (POLYZONIAS) : heliotro-
pism in, 103; heteromorphosis in, 142,
173, 174, 249 ; change of sense of heliot-
ropism in, 266.

SESSILE ANIMALS : heliotropism in, •
90-106.

SEXUALITY : relation of, to heliotropism,
52, 68, 113.

SHARKS : geotropism in, 186.

SIAMESE TWINS : formation of, 304 ff.

SNAILS : heliotropism in, 73.

SODIUM CHLORIDE : poisonous character
of solution of, 544 ff .

SPERMATOZOA: stereotropism of, 23, 111.

SPHINX EUPHORBIA: heliotropism in,
38 ff.

SPIROGRAPHIS SPALLANZANII : heliotro-
pism in, 90-100; movements of, not in-
stinctive, 107 ; change of sense of heli-
otropism, 266.

STENTOR VIRIDIS: influence of oxygen
on, 15.

STEREOTROPISM: definition of, 23; in
Porthesia chrysorrhoaa, 34; in Lepi-
doptera, 54; in plant lice, 54; in ants,
55 ; in June-bugs, 71 : relation of, to in-
stinct, 109, 111; in Tubularia mesem-
bryanthemum, 121, 124; in Aglaophe-
nia pluma, 135; in Plumularia, 1:18; in
Sertularia, 143: in Hydroids 174; in
Antennularia antennina, 251; in Lum-
bricus foetidus, 360.

STIMULATION: in heteromorphosis, 140;
threshold of, 219: latent period of, 221.

STRONGYLOCENTRATUS FRANCISCANUS :
artificial parthenogenesis in, 626, 638,
644.

STRONGYLOCENTRATUS PURPURATUS : ar-
tificial parthenogenesis in, 626, 638, 644.

SUNLIGHT: effect on Lepidoptera, 48;
effect on Polygordius, 277.

782

STUDIES IN GENEBAL PHYSIOLOGY

TEMORA LONGICORNIS : change of sense
of heliotropism in, 282.

TEMPERATTJBE : effects of, on Porthesia,
36; on Musca larvae, 65; on Polygor-
dius, 274 ; on depth-migrations, 293.

TENEBRIO MOLITOR: heliotropism in, 70;
stereotropism in, 71.

TENTACLES : formation of, in Cerianthus,
145-152, 160.

THYSANOZOON BROCCHII : regeneration
in, 221 ; brain physiology of, 347 ff .

TOXOPNEUSTES : natural parthenogene-
sis in, 684.

TRADESCANTIA : influence of oxygen on
formation of cell wall, 384.

TRANSFORMATION OF ORGANS: in Hy-
droids, 627 ff. ; basis of, 634 ff .

TUBES : formation of, in Cerianthus, 164.

TUBULARIA: heteromorphosis in, 118,
215; stereotropism in, 174; irritability
and organization of, 211; regulation
and concentration of sea-water in,
222 ff. ; effect of salts on growth of, 230,
242 ff. ; regeneration of, 336.

TUBULARIA CROCEA : organization of, 214.

TUBULARIA INDIVISA : growth of polyps
in, 127.

TUBULARIA MESEMBRYANTHEMUM : heter-
omorphosis in, 120-27; morphology of,
121; laws governing reactions of, 125;
conditions of growth of polyps of, 127 ;
internal causes of organization in, 205 ;
stereotropism in, 213.

TUNICATES : heteromorphosis in, 627.

TURGOR: importance of, in growth, 162
ff., 175.

VARIATION : in irritability, 40.
VASCULAR SYSTEM : development of, 297.
VIOLET RAYS: influence of, on growth,
426 ff.

VITAL FORCE: anthropomorphic idea,

VOLUNTARY MOVEMENTS: influence of
physical laws on, 107, 109, 110.

WASPS : Lubbock's experiments on, 11.
WATER FLEAS : effect of light on, 8.
WATER RIGOR: influence of dilute sea-
water on, 327.

WILL: subject to physical influences,

107, 109. 110.
WORMS : brain physiology of, 345 ff .

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