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lUiarine Biological Laboratory Library

Woods Hole, Mass.

Presented by

Marion Irwin Osterhout
February 1966

83

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SOME FUNDAMENTAL PROBLEMS OF
CELLULAR PHYSIOLOGY

PUBLISHED UNDER THE AUSPICES OF THE

YALE SCHOOL OF MEDICINE

ON THE FOUNDATION ESTABLISHED IN MEMORY OF

WILLIAM CHAUNCEY WILLIAMS, M.D.,

OF THE CLASS OF 1 822, YALE MEDICAL SCHOOL

AND OF

WILLIAM COOK WILLIAMS, M.D.,

OF THE CLASS OF I85O, YALE MEDICAL SCHOOL

The Third
William Thompson Sedgwick Memorial Lecture

For the purpose of commemorating the services of Wil-
liam Thompson Sedgwick to the cause of Biology and
Public Health there has been established a Memorial Lec-
tureship in the department of the Massachusetts Institute
of Technology which he created. The desire of the
founders is that the Sedgwick Memorial Lectures shall be
given from year to year by men of distinguished eminence
in any one of the subjects comprehended within the general
scope of Biology and Public Health in order that it may
fittingly express the deep and broad sympathy of the man
whom the Lectureship is designed to honor.

SOME
FUNDAMENTAL PROBLEMS

OF

CELLULAR PHYSIOLOGY

OH
074

BY

W. J. V. OSTERHOUT

Member of The Rockefeller Institute for Medical Research
Formerly Professor of Botany in Harvard University

Vvfe)^

uj| library

New Haven, Yale University Press

London^ Humphrey Milford^ Oxford Univsrsity Press

1927

Copyright, 1927, by Yale University Press
Printed in the United States of America

THE
WILLIAMS MEMORIAL PUBLICATION

FUND

The present volume is the ninth work pub-
lished by the Yale University Press on the Wil-
liams Memorial Publication Fund. This Foun-
dation was established June 15, 19 16, by a gift
made to Yale University by George C. F. Wil-
liams, M.D., of Hartford, a member of the
Class of 1878, Yale School of Medicine, where
three generations of his family studied — his
father, William Cook Williams, M.D., in the
Class of 1850, and his grandfather, William
Chauncey Williams, M.D., in the Class of
1822.

SOME FUNDAMENTAL PROBLEMS OF
CELLULAR PHTSIOLOGT

I SHALL not try to tell you how highly I
value the privilege of speaking on this occa-
sion. Nor shall I attempt to pay a tribute to him
whose memory we recall today, for this has been
done by others far better than I could hope to do
it. But perhaps I may be permitted to say that in
all the years of our personal acquaintance he
seemed to me as one who worked in the spirit
of Pasteur, with keen interest in both theoretical
and practical researches, and in deep sympathy
with every attempt to study the foundations
upon which all applied science must rest. In con-
sequence I feel it appropriate to dwell today
upon some fundamental aspects of biology.

Two things of note in the recent development
of biology may serve as my text. One is the in-
creasing attention paid to the fundamental
activities of the cell. The other is the growing

CELLULAR

conviction that these activities may be largely
determined by the permeability of the proto-
plasm. Hence the study of permeability has
acquired especial importance not only in theo-
retical respects but in many practical problems,
as, for example, the absorption of food, the ap-
plication of drugs, and the physiology of excre-
tion and secretion.

Let us consider for a moment certain activities
which are common to all living things. All cells
synthesize certain foods which exist in soluble
form. What prevents these substances from dif-
fusing out of the cell into the surrounding
liquid, thereby depriving it of materials essen-
tial to its existence? Experiments have shown
that the protoplasm is sufficiently impermeable
to such materials to prevent serious loss and that
it also possesses the power to exclude various
substances, some of which are deleterious. At the
same time it must admit raw materials out of
which it manufactures food. Hence the proto-
plasm has selective permeability, permitting
certain things to go in and out while barring the
passage of others.

PHTSIOLOGT

In order to understand how materials are ab-
sorbed and given out, we may consider first of
all the structure of the cell. A typical plant cell
consists of a cell wall (of non-living material)
within which is a layer of living protoplasm,
often so thin as to be little more than a delicate
membrane. Within this is a large central vacuole
filled with cell sap, and in many cases plastids
(such as the chloroplasts) are present. A simple
animal cell differs in several respects: of especial
significance for our present purpose is the ab-
sence of the cell wall and of the central vacuole.

When a plant cell absorbs water the proto-
plasm expands and the cell wall is stretched. If
water is withdrawn the cell wall shrinks j this
shrinking soon stops but the protoplasm may
continue to contract until it is drawn away from
the wall. If it is allowed to reabsorb water it
expands, stretching the cell wall with consider-
able force (strikingly manifested in growth,
when mushrooms raise flagstones, or ferns push
up through concrete sidewalks^.

The manner in which this force is generated
may be illustrated by tying a membrane to a

CELLULAR

piece of glass tubing. If we select a membrane
permeable to water but not to sugar, placing
inside of it a solution of sugar and outside of it
pure water, we find that as water is absorbed the
level rises in the tube 5 if we endeavor to pre-
vent this by means of a weighted piston placed
in the tube, we find that considerable force is
requisite. The pressure produced within the
apparatus is called osmotic pressure.

It is supposed that pressure is produced in
much the same way in the living cell. Here it is
not the cell wall but the protoplasm (or its outer
layer) which corresponds to the membrane in
our experiment, and, like the membrane, it is
permeable to water but is more or less imperme-
able to sugar and certain other substances. Such a
membrane, permeable to some substances but
not to others, is called semipermeable.

The discovery of these facts opened up a field
of great interest but no great progress was made
until quantitative methods began to be applied j
as soon as this happened developments of the
highest importance took place. The quantitative
era may be said to begin with some observations

PHTSIOLOGT

made in 1873 by Pfeffer, in his studies on the
movements of irritable plant organs. He in-
vestigated particularly the behavior of certain
stamens which, when touched by an insect,
suddenly shorten, thereby depositing pollen
upon the insect. The shortening is due to the
giving out of water by the cells of the stamen j
the water moves into the intercellular spaces,
from which it is reabsorbed by the cells during
the subsequent process of recovery. This reab-
sorption stretches the cell walls, thereby length-
ening the stamen. To accomplish this a consider-
able force is required, which Pfeffer estimated
at from two to four atmospheres.

At that time no one could explain how so
much pressure was produced. Pfeffer sought to
throw light on the question by constructing a
model which would act as nearly as possible like
the living cell. He prepared an unglazed porce-
lain cup in the pores of which he produced a
deposit of copper ferrocyanide (after the
method of Traube) : this was closed by a stopper
in which a manometer was fixed. The membrane
of copper ferrocyanide is permeable to water but

CELLULAR

not to sugar: if a sugar solution is placed inside
and the cup is then immersed in water the latter
is absorbed and the manometer registers an in-
crease of pressure.

Pfeffer regarded the copper ferrocyanide
membrane as analogous to the protoplasmic sur-
face of the cell, the sugar solution in the interior
corresponding to the solution of sugars, and
other food substances, contained in the central
vacuole.

The question with which Pfeffer started out
was apparently answered. Since the artificial
model could produce as much pressure as the
plant cell the mechanism might be regarded as
satisfactorily explained. Pfeffer, however, went
further, investigating the effects of concentra-
tion and temperature and coming to the con-
clusion that the pressure is directly proportional
to the concentration, and that it increases about
1/273 when the temperature rises 1° C.

In the meantime van't Hoff became interested
in the attraction between water and other sub-
stances, a problem which is intimately connected
with osmosis. One day as he left his laboratory,

PHTSIOLOGT

thinking deeply on this question, he met de
Vries, who told him the results of Pfeffer's
observations. Seldom has an interchange of
scientific ideas been more fruitful than this his-
toric interview. It is a classic example of cross
fertilization among the sciences. Not only did it
initiate a new era in physical chemistry, but it
was destined to be of fundamental importance
to biology.

Pfeffer's results led van't Hoff to the revo-
lutionary notion that molecules of dissolved sub-
stances obey the laws of gases. At that time it
seemed possible that the application of these
laws might clear up at one stroke most of the
difficulties which had made the study of solu-
tions so puzzling. This idea had far-reaching
results.

An important difficulty remained to be ac-
counted for. Inorganic salts failed to obey the
general law which governs the osmotic behavior
of sugar and many organic substances, de Vries
had observed this in experiments on plant cells
and had expressed the deviations from the law
by means of the so-called "isotonic coefficients."

CELLULAR

There was, however, no clue to the cause of this
deviation. In general the inorganic salts were
found to give too much osmotic pressure.

•The difficulty seemed to be cleared up by the
theory of Arrhenius, which states that all the
exceptional substances have the power to con-
duct the electric current in solution and that
they owe this property to the splitting of their
molecules into electrically charged ions. This
explains why they have abnormally high osmotic
pressures, for each ion produces as much osmotic
pressure as the molecule itself.

There remained the problem of finding a
generally useful method of measuring the os-
motic pressure of living cells, de Vries took this
up. Instead of allowing the cell to absorb water,
as in Pfeffer's experiments, he employed the
opposite method, withdrawing water from the
cell. If the cell absorbs water with a certain
force we may oppose to it an equal force so that
no absorption takes place. If we increase the
opposing force, water will flow out of the cell,
and this may cause the protoplasm to shrink
away from the cell wall.

PHTSIOLOGT

If a 5 per cent sugar solution extracts a little
water from the cell its osmotic pressure must be
a little greater than that of the solution in the
cell. Since the osmotic pressure of a 5 per cent
sugar solution can be determined, the osmotic
pressure within the cell can be estimated.

In this way de Vries not only measured the
osmotic pressure of a large number of plant cells
but he performed the remarkable feat of de-
termining by means of such cells the molecular
weight of a compound which baffled the best
efforts of chemists. This substance is a sugar,
raffinose, for which three different formulae
were proposed. Although the chemists were
unable to decide which formula was correct it
was for de Vries a simple matter. The three
formulae gave the molecular weights 396, 594,
and 1 1 88. If raffinose has the same molecular
weight as cane sugar (342) a 5 per cent solution
will extract water from the plant cell to the
same extent as a 5 per cent solution of cane
sugar. But if its molecular weight is only half as
great the 5 per cent solution will contain twice
as many molecules and its power to draw out

lo CELLULAR

water will be twice as great. This follows from
the fact that the osmotic pressure is proportional
to the number of molecules present (since sugar
molecules do not split up in solution, as do mole-
cules of inorganic salts), de Vries found that a
5 per cent solution of raffinose had less osmotic
pressure than a 5 per cent solution of sugar,
hence it must contain fewer molecules and its
molecular weight must be greater than that of
cane sugar. His calculations gave a molecular
weight of 596, which differs by only a third of
I per cent from one of the molecular weights
(594) proposed by the chemists.

Much of the work of de Vries in this field
preceded the investigations of van't Hoff and
Arrhenius and helped to lay the foundation for
them. This was particularly the case with regard
to the substances which produce unexpectedly
great osmotic pressure, de Vries rendered an in-
dispensable service by calling attention to these
cases and by carefully measuring the pressure
produced, thus paving the way for the work of
Arrhenius.

About this time important studies on the

PHTSIOLOGT 11

osmotic pressure of animal cells were made by
Hamburger, Griins, Hedin, and others.

There remained another problem of great in-
terest, namely, the nature of the semipermeable
surface of the cell. It is evident that this deter-
mines what substances can enter the cell, and
that on its composition the whole metabolism of
the cell may depend. PfeflFer and others had
suggested that it consists of protein, but the first
systematic investigation of its nature was made
by Overton. It had been suggested by Quincke
that the outer layer of the cell consists of a film
of oil: Overton came to a similar conclusion,
substituting for "oil" the term "lipoid," which
includes such substances as lecithin and choles-
terin.

Overton concluded that no substance can enter
the cell unless it is soluble in lipoid. The most
important evidence which he brought forward
may be summarized as follows :

1. Salts (which are as a rule insoluble in lip-
oid) are unable to enter the cell.

2. Dyes which are insoluble in lipoid do not

12 CELLULAR

enter the cell, while lipoid-soluble dyes pene-
trate readily.

3. Organic substances (including such anes-
thetics as ether and chloroform) penetrate and
affect the cell the more readily the more soluble
they are in lipoid.

Overton's hypothesis has been a very valuable
stimulus to investigation but it has become in-
creasingly evident that satisfactory progress is
impossible without more accurate measurements
and a number of attempts have been made to
provide suitable methods. It is evident that the
best procedure is to expose the cell for a suitable
time to a solution and then to analyze the cell-
contents to discover what has penetrated. This is
beset by difficulties which investigators have
sought to overcome in various ways.
''^ Some have made analyses of tissues, but it is
evident that these must include too much inter-
cellular material to be satisfactory. Analysis of
the solution bathing the tissue in order to deter-
mine what is absorbed, is open to the objection
that substances collect on the surfaces of cells, as
well as in the cell walls and in the spaces be-

PHTSIOLOGT 13

tween them, so that it is impossible to say what
actually penetrates the protoplasm. Others have
sought to analyze the cell sap. Plant cells are
most favorable for this purpose, since, as a rule,
they contain vacuoles filled with sap. In general
the method has been to crush the tissues and ex-
press the sap, but this procedure involves many
possibilities of error, such as contamination of
the cell sap by substances present in the cell walls
or intercellular spaces and chemical reaction be-
tween the cell sap and the crushed protoplasm or
the cell walls. (The degree of pressure used in
expressing has a marked influence on the concen-
tration of the sap.) The investigation of blood
and other body fluids is open to the objection
that we do not know to what extent substances
penetrate between the cells in reaching these
fluids. In many of these cases penetration seems
to present very special features.

The taking up of dyes has been extensively in-
vestigated but this method is beset by many pit-
falls. To a great extent the coloration of the cell
by a dye shows the extent to which the dye can
combine with the substances within the cell

14 CELLULAR

rather than the permeability. Some cells contain
substances which combine with the dye so that
it becomes far more concentrated within the cell
than in the external solution. Unless the cell has
this power it often fails to appear colored even
though it may contain the dye in the same con-
centration in which it exists outside. In such cases
it may sometimes be detected by plasmolyzing
the cell and thus concentrating the dye. A fur-
ther complication is that a cell may appear to
have taken the dye into its interior when in re-
ality only the surface or the cell wall is stained.
There are many other difficulties, which need
not be discussed here, such as toxic action and
changes in the dye (including decolorization as
it enters the cell).

In some cases the penetration of acids and al-
kalies has been studied by means of organisms
containing natural indicators or by introducing
indicators into the cell. Use has also been made
1 of the fact that the penetrating substance may
cause a visible precipitate within the cellj this is
especially the case with alkaloids. Furthermore
the absorption of calcium has been detected by
V

PHTSIOLOGT 15

observing the formation of crystals of calcium
oxalate within the cell. It is evident, however,
that these methods have but limited application,
and that in many cases they are open to the ob-
jection that the penetrating substance injures the
cell.

The penetration of a substance may some-
times be demonstrated by observing its effect
upon metabolism, but this method is inadequate
from a quantitative standpoint. Some investiga-
tors contend that substances may produce effects
on metabolism by their action at the surface,
without actually penetrating the cell.

Owing to these difficulties a number of indi-
rect methods have been employed. Of these
plasmolysis has been the most popular. The
principle is very simple. If to the fluid surround-
ing the cell a substance be added in sufficient con-
centration it will cause the withdrawal of water
and shrinkage of the protoplasm, but if this sub-
stance subsequently penetrates, the osmotic pres-
sure inside will increase and water will be taken
up. When the concentration of the substance in-
side equals that outside, the cell will contain ap-V /

I 6 CELLULAR

I proximately as much water as before the sub-
j stance was added. The cell in consequence
recovers its normal appearance. The time re-
quired for such recovery is commonly regarded
as an approximate index of the rate of penetra-
tion of the substance in question.

Even under the most favorable conditions
this method is more or less injurious. When the
protoplasm is torn away from the wall of a plant
cell its surface is usually altered and is often
torn so that detached bits of protoplasm remain
adhering to the wall. Even if the cell subse-
quently recovers after such treatment we can-

/ not be sure that during the experiment it was

( entirely normal.

Another method is by measuring electrical re-
sistance. In earlier experiments it appeared pos-
sible to measure the permeability of normal cells
to ions by this method. In measuring the electri-
cal resistance of the marine plant Laminaria by
means of an alternating current it was assumed
that the current passed in part through the pro-
toplasm and in part through the spaces between
the protoplasmic masses. Hence by making al-

I

PHTSIOLOGT 17

lowance for the latter part it seemed feasible to
determine to what extent ions entered the proto-
plasm and carried the current through it.

In the case of Laminaria similar results are
obtained whether we use direct or alternating
current. But recent experiments on very large
(multinucleate) cells, carried out in my labora-
tory by Mr. Blinks, indicate that the resistance
of protoplasm to a direct current is very much
greater than to an alternating current. It there-
fore seems probable that the surface of the cell
acts as a condenser and transmits an alternating
current without actual passage of ions. But meas-
urements of resistance to an alternating current
may nevertheless be very useful in detecting
changes in permeability to ions. If the normal
cell exhibits a high resistance and if this is less-
ened by a toxic agent, it shows that its permea-
bility to ions has been increased. As a matter of
fact determinations of electrical resistance by the
means of alternating current show a progressive
increase of permeability during the process of
death and this is borne out by a variety of other
evidence, such as the increased permeability to

I 8 CELLULAR

dyes (and other substances) which is observed in
dead or dying cells.

An advantage of the electrical method is that
it gives us time curves of the process of death
which can be analyzed mathematically and
which enable us to make quantitative predictions
regarding the behavior of protoplasm under
various conditions. It may also enable us to place
upon a quantitative basis such fundamental con-
ceptions as normal vitality, injury, recovery, and
death, which have hitherto been vaguely de-
fined.

We find, for example, that when the normal
electrical resistance has been ascertained it is a
simple matter to test material as it comes into
the laboratory and to tell whether it is in normal
condition or not. If it is not normal we can de-
termine just how far it deviates from normality
and predict with considerable accuracy how long
it will live.

As an illustration of the method we may con-
sider what happens when the marine plant,
Laminariay is taken out of its normal environ-
ment of sea water and placed in a solution of

PHTSIOLOGT 19

pure sodium chloride. We find that it is at once
injured, and if the exposure be sufficiently pro-
longed it is killed. During the whole time of ex-
posure to the solution of sodium chloride its
electrical resistance (measured with an alternat-
ing current) falls steadily until the death-point
is eventually reached j after this there is no fur-
ther change. A study of the time curve of this
process shows that it corresponds to a monomo-
lecular reaction (slightly inhibited at the start).
This may be expressed in the form of an equa-
tion which can be utilized to predict the curve of
death under various conditions. We find that in
testing these predictions we must ascertain when
the death process reaches a definite stage (i.e.,
when it is one-fourth or one-half completed).
This can be determined experimentally with a
satisfactory degree of accuracy.

We can therefore follow the process of death
in somewhat the same manner that we follow
the progress of a chemical reaction in vitro; in
both cases we obtain curves which may be sub-
jected to mathematical analysis, from which we
may draw conclusions regarding the nature of

20 CELLULAR

the process. This method has been fruitful in
chemistry and it is possible that it may prove
equally so in biology.

Studies undertaken from this point of view
lead us to look upon the death process as one
which is always going on, even in a normal, ac-
tively growing cell. In other words we regard
the death process as a normal part of the life
process, producing no disturbance unless unduly
accelerated by an injurious agent which upsets
the normal balance and causes injury so that the
life process comes to a standstill.

The process of death which occurs in a solu-
tion of sodium chloride may be checked by add-
ing a little calcium chloride to the solution. In
this case we speak of antagonism between so-
dium and calcium. When the calcium is added in
the proper proportion the fall of resistance is
very slow and the tissue lives for a long time.
Any deviation from this optimum proportion
hastens death.

When the plant is injured and the resistance
falls, we may consider that the loss of resistance
gives a measure of the amount of injury. This

PHTSIOLOGT 21

enables us to place the study of injury upon a
quantitative basis. As the result of this we are
able to formulate a definite conception of the
mechanism of recovery. We find that if injury
in a solution of sodium chloride amounts to five
per cent, the tissue recovers its normal resistance
when replaced in sea water. But if the injury
amounts to twenty-five per cent, recovery is in-
complete j instead of returning to the normal
the resistance rises to only ninety per cent of the
normal. The greater the injury the less complete
the recovery. When injury amounts to ninety
per cent there is no recovery.

This is of especial interest, since in physio-
logical literature it seems to be generally as-
sumed that when recovery occurs it is always
complete, or practically so, as if it obeyed an "all
or none" law. But it is evident that partial re-
covery may be easily overlooked unless accurate
measurements can be made. This fact may serve
to illustrate the importance of quantitative
methods in the study of these fundamental
problems.

The significance of this method is further

22 CELLULAR

shown by the fact that it has led to the develop-
ment of equations which enable us to predict
with a satisfactory degree of accuracy the recov-
ery curves which are observed under a great va-
riety of conditions.

As the result of these investigations we are
led to look upon recovery in a somewhat differ-
ent fashion from that which is customary. While
recovery is usually regarded as due to the re-
versal of the reaction which produces injury, the
conception here developed is fundamentally
different. It assumes that the reactions involved
are irreversible (or practically so) and that in-
jury and recovery differ only in the relative
speed at which certain processes take place.

These experiments lead to the view that life
depends upon a series of reactions which nor-
mally proceed at rates bearing a definite relation
to each other. If this is true it is clear that a
disturbance of these rate-relations may have a
profound effect upon the organism, and may
produce such diverse phenomena as stimulation,
development, injury, and death. Such a dis-
turbance might be produced by changes of

PHTSIOLOGT 23

temperature (if the temperature coefficients of
the reactions differ) or by chemical agents. The
same result might be brought about by physical
means, especially where structural changes
occur which alter the permeability of the plasma
membrane or of internal structures (such as the
nucleus and plastids) in such a way as to bring
together substances which do not normally react.

The results obtained lead to a very simple
conception of life processes, namely that they
consist of a series of consecutive reactions. By
varying the speed of the different members of
the chain we can, without introducing any new
substance, profoundly influence the course of
events. In this way the effects of the most di-
verse stimuli on such processes as growth, differ-
entiation, reproduction, and irritable response
might be accounted for.

The results obtained by the electrical method
have been confirmed by a variety of other
methods, as, for example, by the passage of
substances through diaphragms of living tissue.
In this case diffusion takes place principally be-
tween the cells, but as permeability increases it

24 CELLULAR

takes place to an increasing degree through the
protoplasm and this increase can be measured.

In order to determine what substances enter
under normal conditions we require accurate de-
terminations of actual penetration by direct
analysis of the contents of the cell. I have been
so fortunate as to find plants in which this is
possible. These have large multinucleate cells
from which the sap can be squeezed out without
danger of contamination and without any of the
handicaps which rendered previous attempts,
made with cells of ordinary size, unsatisfactory.

The fresh water plant Nitella produces
multinucleate cells up to six inches in length and
a thirty-second of an inch in diameter. Within
the cellulose wall is a delicate layer of proto-
plasm containing nuclei and chloroplasts. The
chloroplasts have a regular arrangement which
is disturbed if the cell is slightly injured 3 at the
same time the chloroplasts become more opaque.
These are valuable criteria of normal condition.

Within this layer of protoplasm is the large
central vacuole filled with sap in which float
globules of protein, the whole being as a rule

PHTSIOLOGT 25

in constant motion, flowing spirally up the cell
in one direction and down in the other, so that
the motion is opposite on the two sides.

By cutting one end of the cell and applying
gentle pressure the sap can be squeezed out, free
from protoplasm or chloroplasts. In making
experiments the cell is placed in a solution of
the substance whose penetration we are studying,
removed after a suitable exposure, rinsed, and
dried on the surface by means of filter paper.
The sap may then be squeezed out without
danger of contamination, since it is not allowed
to come in contact with the surface except for an
instant in the immediate neighborhood of the
cut, and even this contact may be avoided by
piercing the cell with a fine glass capillary and
collecting the sap by means of gentle suction or
pressure.

Analysis of the sap reveals a most interesting
series of facts. Nearly all of the substances con-
tained in it exist at a much higher concentration
than is found outside. There must be a mechan-
ism which traps these substances^ and causes them
to accumulate. In order to discover what this

z6 CELLULAR

mechanism is, it is necesary to experiment with a
substance which accumulates and to analyze the
process step by step.

The first of such quantitative investigations
on Nitella were made by Miss Irwin, using a
basic dye, brilliant cresyl blue. The technique is
very simple: after suitable exposure of the cell
to the dye the sap is drawn up into capillary
glass tubes whose color is compared with that of
similar tubes filled with dye of known concen-
tration. The accuracy of the method is satis-
factory, since it is possible to distinguish between
concentrations of .000017 and .000024 molar.
This procedure avoids all the difficulties pre-
viously mentioned in connection with experi-
ments on dyes.

The penetration of the dye follows the time
curve of a reversible monomolecular reaction. It
is possible that the dye combines with some con-
stituent of the cell, giving a reversible reaction
which appears to be monomolecular because the
concentration of the uncombined dye is constant.

The idea of chemical combination is sup-

PHTSIOLOGT 27

ported by the fact that the temperature coeffi-
cient is high.

On the other hand it may be assumed that
the dye enters as a free base which dissociates
more in the acid cell sap than in the more alka-
line external solution and so accumulates in the
fashion discussed later on in connection with the
penetration of hydrogen sulphide into Valonia.

Of especial interest is the fact that as the alka-
linity of the external dye solution increases the
rate of penetration increases and the excess of
concentration of the dye inside over that outside
also increases. This indicates that the penetrating
substance is the free base of the dye whose con-
centration would wax with increasing alkalinity.
This idea is also in harmony with the experi-
ments on exosmosis which show that when a
stained cell is placed in a solution destitute of
dye, the dye comes out more rapidly the
greater the acidity of the external solution. We
might explain this by saying that as the free
base comes out of the cell the concentration
gradient is greatly increased if the free base
is at once changed into another form 3 this would

28 CELLULAR

take place the more readily as the external solu-
tion becomes more acid.

While these studies were being made on
Nitella The Rockefeller Institute for Medical
Research very generously made it possible to
carry on investigations on the marine alga Va-
lonia. This forms large multinucleate cells,
shaped somewhat like a balloon, containing up
to 10 cubic centimeters of sap. The sap is desti-
tute of the motion observed in Nitella; it is sur-
rounded by a delicate layer of protoplasm con-
taining chloroplasts and numerous nuclei: this
is surrounded by a cellulose wall.

The use of large cells enables us to find out
what goes on inside to a greater extent than was
previously possible. As a result of these studies
I have formed quite different conceptions of the
behavior of the interior of the cell from those
which I previously entertained. It has become
apparent that we must now restate our problems
and begin a fresh attack upon the whole subject.
Although it is too soon to try to picture these
new conceptions with any degree of complete-

PHTSIOLOGT 29

ness a brief outline of some of them may not be
amiss.

Consider first of all the astonishing difference
which may exist between the inside and the out-
side of the cell. Valonia macrofhysay which
grows in sea water, excludes certain substances
absolutely as long as it remains in a normal con-
dition j this is the case, for example, with mag-
nesium and with sulphate. If it admits calcium
at all it is only in traces. Sodium is admitted but
its concentration remains far below that in the
sea water. On the other hand, potassium is stored
up within the cell in much greater concentration
than is found in sea water. It is evident that the
cell possesses a trapping mechanism for accumu-
lating certain substances and also a means of
excluding certain other substances either wholly
or in part.

As a consequence the inner and outer surfaces
of the protoplasm may be in contact with very
different solutions. Is it necessary for the life
of the cell that they should be different? What
will happen if they are made identical? To
answer this question sap was extracted and living

30 CELLULAR

cells were placed in it, thus bringing the inner
and outer surfaces of the protoplasm in contact
with identical solutions (since the sap can be
extracted without being altered). Most of these
cells lived but a short time (as a rule less than
a week), while under the same conditions in sea
water the majority live for several months.
This does not seem to be due to bacterial action
for the cells died promptly in sap which had
been boiled and filtered.

Normally there is considerable difference be-
tween the acidity of the sap and the sea water
(the pH values of sap and sea water are 5.8 and
8.2 respectively). When cells are placed in their
own sap this difference is abolished. Is this re-
sponsible for their rapid death? To answer this
question the acidity of the external sap was
varied j the results showed that acidity is not the
primary factor involved although cells die
much more quickly at pH 5.8 than at 8.2.

The important factor might conceivably be
the maintenance of differences in electric poten-
tial between the inside and outside of the proto-
plasmic layer but this does not seem to be the

PHTSIOLOGT 31

case, as measurements show that practically no
such difference exists.

The primary factor seems to be a lack of cer-
tain elements in the external medium which are
needed to make a "balanced solution." It is
evident that the sap is not a balanced solution in
the ordinary sense and this raises the question
whether balanced solutions are in general neces-
sary for the interior of the cell.

There is some ground for believing that the
maintenance of differences between the interior
and exterior of the cell is essential for carrying
on vital processes in general.

The fact that juices from the interior of the
cell may be toxic when applied to the exterior
may find a parallel in certain observations on
animals. Many investigators believe that when
tissues are burned or crushed, toxic substances
are set free. It may be that these substances are
due to reactions which never take place except
as the result of injury, but it is possible that
some of these substances are normally present in
the interior of the cell, in which case we should
have an analogy to what is observed in Valonia.

32 CELLULAR

The analysis of the sap of Valonia macro-
fhysay which was collected at Bermuda, agrees
fairly well in general with that of the sap of
F. utricularis collected at Naples. It is therefore
very surprising to find that another species, V,
ventrkosay collected at Bermuda, differs in toto
from V. macrofhysa in the composition of its
sap. This is shown by Table I. It is evident that
F. ventricosa has a sap whose composition is
much like that of sea water.

TABLE I

Molecular C omfosltion Expressed as Per Cent of

Halide (Cl+Br).

Sap of Sap of

Bermuda Valonia Valonia
sea water tnacrofhysa ventricosa

CI -"1- Br loo.oo loo.oo loo.oo

Na 85.87 15.08 92.80

K 2.15 86.24 2.58

Ca 2.05 0.288 1.36

Mg 9.74 Trace? 2.49

SO^ 6.26 Trace? Trace?

Organic matter parts

per thousand .... 1-433 i.oc)

PHTSIOLOGT 33

These striking differences between two species
of Valonia raise the question whether it is justi-
fiable to assume, as is often done, that close
relationship involves similarity in chemical
composition and metabolic processes. The impli-
cations of this question are far-reaching. If it is
possible for nearly allied forms to differ so
profoundly, it is evident that we have as yet no
satisfactory conception of the nature of the
variables involved in differentiation or in the
development of specific characters.

These differences raise another question of
considerable importance, whether sodium or
potassium predominates in the sap of plant or
animal cells. For the study of this question it is
desirable to employ cells whose sap can. be ob-
tained without alteration, as in the case of Va-
lonia and Nkella. Nitella from Woods Hole
shows approximately equal amounts of sodium
and potassium j in a species collected near Cam-
bridge there is a little more potassium than
sodium. Hoagland and Davis state that in Ni-
tella clavatay taken from pond water, the pro-
portion of potassium to sodium in the sap is as

34 CELLULAR

5.43 to I. In the case of large cells in tap water,
however, they report that sodium and potassium
were more nearly equal, and in small cells in
culture solution sodium predominated over
potassium.

In these cases the sap could be extracted with-
out contamination or alteration. Where this is
not possible we cannot be sure of its composition.
Ordinary ash analyses, made without extracting
sap, do not tell us whether substances are present
in soluble or insoluble form, whether they are
located within the cell or in intercellular spaces.
But such analyses may nevertheless give us some
idea of the general situation.

Ash analyses show a decided predominance of
potassium over sodium throughout the flowering
plants and mosses. In the case of marine algae
such analyses frequently show a preponderance
of sodium over potassium, but this might be due
to sea salts held in the cell walls or adhering to
the surfaces of the plant.

The analyses of animals are less consistent
than those of flowering plants but in the major-
ity of cases potassium seems to predominate over

PHTSIOLOGT 35

sodium. Erythrocytes are of special interest. In
some cases they contain little or no sodium and
there is reason to believe that the large amount
of potassium which is present exists in solution
as an inorganic salt. On the other hand there are
species whose erythrocytes contain mostly so-
dium with very little potassium.

It is therefore apparent that we are not war-
ranted in concluding that potassium invariably
predominates over sodium in the sap of living
cells.

It may be noted that by adding ammonium
chloride to the sea water it is possible to substi-
tute ammonium to a certain extent for the so-
dium and potassium of the sap without appar-
ently injuring the cell.

Another interesting problem is suggested by
the fact that the dissimilarity in composition of
the two Valonias bring about a marked difference
in their behavior in that V. fnacrofhysa sinks in
sea water while V . ventricosa floats. It may be
said in this connection that it would seem that
the flotation of living cells may be brought about

36 CELLULAR

m a variety of ways, and that the subject de-
serves more careful investigation.

Another problem which has been attacked by
the use of these large cells is whether proto-
plasm is permeable to ions. This question, an-
swered in different ways by opposing schools,
has become a center of controversy. Each side
has assembled an imposing array of facts on
every point of importance but the evidence re-
mains conflicting and the interpretation doubt-
ful. It is evident that the most satisfactory way
of attacking the problem is to study the pene-
tration of substances which are partly ionized
and to determine by direct analysis whether the
concentration of such a substance inside the cell
corresponds to the ionized or to the non-ionized
part of the substance in the external solution.

A suitable material for this purpose is hydro-
gen sulphide, which penetrates readily, is easily
measured, and is only slightly toxic at low con-
centrations.

The experiments show that when a living cell
of Vdonia is allowed to take up hydrogen sul-
phide until the sap comes into equilibrium with

PHTSIOLOGT 37

the sea water in respect to this gas, the concen-
tration inside is always less than outside (except
in quite acid solutions), and as the alkalinity of
the external solution increases, and the hydrogen
sulphide becomes more completely ionized, its
concentration in the sap becomes less. This is
shown in Fig. i, in which the concentration of
total hydrogen sulphide in the sap is designated
by the symbol (O) and the concentration of un-
dissociated hydrogen sulphide in the sea water
is designated by the symbol {^) when calculated
from the dissociation constant, by the symbol
(A) when determined by the method of vapor
tension, and by the symbol ( X ) when measured
by the rate of evaporation.

Not only the inside concentration but also the
rate of penetration corresponds closely to the
concentration of undissociated substance outside.
This indicates that under normal conditions
hydrogen sulphide cannot enter the cell very
rapidly except in the form of undissociated
molecules and that it does not dissociate to any
great extent after entering the cell. Precisely

38 CELLULAR

the same conclusion is reached as the result of
studies on the penetration of carbon dioxide.

It is true that, as far as final concentrations
are concerned, we might expect a similar result

Fig. I demonstrates that the total sulphide in the sap corre-
sponds with the undissociated HgS in the external solution.
Ordinates represent undissociated H2S in the case of sea water
and total sulphide (HgS + HS' + S") in the case of sap.
Abscissae represent pH values. The concentration of total
sulphide (H2S + HS' + S") in the cell sap (O) is expressed
as per cent of the total sulphide in the outside solution. The
values for the concentration of undissociated HgS in the sea
water as calculated from the dissociation constant (D) and
as determined from the vapor tension (A) and from the rate
of evaporation (X) at various pH values of the external
solution are expressed as per cent of the corresponding values
in the range pH i to 3 where all the HgS is regarded as
undissociated. Each point represents one determination.

PHTSIOLOGT 39

if the ions of these substances entered and if a
Donnan equilibrium were set up in a certain
way, but in that case we might expect to find a
diflFerent behavior in respect to rate of penetra-
tion. We may expect the rate of penetration to
rise as the concentration of the penetrating sub-
stance increases. If it is the undissociated mole-
cules which enter, this is what we actually ob-
serve, for as the acidity of the sea water increases
the concentration of undissociated molecules and
the rate of penetration likewise increase. If,
on the other hand, we assume that it is the ions
which enter, we should be obliged to conclude
that as their concentration increases (with in-
creasing alkalinity of the sea water) the rate of
penetration falls off.

It is evident that (except when there is an
ionic exchange in opposite directions) a cation
cannot pass through the surface without a cor-
responding anion, so that in general a cation can
pass only when it strikes the surface at the same
time as an anion. Hence in many cases a cation
must strike the surface without entering but in
the case of an undissociated molecule every one

40 CELLULAR

which Strikes the surface may penetrate provid-
ing there is no other hindrance except the elec-
trical forces. On this basis we should expect
undissociated molecules to enter more rapidly
than ions.

In this connection it may be noted that the
work of Loeb, Harvey, Crozier, Haas, Jacobs,
M. M. Brooks, Smith, Clowes, and Beerman
(on various weak acids) indicates that undis-
sociated molecules penetrate, although the
methods employed do not enable us to decide
positively whether ions enter or not. Those who
have concluded that ions cannot penetrate have
done so on purely theoretical grounds or as the
result of indirect evidence. This also applies to
a large extent in cases where the opposite con-
clusion has been reached.

If it should turn out to be generally true
that ions are unable to penetrate except very
slowly how shall we regard the evidence for the
contrary view? This evidence rests chiefly on
experiments with plasmolysis and electrical con-
ductivity. It is found that many cells recover
after plasmolysis when left in the plasmolyzing

PHTSIOLOGT 41

salt solutions (provided they are not too concen-
trated). Since these salts are largely ionized
this may be regarded as evidence of permeability
to ions. It is, however, quite possible that in
these experiments the cells are readily per-
meable to ions only because they are abnormal.
It is well known that plasmolysis produces in-
jury and that injury is accompanied by changes
in permeability. It is also possible that the cells
may subsequently recover from such injury
and appear to be normal j in this case the per-
meability to ions would be only a temporary one.
Injury might affect only a portion of the cell
surface (possibly numerous small areas). Ex-
periments on large multinucleate cells ( Valonia,
Nitellay Caulerfay Bryofsis) indicate that a por-
tion of the cell surface may be greatly altered
while the remainder remains in normal condition
for a long time afterward.

If recovery from plasmolysis in salt solutions
depends on alterations of permeability we
should expect the rate of recovery from plas-
molysis to correspond somewhat with the
amount of alteration. If the alteration goes too

42 CELLULAR

far the cell may become so permeable that no
recovery is possible, but up to a certain point
increase in permeability would increase the rate
of recovery from plasmolysis if exosmosis were
not greater than endosmosis. From this stand-
point we might expect the recovery in sodium
chloride to be more rapid than in a balanced
solution of sodium and calcium chlorides (or
in sea water) since alterations in permeability
would be more rapid in sodium chloride. This
seems to be the case. When recovery occurs in
balanced solutions it is possible that it is also due
to alterations in permeability, since it is well
known that hypertonic balanced solutions may
cause injury.

If ions are unable to penetrate normal proto-
plasm how are we to regard the experiments
which indicate that marine plants bathed in sea
water allow ions to enter the protoplasm and
thus conduct the electric current under condi-
tions which seem to ensure that the cells are in a
normal state?

In the first place it is possible that if the cell
normally opposes a high resistance to the

PHTSIOLOGT 43

passage of ions this resistance may be overcome
under electric stress so that ions may be forced
through the surface of the protoplasm, although
they would not enter if the electric potential
were absent. In this case the measurement of
electrical conductivity would reveal changes in
resistance to the passage of ions brought about
by various conditions, but the passage of the
electric current would not mean that ions could
penetrate to an appreciable extent in the absence
of an applied potential. From this standpoint we
may say that the general conclusions derived
from electrical experiments would not be
changed except that the normal cell would not
be regarded as permeable to ions. The measure-
ment of changes in resistance to the passage of
ions brought about by abnormal conditions and
the conclusions drawn from these measurements
would still be valid.

In the second place it is possible that the meas-
urements of conductivity do not indicate the
passage of ions through the protoplasm, as has
been supposed. If the cell acts as a condenser an
alternating current may pass without actual

\^l LIBRARY 1?

44 CELLULAR

transfer of ions through the protoplasm as
indicated by some older experiments and more
recently made highly probable by investigations
carried out in my laboratory by Mr. L. R.
Blinks. In that case the increase in conductivity
(as measured by the alternating current) which
occurs when a cell is injured may be regarded as
analogous to the change by which a condenser
becomes a conductor. If the cell surface is
covered with a non-conducting substance injury
might alter this substance in certain places, so
that the conductivity would increase. If this view
should turn out to be correct we should never-
theless continue to regard the measurements of
the electrical conductivity of living tissues by
means of the alternating current as of great
value in detecting changes of permeability. The
reasons for this have been given in discussing
the electrical method.

If we adopt the hypothesis that ions can enter
normal protoplasm only very slowly it is evident
that injury and death are accompanied by in-
creased permeability to ions. There is good
evidence that this is the case.

PHTSIOLOGT 45

Although undissociated molecules appear to
enter much more rapidly than ions it is not to be
expected that all undissociated molecules enter
the protoplasm with the same readiness and in
many cases it appears as though they cannot
penetrate into the sap unless they can combine
chemically with some constituent of the proto-
plasm.

Some such considerations may possibly explain
the excluding mechanism whereby, for example,
Valonia macro fhysa prevents more or less com-
pletely the entrance of undissociated molecules
of such substances as phenol, glucose, lactose,
sucrose, and many others, while admitting more
or less freely undissociated molecules of hydro-
gen sulphide, carbon dioxide, ammonia, ethyl
alcohol, dimethyl-glyoxime, paraphenylene-
diamine, and urea.

It would therefore seem as if the surface
forms a barrier which more or less completely
prevents the passage of ions, and as if undis-
sociated molecules can enter the sap only by
passing through a layer which excludes such sub-

46 CELLULAR

stances as are insoluble in it or unable to unite
with it chemically.

In this connection we may recall the fact
that a monomolecular film on the surface
of water does not prevent the evaporation of
water. It is evident that water molecules pass
through the oil. In this case the escape of water
may not be governed by the solubility of the
water in the oil but by the size and speed of the
water molecules and the direction in which they
strike the surface. If we extend this idea to other
kinds of molecules we must also consider that
the shape of the molecules may be of importance
in some cases. It is of course quite possible that
the surface of the protoplasm is a non-aqueous
monomolecular layer.

Let us now endeavor to picture a mechanism
by which certain substances may accumulate in
the cell. Suppose that undissociated molecules
of a weak base penetrate until their concentration
is the same inside and outside (the acidity and
other conditions being the same inside and out-
side) . If now the cell produces acid, some of the
molecules of the weak base will dissociate and

PHTSIOLOGT 47

more will diffuse in until the concentration of
undissociated molecules again becomes the same
within and without. This process may continue
until the inside concentration of total weak
base (ionized plus non-ionized) becomes much
greater inside than outside. A weak acid would
accumulate in the cell in the same way if the
cell produced alkali. We may extend this hy-
pothesis to include not only weak acids and bases
but all other substances, organic or inorganic,
which are able to change with changes in the
concentration of hydrogen ions. Such changes
(including tautomerism, formation of complex
salts, hydration, hydrolysis, etc.) may affect sub-
stances to a greater extent than is at present sus-
pected.

It may seem possible in some cases that accu-
mulation may be due to a Donnan equilibrium.
This exists when one or more non-diffusible ions
are present in the cell or in the external solution
and it requires that the following ratio should
exist for any pair of diffusible ions:

cone, cation inside cone, anion outside
cone, cation outside cone, anion inside

48 CELLULAR

If we have ten times as many hydrogen ions
inside as outside and if potassium ions are pres-
ent inside we should expect ten times as many
potassium ions inside as outside providing both
these cations are diflFusible. This might seem at
first sight to afford a means of explaining the
accumulation of potassium when the sap is more
acid than the external solution, as is normally
the case with Nitella and Valonia. But it also
requires that the accumulation of all other dif-
fusible cations should take place to the same
extent and this is not the case.

We see, therefore, that we may explain accu-
mulation in many cases where a substance exists
in two forms whose relative proportions vary
with the differences in acidity, and it is possible
that this explanation applies to more substances
than is at present suspected. It may be objected
that this would require that all cells having
equally acid sap should behave in the same way
in regard to accumulation. This, however, would
not necessarily be true, since differences in per-
meability may bring about differences in the
relative proportions of electrolyte in the sap.

PHTSIOLOGT 49

Although the protoplasm might not change the
final equilibrium it might prevent a true equili-
brium being reached by causing some substances
to enter so slowly that no true equilibrium would
be reached during the period of growth.

The problem is a very complicated one and it
seems clear that we cannot hope to attack it suc-
cessfully without the help of careful quantita-
tive work. The studies here described are a
preliminary attempt in this direction.

These studies lead me to look upon the semi-
permeable membranes of the cell as even more
important than I supposed. In certain cases it
seems quite possible that in a cell immediately
after death the same substances may be present
and the same reactions go on as just before it
was killed. But the difference between the living
and the dead state is very marked in respect to
the semipermeable surfaces: after death they
lose their selective power and the internal and
external solutions begin to mingle. When the
semipermeable surfaces no longer safeguard the
privacy of the chemical processes of the cell the
vital activities are unable to continue in. normal

50 CELLULAR

fashion. The diflPerence between a cell imme-
diately before and just after death might there-
fore be essentially a difference in the structure
and chemical composition of the semipermeable
surface.

It may be added that such semipermeable sur-
faces are not confined to the exterior of the cell
but exist also at the boundaries of nuclei, plas-
tids, microsomes, and other structures in the cell.
It may be that the chief advantages of cell divi-
sion (as well as of the differentiation of cell
organs) is to provide such surfaces and thereby
to segregate various vital activities.

In this connection we may recall that Loeb
has insisted that the colloidal properties of
matter are manifested only where semiperme-
able surfaces exist. A study of such surfaces is
important for chemistry as well as for biology
and it is possible that further investigations in
this field will lead to contributions to chemical
theory just as did the work of Pfeffer at an
earlier period.

These and other studies now in progress have
changed our point of view and are bringing a

PHTSIOLOGT 51

wealth of fresh problems. Although some of
these may be insoluble in the present state of
science, we ought at least to see how far we can
go. The whole subject is so important that we
can afford to neglect nothing which may help us
to understand the mechanism of these funda-
mental activities of the cell.

Since this lecture was given additional facts
have come to light which have necessitated some
changes in viewpoint.

The investigations of Blinks and Howe indi-
cate that what is here called Valonia ventricosa
is in reality a species of HalicystiSy which puts a
different face upon a part of the discussion. The
accumulation of dye appears to be due to its be-
havior as a weak base which enters the sap and
dissociates rather than to its combination with
the proteins of the sap {cf. Irwin, M., /. Gen,
Physiol.y 1 926-1 927, Xj 75). It seems possible
that the accumulation of potassium can be ex-
plained by assuming that it enters as potassium
hydrate, is converted to potassium carbonate and

52 CELLULAR PHTSIOLOGT

bicarbonate, and becomes potassium chloride by
exchange of the carbonate and bicarbonate ions
produced inside the cell for chloride ions coming
in from the sea water. The distribution of chlo-
ride ions would be determined in this case by the
Donnan principle (cf. Osterhout, W. J. V.,
Proc. Soc, Exfer, Biol, and Med.y 1926, xxiv,
Dec. No.).

The fact that the electrical resistance of the
protoplasm is so high might be explained, as
stated above, by supposing that the outer layer
of the protoplasm consists of a layer which is
only slightly permeable to ions and which acts
to a considerable extent as a condenser. Later in-
vestigations by Dr. Blinks indicate that (due to
the polarization capacity) much the same effect
would be produced if ions of one sign entered
the protoplasm much more readily than those
with the opposite charge. Further investigation
will be needed in order to clear up this question.

INDEX

Absorption of food, 2

Accumulation, 46-49

Acidity, 30, 39

Acids, 14, 40

Algae, marine, 34

Alkalies, 14

Alkaloids, 14

"All or none" law, 21

Alternating current, 16, 17,

44
Ammonium, 35, 45
Anesthetics, 1 2
Animals, 34
Antagonism, 20
Arrhenius, 8, 10
Ash analyses, 34

"Balanced solution," 31
Beerman, 40
Bermuda, 32
Blinks, 17, 44, 51, 52
Brilliant cresyl blue, 26
Bromide, 32
Brooks, M. M., 40
Bryofsisj 41
Burned tissues, 31

Calcium, 14, 32

Calcium chloride, 20, 42

Calcium oxalate, 15

Cambridge, 33

Cane sugar, 9

Carbon dioxide, 38, 45

Caulerfa, 41

Cell sap, 13, 24, 25, 28, 31,

33-38
Cell wall, 3
Chloride, 32
Chloroform, 12
Chloroplasts, 3, 24, 25, 28
Clowes, 40

Colloidal properties, 50
Condenser, 17, 52
Conductivity, 43, 44, 52
Copper ferrocyanide, 5, 6
Crozier, 40
Crushed tissues, 31

Davis, 33

Death, 18-20, 22, 44, 49

Development, 22

Diffusion, 23

Dimethyl-glyoxime, 45

Donnan, 52

Donnan equilibrium, 39, 47

Drugs, 2

54

CELLULAR

Dyes, II, 13, 14, 18, 26, 27

Electrical resistance, 16-19

Endosmosis, 42

Erythrocytes, 35

Ether, 12

Ethyl alcohol, 45

Excretion, 2

Exosmosis, 27, 42

Flotation, 35
Flowering plants, 34

Glucose, 45
Griins, 1 1
Growth, 3

Haas, 40

Halicystisy 51

Hamburger, 1 1

Harvey, 40

Hedin, 1 1

Hoagland, 33

Howe, 5 1

Hydrogen sulphide, 27, 36-

38, 45

Indicators, 14
Injury, 18-22, 41, 44
Ionization, 8, 36-46

Irritability, 5
Irwin, 26, 51
"Isotonic coefiEcients," 7

Jacobs, 40

Lactose, 45
Laminaria^ 16-18
Lipoid theory, 11
Loeb, 40, 50

Magnesium, 29, 32

Microsomes, 50

Molecular weight of sugars,

9, 10
Monomolecular film, 46
Mosses, 34

Naples, 32

Nitellay 24, 26, 28, 41, 48

Nuclei, 23, 24, 28, 50

Organic matter, 32
Osmotic pressure, 4, 9-1 1
Overton, 11, 12
Oxalate, 15

Paraphenylenediamine, 45
Pasteur, i

PHTSIOLOGT

55

Permeability, 2, 23

to ions, 36-46, 52
Pfeffer, 5-8, 11, 50
Phenol, 45

Plasma membrane, 23
Plasmolysis, 8, 15, 40-42
Plastids, 3, 23, 50
Potassium, 29, 32-35, 51

Quincke, 11

Raffinose, 9, 10
Recovery, 18, 21, 41
Relationship, 33
Rockefeller Institute, 28

Salts, II

Sap, 13, 24, 25, 28, 31, 33-

38
Sea water, 32
Secretion, 2

Selective permeability, 2
Semipermeable, 4
Smith, 40

Sodium, 29, 32-35

Sodium chloride, 19-21, 42

Stamen, 5

Stimulation, 22

Sucrose, 45

Sugar solution, 4, 6, 9, 45

Sulphate, 29, 32

Temperature, 23
Toxic substances, 31
Traube, 5

Urea, 45

Vacuole, 3

Valonia, 27-29, 31-33, 35>

41, 45) 48, 51
van't Hoff, 6, 7, 10
Vitality, 18
Vries, de, 7-10

Weak acids, 40, 47
Woods Hole, 33