-NRLF

Dr.

Gift of
James Hopper, Jr.

HYDRATION AND GROWTH

BY
D. T. MACDOUGAL, PH. D., LL. D.

Director of the Department of Botanical Research
Carnegie Institution of Washington

PUBLISHED BY THE CARNEGIE INSTITUTION OF WASHINGTON
WASHINGTON, 1920

CARNEGIE INSTITUTION OF WASHINGTON
PUBLICATION No. 297

PRESS OF GIBSON BROTHERS, INC.
WASHINGTON, D. C.

I \

PREFACE.

Three main conceptions concerning growth and its developmental
aspects in plants are to be met in the history of physiology in the half
century beginning with the researches of Sachs and his school. The
first or earliest, that of special stuffs or substances necessary for the
initiation of growth and differentiation of various organs, especially
for the origination and development of reproductive organs, is now
giving way to the modern conclusion that "formative" material as
such has no actual existence in fact and no good basis in theory. The
present trend of thought leads to the assumption that growth
proceeds from and depends upon states or combinations of material
or accumulations in connection with living matter rather than upon
any special constructive stuff or substance. This may also be held
to apply to hormones, vitamines, and other symbolic expressions for
combinations of material necessary for initiating and maintaining de-
velopment, reproduction, or growth.

The second aspect of the subject is that which deals with the incor-
poration of new material in the cell and its subsequent distention by
an osmotic mechanism, upon the basis of researches of Pfeffer and
de Vries. The protoplast is dealt with as a sac. The products of the
metabolic processes converge in the vacuole, which in consequence
becomes the seat of osmotic forces and the center of the mechanism of
distention. An important feature of this scheme of operation is an
ideal "semi-permeable" membrane, not morphologically identifiable,
internal to the cellulose wall or other durable and visible integument.
The exploitation of the theory of permeability of this membrane has
been carried out in such manner as to place undue emphasis on the
action of the external layer of the protoplasm. The basic conception
of the diffusion of material into the vacuole remains sound, and the
differentiating action of protoplasm by which the increase in the con-
tents of the vacuole sets up an internal pressure expressed as turgidity
is undeniable. But the attempt to base all features of water-relations,
turgidity, and growth upon the action of solutions has been proved
inadequate and has resulted in an obvious neglect of the play of molec-
ular forces in surface tensions, in imbibition, and other activities of
matter in a colloidal state.

The third group of inquiries has been directed toward measurement
for the purpose of establishing the physical constants of growth.
Auxesis or developmental enlargement in living things has been mis-
takenly dealt with as a unified process, or as a series of successive
reactions in studies of temperature effects, by many writers, and
coefficients of some apparent validity within a small part of the range
within which growth takes place have been found. Growth is a con-
stellation of activities and the rate of one of these dependent on tem-
perature may be the determining one when the particular process forms
either the retarding or leading agency. At other times the relative

rv Preface.

rates of metabolism, respiration, hydration, and diffusion may coincide
in such manner as to make possible the application of a simple formula
for the effects within a range of 15° or 16° C. The relation of growth
to temperature for any plant between 10° and 50° C. is not to be
expressed by any simple formula. The same general statement may
be made concerning light and other agencies, none of which has received
more than a fraction of the amount of attention which has been paid
to temperature effects.

The assumption as to the general identity of protoplasm in plants
and animals, or even in plants as a group, is one which operates to stifle
analytical investigations in a subject of this kind. The relative
amounts of proteins, carbohydrates, lipins, and salts in the two groups
differ widely. In addition to the capacity of the plant to synthesize
carbohydrates, amino-acids, etc., which the animal can not, the res-
piration and metabolism of the plant are predominantly carbohydrate,
while those of the animal are proteinaceous to a much larger extent.
It would seem obvious that a protoplasm rich in fats, high in proteins,
and permeated with their derivatives would display an imbibition and
growth different from living matter in which the base is chiefly the
comparatively physiologically inert pentosan groups and which neces-
sarily adsorb the salts and acids in a characteristic manner. The
unities or general properties of the protoplasm of widely different
organisms do not rest upon the presence or proportion of elements or
compounds so much as upon the manner in which the necessary con-
stituents are brought together. This indispensable condition of life
is the colloidal state, in which the substances of living matter, form a
semi-solid or elastic gel consisting of over 90 parts water. The mole-
cules are large, slow-moving, and adhere to form aggregates as con-
trasted with the separation of molecules in the water of solutions. This
colloidal structure may be profitably likened to that of a house or fac-
tory, serving simply as the scene of metabolic processes which take
place under special conditions of surface tension. The colloidal labora-
tory may be in the form of an emulsion, a reticulum, a sponge, a crystal-
line or lamellar structure, with corresponding effects on metabolism,
while the products of respiration may in turn cause alterations in the
chambers in which it takes place.

The purpose of the present work has been to study growth upon the
basis of a more inclusive conception than that usually implied in
osmosis. The total absorbing capacity of a cell or mass of protoplasm
for water is regarded as being exercised in the process of hydration.
The source of energy in growth and swelling is the unsatisfied attrac-
tion of molecules, or particles or ions bearing an electrical charge.
Substances made up in this manner may unite with definite propor-
tions of water which becomes part of a symmetrical chemical structure,
the union being known in classical chemistry as hydration. In addi-
tion, however, it is known that such particles may also adsorb and hold
in combination additional molecules of water, an action especially

Preface. v

characteristic of swelling in colloids, and the term hydration is used in
the present work to include the entire range of action.

The method of study employed has been one in which biocolloids
have been compounded from pentosans and proteins in proportions
simulating those of the plant, and the total range of swelling of thin
plates of this material has been measured by the auxograph which has
been developed for this purpose. Series of measurements of such
material have been arranged to run parallel with measurements of the
unsatisfied hydration capacity of living cell-masses and of dehydrated
tissues.

The acids and salts have been employed in concentrations mostly
within the range of the biological possibilities. It follows, therefore,
that these substances have been applied in solutions in which complete
or nearly complete dissociation has taken place.

It was deemed of the greatest importance to traverse a wide field of
possibilities, which made the use of simple methods advisable, and
solutions have therefore been applied in terms of molar or normal con-
centration, and acidity has been determined by titrations. The im-
portance of determinations of the acidity, especially of the cell-sap,
and its expression in terms of hydrogen- or hydroxyl-ion concentration
would be greater in any more critical study of the features of colloidal
and protoplasmic action discussed, although it is not to be taken for
granted that this is the dominant or determining factor in all cases.

The use of simple methods has served to reveal the general hydra-
tion relations of plant protoplasm, the influence of acidity and tem-
perature upon growth and swelling, and to uncover the special effects
of the amido-compounds upon hydration and their suggested possi-
bilities in affecting growth.

The significant water-relations of the cell-colloids are not entirely
included in direct reactions of the kind mentioned, however. As will
be described in Chapter VII, the exigencies of plant life include condi-
tions under which dehydration of the plasmatic colloids may reach
such a degree that the nature of some of the sugars in growing cells
may be affected, and one of these changes is the conversion of poly-
saccharids with a low hydration coefficient to pentosans with a high
hydration capacity, with the resulting succulency or xerophily of the
tissues in which this takes place.

I am indebted to my colleagues for suggestions and assistance both
in the experimentation and in the preparation of the manuscript,
especially to Dr. H. A. Spoehr, who has collaborated in previous papers
and who has given continued cooperation and valuable advice on
various phases of the work presented here.

DESERT LABORATORY, D. T. MACDOUGAL.

Tucson, Arizona, 1919.

CONTENTS.

PAGE.

1. Growth and colloidal reactions 1

II. Fundamental features of phytocolloids 11

III. The constituents of biocolloids which affect hydration and growth 27

IV. The effect of salts and acids on biocolloids and cell-masses 37

V. The effects of organic acids and their amino compounds on hydration and

growth 54

VI. Reactions of biocolloids and cell-masses to culture solutions, bog, swamp, and

ground water, and other solutions 65

Vll. Fluctuating or alternating hydration effects. Basis of xerophily and succu-
lence 78

V11I. Water deficit, or unsatisfied hydration capacity 92

IX. Temperature and the bydration and growth of colloids and of cell-masses. ... 110

X. Imbibition and growth of Opuntia 128

XI. The hydration reactions and growth of Mesembryanthemum, Helianthus, and

Phaseolus 145

XII. Water-content, dry weight, and other general considerations 161

Literature cited 174

VI

HYDBATION AND GROWTH.

BY D. T. MACDOUQAL.

I. GROWTH AND COLLOIDAL REACTIONS.

Growth consists in increases in volume of masses of living matter,
usually but not invariably accompanied by accretions of material
other than water to the colloids of the protoplasm. Auxetic changes
in members, organs, or cells of the larger plants may be readily deter-
mined by external measurements, and the greater part of the available
information concerning the subject has been obtained in this manner.
Many generalizations, however, rest upon data secured by taking the
gross weight of organisms; in other cases the dry weight is used as a
criterion, a method which obviously may be used only in securing end
or total results. A count of the number of individuals may afford a
reliable basis for the estimation of the rate of growth and multiplica-
tion of unicellular organisms such as bacteria, in which the limits of
enlargement of the individual are quickly reached. Much of the value
of the results presented in the present volume is to be attributed to
methods by which the varying dimensions of organs and of individual
plants were followed not only through the entire period of develop-
ment, but alterations accompanying maturity were measured with
some precision. The information thus secured made it possible to
interpret the effects of the ever-changing daily complex of environic fac-
tors and to evaluate to some extent the effects of previous experience
upon the behavior of a growing organ at any stage of its development.

Thus, for example, the action of a cell-mass at any given tempera-
ture is influenced not only by the degree of the temperature and other
environic conditions at that tune, but also by the previous experience
with these factors, particularly temperature. This " memory ' ' of ante-
cedent impressions is not psychological in any sense, but rests upon
definite properties of colloids which are known or are measurable.

The diversity of constitution and consistency and variation in col-
loidal condition of living matter is so great as to evade exact or detailed
description. But the general composition of protoplasm, the character
of its activities, the mode or manner of changes in its colloidal states,
and a measure of the factors affecting its activities may be compre-
hended without leaning upon vitalistic conceptions or resorting to
mysticism in any form. The fundamental and ultimate structure or
architecture of protoplasm is a result of the force of surface tension and
is a gel in which the solid material occurs in two main states or phases

2 Hydration and Growth.

with water. In the more liquid phase the molecules of the substance
are associated with such a large proportion of water as to be in a sus-
pended condition, while in the more solid phase the proportion of
water is much less. This phase has a distinct architecture which has
been likened to that of a mesh, felt, foam, or honeycomb, in which the
denser phase forms the framework and the fluid fills the interstices.
Under certain conditions the phases may be reversed, and the solid
particles be rounded into globules entirely surrounded by the fluid.
These structures are far too minute to be visible under the microscope,
as the particles which are dispersed in the liquid or are aggregated in the
denser structure ma each consist of a few molecules only. In addi-
tion to the material in the actual sponge of protoplasm, some of the
same or other substances may be present in dispersions or solutions in
cavities and spaces in the cell, which result from morphological or
mechanical action of the protoplast (see p. 21).

The aggregation of molecules of a substance in a colloidal condition
such as that noted above is a more complex matter than that of the
solution of a crystalloidal compound, as, in addition to the forces of
chemical combination, surface tension results in adsorption or union
of substances in indefinite proportions.

Four main groups of substances make up the protoplastic engine —
carbohydrates, proteins and their derivatives, the lipins, and the salts.
Perhaps all carbohydrates may exist in a colloidal condition, but the
group polysaccharids, including the pentosans, are the most important
in the architecture of the protoplasmic mesh, as these substances with
proteinaceous compounds appear to determine the water-relations of
living matter, and to contribute to the design of a machine in which
metabolism takes place. The proteinaceous substances may in plant
protoplasm form a widely varying proportion, generally very low, but
sometimes ranging as high as 90 per cent of the entire dry weight of
the protoplasm, with very important consequences as to imbibition.
The enzymes are included with these substances, and as the metab-
olism, including respiration of plants, is predominantly a complex of
transformations in carbohydrates, the possibilities of variation in this
feature are very great. The nature of the pentose derivatives present
in the protoplasm may also be a feature of considerable importance in
metabolism and water-relations, as suggested by the differential be-
havior of various gums and mucilages when compared with that of
agar. The. colloidal carbohydrates, or those which enter into the
make-up or design of the living machine and the proteinaceous sub-
stances, are theoretically mutually nondiffusible, so that the gelation
or solidification of a 10 per cent solution of agar and gelatine or starch
and gelatine according to Beijerinck1 would result in a mechanical

1 Beijerinck, M. W. Ueber Emulsoidenbildung wasseriger Losungen gewisser gelatinierender
Kolloide. Zeitsch. f. Kolloid-Chem., 7:16. 1910.

Growth and Colloidal Reactions. 3

mixture of the two substances in which the two would exist separately
in their characteristic emulsion. The ammo-acids, on the other hand,
diffuse readily into the colloids, and these may be visualized as being
aggregated with the carbohydrate colloid, in both phases, and, as may
be seen by reference to Chapter II, they set up water-relations differ-
ent in some features from those determined by the hydrogen and
hydroxyl ions.

The place of the lipins in the hydration mechanism can not at present
be made the subject of profitable conjecture. While lecithin, for
example, is known to adsorb water, its part in the living matter of the
plant must be all but negligible, as it by no means bulks as large here
as in the animal.

The general effect of the salts is to lessen the imbibition capacity of
agar-protein mixtures when in simple solutions from a concentration
of 2 N to 0.00005 N. Set in action with acids or in antagonistic rela-
tions, other effects are produced in sections of living plants. Although
one of the subjects receiving the most attention in colloidal physics,
and although extensive experiments with tissues and cell-masses of
plants and animals dealing with the matter have been made, it is not
yet definitely determined whether or not the action of a salt upon a
biocolloid may be expressed by the algebraic summation of its acid and
base as originally proposed by Pauli for gelatine, or whether the effect
is due partly to other factors.

The extent to which protoplasm may be made up of dense particles
of substances of a single category, such as protein or globulin granules,
starch grains, or of minute masses of more highly hydrated strands or
globules of albumin or of carbohydrates, or lipins, or of combinations
such as those of lipin and protein in the mitochondria, can not be
visualized. Neither is it possible to say whether the carbohydrate
and protein molecules are equally aggregated in both the continuous or
external phase and the discontinuous or internal phase of the gel, or
whether one predominates in each phase according to the proportions
in which they are combined. In any case phase reversals are possible.
The imbibitional reactions of the living matter of plants are seen, how-
ever, to be parallel to those of a salted carbohydrate-protein gel com-
bined in a high state of dispersion and questions as to systemic arrange-
ment must be left in abeyance for the present. So far as known, it is
the actual composition and relative proportions of the substances of
the main organic groups and the amount, the stage, and sequence of
incorporation of the infiltrated salts that constitute variables of the first
order of importance in the determination of the behavior of the mass.

The chief distinction between the protoplasm of plants and that of
animals may be taken to lie within the play of these major features.
Living matter in animals includes lipins and consists predominantly of
nitrogenous material, which displays maximum hydration capacity in

4 Hydration and Growth.

a hydrogen-ion concentration above the iso-electric point, or, as com-
monly expressed, when in a state of acidosis, in consequence of which
many sweeping premature generalizations have been made as to the
relations of electrolytes to protoplasm. Plant protoplasm, in so far as
the higher forms are concerned, is poor in lipins, is usually characterized
by a major proportion of carbohydrates, although in such simple forms
as the bacteria the protein content may be very high. The water-
relations of a cell-mass in plants will naturally be determined by its
protein-carbohydrate ratio, with the implied corollary that a varying
hydration capacity is displayed which may reach its maximum in a
condition of acidosis in forms rich in nitrogen, and in a neutralized,
relatively salt-free condition in those in which the proportion of col-
loidal carbohydrate is relatively great.

Cytological science recognizes that homogeneous states of the col-
loids do not prevail throughout the cell and a vast literature has grown
up concerning the masses of unlike composition, structure, and form,
some of morphological value, which make up the cell-body. Attention
has naturally been concentrated on the more readily visible, durable,
and measurable bodies, some of which are indubitably the scene of per-
formances of the first rank, and form the chief mechanism in genetics.

It is not to be forgotten, however, that the diverse mixtures of gels
and sols constituting the greater part of the protoplasmic mass, the
structure of which may not be resolved by direct microscopical methods,
is the ultimate colloidal machine in which the organs of the cell are
built up, torn down, and metamorphosed. The study of some of the
solid bodies in the protoplasm may yield the same comprehension of
the play of chemical energy and surface tension of living matter as
might be gained of the cyclonic forces of a storm by a measurement and
dissection of hailstones. The greatest possibilities in cell mechanics are
those which lie in the changes in viscosity, volume, and water-relations
of cell-organs as determined by the composition and arrangement of
the colloidal emulsion or mesh and the nature of the metabolism which
goes on in its sols and gels.

It is also to be emphasized that it is not only untrue but unprofitable
to assume that the living matter of plants and animals have the same
general chemical properties. The difference in the occurence and r61es
of lipin in the two groups is fundamental, and, in addition to the water-
relations, the metabolisms of the two present differences not attributable
simply to the carbohydrate-protein ratio in their composition. Thus
the living matter of plants includes within its metabolic cycles such
features as the synthesis of the carbohydrates and of the amino-acids,
the last-named capacity being exhibited only to a very limited extent
by animals, which, in the main, are characterized by a mstabolism of
sugars notably different from that of the plant.

Growth and Colloidal Reactions. 5

Available experiences with protoplasm lead to the conclusion that
it may be considered as a system of gels and sols in which the com-
ponent material is found in different conditions with respect to the pro-
portion of water combined or associated with it (see Chapter II). The
more fluid parts of the cell owe their liquidity to the fact that in such
material water containing a small proportion of the colloidal material
forms a medium or continuous element in which aggregations of mole-
cules or submicrons combined with a smaller proportion of water are
dispersed and may move about more or less freely. This condition
may be predicated of the contents of the vacuolar cavities and of the
regions in the cell which appear clear or vacant in living material or in
cytological preparations. The denser parts of the protoplast would
be composed of a much larger proportion of aggregated matter sepa-
rated by much thinner or more attenuated layers of the more fluid phase.

Some writers assume that the submicrons of colloidal material aggre-
gate to form a continuous framework or structure which has been
likened to a fine sponge, network of fibers, or honeycomb. The more
liquid colloid fills the cavities or interstices of the framework. This
condition may not be so completely the reverse of the preceding as to
bring the more fluid colloid into complete discontinuity. It may be
safely assumed, in fact, that almost any mass of active protoplasm in-
cludes both conditions, and in a very fluid portion of the living matter
small fragments of gel may be carried, while even in the denser newly
separated embryonic cell-regions minute cavities may be formed by
syneresis in which the colloid is in its extreme disperse condition. Such
syncretic cavities may well be the beginning of the vacuoles, in contra-
distinction to the view which assigns a definite morphological entity
to these features.

The formation of these syncretic cavities, the size of the molecular
aggregates, and many other features of a colloidal mass are affected
by the dilution or dispersion of the original material, the rate of dehy-
dration and gelation, and even such fundamental characters as the
relations of the two phases may be affected by the origin and rate of
deposition of the material. In addition to these very fertile sources
of variations in living matter, the cell at most times carries inclusions,
such as starch grains, crystals, and protein granules which are com-
paratively inert, partly by reason of their small surfaces, and may not
exercise much influence upon the surrounding gel. Oxidation, proteo-
lysis, hydrolysis, or solution of these bodies may set free or split the
compounds included, and these, quickly diffusing through the colloidal
mass, may play an important part in the morphological crises of the cell.

Many mistaken attempts have been made to compare the growth of
organisms and the formation of crystals directly, and to establish their
identity or continuity. The results of such efforts serve to bemuse the
mystic, to divert the philosopher, and to furnish poetical conceptions

6 Hydration and Growth.

to writers who view matter and all material conceptions from a remote
distance. Colloids, with their electrical charge, absorptive and adsorp-
tive properties, and molecular arrangement, display a series of char-
acters fundamental to organic growth, of which swelling as a result of
hydration is one of the most noticeable, which do not extend to crystals.

Furthermore, the colloidal systems which are exemplified in living
or organic material are rarely at rest in the sense of which this may be
said of crystals. It is true, of course, that substances or formations
may occur in nature or in the laboratory which are made up of both
crystalline and colloidal material, and it is also true that some com-
pounds may pass from one condition to the other, but the action by
which a crystal is formed is not one coincident with colloidal reactions,
nor does the perfect crystal behave like a mature cell, organ, or organ-
ism. In fact, the more perfect a crystalline structure may be, the
farther does it depart from the state in which it might display activities
or enlargements similar to those of growth of living matter.

The essential feature of an idealized growth is the accretion or addi-
tion of water and material to the mass of colloid constituting the cell.
The actual mechanism of incorporation is not easily delineated. If
protoplasm consisted of a system of colloidal structures such as those
of the pentosans and the proteins interwoven but not diffusing into
each other, the more solid material which lowers the surface tension to
the greatest extent, having the least attraction for water-molecules,
would tend to usurp the position of the surface layer. Furthermore
the solid phase, whether it be in the form of globules or in the continu-
ous element, would tend to increase and crowd together with a lessen-
ing of the more liquid phase. This would imply that when gelatine in
small proportion is mixed with agar or starch in the larger proportion
that the carbohydrate would form the colloidal framework or mesh as
well as the external layer of the mass.1

The separate colloidal masses where they do exist have, of course
definite boundary layers, as are formed wherever two colloidal phases
meet. Protoplasm may not be regarded, however, as altogether a
mechanical admixture of minute strands of material of different com-
position. Much of it, including the more fluid portions, must consist
of molecules of carbohydrates, proteins, salts, and even lipins aggre-
gated to form submicrons in the disperse phase or in the denser, more
solid fibers, mesh, or honeycomb of the structure. The external layer
formed might well be in a sense a mosaic, but it is to be noted that no
actual proof of such a condition is at hand. Both absorption or imbibi-
tion and osmosis, including differentiated diffusions, would be affected
by the composition and relations of the two phases of the colloids in this
outer layer, and it seems highly probable that an adequate interpre-

1 Free, E. E. A colloidal hypothesis of protoplasmic permeability. The Plant World, 21 :
141. 1918.

Growth and Colloidal Reactions. 7

tation of permeability will be obtained by a study of these features.
Meanwhile no general agreement as to the nature of the "membrane"
or its action is to be expected until many widely current assumptions
are discarded. The external layer of a protoplasmic unit is in every
case a product of the surface energy of the mass or systems of living
material internal to it and of the medium, and has no other permanent
or morphological value. Its constitution must necessarily vary widely,
as does that of the living protoplasm.1

This aspect of the external layer is one which finds recognition among
writers on biophysics in various ways. Mathews assumes conditions
in the protoplasm which are not valid in plants when he says :

"Thus it is suggested that in the surface of contact of protoplasm with
water, lipin substances will accumulate and thus make a kind of intermediate
layer of a lower surface tension and of a fatty nature. But, inasmuch as the
whole substratum of the cell is of a fatty or lipin nature, it is difficult to see
how the surface tension of the junction of fat and water could be changed by
the passage of more lipin into the film; and, as a matter of fact, there is no
good evidence that there is such a layer about the protoplasm."1

McClendon recognizes a wider range of facts,3 as follows :

"The composition of the plasma membrane remains a mystery. It seems
logical to assume that its building stones are selected from the chief constitu-
ents of cells, proteins, fats, lecithin, cholesterin, and carbohydrates. It is a
very unstable structure, as will be shown later."

An admirable presentation of the matter is to be credited to D'Arcy
W. Thompson, the keynote of which lies in the sentences:4

"The adsorbed material may range from the almost unrecognizable pellicle
of a blood-corpuscle to the distinctly differentiated 'ectosarc' of a protozoan,
and again to the development of a fully formed cell-wall, as in the cellulose
partitions of a vegetable tissue. In such cases, the dissolved and adsorbable
material has not only the property of lowering the surface tension, and hence
of itself accumulating at the surface, but also has the property of increasing
the viscosity and mechanical rigidity of the material in which it is dissolved
or suspended, and so of constituting a visible and tangible 'membrane'."

In addition to the external layer of the highly hydrated protoplasm,
this living material is usually separated from the surrounding medium
by walls or coats of specialized character, variously formed, and which
may be composed in part or altogether of material originating outside
of the masses which they inclose, which may modify the diffusion of
liquids into the colloidal mass in a very important manner.

If the swelling is one of simple hydration, the entrance of additional
water would result solely in an increased dispersion of both phases of

1 See Stiles and Jorgensen, Quantitative measurement of permeability. Bot. Gazette, 65 : 526. 1918.

2 Mathews, A. P. Physiol. Chem., 2d ed., p. 211. 1916.

3 McClendon, J. F. The physical chemistry of vital phenomena, p. 95. 1917. Princeton Univ.

Press.

4 Thompson, D'Arcy W. Growth and form, pp. 281 and 282. 1917. Cambridge.

8 Hydration and Growth.

the colloid. If dissolved salts are carried by the water, these substances
might unite chemically with the material in the molecular aggregates
in both the more liquid and the more solid phases of the colloid and
cause changes in the water-relations of the mass, or the dissolved sub-
stances entering with the water might form adsorption compounds by
the uniting in indefinite proportions with the colloidal material in
which the water-relations might be changed in another way. Such
changes would, of course, be followed by variations in volume. It is
to be added that water itself may enter into both relations with the
colloidal material and that the initial swelling of a dried colloid prob-
ably includes such a chemical combination of water with its molecular
aggregates. The action of salts or of acids brought into the mass with
water may be such as to carry the dispersion or solvation to the stage
in which the mass assumes a liquid condition.1

In that type of growth in which carbohydrates or proteins are carried
into the mass by water, it may be seen that the accumulation of the
additional material in the more liquid phase would by the action of the
forces of surface tension result in the aggregation of new masses of
material. Such formation of additional elastic gel structure might
occur theoretically throughout the entire mass of the cell, but in actual-
ity would be modified and controlled at every point by the factors
which affect hydration. Aggregation of material in syncretic cavities
may be taken to present possibilities of the formation of specialized
protoplasmic masses or cell-organs or of secretions. Writers with a
keen historical sense may be disposed to see in the conceptions outlined
above a modernized statement of the micellar hypothesis of growth of
Naegeli, but no great interest may be attributed to any such forced
parallelism.

The measurements described afford a reliable basis for the conclusion
that the extent and character of the swelling of gels compounded of
carbohydrates and proteins or protein derivatives depends in great
degree upon the proportions of the main constituents, not only with
respect to pure water, but in solutions of salts and electrolytes in
general. The general effect of a salt on hydration depends upon its
concentration and whether it is already present in the colloid in chem-
ical union or in adsorption with the colloidal material, or whether it
enters with the solution or water of hydration ; also upon the character
of the salts adsorbed. The extension of the observations upon which
these conclusions rest to living cell-masses and to desiccated and dead
material from plants demonstrates that colloids may be compounded
which may simulate with fair parallelism cell-colloids with varying
carbohydrate-protein ratio, salt-content, and acidity. In no feature
is this more striking than in the temperature relations. The rate and
amount of swelling of plants, and of colloidal mixtures which simulate

1Ostwald and Fischer. Theoretical and applied colloid chemistry, p. 101. 1917.

Growth and Colloidal Reactions. 9

them, in water or in some solutions may increase from temperatures
near the freezing-point to 39° to 46° C. and then fall off above this
region, or in acid solutions of a concentration normal to the plant both
biocolloids and sections of living and dried plants may show decreased
hydration as the temperature rises above 17° or 18° C.1

Nowhere is metabolism more active than in the embryonic growing
cell. The dissociations|which are usually included in the conception
of respiration may be taken to concern molecules of material already
present in the more liquid phase of the colloid or newly introduced.
The splitting of the sugars results in the formation of acids as one stage
of the process, and if the succeeding stages are impeded such material
accumulates, acidosis results, with new temperature relations which
may affect imbibition and the enlargement constituting growth in a
profound manner. Other actions will depend upon the composition
of the cell with respect to its carbohydrate and proteinaceous constitu-
ents. If it is high in albuminous material, its capacity for absorbing
and swelling may be greatly increased, while on the other hand this
effect will be greatly lessened if a large proportion of pentosans are
present, especially in the presence of salts. No further recapitulation
of detail is necessary to emphasize the fact that the products of the
reactions within the cell may be responsible for many of its most marked
changes in behavior. Such changes do not in any manner give support
or approval to vitalistic theories as to the constitution or activities of
living matter.

The other soluble carbohydrates, including the hexoses — sucrose,
dextrose — do not occur in the cell in such concentration as to affect the
enlargement of the protoplasmic mass directly, but in the vacuoles they
may exert an osmotic effect additive to that of the amino-acids which
may accumulate in these cavities. It is to the osmotic activity of
these substances in the vacuoles that turgidity is due, and a by no
means unimportant part in the maintenance of the rigidity of organs
and other features is to be ascribed to these turgor stresses and ten-
sions. That osmotic pressure may also play an important part in the
enlargement of the plant cell may well be concluded from the fact that
in the stage following the initial swelling of the embryonic cell a large
share of the increase in volume is due to the increase of the vacuoles.
The inadequacy of osmotic phenomena and of the conception of the
semipermeable membrane to provide a mechanism for the trans-
location of complex material from cell to cell, and the incorporation of
new material in a growing mass has long been recognized. It would be
a mistake to conclude that the vacuole is simply a sac charged with
electrolytes, as these cavities invariably hold proteins and carbohy-
drates in a colloidal condition in which the degree of dispersion may

1 MacDougal, D. T. The relation of growth and swelling of plants and of biocolloids to temper-
ature. Proc. Soc. for Exper. Biol. and Med., 15, No. 3, p. 48. 1917.

10 Hydration and Growth.

vary widely, but still absorb water. A correct delineation of the man-
ner in which osmosis and imbibition interlock in growth is one of the
tasks demanding the immediate attention of the physiologist.

It has been assumed in the present work that when the chief con-
stituents of protoplasm are brought together in a colloidal condition,
approximating that of living matter, the behavior of this material would
furnish data fundamental to the physics of growth. The justification
for this assumption is to be found in the following pages, in which are
described the reactions of biocolloids, of dead sections, and of organs
of living plants in hydration and growth as affected by solutions, cul-
ture media, the products of metabolism, and environmental agencies,
especially temperature.

II. FUNDAMENTAL FEATURES OF PHYTOCOLLOIDS.

It became evident in the earlier stages of the studies described in the
present work that the swelling of gelatine does not afford a parallelism
to the action of vegetative cell-masses of the higher plants, and that
only in certain reproductive elements or in some of the lower forms is
the proportion of nitrogenous material sufficiently great to give reac-
tions similar to those of gelatine. Experimental demonstrations of the
general character of the cell colloids was first made with sections of
joints of Opuntia. The results of an extended series of analyses of
these plants made by Dr. H. A. Spoehr covering all of the seasonal
changes were available, and from his results it can be seen that their
general composition is about as shown in table I.1

TABLE 1.

Constituents.

Young
joints.

Old

joints.

Water

p. ct.
95

p. ct.
75

Crude protein

0 5

1 0

Carbohydrates hydrolyzable with
1 per cent HC1

1.0

10.0

Cellulose

1.0

3.0

Crude fat

0.25

0.5

Ash

1.0

3.5

The hydration of an organ or cell-mass with a composition similar
to that shown in table 1 would of course be determined by the hydro-
lyzable carbohydrates and proteins and affected by the salts.

The first experiments were directed to ascertaining some of the
reactions of the carbohydrates which are known chiefly or entirely in
the colloidal form and which might be a constituent of the plasmatic
gels. The most readily available representative of these substances
was agar. Strands of the material were liquefied in water at 60° or
70° C., poured into shallow molds with the area of a postal card, and
allowed to desiccate to plates from which pieces 3 by 5 mm. were cut.
Swelling was measured by the auxograph, using an improved form, the
essential part of which consists of a compound lever, the members of
which are pivoted in adjustable bearings in a rigid brass frame.2 The
bearing lever has a forked free end suitable for the attachment of a
counterpoise and of a vertical swinging arm of twisted brass wire.
The free portion of this vertical lever is sheathed with a section of

1 MacDougal and Spoehr. Growth and imbibition. Proc. Amer. Phil. Soc., 56 : 335. 1917.
Philadelphia.
*Ibid., 327.

11

12 Hydration and Growth.

glass tubing with thin walls, drawn to a point and sealed hi a flame.
The pointed glass tip fits into a hole in the center of a thin glass plate
resting on the sections to be swelled. The attachment of the swinging
lever to the free end of the bearing lever may be adjusted to give an
amplification of the swelling, which is recorded by the pen tracing an
inked line on a sheet of paper 8 cm. wide ruled to millimeters. The
paper is coiled inside a brass cylinder and issues through a slit passing
to the drum of a clock of the same pattern as used on standard thermo-
graphs in such manner as to present a uniform plane surface to the
action of the pen in all parts of its arc. The tautness of the paper may
be varied by altering the position of the slit in the brass cylinder and
clocks may be employed which give the paper a motion of 28 cm. in
24 or in 168 hours. The two levers are connected with a short length
of jeweler's chain to minimize friction, and the base of the frame carry-
ing the levers is seated on the top of a rack-and-pinion column with a
vertical motion of 12 cm. and is capable of being fastened rigidly at
any height within its range of 10 to 12 cm. (fig. 1).

The dishes finally selected for containing the sections to be immersed
in solutions were of the Stender type, 5 cm. in diameter and 24 mm.
deep. It was found advisable to have the surface of the bottdm inside
the dish ground plane in order to avoid slipping and movement of the
swollen sections and of the delicate jellies formed by the biocolloids
when in states of extreme hydration. For similar reasons it was neces-
sary to place the entire preparation on a concrete pier, or still better
upon a slab of marble, granite, or concrete, " floated" in a large box
of sandy loam which had a direct bearing on the ground. The dishes
containing the sections were seated on various supports, the best form
being that of an iron or concrete cylinder about 12 cm. in height and
10 cm. in diameter. Three soft-metal studs were let into the basal
end of this cylinder to give it a three-point bearing on the marble slab.
With preparations made in this manner and with the counterpoise
arranged to give the least weight upon the swelling objects compatible
with a steady following movement of the pen, it was possible to obtain
valuable records of the velocity and course of swelling of sections of
plants and of various colloidal substances. It was soon learned that
more reliable results might be obtained with thin sections, in which
the coefficient of expansion would be high and complete hydration
would be attained quickly and with less dispersion of the colloid in
the liquid.

No records of temperature in the earlier tests were kept, but any set
of swellings, generally of three or four different solutions, were carried
on simultaneously and under the same conditions. It is to be under-
stood, therefore, that in tabulated data, as table 2, the relative swell-
ing of sections of one sample in the different solutions at the same time
only may be compared. The experiments had not been carried very

Fundamental Features of Phytocolloids. 13

far before it became apparent that the temperature of the solution in
which the swellings were made was an indispensable feature of the
control, and as but few contemporary workers have recorded this con-
dition, it is not possible to cite their results with profit.1 Some inter-
esting results are illustrated by the figures obtained without tempera-
ture control, which derive their value from the fact that all of those in

FIG. 1. — Auxograph arranged for recording changes in thickness of trio of sections of Opuntia and
of biocolloids. The vertical arm, which is set in position on horizontal arm to give an ampli-
fication of 20, rests on a triangle of glass laid on top of the sections. The dish containing
the sections rests on an iron cylinder to secure stability and a weight is placed on the T base
of the instrument. The record sheet is ruled to millimeters (not shown) with heavier hori-
zontal lines 1 cm. apart. The heavy curved lines represent hour intervals. The space is
ruled to 15-minute intervals (not shown). Height of clock and lever supports adjustable.

1 MacDougal, D. T. The relation of growth and swelling of plants and biocolloids to tempera-
ture. Proc. Soc. Exper. Biol. and Med., 15 : 48. 1917.

14

Hydration and Growth.

any given line have been obtained under identical conditions. One of
these comparative series made in 1916 may be cited as an example of
the relative behavior of agar and gelatine to water, acids, and alkalies.

TABLE 21.

Swelling of
agar.

Swelling of
gelatine.

Sodium hydroxid (hundredth molar) . . .
Hydrochloric acid (hundredth molar) . . .
Water

p. ct.
124
113
197

p. ct.
250
382
83

The next departure in the experimentation was to make a mixture
of these two substances as representing the carbohydrates and proteins
of the plant, and this was done in a series of plates in which the two
elements entered in proportions from 1 or 2 to 9 parts in 10. The
diverse results which were obtained gave ample promise of affording
many useful comparisons with the action of plants.

It was, of course, not taken for granted that the ammo-acids used
duplicate those which are found in the plant or that such compounds
afforded all of the more important factors affecting water-relations.
The next step in the making of a colloidal mixture which might imitate
the action of the plant in relation to water was to use various albumin-
ous compounds to furnish the nitrogenous element in the biocolloids.

According to Beijerinck and others, combination of agar and gelatine
or gelatine and starch in a 10 per cent solution would result in a simple
admixture of the colloidal masses of the two substances in the form of
minute masses or strands.2 Such mixtures would be more intimate and
present greater surfaces than those made up from the powdered mate-
rial brought together with little water and at low temperatures. Pro-
gressively finer subdivision of the materials and more perfect dispersion
would finally reach a point near the limits of gelation where the sub-
microns of agar and starch, for example, might come together in the
walls or fibers and in the more liquid part of the two-phase system of
colloids, and the substances in the parts remaining to fill cavities or
vacuoles might be in various groupings, according to one view. On
the other hand, very weighty theoretical considerations lead to the
conclusion that the relations of the carbohydrate-protein substances
in such a system would be determined quantitatively. Thus a mixture
of 8 or 9 parts of agar and 1 or 2 parts of gelatine or albumin at a high
degree of dispersion would be followed by a gelation in which the pre-
dominating substance, agar, would form the external or continuous

1 MacDougal, D. T. Imbibitional swelling of plants and colloidal mixtures. Science, 44 : 502.
1916.

2 Beijerinck, M. W. Ueber Emulsoidenbildung bei der Vermischung wasseriger Losungen
gewisser gelatinierendern Kolloide. Zeitsch. f. Kolloid-Chem., 7 : 16. 1910.

Fundamental Features of Phytocolloids. 15

phase, and the protein the internal discontinuous or globular phase.
This conception is a very attractive one because of the possibilities
implied. Included among these would be the play of osmotic forces
in the inclosed and non-diffusible gelatine, which might be a contribu-
tory factor in the high swelling coefficients displayed by biocolloids.1

The sections and plates of agar and proteins, amino-acids, etc., used
in the accompanying experiments probably included the materials in
many possible arrangements, but as the method of preparation was
uniform, the relative value of the results obtained from them remains
unimpaired. This heterogeneity is a direct resultant of the varying
history and unequal disposition of the material which enters into the
colloidal mass, and would find direct parallel in living matter, which is
practically never homogeneous as to composition or uniform as to
architecture throughout any measurable mass, and hence its morpho-
logical units are not isotropic as to action when measured with com-
mensurate accuracy.

The experimenter dealing with the hydration of these elastic gels
does not proceed far before he becomes aware that the method of com-
pounding, melting, drying, temperature, and other features of the
experience of the biocolloids influence the behavior of the thin plates
which may be made from them. It will be important, therefore, to
describe the preparation of the sections which were used in these and
other tests.

The agar and the proteinaceous material should all be from one
source and if possible from a single lot in any comprehensive series of
tests where close comparisons are desirable, as it can by no means
be assumed the composition of separate lots will be identical as to
salt or nitrogen content. The variations in gelatine are not so easily
apprehended. Both agar and gelatine, or other proteinaceous com-
pound used, should be first soaked in distilled water at some tempera-
ture between 15° and 20° C. for a period of a half hour. The agar is
now heated with an amount of distilled water over a water-bath that
will bring it to a 2.5 per cent solution or suspension. The suspension
of the agar may be accomplished more quickly by the use of an auto-
clave. When this is completed, and it is difficult to bring the last
particles into liquid form, it should be filtered hot into a flask to obtain
a clear solution. If the biocolloid is to be made by the addition of an
amino-acid, any one of these substances may be added at temperatures
between 50° and 80° C., but when albumins are used the agar solution
must be cooled in a warm water-bath until it comes down to a tempera-
ture below the coagulation-point of the latter substances. This will
be found somewhere below 40° C., and the protein solution should be
poured in with sufficient stirring to procure good admixture, or the
same end reached by a vigorous shaking.

1 See discussion in Robertson, T. B., The physical chemistry of proteins, pp. 294-350. New
York and London. 1918.

16

Hydration and Growth.

The most useful amount of material for making dried plates of a
thickness of 0.3 mm. or less is that which includes 10 grams of dry
material made up to a 2 per cent liquid or solution. This amount will
form two plates about 8 by 15 cm. which will come down to the
desired thickness when dried at 15° to 20° C. Two methods of casting
such dried plates may be used, according to the composition of the
colloid that is being manipulated. Gelatine, or plant mucilages, or
mixtures in which these substances compose half or more of the whole,
must be poured directly on glass plates or on sheets of gold or plati-
num foil.

A sheet of plate-glass with a good surface is set in a level position
after the surface has been well cleaned and polished. A cell about
8 mm. in depth and 10 by 15 cm. is now made on it by glass strips fitted
together so closely than when the warm material is poured onto the
glass it will not leak out at joints or corners. After the mixture has
cooled sufficiently for the gel to set, the plate is placed in the desiccator
and drying should be carried on at such rate that no further loss of
weight occurs after about 40 hours. The dried plate is now worked
free from the glass at one margin by an instrument with a chisel edge
and then stripped free, after which it should be placed in a closed glass
dish to keep it free from dust and undue desiccation, which would
produce buckling or warping.

FIG. 2.

Drying frame. A sheet
of wire gauze of 1 mm.
mesh stretched on a
heavy wooden frame,
being fastened secure-
ly in place by a tightly
fitting strip of wood
which carries the mar-
gin of the netting down
into a groove in the
frame, as shown in the
smaller detail drawing.
A, molding form of
brass bars; B, margin
of wire netting and
clip fastening it in
place; C, wire netting
as it clears frame. The
wire netting and brass
bars are coated with
fine shellac.

Many plates are ruined in the method described, and when possible
the following devices will be found useful: A sheet of rustless wire
screen with a mesh less than 2 mm. is stretched on a heavy wooden
frame (see fig. 2), so as to offer a good plane surface. A mold of four
brass bars is laid on the surface and a sheet of hard filter-paper, such
as Whatman No. 40, is fitted into this cell. The frame is placed in a
level position and the mixture poured into the shallow cell to a depth
of about 8 mm. (fig. 3) . After it has cooled and set, the brass members

Fundamental Features of Phytocolloids.

17

of the shallow cell are removed and the filter-paper is peeled from the
margins and the free flaps are fastened by paste to the edges and sur-
faces of the wooden frame so as to be perfectly taut and so firmly that
when the colloid dries it may not shrink in width or length. Addi-
tional care should be taken to see that the material does not tear loose
from the paper at this time, and if it does it should be secured by using
some fresh material as a paste to fix it to the paper. The margin dries
most rapidly, and if securely attached to the paper holds the plate in
place throughout (see fig. 4).

FIG. 3.

Drying-frame with sheet
of hard filter-paper
fitted in place ready to
receive liquid colloidal
mixture which is to be
poured on the'paperto
ajjtdepth of about
8 to 10 mm.

The drying-frame is now placed on a rack in a desiccator which
consists of an inclosed chamber in which is placed an electrically driven
fan and a large, shallow pan of water. The best results are secured by
a rate of drying which results from having air with high humidity
driven over the plates constantly without dehydrating the surface
layers too rapidly (fig. 5). Drying will occupy about 40 hours, during
part of which time it may be advisable to stop the fan. As soon as
the sheet appears to be dried to a flexible, leathery consistency the
paper may be freed from the frame and then stripped from the plate
of material, which should remain plane, with but little curling or buck-
ling. The rough and uneven margin should be cut away with scis-
sors, the data as to composition, etc., written on one end with common
ink, and then placed in a closed dish for preservation until used.

The precautions described are necessary in any effort toward accuracy
in the measurement of the swelling of a colloid, as the increase will,
among other factors in its experience, reflect most strongly the method
in which it was laid down, deposited, or dehydrated. A dried section
of a colloid of the kind used in these experiments tends to return to the
dimensions which it had when the gel set or cooled. Standardization

18

Hydration and Growth.

of material for measurement of swelling under the influence of various
reagents or agencies would therefore require that the shrinkage of the
material as it dries should be controlled. This may be done in many
cases in the manner described. Thus in the case of biocolloids con-

Fio. 4. — Drying-frame with plate of colloid, ready to be put into the desiccator. The molding-
bars A have been removed and the free portions of the filter-paper have been carried down
over the side of the frame and fastened securely in place with paste.

a

FIG. 5. — Sectional views of desiccator for drying plates of colloids, a, frame with drying-plate
lying on a sheet of stretched filter-paper, which in turn is placed on a shelf of slats; 6, metal
pan containing water to maintain relatively high humidity; c, electric fan arranged to keep
current of air moving over the suiface of the water, the plate of colloid, and in circulation in
the chamber; d and e, hinged lids which may be raised to control ventilation and the rela-
tive humidity of the chamber. A wooden chamber about a meter in height, over a meter in
length, and 80 cm. in width.

sisting of 9 parts agar and 1 part bean albumen a plate cast in this
manner came down so perfectly that a strip 30 mm. in length placed in
distilled water swelled over 2,000 per cent in thickness, but did not
increase any measurable fraction of a millimeter in length. A strip

Fundamental Features of Phytocolloids.

19

cut from the extreme margin of the plate would doubtless have shown
some elongation.

The marginal strip of a plate consisting of 6 parts agar, 3 parts gum
arabic, and 1 part gelatine which had a length of 15 cm. increased to
15.5 cm. in 45 hours. To be compared with this elongation of 3 per
cent is that of the increase of a pile of sections 2 cm. in height cut from
the same plate, which rose to 15 cm. in 24 hours, showing an increase
of 750 per cent. The proportion would doubtless have been still
greater had the swelling of one section been measured alone, as trios
of sections cut from the middle of the plate showed swellings of 1,141
per cent at 14° to 17° C.

FIG. 6. — Demonstration of the swelling of sections of plates of agar 4 parts, opuntia mucilage
4 parts, gelatin 1 part, bean protein 1 part, in thickness with but little increase in length, due
to the manner in which the moist colloid was held while drying.

Another test of the same kind is illustrated by figure 6. Sections of
a plate consisting of 4 parts agar, 4 parts opuntia mucilage, 1 part gela-
tine, and 1 part bean protein were placed in a frame of glass rods and
immersed in a vessel of distilled water, swelling from a total thickness
of 15 mm. to a total of 180 mm., while a strip cut from this plate which
had an initial length of 8 cm. had elongated to 8.5 cm. The increase
in thickness was 1,200 per cent, while that in length was but 6 per cent.

Plates of gelatine invariably showed a greater increase in areal
dimensions than that of biocolloids such as those mentioned above, and

20 Hydration and Growth.

no plate was made in which this was entirely eliminated. Thus a plate
which would yield increases in thickness of 6 to 800 per cent in trios of
sections in distilled water reached a length as much as 50 per cent
greater than the original under the same conditions.1

A unilateral action such as that described is one which appears to
rest upon the supposed honeycomb structure of the colloid. Dehydra-
tion would lessen the volume of the mass, and as the sheets or strands
of denser material are held in a plane parallel to the surface, the spaces
containing the more discontinuous, more liquid element would be
deformed and their vertical diameter decreased. Accession of liquid
or of water would be followed by the partial resumption of the original
form and dimensions. Experience in dealing with a large number of
plates leaves the impression that the swelling does not bring the sec-
tions back to the thickness of the cooled gel as it was originally in the
mold.

The fact that colloids such as those present in living matter may
retain a shearing strain was recognized by Butschli and was the subject
of some experimentation by Hardy2 in a study of coagulation phe-
nomena, who concludes that "shearing a colloidal mass, fluid or solid,
actually does produce heterogeneity or, simply, structure, which is
fixed by the process of coagulation." Such sheared masses of colloid
are doubly refractive. Klocke demonstrated the acquisition of such
double refraction by sheets of gelatine which were dried on frames
covered with tin-foil. It is to be noted that the plates of gelatine
which were dried without superficial shrinkage in my own experiments
when hydrated showed some extension, while those of the agar-protein
mixture did not. The hydration in both cases presumably removed
the strain as the structure produced by the stress disappeared. Great
cytological interest attaches to the simple experiment by Hardy, in
which a small quantity of a colloidal solution is drawn along a glass
slide by the point of a needle, after which it is "fixed" by the methods
of the cytologist, with the result that the mass appears to consist of a
number of fibrillaB " * * * so striking that they look as if one might
isolate them by teasing."

Much interest also attaches to some recent work of Miss C. L. Carey
of Barnard College upon the structure of agar films. 2.5 per cent
gels of this substance were prepared by a method similar to that
described on page 16 for preventing superficial shrinkage. When such
plates were dried at 45° to 70° C. and again placed in water the
rehydrated plates yielded drops of water so readily that an examination
of thin sections under the microscope was made, revealing cavities

'For the original notice of increase in thickness and not in length, see MacDougal and Spoehr,
Growth and Imbibition, Proc. Am. Phil. Soc., 56 : 343, 344. 1917. Philadelphia.

1 Hardy, W. B. On the structure of cell protoplasm. Journal of Physiol., 24 : 158. 1899.
See especially pp. 187-190. London.

Fundamental Features of Phytocolloids.

21

with their long axes parallel to the surface, as shown in figure 7, which
was furnished by Miss Carey in response to the request of the author.

The extraction and preparation of a number of substances, includ-
ing protein from oats, albumin and globulin from beans, the total pro-
tein of beans, asparagin, aspartic acid, etc., was undertaken by Dr.

FIG. 7. — Longitudinal section of an agar plate dried at 70° C. without superficial shrinkage, with
development of elongated spaces or cavities which are found when the film is hydrated
Drawn with camera lucida by Miss C. L. Carey. X78.5 diam.

FIG. 8. — Scale designed for measuring thickness of paper and suitable for
determiningjjthickness of sections of plates of biocolloids. Sheets of a
thickness of 0.001 to 0.11 inch(= 2.8 mm.) may be measured (see fig. 23
for calipers used in measuring larger objects).

Isaac Harris, of|Squibb & Sons' laboratories, New Brunswick, New
Jersey, while Mr. E. R. Long furnished preparations of such sub-
stances as zein, which made it possible to make up plates of biocolloids
entirely from products of plants.

The swelling of mixtures of agar and of the protein extract of bean
in plates 0.3 to 0.4 mm. in thickness were as shown in table 3.

22

Hydration and Growth.

The greatest capacities for hydration encountered were those in
which plant proteins, such as those of oats, were added to agar in the
proportion of about 1 in 10, a ratio which finds its equivalent in the
constitution of many of the higher plants. The maxima exhibited by
such mixtures are not duplicated by the plant, in which the presence of
salts in the colloids and the morphological structure operate to limit
the amplitude of the swelling.

TABLES.

Dist. water.

Hydrochloric
acid,
0.01 M.

Sodium hydrox.,
0.01M.

Gelatine 90, protein 10 I
(Phaseolus) ]

p. ct.
585
486

p. ct.
1,401
1,200

p. ct.
942
704

386

800

Averages

485

1,300

817

Gelatine 75, protein 25 f
(Phaseolus) \

696
500

818
1 , 060

621

848

Averages. .......

598

939

734

Agar 90, protein 10 f
(Phaseolus) \

800
800

50
75

150
150

Averages

800

62

150

Agar 99, protein 1 f
(Phaseolus) \

1,080
800

300
360

220
240

Averages

940

330

230

The method of admixture of the carbohydrate-protein-saline con-
stituents of the biocolloids consisted mainly in the use of temperatures
which would bring all of the substances into a liquid condition in which
they might be as intimately united as possible, and the plates formed
appeared translucent but uniform throughout, although it is not to be
assumed that the components in this case or in any preparation, or in
protoplasm, are mutually interdiffused. It seemed desirable to attempt
to make mixtures in which the carbohydrate-protein elements would
be less intimately united, and to that end some simple experiments
with powdered agar and powdered gelatine were carried out.

Powdered gelatine and agar which would pass the millimeter mesh
of a screen were used to ascertain what degree of expansion would be
registered on the auxograph when these were simply placed in a layer
in the dish and subjected to the action of solutions. Whatever the
arrangement of the material in these particles, their separate action
in swelling and in dispersion or solution would be free from the action
resulting from structures such as those presented by plates held rigidly

Fundamental Features of Phytocolloids. 23

while being dried. When a layer of powdered gelatine was placed in
the bottom of a Stender dish to a depth of 1.5 mm. and covered with a
perforated glass triangular plate which would go into the dish readily,
a swelling of 270 per cent in water at 18° C. was registered by the auxo-
graph. Furthermore, this increase was not the rapid swelling of a
mass with subsequent relaxation, but lasted over 5 days; the greater
part of the swelling occurred during the first half hour, then continued
at a decreasing rate for the period mentioned.

A similar experiment was made with powdered agar, the particles of
which were probably of a much smaller average size. The swelling
was of the same kind, but was complete in 4 days, although reaching
the higher total of 317 per cent. This higher hydration capacity is
characteristic of agar as compared with gelatine.

It is of course to be expected that when two colloidal substances in a
two-phase system are combined and the resulting material is subjected
to agencies that will coagulate or neutralize one of them, the section
would then show the relations of the one still in the colloidal state.
This was demonstrated with some completeness by a mixture of agar
and milk albumin.

Preparations in which 1 part albumin from milk was stirred into
9 parts melted agar at 40° C. and under, thus remaining active and
suspended, showed swellings in the form of dried plates 0.1 to 0.15 mm.
in thickness at 16° C., as shown in table 4.

TABLE 4.

p. ct.

Water 1,792

Citric acid, 0.01 N 333

Sodium hydroxid, 0 . 01 M 386

The high swelling in water, the increased imbibition in acid, and the
equalization of the acid and alkali effects are characteristic of agar-
protein mixtures and are in contrast with the reactions of the following
test, in which the albumin was coagulated. As a result it no longer
intermeshed with the agar in the gel, but aggregated as small particles,
indifferent to the presence or proportion of water. A mixture of agar
95 parts and milk albumin 5 parts was prepared, in which the last-
named substance dissolved in water was added to the melted agar at
a temperature near 100° C., at which coagulation followed. The mix-
ture, however, when poured on a glass plate, dried into a film about
0.13 mm. in thickness, which had a leathery texture and was trans-
parent and appeared homogeneous. Sections swelled under the auxo-
graph at 16° C. increased 261.5 per cent in distilled water, 346.2 per
cent in hundredth-molar sodium hydroxid, and but 191.2 per cent in
hundredth-normal citric acid. The proportions are in general accord
with those obtained by swelling of agar alone, suggesting that the
neutralized or coagulated albumen has no effect on the imbibition
capacity of agar, in which it may be incorporated.

24 Hydration and Growth.

Other substances were next tested which are insoluble in water and
hence might not be expected to enter into the two-phase system with
agar. Mr. E. R. Long prepared some zein, a protmaine derived from
Zea mais, at his laboratory at Seattle, Washington, early in July 1917,
and this was made up with 1 part of zein to 9 of agar.

The fine, iregular particles, after being wetted, were stirred into the
melted agar and the mixture was poured onto a glass slab and dried
down to a thickness of about 0.3 mm. The plate was rough to the
touch, the granular particles of the zein being distinctly visible as
opaque masses. Sections tested under the auxograph gave the meas-
urements as follows:

TABLE 5.

p. ct.

Water 1,033.3

Citric acid, 0.01 N 400

Sodium hydroxid, 0.01 M 350

The presence of the zein could not be said to be entirely without
effect, as these measurements show some departure from agar in the
relatively high swelling in acid. The imbibition in the hydroxid solu-
tion was extremely slow. Saturation was reached in 24 to 30 hours in
water and acid, but enlargement continued for twice this period in
hydroxid. All liquids were renewed at 36 hours. An acceleration ensued
in hydroxid and the swelling was still in progress at the end of 48 hours,
at which time the measurements were as above. Some of the equaliza-
tion or increase in the swelling of the biocolloid in acid and its con-
tinued swelling in hydroxid is probably due to the fact that zein is
slightly soluble in both acids and alkalies.

The addition of 1 part globulin from beans to 9 parts agar resulted
in the formation of dried plates much like those of agar-zein. The
globulin, not being soluble in water, was incorporated as small globular
masses. Swellings of sections 0.2 mm. in thickness were exhibited as
shown in table 6, at 16° C.

TABLE 6.

p. ct.

Distilled water 875

Potassium nitrate, 0.01 M 575

Potassium nitrate, citric acid, 0. 01 N 550

Citric acid, 0. 01 N 525

Potassium nitrate, potassium -hydroxid, 0.01 M 450

Potassium hydroxid, 0.01 M 300

The proportionate imbibition in water, acid, and hydroxid is one
characteristic of agar with a small proportion of protein. The solubility
of globulin in salt solutions would lead to the expectancy that its presence
would result in a modification of the swelling of agar in saline solutions.1

The " bean-protein" which has been used so extensively in these
experiments is, as noted elsewhere, an extract with water in which the

1 See Zsigmondy, Behavior of globulins, in chemistry of colloids, p. 222. 1917.
Robertson, T. B. The physical chemistry of proteins. 1918. New York.

Fundamental Features of Phytocolloids. 25

albumin, and some of the globulin, is dissolved in the water, which also
contains the salts of the bean. The effects of albumin were tested sepa-
rately, following the measurement of globulin effects, and the swellings
of thin plates of 9 parts agar and 1 part albumin were as follows :

TABLE 7.

p. ct.

Distilled water 1 , 158

Potassium nitrate, 0.01 M 947

Potassium nitrate, citric acid, 0 . 01 N 500

Citric acid, 0.01 N 421

Potassium nitrate, potassium hydroxid, 0.01 M 421

Potassium hydroxid, 0.01 M 316

The comparison of the above data with those obtained from the agar-
globulin reveals the fact that while the globulin does not appear to
increase the imbibition capacity of agar very much, the albumin does
exercise such positive effect, the mixture showing a capacity three
times as great as in acid or alkali. The swelling in acids is slightly
greater than in alkalies, in accordance with ti e action of other mixtures
of albumin. Imbibition by the globulin mixture in potassium nitrate is
relatively high compared to water-effects, while it scarcely rises above
that in acids. The swelling of the agar-albumin in this salt is more
than twice that of acidified and alkaline salts, acids, and alkalies.

The presence of insoluble inclusions is of course the normal and usual
condition in the cells of plants during extended periods, and it was
desirable to ascertain whether or not material wholly neutral in biocol-
loidal plates would affect hydration. The first trial was made with a
cotton lace about 1 cm. in width and 0.5 mm. in thickness. This was
well softened, and when laid in the mold the warm colloidal mass was
poured over it, accomplishing an intimate penetration among the
smaller fibers of the threads.

The portion of a plate of agar and oat protein free from the webbing
dried down to a thickness of 0.18 mm., and sections of this swelled
2,111 per cent in distilled water, while the increase in swamp water
was 1,277 per cent. Sections containing webbing swelled 1,583 per
cent in bog water and the same amount in distilled water, and 1,195
per cent in swamp water, calculated on the basis of the thickness of
biocolloid noted above. It is by no means certain, however, that the
colloid does dry in equal mass on the webbing. (See Chapter VI for
discussion of bog water.)

Calculated in terms of actual thickness, the swelling of the webbed
sections was 491 per cent in bog water and in distilled water, while it
was but 371 per cent in swamp water. The presence of the webbing
appears to diminish the proportion of swelling in distilled water and
bog water, but not that in swamp water. Swamp water (see p. 68)
contains an amount of calcium salts which notably affects swelling in
clear sections. This operated to mask any effect due to the presence
of the cotton fibers in colloidal sections.

26 Hydration and Growth.

Still another type of inclusion was tested by the incorporation of
spores of Lycopodium in liquefied agar. These spores are not readily
wetted, and hence they could be worked into a colloidal mixture only
at low temperatures, when it was in a stage nearing gelation.

The first attempt was one in which 2.5 parts by weight of spores were
mixed with 40 parts by dry weight of agar. The agar was liquefied in
the usual manner, and when it had come down to a temperature of
about 40 was strained through cheese cloth into a beaker. The quan-
tity of spores given above was now placed on the surface and the whole
was vigorously stirred for several minutes with an ordinary revolving
egg-beater. The agitation was continued until the temperature fell
to 35° C., when the whole was cast as a plate in the usual manner. The
dried plate was 0.3 mm. in thickness and showed the spores and
numerous clumps of spores embedded in it, with very few really coming
to the surface. When sections of the ordinary size were cut and swelled
at 18° C. they showed some buckling. The swellings hi distilled water
and in asparagin 0.05 M were equivalent, being 850 per cent in 24 hours,
with some increase still in progress, the rate being greater in distilled
water.

The second test was arranged with sections 0.27 and 0.28 mm. in
thickness, which swelled 1,125 per cent in 0.01 M asparagin and 667
per cent in acetic acid 0.01 N, both pairs of tests showing an expansion
far less than might be expected from the agar alone. It seems quite
safe to conclude that inclusions such as bodies of zein, globulin, coagu-
lated albumin, fine threads of glass, cotton fibers, and spores lessen the
hydration capacity of the gel in which they may be embedded. As
their effects are due directly to the area of surface and radius of curva-
ture, the action of a comparatively small amount of finely divided
material would be very much greater in the cell. The foregoing results
are in accordance with -those of Hardy, who found that solids included
in a colloid before fixation may influence the structure of films in a
material manner. Grains of carmine incorporated in liquid colloids
modified the mesh and the thickness of the plates or bars or more solid
material, and the prevalence of insoluble particles in plant cells renders
such observations of great interest.1 *

1 Hardy, W. B. On the structure of cell protoplasm. Journal of Physiology, 24: 158. 1899. See
p. 186.

III. THE CONSTITUENTS OF BIOCOLLOIDS WHICH AFFECT
HYDRATION AND GROWTH.

Some organs and cell-masses of plants as well as of animals display
swelling reactions similar to those of gelatine with respect to acids and
salts, and a great deal of discussion of the colloidal action of proto-
plasm has been based on the assumption that this parallelism runs
throughout. That investigation should have taken this course is
natural when it is recalled that gelatine, in common with proteins and
many protein derivatives, is amphoteric, and may dissociate either as
an acid or as a base, being stronger as an acid than as a base. In a
condition of neutrality or at its iso-electric point its hydrogen-ion con-
centration is represented by the symbol pH = 4.7. It is to be seen
that the diversity of hydration reactions which such substances may
display might well give rise to the assumption in question. Further-
more, it is to be granted that some organs and cell-masses of animals as
well as of plants are so high in nitrogenous compounds as to be charac-
terized by the reactions of amphoteric colloids. Thus, for example, bac-
teria may contain so much nitrogenous material that the dried remainder
obtained from them may appear to consist principally of albumin.1

It would be a mistake, however, to assume a close identity of the
protoplasmic machine as to its colloidal components. The results
described in the present work make it evident that it is a heterogeneous
gel, which, in plants at least, is largely composed of inert or neutral
carbohydrates of the pentosan group, of which agar, gum arabic, and
mucilages are examples. The swelling or hydration of such gels is
modified by the action of the proteins, amino-acids, and other nitro-
genous substances which may be incorporated with them, but through-
out all of my experimentation it was evident that the water-relations
of growing plants were more of the character of pentosans than of
gelatines.

Reproductive cells and elements of all kinds may be expected to
prove high in nitrogen, and hence would show swelling reactions of the
general nature of gelatine.2 So well is this established that is is pos-
sible to predict the main facts as to nitrogen-content upon the basis of
a series of swelling tests with distilled water, acids, and alkalies.

The relation of the nitrogen-content to swelling is well illustrated by
some reactions of red algae of the Pacific Coast which were studied at
the Coastal Laboratory, Carmel, California, in July and August 1917,
by Dr. J. M. McGee.3

1 Thompson, D. A. W. Growth and form, pp. 40 and 41. 1917. Cambridge Univ. Press.

1 Lloyd, F. E. Colloidal phenomena in the protoplasm of pollen tubes. Report Dept. Bot.
Research, Carnegie Inst. Wash, for 1917 (Year book No. 16). 1918.

3 McGee, J. M. The imbibitional swelling of marine algse. Plant World, 21 : 13. 1918. Balti-
more.

27

28 Hydration and Growth.

Trios of sections of the laminae were swelled in various solutions and
their increase registered by the auxograph. These marine algae have a
normal balance enabling them to exist in sea-water which contains
about 3.50 per cent total salts. The effect of the various substances
on imbibition in these plants was therefore obtained by adding them to
sea-water in such quantities that they formed hundredth-molar solu-
tions. The results with Iridcea laminarioides were as follows at 16° C. :

TABLE 8.

Thickness, 0.4 mm. p. ct.

Sear-water + NaOH, 0.01 M 25

Sea-water + HC1, 0.01 M 31

KNO3 + citric acid, 0.01 N 175

Young fronds of Gigartina exasperata gave average swellings as
below at 16° C.:

TABLE 9.

p. ct.

Sea-water, sodium hydroxid, 0 . 01 M 28

Sea-water, hydrochloric acid, 0.01 M 38

Potassium nitrate, citric acid, 0 . 01 N 142

Such reactions are indicative of a high proportion of amino-acids,
which probably fell off toward maturity, and which may have been
extracted by washing as sections which had been treated with distilled
water, a treatment which would result in the extraction of some of the
salts and the amino-acids. Such sections when dried to a thickness
of 0.5 mm., gave swellings at 16° C. as follows:

TABLE 10.

p. ct.

Distilled water 4,331

Hydrochloric acid, 0. 01 M 2, 967

Sodium hydroxid, 0.01 M 2, 756

The analysis of the washed and dried material showed that it con-
tained 68 per cent carbohydrates, 18 per cent gelatine-like material,
and 14 per cent of salts. It is of interest to note that these algse, which
inhabit the shore, display a course of acidity through the day generally
similar to that of other thick and succulent plants by which the acidity
is highest in the morning, decreasing toward the end of the day, but
sometimes rising before night.1

The vacuolar fluid of the plant-cell may be taken to carry minute
quantities of the carbohydrates which enter into the protoplastic gel
at all times and, in addition, the sugars which figure so prominently in
the metabolism of the plant. The mucilage and pentosans in general
change but slowly and are to be considered as being of importance
chiefly by reason of their properties and effects as constituents of the
colloidal structure. The presence of sucrose and dextrose of course
modifies the osmotic properties of the cell, and as these substances are

1 Clark, Lois. Acidity of marine algse. Puget Sound Marine Sta. Publ. 1, No. 22. 1917.

Constituents of Biocolloids Affecting Hydration and Growth. 29

dissolved in the fluids which are imbibed by the gels, their probable
influence is a matter of some importance. An examination of the
effects of sucrose and dextrose on agar and gelatine and mixtures
of these two substances was made by Dr. E. E. Free at the Coastal
Laboratory, Carmel, California, in September 1916, and his results
show no certain effect upon the hydration of gelatine, agar, and of
mixtures of the two substances from water solutions containing as much
as 25 per cent of sucrose or dextrose.1 More recently, E. A. and H. T.
Graham have found that glucose, saccharose, and lactose, when added
to gelatine, retard the diffusion of such acids as hydrochloric, nitric,
sulphuric, phosphoric, lactic, formic, acetic, and butyric, with an accom-
panying effect on the swelling. Diffusion was also found to be retarded
by sodium chloride.2 The universal presence of both sugars and salts
in the plant cell gives great importance to the relations indicated.

Mucilages or pentosans are present in varying proportions in all
plant cells, and it is the character and relative amounts of such com-
pounds that largely determine the hydration reactions of the proto-
plast. Of these substances agar was used most generally throughout
the experiments, because it goes into the disperse condition very slowly
and in this particular is identical with protoplasmic gels.

According to information furnished by Mr. H. Nakano, of the Botan-
ical Garden of Tokyo, agar is prepared chiefly from the algae Gelidium
amansii Lamour, G. pacificum Okam, G. linoides Kiitz, Pterocladia
capillacea Born, et Thur., while some material of Gelidium subcostatum
Kiitz, Ceramium boydenii Gepp., Campylcephora hypenaloides Y. Ag.,
Acanthopeltis japonica Okam, etc., may be included. The process
includes washing in fresh water, decoloration in the sun, milling, boil-
ing, filtering, maceration in sulphuric or acetic acid, freezing, and
drying. Modernized methods simplify this treatment somewhat.
Salts and nitrogen are present in the final product in minute quan-
tities insufficient to affect hydration.3

Other gums and mucilages of this group which were tested included
gum arabic or acacia, cherry gum, prosopis gum, tragacanth, and
opuntia mucilage, all of which are more readily dispersible in water,
but which do not go wholly into suspension even in prolonged im-
mersion. The mucilages of the cacti are pentosans, or substances
which yield hexose and pentose sugars on hydrolysis with dilute acids.
Substances of a similar character formed by the condensation from
simpler sugars may be taken to be universally present in plant cells,
being aggregated in the plasmatic mesh, in which condition the muci-

1 Free, E. E. Note on the swelling of gelatine and agar gels in solutions of sucrose and dextrose .

Science, 46 : 142. 1917.

2 Graham, E. A. and H. T. Retardation by sugars by diffusion of acids in gels. Jour. Amer .

Chem. Soc. 40 : 1900. 1918.

3 For further information concerning the origin and preparation of similar products, see

Swartz, M. D. Nutrition investigations on the carbohydrates of lichens, algse, and re-
lated substances. Trans. Conn. Acad. of Arts and Sciences, 16:247-382. Apr. 1911.

30

Hydration and Growth.

lages and gums elude microchemical tests. It has already been pointed
out that it is in this condition that they produce the peculiar hydration
properties of living matter which are those of an agar-protein gel.1

Generally the mucilages originate in minute quantities in numerous
places in the protoplasm, but when such structures as starch-grains or
layers of wall material are transformed, the gels so formed largely re-
mains in place, and as they swell to occupy a much larger space than

TABLE 11. — Hydration of sections containing gums and mucilages.

Distilled
water.

Citric
acid,
0.01 N.

Sodium
hydroxid,
0.01 M.

Potassium
nitrate,
0.01 M.

Agar (17° C.)

p. ct.
2,420

1,760
fl ,141
\1,072
/ 750
\1,278
1,417
2,020
1,684
2,178
/ 846
\1,038
900

p. ct.
1,300

1,182
957
572
889
912
1,050
1,100
947
1,367
1,500
1,500
650

p. ct.
602

824
478
458
639
556
750
520
474
778
731
731
350

p. ct.
1,700

Agar 6, prosopis gum 2, gel. 1, bean
protein 1 (25° C.)

Agar 6, gum arabic 3, gel. 1 (14-
17° C.)

1,250
1,389
1,082
1,100
1,268
1,347
1,725
1,346
1,200
300

Agar 8, cherry gum 2 (16° C.)

Agar 6, cherry gum 3, gel. 1 (16° C.).
Agai 8, cherry gum (precip.), 2. ...
Agar 8, gel. 2 (16° C.)

Agar 8, tragacanth 2 (15° C.)

Tragacanth (15° C.)

Opuntia mucilage (15° C.)

Water.

Hydrochloric
acid,
0.01M.

Sodium
hydroxid,
0.01M.

Agar 6, opuntia mucilage 2, bean protein 1,
gel. 1 (26-27° C.)

p. ct.
1,780
2,400

500
425
400
387

p. ct.
780

900
806
762
806
706

p. ct.
1,060

950
562
531
575
612

(22° C.)

Gelatine 90, cactus mucilage 10

Average

428

329
431

770

850
789

557

685
431

Gelatine 100, agar 5, averages

Gelatine 80, agar 20, averages

that occupied by the bodies from which they were formed, the resultant
masses may be so large as to crowd the protoplasm into a small com-
pass.2 Their hydration offers such indeterminate features as to make

1Spoehr, H. A. Carbohydrate economy of the cacti. Carnegie Inst. Wash. Pub. No. 287

pp. 44-47. 1919.
2Stewart, E. G. Mucilage or slime formation in the cacti. Bull. Torr. Bot. Club 46: 175.

1919. Lloyd, F. E. The origin and nature of the mucilage in the cacti and in certain

other plants. Amer. Jour, of Bot., 6:156. 1919.

Constituents of Biocolloids Affecting Hydration and Growth. 31

it impossible to secure measurements by the methods which may be
used with agar and with gelatine. Information as to their effects can
only be obtained by observations on the action of mixtures of which
they form a part. Table 11 includes some of the data as to the swell-
ing of colloids, including pentosans secured in this laboratory.

It is also obvious that the addition of any of these gums or mucilages
to agar tends to lessen swelling in water and to equalize the imbibition
in water and in acids. Their general effect, however, when combined
with nitrogenous substances, is to make a colloid which has a higher
coefficient of swelling in water than in organic acids, although, as may
be seen later, a special relation is sustained to the amino-acids.

The vacuolar fluid of the plant cell probably always contains some
protein or its derivatives in the form of amino-acids, while various
nitrogenous compounds have been identified in the nucleus and other
bodies of morphological rank. The formation, disintegration, and
migration of these substances from one part of the cell to another
offers a most inviting field for the researcher concerned with the physics
of the cell.

The proteinaceous substances are of course invariable constituents
of the biocolloids of the plant protoplast. The varying reactions of
such material to the hydrogen-ion concentration or acidity of solu-
tions and to salts are exemplified in nearly every section of this work.
Combinations of agar with protein extracts, with albumins, peptones,
gelatine, and amino-acids were tested to such an extent that it is
possible to say that the highest coefficients of hydration in water alone
are exhibited by pentosan-albumin mixtures in which the substances
of the first group form the greater part of the mixture. All such trials
were with materials with possible physiological significance, especially
in plants.

Many of the nitrogenous compounds used in making biocolloids in
our tests are known to be actually present in the cell. The presence
of one of them, peptone, in the nucleus is definitely established. A
characteristic behavior of the mixtures containing such substances has
already been noted (see MacDougal and Spoehr, The effects of acids
and salts on biocolloids, Science, 46: 269. 1917). Increases of nearly
3,200 per cent in distilled water, 567 per cent in hundredth-molar
hydrochloric acid, and the superior and long-continued swelling in
hundredth-molar potassium hydroxid, which sometimes reached a
total of nearly 1,700 per cent at room temperatures (20° to 28° C.), were
the characteristic features. These figures were obtained by the use of
sections consisting of 90 parts agar and 10 parts Witte's peptone.

The tests were repeated, using "Diffco" peptone in the same pro-
portion and with temperature kept strictly at 15°,C., and the records
given below are all at the close of 22 hours. The measurements were
as follows:

32 Hydration and Growth.

TABLE 12.

p. ct.

Water 1,400

Potassium nitrate, 0.01 M 1 , 200

Potassium nitrate, citric acid, 0.01 N 900

Citric acid, 0.1 N 675

Potassium nitrate, potassium hydroxid, 0.01 M 575

Potassium hydroxid, 0.01 M 400

The hydration in all the solutions was less than in the earlier tests,
partly due, no doubt, to the difference in the peptones used. The
increase in hydroxid is small; no direct comparison can be made as to
the acid, as a hundredth-normal solution was used in the other tests,
while the swelling in acidified salt solutions is relatively large. Such
results emphasize the fact that material standardized for cultural
and chemical purposes may present differences in colloidal action of a
serious character.

Nucleinic acid is a substance of interest in connection with its
occurrence in the nucleus and its direct effect in simple combination
was tested. A mixture of 90 parts agar and 10 parts nucleinic acid
was dried into plates 0.2 mm. in thickness. Two series of sections
were swelled in a dark room at 16° C., with the following results:

TABLE 13.

p. ct. p. ct.

Water 1,400 1 ,025

Potassium nitrate, 0.01 N 900 800

Potassium nitrate, citric acid, 0.01 N 650 675

Potassium nitrate, 0.01 N 850 750

Potassium citrate, citric acid, 0.01 N 725 ....

Citric acid, 0. 01 N, 700 625

Sodium hydroxid, 0.01 M 1,000 925

The action of this mixture fixes attention by reason of the extra-
ordinarily high amount of swelling in the alkaline solution. The
amount of swelling in acid does not average more than half that in
distilled water. The behavior of nucleinic acid and of peptone when
mixed with agar is such as to suggest that a study of the action of
these substances when combined separately and together with agar
would be of interest in connection with interpretations of nuclear
phenomena.

The series of tests now including some data from mixtures of most
of the albumin and protein derivatives which were available, it was
deemed advisable to introduce more than one nitrogenous compound
into the biocolloid. The first mixture contained 90 parts agar, 3 parts
nucleinic acid, 3 parts peptone, and 4 parts of asparagine. The plates
dried to a thickness of 0.2 mm. and sections were swelled in a room
at 15° C. with the results as given in table 14.

The outstanding features in table 14 are the comparatively high
amount of swelling in the salts. The expected high hydration in acid
solutions is not exhibited.

Constituents of Biocolloids Affecting Hydration and Growth.

33

TABLE 14.

Water.

p. ct.
1,025

Potassium nitrate, 0.0 M 950

Potassium nitrate, citric acid, 0.01 N 675

Citric acid, 0.01 N 650

Potassium nitrate, potassium hydroxid, 0.01 M 775

Potassium hydroxid, 0.01 M 575

The comparative test of the result of the inclusion of aspartic acid
and of its amine, asparagine, in the biocolloid is important, because the
amine is a noticeable constituent of plant cells, in which it is frequently
abundant. The acid appears to be only sparingly soluble and in a
plate of agar 90 parts and 10 of this acid aggregates as whitish lumps in
the plate or as an efflorescence on the surface. Much of the last form-
ation comes off, so that the proportions given above do not hold for the
dried material. The asparagine forms clear plates with the agar.
The swellings were as follows :

TABLE 15.

Citric

Sodium

Water.

acid,

hydroxid,

0.01 N.

0.01 M.

p. ct.

p. ct.

p. ct.

Agar, asparagine. . . .

640

300

402

Agar, aspartic acid .

295.5

250

625

The asparagine mixture shows swellings in water, acids, and alkali
not widely different in proportion from those hi which proteids of the
bean are used. The aspartic acid, in accordance with expectation,
shows an amplitude of swelling characteristic of organic acids in the
solution in both acid and distilled water as reagents. Neutralization
by hydroxid, and the renewal of this reagent, was followed by greater
swellings.

The introduction of a fat into a biocolloid was attempted with a
preparation of lecithin (Merck) from eggs. An amount which would
give a proportion of about 0.5 per cent was smeared as a coating on a
glass stirring-rod. After half the contents of a flask containing a
mixture of agar 90 parts and milk albumin 9.5 parts had been poured
on a glass plate for drying, the remainder was stirred until all of the
lecithin had dissolved from the rod. It was then poured on a glass
slab to cool. Separate particles could not be distinguished with a
hand lens, but the mixture had a brownish tinge. When the film had
dried to a thickness of 0.2 mm. it was tested under auxographs at 16°
to 18° C. and the swelling measurements given in table 16 obtained.

No distinct effect of the fatty substance can be detected in these
reactions, nor were any departures discernible in another preparation
containing 90 parts of agar, 9 parts of bean protein, and 1 of lecithin.
The bean protein is a water extract of Phaseolus vulgaris containing
the albumins and also the other proteins soluble in the salts present.

34

Hydration and Growth.

When such material is stirred into distilled water a clear solution is
obtained. The mixture was added to the melted agar at about 30° C.
An amount of lecithin (Merck) from eggs, supposed to be about a gram,
was smeared on the outer wall of a thin vial. The vial was dropped
into the warm mixture and shaken until it had nearly all passed into

TABLE 16.

Water.

Citric
acid,
0.01 N.

Sodium
hydroxid,
0.01 N.

p. ct.
1,923
1,150

p. ct.
423
200

p. ct.
250
423

Average

1,536

311

336

Averages of swelling
of mixture lack-
ing lecithin

1,791

333

336

the solution, giving it a brownish tinge. By another method the
lecithin was smeared on the inner surface of a warmed flask. The
agar-protein mixture was poured in at a temperature of about 45° to
50° C. and shaken until all of the lecithin had been taken up. Dried
plates prepared in this way showed no important departure from the
behavior of mixture without lecithin. A series of such swellings with
a plate 0.47 mm. in thickness at 16° to 18° C. gave the following:

TABLE 17.

p. ct.

Water 1, 106

Citric acid, 0.01 N 329

Sodium hydroxid, 0.01 M 436

It is obvious that these crude tests by no means constitute an ade-
quate trial of the effects of fats or lipins in hydration of living matter.
The prominence of the lipoid theory of the cell-membrane and the
weight of some of the arguments adduced in its support renders it
highly important that refined methods of experimentation be used in
incorporating a lipin colloid in pentosan-protein mixtures, the hydration
of which might yield results of importance bearing on permeability.

In an effort to make a mixture bearing a closer resemblance to the
general hydration relations of plant protoplasm, the following mate-
rials were assembled:

Agar 4.2 grams, which was liquefied in 160 c.c. of water at tempera-
tures of about 100° C., oat protein 0.18 gram, and oat albumin 0.820
gram, were dissolved by shaking up with 50 c.c. cold water. After this
had been done 0.2 gram of lanolin was put in a vessel with the dis-
solved albumin and warmed to about 35° C., being shaken vigorously
at intervals. The agar was now strained through two layers of cheese
cloth into a beaker and stirred until it came down to a temperature of
about 40° C., when it was placed in an enameled cup suitable for the

Constituents of Biocolloids Affecting Hydration and Growth. 35

action of an ordinary revolving egg-beater. The albumin-lanolin
mixture was now added to the agar and it was stirred vigorously some
time above 35° C., and when it had come down to 33° C. it was cast in
two portions. One was on filter-paper which was stretched in the
usual manner, and the other smaller lot on a glass plate. The mixture
set in a few minutes, the room temperature being about 20° C. The
plates as above came down to an average thickness of 0.2 mm., which
were tested at a temperature of 18° C., swelling 2,100 per cent in dis-
tilled water and but 1,750 per cent in 0.01 M asparagine.

A conception of living matter as simply a two-phase colloid in which
the main elements are distributed between the more liquid and the
denser phases simply according to their physical properties may be
sufficient to interpret certain general reactions, but one does not pro-
ceed very far with the actual mechanics of living matter until it is
realized that specializations of various kinds come in. Attention has
already been called to the varying proportion of proteins and their
probable effect on hydration. According to Kite,1 the vacuolar fluid
of Spirogyra contains some proteins in a dissolved or disperse state,
and this fluid is even higher in nitrogenous material in Char a. A
doubtful amount of plasmatic material may be taken to be hi a dis-
perse condition in plants under ordinary conditions, and the heavier
portions differ widely as to density or viscosity, the outer layer of the
nucleus and of the cell being a gel of greater rigidity than the interior
portions. The fact that "none of the cytoplasm goes into solution
very readily even when cut into very minute pieces," as described by
Kite with respect to Spirogyra, can not be taken to prove that pro-
toplasm may not readily go into the disperse or liquid phase in the
fluids of the cell. The pentosans may move slowly, but during the
growth of the nucleus, water and other substances pass into it from the
surrounding cytoplasm. Again, at other times, material may be seen
to pass from the nucleus to the cytoplasm. Many of these phenomena
may now be explained on known behavior of colloids, and the study
of colloidal action promises to yield much additional information upon
the movement and interaction of the parts of the protoplast.

Most of the variations in composition mentioned are illustrated in
the structure of a single cell, and in the growth and development of
these units the accumulation, migration, and disintegration of these
substances may be definitely connected with the more important
movements in the cell. The localization of salts is a matter which
has been dealt with at great length by MacCallum, and the results
which he has secured with a few of the more important salts afford a
basis for some conception of the heterogeneity of the cell with regard
to this feature.

1 Kite, G. L. The physical properties of protoplasm of certain plant and animal cells. Amer.
Jour, of Physiol., 32 : 146, especially pp. 161 and 162. 1913. See also Conklin, E. G. Effects
of centrifugal force on the structure and development of the eggs of Crepidula. Jour. Exper.
Zool., 22: Feb. 1917. See pp. 356-364.

36 Hydration and Growth.

Migrations of albuminous material from one part of the cell to
another and translocation of the proteins is a subject upon which
nearly all cytologists speak with great reserve because of the lack of
well-grounded observations. An extensive examination of such action
by the use of root-tips of yiciafaba has been made by Professor C. F.
Hottes, of the University of Illinois, and some of his results as yet
unpublished include facts of great possible importance. Dr. Hottes
says1 that seedlings deprived of the cotyledons and grown in the dark
at 20° C., being supplied with nutrient salts and water, continued to
grow for a period of three days to a week. The changes in the nucleus
and the concomitant action of the cytoplasm during this time are very
striking in the root-tips. The nucleolus is enormously reduced in size
and its materials escape into the cytoplasm. Materials from distant
cells, albuminous in nature, are transported through considerable
distance to the meristem of the tip, and these cells remain alive, sustain-
ing a parasitic relation to the cells from which the material has been
derived, and the fundaments of lateral roots are broken down and
translocated in the same manner. The progress of the translocation
may be followed through the strands connecting with the tip meristem.
Such transfer of materials is apparently inhibited at low and high
temperatures which lessen or stop growth. Antipyrine accelerates
exudation and transfer of such proteinaceous material and chloral
hydrate inhibits it. Furthermore:

"In all treatments leading to inhibition of cell activity, I find enlargements
of nucleolus, increase of chromatin without the passage of perceptible amounts
of these materials into the cytoplasm. In cell acceleration the nucleolar
material can be distinctly followed through the reticulum of the nucleus into
the cytoplasm. The chromatin (tropochromata) fluctuates in quantity and
its increase and decrease is concomitant with the absence and presence of
chromatin (chromidia) in the cytoplasm."

As the proteins diffuse sparingly,2 their translocation in living mat-
ter must take place by some other method, and one by which a rela-
tively rapid movement would be possible. So far as plants are con-
cerned, the possibilities offered by the amino-acids may prove to be
of the greatest importance in this connection. These substances pass
through membranes, show a relatively high rate of diffusion, and are
readily derived and combined.

1 Letter to author.

8 Robertson, T. B. The physical chemistry of proteins. New York. 1918. See p. 330.

IV. THE EFFECT OF SALTS AND ACIDS ON BIOCOLLOIDS
AND CELL-MASSES.

A proper supply of certain salts in the substratum is one of the most
important requirements of the plant, and those known to the physiolo-
gist as necessary for growth and development are designated as "nutri-
ent salts," although more properly to be designated as culture salts.

Available analyses show the general proportion of the various sub-
stances present in the organs and tissues of many kinds of plants. The
specialized or localized accumulations in the regions of the cell have
been demonstrated of only a very few substances, of which iron and
potassium seem to be the most notable.1 Chemical unions or precipi-
tations may account for the local concentration in some cases, while in
other structures the surface tensions of the minute masses of gels or
liquid may be responsible.

The heterogeneous character of living matter and the known facts
of its hydration and that of biocolloids by which water, acids, and
salts, etc., enter into combination in both definite and indefinite pro-
portions with the colloidal material, together with the behavior of
cell-masses in imbibition, have made it seem inadvisable to attempt to
express the reactions obtained in terms of hydrogen-ion or hydroxyl-
ion concentrations, and but few measurements of this kind are cited in
the experiments described in the present paper. Although this method
entails a treatment empirical to a certain extent, yet forced parallel-
isms and false explanations resulting from the application of simple
formulae to complex phenomena are avoided. Attention has been
confined chiefly to the study of the action of solutions in which dis-
sociation may be assumed to be complete or nearly so. By following
this simplified procedure it has been possible to explore wide fields of
biological possibilities, the exact mapping of which will need con-
centrated attention upon comparatively narrow problems. This is
especially true of the action of the amino-compounds upon biocolloids,
concerning which certain preliminary results are described in the fol-
lowing pages.

The amount of acid or salts and of water which may be taken up
from a solution and the accompanying swelling is influenced by several
factors. The reader is referred to texts on physics and on colloids for
detailed discussions of adsorption equations and for information con-
cerning the allowable generalizations concerning the relative amounts
of material which may be taken up by a colloid from a solution system.

For the present some results recently obtained by Miss C. L. Carey
and as yet unpublished will be of interest, as the absorption of water

1 MacCallum, A. M. The distribution of potassium in animal and vegetable cells. Jour, of
Physiol., 32 :95. 1905. Also, Presidential address, Brit. Assoc. Adv. Sc., Report for 1910, p. 744.

37

38

Hydration and Growth.

and hydrochloric acid taken from a solution by various materials comes
within the range of this chapter. In these tests 3 to 6 grams of the
colloid or of the plant material were placed in a dish containing about
100 c. c. of the acid solution at 21° C. The results are given in table 18.

TABLE 18. — Water absorbed from hydrochloric acid per gram dry substance.

Material.

Concentration.

N/20.

N/10.

N/5.

N/2.

Agar A

2.312
4.242

2.164

Agar B

3.497
3.570

3.571
3.461

Agar C (only one determination in each
cone, for agar C)

4.275

3.917
2.944

Agar A (48 hours)

Agar A (4 days)

3.693

Agar A (7 days)

2.688

Agar A (10 days) ... .1.

2.678

Agar A (14 days)

2.731

Agar-gelatin

4.520
14.362
1.368
1.096
1.169
.822

5.884

4.065
9.830
1.504
1.163
1.181
.804

5.891

3.821
5.892
1.514
1.112
1.257
.786

5.645

3.913
5.019
1.602
1.079
1.210
.812

5.678

Gelatine (Cox's)

Lupinus albus cotyledens

Vicia faba cotyledons

Phaseolus lunatus cotyledons '. . .

Starch (commercial "corn starch"). . . .
Coconut (commercial shredded, after
removal of oil and sugar)

The amount of acid absorbed was greatest in all cases from the high-
est concentrations used. The amount of hydration which accom-
panied the incorporation of the acids in the colloids is given in
table 19.

TABLE 19. — Absorption of hydrochloric acid and water from solution, per gram dry

substance.

Material.

Hydrochloric acid, grams.

Water, grama.

N/20.

N/10.

N/5.

N/2.

N/20.

N/10.

N/5.

N/2.

Agar A

0.01115
.01177

.01331

0.01591

2.312
4.242

4.275

2.164

3.917
2.944
3.693
2 688

Agar B

0.03168
.03457

0.06990
.06892

3.497
3.570

3.571
3.461

Agar C (only one determination
in each concentration for
agar C)

.02151
.01735
.01913
.01476
.01386

.01373
.02508
.05577
.01700
.01918
.01996
.00442

.03585

Agar A (48 hours), 4 determi-
nations

Agar A (4 days), 2 determina-
tions . .

Agar A (7 days), 3 determina-
tions

Agar A (10 days), 3 determina-
tions

2.678

2.731
4.065
9.830
1.504
1.163
1.181
0.804

5.891

Agar A (14 days), 1 determina-
tion

.03433
.06991
. 02490
. 02636
.02672
.00779

.04789

.07705
.11075
.04173
.03191
.03482
.01434

.08969

4.520
14.362
1.368
1.096
1.169
0.822

5.884

3.821
5.892
1.514
1.112
1.257
0.786

5.645

3.913
5.019
1.602
1.079
1.210
0.812

5.678

Agar-gelatine

.01941
.04130
.00843
. 01269
.01256
.00259

.02344

Gelatine (Coxe's)

Lupinus albus cotyledons . . .

Vicea Faba cotyledons

Phaseolus lunatus cotyledans . .
Starch (commercial starch) ....
Coconut (commercial shredded
after removal of sugar and oil) .

Effect of Salts and Acids on Biocolloids and Cell-masses.

39

Briefly summarized, agar takes up the greatest amount of water in
24 hours from a 0.05 N solution, and the maximum imbibition in
gelatine and gelatine-agar combinations also ensues in this concentra-
tion, which is one duplicated in the cell-masses of the plant. Cotyl-
edons and sections of the plants tested found their maximum at a
concentration of 0.1 N at the temperatures named.

Hydration of dried plates, or of sections of living plants, is, of course,
accompanied by a diffusion or solution out of the contained salts in a
manner determined by a large number of environmental conditions,
inclusive of the proportionate amount of water to which the colloid is
exposed or in which it may be immersed.

Thus, in most of the experiments described in this volume, the sec-
tions having a total initial volume of dried material of about 2 or 3
c. mm. were immersed in dishes containing 33 c. c. of water. The
hydration of material over a period of 24 to 50 hours would necessarily
result in the solution out of a portion of the salt contained, which might
form as much as 18 per cent of the original dried weight of the sec-
tions. On the other hand, swelling in a solution of salt-free colloid,
for example, might result in an accumulation.

A series of tests was made to ascertain the relative amounts of water
which might be taken up by one of the biocolloids used extensively in
this work from a graded series of a salt solu-
tion. Since it showed a maximum hydration
capacity at temperatures of 15° to 40° C., a mix-
ture of agar 90 parts and oat protein 10 parts
was used, and the sections, which ranged from
0.16 to 0.18 mm. in thickness, were measured
as to each set and arranged in trios in glass
dishes. The sections were as nearly uniform as
possible and the average volume of the air-dry
trios of sections in each dish was 12 c. mm.
The testing dishes held 30 c. c. of the salt solu-
tion. Temperatures were taken by means of
small thermometers of the clinical type, and
readings of the temperature of the solution in
the dishes were made several times during the
course of the test. It is to be noted that the
end-point of the swellings would not have been
reached until after 40 to 48 hours in the less-
concentrated solutions, but the amount of ex-
pansion which might have been displayed in

the last 12 hours of this period would not have changed the totals
greatly or the proportions in any important manner. The data given
in table 20 represent the average expansion of sets of 3 sections at
16° to 17° C.

TABLE 20. — Swelling of a
mixture of agar 90 parts
and oat protein 10 parts
in distilled water and
potassium nitrate at 16°
to 17° C.

Potassium nitrate.

2 M

p. ct
445
640
530
695
860
1,165
1,305
1,560
1,670
1,500
1,670
1,720

1 M

0.5 M

.1 M

.05 M

.02 M

.01 M

. 005 M

. 0025 M

.00125 M
.000625 M
.0003125 M...

Distilled water

1,940

40

Hydration and Growth.

TABLE 21.

The range of concentrations examined does not exhaust the possi-
bilities. It has been previously found that biocolloids will take up
some water and swell from the most concentrated mixtures of salts.
On the other hand, the greatest attenuations exerted some influence
on the wTater capacity, although it may be surmised that at a lower
concentration the deviation on the swelling in the salt solution from
that in distilled water would be so slight as to be negligible in all
biological applications of the facts. Living matter is at all times
impregnated with salts to a degree within the range of these tests.

In the continuance of the series to test the
effects of some of the salts of biological im-
portance, plates of agar and oat protein 0.2
mm. in thickness were swelled at 15° C. in cal-
cium nitrate. Two series of measurements are
shown in table 21.

The weakest attenuation used allows a swell-
ing practically equivalent to that of distilled
water. Two series of greatest divergence
from the effects of potassium nitrate are to be found in dilutions of
0.02 M, while distinctly different action is seen to prevail in the con-
centrated solutions above the unimolecular.

The sections of agar-oat protein used for testing the effects of cal-
cium chloride were 0.16 mm. in thickness and the swelling was made
at the same temperature as in potassium nitrate, 15° C. The measure-
ments in 24 hours are given in table 22.

TABLE 22. TABLE 23.

Calcium nitrate.

p. ct.

p. ct.

2 M

975

917

0.2 M. . .

525

722

.02 M..

650

778

.002 M.

1,425

1,555

.0002 M

1,975

Calcium chloride.

Distilled water.
2 M

p. ct.
1,660
273
375
656
907
1,279
1,281
1,469
1,438

p. ct.

1 M

0.1 M

.01 M

. 005 M

.001 M

.0002 M
.00005 M

1,438
1,688

Potassium chloride.

Water.

2 M

p. ct.
468
656
1,031
1,344
1,156
1,906
2,210
1,406

p. ct.

p. ct.

p. ct.
1,781
1,989

1 M

0.1 M
.01 M
.005 M...
.001 M...
.0002 M..
.00005 M.

1,463
1,625
1,719

1,467
1,667
1,533

The chloride of calcium appears to limit swelling to a greater
extent than the nitrate, so far as table 22 is comparable with that
obtained from swelling in the nitrate.

The next trial was made with potassium chloride in solutions of the
same concentration as above. Agar-oat protein was used and the
swellings at 15° C. in 24 hours are given in table 23.

The amount of imbibition in potassium chloride is greater than that
in calcium chloride in equivalent concentrations, while it is noticeable

Effect of Salts and Acids on Biocolloids and Cell-masses. 41

that in the 0.0002 M solutions here, as in some of the trials previously
made, the swelling is very high, probably even higher than that in dis-
tilled water.

Swellings of the agar-oat-protein mixture for 24 hours at 15° C. gave
the results shown in table 24 in di-potassic phosphate (K^HPC^) .

The sections in the 1 M solution were sealed
by the glass triangle, which was pressed too
closely on them, with the result that swelling
progressed very slowly to a defective total.

Di-potassic phosphate.

Distilled water . .

2 M

1 M

0.1 M

.01 M

.005 M

.001 M

.0002 M

00005 M..

p. ct.

156

125

625

806

1,563

1,719

1,889

1,964

If the results of the swelling in the di-potassic
phosphate were plotted as a graph, it would be
seen that the steepest part of the curve would
lie in the region between the concentrations of
0.01 M and 0.005 M. The graph of the potas-
sium chloride would be a much more regular
figure. The steepest part of the graph of the
results with calcium chloride would probably
lie between 0.01 N and 0.005 N, the steepest
part of the graph of calcium nitrate would probably be in the region
between 0.02 N and 0.002 N, and the steepest part of the line express-
ing the falling-off of the retarding action of potassium nitrate would
be between 0.05 N and 0.005 N.

The breaks or discontinuities in the rise of the curve of imbibition
total led to the belief that some errors had crept in, and repetitions
were made with concentrations from 0.01 M to 0.000005 M. The
additional experiments were for the most part symmetrical with each
other, although it is not allowable to contrast the separate items of the
swellings of two different lots of material. The ground at first taken,
that in minute quantities some of these salts might cause a swelling in
excess over that in distilled water, still lacks confirmation. It is a
matter, however, that should be tested with great care, as such re-
actions for sections of plants are included in my records.

The swelling in various concentrations of a salt supposedly depends
chiefly upon the acid ion, although the action of the basic ions is not
actually excluded. The above tests were made in solutions varying
from 4 M to 0.5 M, which are far too concentrated to be of direct
biological interest. A more dilute series of potassium salts in 0.01 M
and 0.001 M solutions was made up and the swelling of sections of
agar 90 parts and peptone 10 parts, 0.22 mm. in thickness, were made
at 15° C. The tests were closed at the end of 24 hours, and although
some slight increase was still in progress, the relations of the various
preparations were identical with those which might be expected of
the end-points. (Table 25.)

According to Hofmeister, as cited by Taylor, swellings of gelatine
in chlorides and nitrates should be greater than in the citrates and

42

Hydration and Growth.

TABLE 25.

sulphates. The differences found in my own tests with the above
mixtures, which, it must be pointed out, are so small as to be very close
to the limit of variation, are of the reverse kind. They are, however,
of such a nature as to warrant the assertion that the greatest swelling
of this biocolloid in the group of substances
named does not take place in the nitrates
and chlorides.

Another aspect of this matter was tested
by arranging a series in which ,an agar-
peptone mixture was swelled in two con-
centrations of sodium acetate, which is re-
puted to retard imbibition in simple col-
loids, and sodium chloride, which is said
to increase the amount of swelling over
that of water. The measurements of such
swellings at 15° C., closed at the end of 24 hours, are given in table 26.

These results are featureless, so far as the above point is concerned.
The lesser concentration of the sodium acetate seems to give a
greater swelling than the higher, but, on the other hand, the sodium
chloride, which should promote imbibition, does not induce swelling
as great as those in the acetate. The sections used were salt-free and
a parallel series was run, using dried sections taken from the median
layer of Opuntia joints, which at 15° C. gave the measurements shown
in table 27.

TABLE 26. TABLE 27.

Concentration.

0.01 M.

0.001 M.

p. ct.

p. ct.

Chloride . .

818

1,136

Nitrate . . .

818

1,205

Phosphate

932

1,136

Citrate . . .

977

1,227

Sulphate . .

J975

1,250

Estimated from 0.007 M.

Concentration.

0.01 M.

0.001 M.

Sodium acetate. .
Sodium chloride .

p. ct.
1,167
1,214

p. ct.
1,262
1,214

Concentration.

0.01 M.

0.001 M.

Sodium acetate. .
Sodium chloride.
Distilled water. . .

p. ct.
650
613
570

p. ct.
613

588

The swelling in both salts is greater at the higher concentration, and
the maximum effect may lie at a higher point. The acetate induces a
higher hydration effect than the chloride. The plant sections are of
course extremely complex as to chemical composition, although their
relations to water are taken to be chiefly determined by the pentosan-
protein ratio, and are modified by the salts already present and by the
residual acidity. It is evident that gelatine and isinglass do not
furnish conditions for swelling analogous to those of the plant, as as-
sumed by so many writers, since the results given above do not coincide
in the main with those obtained by Hofmeister.1 As has been pointed
out elsewhere in this work, the similarity of action of the plant to that

1 Hofmeister, F. Die Betheiligung geloster Stoffe an Quellungs-vorgange.
Pathol. u. Pharm., 27:395, 1890, and 28:210, 238, 1891.

Archiv. f. Exper.

Effect of Salts and Adds on Biocolloids and Cell-masses. 43

of gelatine and of the proteins and their derivatives will depend chiefly
upon the proportions of such substances in the living cell-masses. The
properties of gelatine may illustrate those of protoplasm only in so far
as they are general to the elastic gels, in which class of colloids both may
be included.1

The salt-content of colloids of living matter in all probability changes
very slowly, while the acidity may vary with great rapidity and through
a wide range. A set of tests were therefore arranged in which the
salt-content would remain constant while the solutions contained a
series of acid concentrations. The first series was one in which the
salt was dissolved in the solution of the acid after the manner in which
many measurements have been previously made. Sections of plates
of agar 90 parts and oat protein 10 parts which had an average thick-
ness of 0.18 mm. were cut so that a trio had a total volume when air-
dry of 12 cu. mm. and the dishes in which these were placed held about
30 c. c. of the solution. These measurements were made with the solu-
tions standing at 16° to 17° C. The results were as follows:

TABLE 28.

p. ct.

Distilled water 1 , 722

Potassium nitrate, 0.01 M 1, 250

Potassium nitrate, 0.01 M + citric acid, 0.05 N 472

Potassium nitrate, 0.01 M + citric acid, 0.01 N 628

Potassium nitrate, 0.01 M + citric acid, 0.005 N 667

Potassium nitrate, 0.01 M -j- citric acid, 0.001 N 944

Potassium nitrate, 0.01 M -J- citric acid, 0.0002 N 1,055

Potassium nitrate, 0.01 M + citric acid, 0.00004N 1, 139

The above measurements were taken at the end of 24 hours, at
which time the sections in distilled water and in the two solutions
containing least acid were still slowly expanding, at a rate which
would not have changed the final aspect of the test. These results
are of importance, since it has been found that the range of
acidity in such plants as growing joints of cacti may be practically
equivalent to that from the highest acid-content to the lowest during
the daylight period, coincident with an enormous variation in the
water-capacity of the organ.2 The measurements of plates composed
of 90 parts agar and 10 parts bean protein 0.25 mm. in thickness, in
dark room, at 16° to 17° C., gave the results shown in table 29.

TABLE 29.

p. ct.

Distilled water 1,280

Potassium nitrate 1 , 060

Potassium nitrate, citric acid, 0.01 M 802

Citric acid, 0.01 N 604

Potassium hydroxid, 0.01 M 604

Effects similar to the original are to be discerned in the above. The
combination of acid and salt reduces the hydration capacity of the

1 Fenn, W. O. Similarity in the behavior of protoplasm and gelatine. Proc. Nat. Acad. Sci. ,
2: 539. 1916.

1 MacDougal, D. T., and H. A. Spoehr. The effects of acids and salts on biocolloids. Science,
46:269. 1917.

44 Hydration and Growth.

colloid below that in the salt alone. A wide variety of tests which gave
opportunity for comparisons are described throughout this volume,
but a few may be recorded here which were carried out expressly to
obtain evidence on this point with biocolloids of different constitution.
Nucleinic acid is a constituent of the nucleus, and as the only other
substance from this body which had been introduced into the tests
which could be assigned to the nucleus was peptone, its swelling re-
actions were tested with much interest. The results of swellings of
these substances, when combined in proportion of 10 parts nucleinic
acid to 90 parts agar, are given in table 30.

TABLE 30.

p. ct. p. ct.

Distilled water 1,400 1,025

Potassium nitrate, 0.01 M 900 800

Potassium nitrate, citric acid, 0.01 N 650 675

Potassium citrate, 0.01 N 850 750

Potassium citrate, citric acid, 0.01 N 725

Citric acid, 0.01 N 700 625

Sodium hydroxid, 0.01 M 1,000 925

A second series a week later gave measurements shown in table 31.

TABLE 31.

p. ct. p. ct.

Distilled water 950 1, 100

Potassium nitrate, 0.01 M 650 550

Potassium nitrate, citric acid, 0.01 N 575 500

Citric acid, 0.01 N 575 450

Potassium nitrate, potassium hydroxid, 0.01 N 900 750

Potassium hydroxid, 0.01 M 850 800

This mixture is seen to swell most in distilled water, while the pro-
portionate swelling in hydroxid is very high, being greater than that in
the salts tested or in acid. Next, it is apparent that the two potassium
salts produce or allow an amount of imbibition not very much short
of that in the hydroxid. The acidification of the salts practically
reduces the swelling to the proportion displayed by the acid alone.
This must be taken to apply to this set of combinations only. It may
not be assumed that a similar generalization would hold for calcium.

Following the above, plates composed of 90 parts agar and 10 parts
glycocoll, 0.15 mm. in thickness, were tested in series parallel to the above
in the dark chamber at 16° C. The swellings are given in table 32.

TABLE 32.

p. ct. p. ct.

Distilled water 1,300 1,266

Potassium nitrate, 0.01 M 700 1,000

Potassium nitrate, nitric acid, 0.01 N 900 766

Citric acid, 0.01 N 666 766

Sodium hydroxid, 0.01 M 366 300

The effects of the acidified salt and of the acid are of the kind pre-
viously noted. The most prominent feature of the reactions was the
low hydration capacity in the alkaline solution and the relatively high

Effect of Salts and Adds on Biocolloids and Cell-masses. 45

expansion in acids, the action of the solution being supplemented by
the amino-acid in the sections, in a manner similar to that of such other
amino-acids as aspartic acid, cystin, tyrosin, etc.

A mixture of agar (50 parts) and gelatine (50 parts) poured on
Pratt-Dumas brown filter-paper dried to a total thickness of 0.3 mm.
Swellings of this were made in the dark chamber at a temperature of
16° C., with the results shown in table 33.

TABLE 33.

p. ct.

Distilled water 400

Potassium nitrate 375

Potassium nitrate, citric acid, 0.01 M 350

Citric acid, 0.01 N 300

Potassium hydrate, potassium nitrate, 0.01 M 425

Potassium hydroxid, 0.01 M 325

These results, so far as they may be correlated with the earlier ones,
show an unexpected relation to acid, hydroxid, and water. It is to be
noted in addition, however, that a combination of potassium hydrate
and potassium nitrate gives the maximum effect in the series.

The application of parallel tests to growing tissues is complicated
by the fact that varying quantities of normal salts and acid salts may
be present, giving a buffer effect. The concentration of the hydrogen
ion may be determinable by estimation of the titrable acid and the
dissociated malates, for example, as found by Jenny Hempel, but in
addition there are to be considered the effects of the amino-acids and
amines, which are not easily to be measured.

Leaves of Mesembryanthemum edule, which were not yet fully grown,
were cut into sections about a centimeter long and allowed to dry
in the air. When the greater part of the water had been lost and the
sections had a leathery consistency, one of the angles was removed
with the scissors, leaving a specimen 1.8 mm. thick. The preparations
were by no means uniform. The use of three to obtain each record
would tend to obviate or smooth the discrepancies, but the data given
in table 34 can not be taken as having been obtained from preparations
strictly equivalent.

TABLE 34. — Swelling of sections of Mesembryanthemum.

p. ct.

Distilled water 72

Potassium nitrate, 0.01 M 69

Potassium nitrate, 0.1 M 56

Citric acid, potassium nitrate, 0.01 M 44

Citric acid, 0.01 N 72

Sodium hydroxid, 0.01 M 28

The main interest in this set of reactions is that directed to the
comparison of the swellings in potassium nitrate, citric acid, and the
combination at the same concentration. The coefficient of swelling
in the plant-like sections of agar-oat protein and agar-bean protein was
least in the acidified salt solution, although the hydrogen-ion concen-

46 Hydration and Growth.

tration of the combined solution should be equivalent to that of the
salt alone. The combination of citric acid and potassium nitrate is
open to some objection, but the effects described are similar to those
obtained by the use of potassium chloride and hydrochloric acid.

The total acidity of pure juice of fresh material at Tucson varied
from 0.0280 in the morning to 0.0232 per centimeter 0.01 N of sodium
hydoxid at 4h30m p. m. Probably some of the acid was broken up
during the drying, but the cell colloids would still be decidedly acid.
The hydrogen-ion concentration in another species of Mesembryan-
themum determined by Lakmoid tests and electrometer measurements
by Hempel gave values of pH — 4.8 to 5.2 for the first and 4.61 to 4.84
by the second method.

The matter was given further test by taking sections of young leaves
in a flaccid condition and measuring the total swellings, which are

given in table 35.

TABLE 35.

p. ct.

Water 22

Potassium nitrate, 0.01 M 22

Potassium nitrate, citric acid, 0.01 N 17

Citric acid, 0.01 N 17

Sodium hydroxid 11

The swelling in potassium nitrate agreed with that of the dried
material in being nearly equivalent to that hi distilled water, and less
in acid salt solution, but the swelling in acid is less, the proportionate
swelling in hydroxid being about the same.

The effects of a similar series of reagents were tried upon disks from
growing joints of Opuntia, with the results given in table 36.

TABLE 36.

p. ct.

Distilled water 11

Potassium nitrate, 0.01 M 9

Potassium nitrate, citric acid, 0.01 N 10

Citric acid, 0.01 N 9

Potassium nitrate, potassium hydroxid, 0.01 M 10

The relations here are different in character from those exhibited
by the material previously examined, but the departures are so small
that no safe conclusion may be founded on them.

The incorporation of any salt with a colloid in the disperse phase
would of course allow the formation of adsorption compounds to an
extent and of a kind not possible when the salt enters the hydrating
gel in a solution. The hydration of such a salted colloid might be
expected to take place at a different rate and to a total varying from
that of the unsalted colloid with unsatisfied chemical affinities and
under the conditions of surface tension which would prevail.1

1 Loeb, J. The similarity of the action of salts upon the swelling of animal membranes and of
powdered colloids. Jour. Biol. Chem., 31: 343. 1917.

Effect of Salts and Adds on Biocolloids and Cell-masses. 47

The first tests of the effects of incorporated salts were those in
which the conditions of the plant were simulated, and the most rational
procedure seemed to be one in which the culture salts of plants should
be added to a mixture of agar and bean protein, the proportions being
as follows :

TABLE 37.

gm.

Agar 9

Bean protein 1

Potassium nitrate 0.00506

Di-potassic phosphate .01622

Magnesium sulphate . 03660

Calcium nitrate . 03490

Total 10.09278

This material was reduced to a dried plate 0.18 mm. in thickness,
which was swelled under the auxograph at a temperature of 15° C.,
giving increases of 1,400 to 1,500 per cent in distilled water, as might
be contrasted with 2,100 to about 2,600 per cent in the reactions of
similar sections free from salts.

A second preparation was made, but with ten times the amount of
salt used in the first one, the salts forming nearly 9 per cent of the
dry weight in one case and 0.85 per cent in the other. The swellings
of the biocolloid with the higher salt-content are given below:

TABLE 38. p. ct.

Distilled water 958

Citric acid 361

Potassium hydroxid, 528

Potassium nitrate, citric acid, 0.01 N 389

Potassium nitrate, 0.01 M 694

Potassium hydroxid, potassium nitrate, 0.01 M 472

The large proportion of salts is seen to hinder swelling in a notable
manner. Temperature effects are of great importance in this con-
nection, as it was found that sections of the plates which contained the
lesser proportion of culture salts and which increased 1,325 per cent
in distilled water at 15° C., swelled 2,666 per cent at 48° to 40° C. (See
Chapter IX for a fuller discussion of temperature effects.)

Another set of dried plates was made for the purpose of obtaining
comparisons in two directions. The colloidal constituents of the
mixture were extended to include agar 70, dextrose 5, gelatine 5, pep-
tone 5, asparagine 5, nucleinic acid 5, and bean protein 5 parts, and a
set of dried plates was made up as above in distilled water. A second
set was made up in the culture solution, in which the salts amounted
to 0.85 per cent of the dry weight. The swellings were made on two
successive days in a chamber constant at 15° C. It is to be noted that
in any inspection of these results rigid comparisons may not be allowed
between the swellings of the two kinds of plates in any solution. The
basis of all comparisons must be the ratio of the swelling of each plate
in any solution to its swelling in distilled water. (See table 39).

Hydration and Growth.
TABLE 39.

Salt-free.

Salted.

Distilled water

p. ct.
1,250

p. ct.
500

Potassium nitroxid, 0.01 M

875

550

Potassium nitroxid, citric acid, 0.01 N

535

425

Citric acid 0.01 N

446

425

Potassium hydroxid, potassium nitrate, 0.01 M
Potassium hydroxid, 0.01 M

750
535

550
525

The relative swellings of the biocolloid without the culture salts
presents the general features of such mixtures, being highest in dis-
tilled water, next in potassium nitrate, less hi acidified potassium
nitrate, less in citric acid, and varying in the relations of the hydroxid
and the alkaline salts.

The plates in which the culture salts were incorporated showed
relative swellings which did not differ widely from the expectancy,
except in the swelling in alkali and alkaline salts. The addition of the
dextrose could not be seen to exert any definite action. The outstand-
ing fact is the general retarding effect of salinity on the hydration
capacity, a fact of possible enormous importance in the organism.

A mixture including 4 parts of agar, 5 parts of gum arabic, and 1
part of gelatine was made and sufficient potassium chloride was added
to make it 0.01 M of this compound. Swellings at 18° to 20° C. were

as given in table 40.

TABLE 40. p. ct.

Distilled water 612

Citric acid, 0.01 N 465

Sodium hydroxid, 0.01 M 214

Hydrochloric acid 0.01 M 535

Hydrochloric acid, potassium chloride, 0.01 M 419

A general restriction of nearly all of the swelling reactions is illus-
trated by the measurements in table 40, while the relative increase in
acids is high.

As a further combination of two forms of carbohydrate, albumen,
amino-acids, and of the salts which are found in plants, a mixture was
made which contained the following material :

TABLE 41. gm.

Agar 6

Acacia 2

Gelatine 1

Albumin (Phaseolua) 1

Potassium nitrate 0 . 0058

Potassic phosphate, dibasic 0185

Magnesium sulphate (7H2O) 0418

Calcium nitrate (4H2O) 0398

Total colloid material 10

Total nutrient salts ... . 0105

Effect of Salts and Adds on Biocolloids and Cell-masses.

49

I2p.m. m. 12p.m. m. Igp^m.

I2p.n

I2p.m. m. 12p.m.

Dried plates were made up in the usual manner and freed from
water in a special chamber with fan at a temperature of about 16° C.
Swellings at 14° to 17° C. were as shown in table 42.

TABLE 42. p. ct. p. ct.

Distilled water 2,200 2,240

Citric acid 0.01 N 802 880

Sodium hydrate, 0. 01 M 602 680

Potassium chloride, hydrochloric acid, 0.01 M 700 640

The notable feature is the high swelling in water and the fact that
the increase in the acid solution is less than in the alkaline, facts
which are probably due in part to the action of gum acacia. This
set of swellings has unusual interest because of its composition, in
which the categories of substances in the plant are represented with
some fairness and adequacy (fig. 9). FIG. 9.

Tracing of auxographic
records of swelling of
sections of plates
consisting of 6 parts
agar, 2 parts gum ar-
abic, 1 part gelatine,
1 of bean albumin
and .05 nutrient salts
in water. A, citric
acid. 01 N. .B, sodium
hydrate. 01 M. C, po-
tassium chloride and
hydrochloric acid
.01 M. D, at 14° to
17° C. Downward
course of pen tracing
denotes increase as
indicated by numer-
als on margin.

The results*obtainedTin*some'*of the foregoing experiments indicated
that the treatment of the biocolloid with salts before acid solutions
were applied might show some features of importance, and this was
also supported by the alternating effects described in detail elsewhere
in this volume. Plates had been made of agar 90 parts and bean
protein 10 parts in two sets. In one a culture solution was used in
such concentration that the included salts formed 0.85 per cent of the
dry weight of the sections. In the other the concentration of the
salts was about ten times this amount. Both showed opaque dots or
minute regions, supposedly insoluble globulin.

Swellings were made at temperatures of 16° to 17° C. on October 12
and 13, 1917, and the measurements obtained from the sections con-
taining the larger proportion of salts are as given in table 43.

TABLE 43.

p. ct.

Distilled water 522

Citric acid, 0.05 N 583

Citric acid, 0.05 N 413

Citric acid, 0.0005 N 348

Citric acid, 0.00005N 500

\\

50 Hydration and Growth.

The measurements in table 43 were taken at the end of 30 hours,
at which time expansion was not complete, although further swelling
would not materially alter the relative values. The proportion of
salts actually present was about one-twelfth of the biocolloid, which in
volume amounted to about 12 c. mm. The dishes held 30 c. c., and
from these data it may be possible to calculate proportions of salts
and acids for comparison with the cases in which the salts are applied
in solution.

A second test was made with the same biocolloid as above, but to
which had been added but one-tenth of the foregoing proportion of
culture salts. The plates were thinner, but the swellings were made
at the same temperatures and under approximately the same con-
ditions, with results as follows:

TABLE 44.

p. ct.

Distilled water 1,667

Citric acid, 0.05 N 667

Citric acid, 0.005 N 899

Citric acid, 0.0005 N 1 , 139

Citric acid, 0.00005 N 1,500

The swelling of these plates, which were 0.18 mm. in thickness, was
carried out at a temperature of 16° to 17° C., and the expansion in
distilled water shows the retarding effect of the salt alone when com-
parison is made with agar-bean protein mixtures not treated with the
nutrient solution. The citric acid in its heaviest concentration is
equivalent to the strongest solution encountered in plants in this work,
at which it is seen to retard swelling very much. Reduction of the con-
centration of the salts seems to be followed by a proportionate increase
of water-capacity, in a fairly regular manner. The discrepancy in the
swelling in 0.005 N acid in the more heavily salted plates is probably
an instrumental error and will be so considered until confirmed. In
the most attenuated solution of acid the swelling approaches that of
distilled water, in which the salt effect alone is apparent.

The series of increases of a biocolloid given on page 48 present the
general differences and relations of sections from plants, and these,
rather than the one in table 44, which has such a high swelling coeffi-
cient in water, are of the character more usually encountered in cell-
masses. It is to be recalled, however, that the composition of the
biocolloid, including a mucilage, albumin, and amino-acids, is one
which may well be duplicated in the plant, and it may be the recurrence
of such combinations which would furnish the phenomena so prominent
in the involutions of the cell.

The principal deductions of the present work support the conclusion
that agencies or conditions which increase the hydration capacity of
protoplasm accelerate growth, and any factor which tends to lessen
either the rate of absorption or the total hydration capacity of living

Effect of Salts and Adds on Biocolloids and Cell-masses. 51

matter retards and limits growth and development, or may have such
special effects as, for example, the condensation of chromatin into
special masses or chromosomes in the course of cell division.1 Now,
protoplasm parallels the colloidal action of gelatine only in so far as it
is composed of protein or protein derivatives, and the proportions of
these substances and of the associated carbohydrates vary from organ
to organ and with the course of the seasons or the stage of develop-
ment. Furthermore, the biocolloids of the cell are acidified or salted,
and their behavior toward any reagent externally applied will of
course be determined or modified by all of the chemical and adsorptive
relations implied. Lastly, the residual acids of respiration vary from
hour to hour under ordinary circumstances.

It might be expected, therefore, that tests which are planned to
determine the influence of acids or bases on growth would bring out a
diversity of results.

G. A. Borowikow (Borovikov), a Russian working at the University
of Odessa, used seedlings of Helianthus 6 days old for testing the
effects of acids and salts upon growth.2 The roots of the seedlings
were immersed in water and solutions in glass jars and the effects
upon growth derived from measurements of the length of the plant-
lets. Acceleration and final maxima were obtained by the use of
hydrochloric, sulphuric, nitric, acetic, and boric acids, in the order
named, that of hydrochloric being the greatest as compared with the
growth of plants in distilled water. It was also noted that the addi-
tion of salts to the acids influenced the rate and final effect, according
to the character of the base. Salts with weak, easily hydrolyzable
bases affected growth almost solely according to the concentration
of the hydrogen ions, but the stronger bases exercised a definite
effect, which in this author's work was to retard growth. It is clear
that the growing cell-masses of Helianthus are not identical in their
action with such amphoteric colloids as gelatine.

Sections of growing internodes of Helianthus in my own work did
not show their greatest swelling in simple acid solutions, but in those
to which some salt of the same molecular concentration had been
added. It was also found that the hydration capacity of artificially
mixed biocolloids, dried and living sections of plants showed a decrease
in hydration capacity with rise in temperature above 16° to 18° C. in
acid solutions. On the other hand, it has been shown that in the
petiole of the calla (Richardia} , which attains a length of over half
a meter and continues to elongate at varying rates throughout its
entire length, the greatest acidity is in the region of most rapid growth.
It is evident that interpretations must take into account a wider

1 Mathews, A. P. Physiol. Chem., 2d ed., p. 235. 1916.

2 Borowikow, G. A. Ueber die Ursachen dea Wachstums der Pflanzen. Biochem. Zeitschr.,
48: 230, and 50: 119. 1913.

52 Hydration and Growth.

range of conditions than those presented by the swelling of simple
gelatine in electrolytes, especially at uncontrolled or unrecorded tem-
peratures.

Professor F. E. Lloyd, working under the equable temperature con-
ditions of the Coastal Laboratory at Carmel, California, measured
the growth of pollen-tubes of Phaseolus odoratus in acids (hydro-
chloric, acetic, malic, citric, formic, and oxalic) at concentrations
N/200 to N/25,600 in association with cane sugar in concentration
of 40 per cent. In these solutions no growth occurs at concentra-
tions at or above N/3,200 of the acid component. Below that limit
the rate of growth is inversely as the concentration. The rate and
total amount of growth possible for any concentration varied with
the acid, it being least at the higher concentrations for formic and
oxalic and highest for acetic.1

No temperature records are cited in any of these cases or in the other
work described by Long or Dachnowski.

The measurements of E. R. Long brought out the fact that growth
in Opuntia was greater in culture salt solutions than in any other
medium tested, while that in alkaline solution was more than in either
malic or hydrochloric acid, these substances being used in concentra-
tions of N/50, while the solutions of Borowikow were N/100 or weaker.2

A. Dachnowski measured the swelling and water-relations of seeds
of beans and corn and cuttings of tomato shoots and obtained some
facts of great interest.3 Beans were found to absorb and retain less
water in acid solutions (N/800) than in equi-nonnal alkaline solutions.
The cations Ca and Na were more active than potassium in limiting
imbibition hi beans, a relation which is reversed hi corn grams, in
which the greatest swelling took place in calcium, a lesser amount in
potassium, and a minimum in sodium. Cuttings of tomato plants
were found to function better hi an alkaline medium than an acid, and
best of all in water in absorption and transpiration. Sulphuric acid
at N/3,200 and potassium hydroxid at N/6,400 furnished exceptions to
these conclusions. The effect of any salt on the water-relations of the
plants used was the sum of the constituent ions, a conclusion confirm-
ing the work of Borowikow.

The well-known cultural conditions required by many bacteria and
fungi furnish still further exemplification of the diversity of behavior
of the biocolloids in the hydration necessary for growth. The plasma
of bacteria is high in albumin and many bacteria show the highest
velocity and greatest development in a medium containing the soluble
proteins combined with sodium chloride and brought to an alkaline

1 Lloyd, F. E. Collodial phenomena in the protoplasm of pollen tubes. Kept. Dept. Bot-
Res., Year Book Carnegie Inst. Wash., 1917, p. 63.

*Long, E. R. Growth and colloid hydratation in cacti. Bot. Gazette, 59: 491. 1915.

1 Dachnowski, A. The effects of acid and alkaline solutions upon the water-relations and the
metabolism of plants. Amer. Jour. Bot., 1 : 412-439. 1914.

Effect of Salts and Adds on Biocolloids and Cell-masses. 53

condition with sodium bicarbonate. The sodium albuminates which
would be formed in the medium are highly dissociated, as would be the
biocolloids of the cell, and both diffusion and hydration would be
accelerated. This supposition would be based on the inference that
the amphoteric cell-proteins were stronger acids than bases. A further
result of the conditions of bacterial action is to be seen in the fact that
the growth of bacteria in cultures containing carbohydrates is accom-
panied by the production of an acidity which checks then1 growth and
retards their action upon proteinaceous substances present. On the
other hand, many molds grow best in relatively high concentrations
of acids, the cycle of fermentations in milk furnishing a striking example
of the differential action of these organisms.1 The first stage is charac-
terized by the action of the lactic-acid-producing bacteria, which con-
tinue until their products reach an inhibitory concentration. The
soured milk now becomes a suitable medium for Oidium and Penicil-
lium, which may thrive in a solution containing as much as 1.25 per
cent lactic acid, and continue to grow until the acid is exhausted, and
then the neutral or alkaline solution again becomes a suitable habitat
or bacteria.

German, N., and L. F. Rettger. The influence of carbohydrate on the nitrogen metabolism of
bacteria. Jour, of Bacter., 3: 389. 1918.

V. THE EFFECTS OF ORGANIC ACIDS AND THEIR AMINO
COMPOUNDS ON HYDRATION AND GROWTH.

The biocolloids of the plant are pentosan-protein mixtures in which
the substances of these two main groups vary widely in their propor-
tions, with a smaller proportion of lipins probably more or less localized.
The variables are so large that generalizations concerning the action
of the plasmatic mass are not easily to be founded. Of the more im-
portant assertions concerning the action of protoplasm, the earliest
and most widely used, that protoplasm undergoes hydration like an
amphoteric colloid, and is exemplified by swelling gelatine, has long
since failed to satisfy the experimental conditions or to offer parallels
to the action of cell-masses of the higher plants.

It obviously follows that the assumption adopted by many writers
that any conditions which facilitate the ionization of the proteins
accelerate growth is not tenable, since the effect of acidity is to lessen
hydration of the pentosans or pentosan mixtures or cell-masses when
acting directly or in the presence of salts. The extensive use of agar
to represent the pentosan element in biocolloids in my experiments
does not imply that this substance or any other body presenting all
of its main physical characters are invariably present in the cell. The
gums from acacia, tragacanth, Opuntia, and from Prosopis and the
cherry-tree in all probability represent types of pentosans which may
really be the most abundant in plants. These gums or mucilages are
readily dispersible and have an indefinite hydration capacity which
soon passes beyond the limits of measurement by the auxograph
Their water-absorbing capacity would be none the less important,
when inclosed in the cell-sacs. The solubility of protoplasm has
formed the subject of some discussion among cytologists, and it would
seem highly probable that valid observations of both extremes may
have been made, the matter depending chiefly on the nature of the
pentosan, gum, or mucilage which entered into the plasmatic colloids,
together with the character of the more liquid phase, or the cell-sap.

The conclusions of Loeb1 to the effect that it is not possible for an
amphoteric colloid to be acted upon by both ions at the same time,
if correct, would add still further proof to the fact that protoplasm
does not behave like an amphoteric colloid in its hydration relations,
except in so far as it may be predominantly composed of such material.

Data for a critical review of this matter are not available at present,
but it is known that bacteria are high in albumin, and similar richness
of proteins is exhibited by fungi, and that certain algae show a large
proportion of amino-acids. In addition, proteins are abundant in

1Loeb, J. Amphoteric colloids. Chemical influence of the hydrogen-ion concentration.
Jour. Gen. Physiol., 1: 39. 1918.

54

Effect of Certain Organic Acids and Amino Compounds. 55

reproductive elements, while voluminous notices of these substance
and their derivatives are found in cytological literature, many of which,
however, need verification and analysis to make them of value.

If protoplasm were entirely or dominantly proteinaceous, the actual
acidity or hydrogen-ion concentration of the sap might be taken as the
chief factor in maintaining the rate and determining the course of
hydration and growth. The predominance of the pentosans in plant
cells, however, offers a set of conditions much more complex than that
of the comparatively simple ionization of gelatine, for, as has been
noted, the conditions which facilitate the action of protein gels retard
and limit the hydration of the carbohydrate gels to an extent and in a
manner which depend upon the structure and character of the pen-
tosans present.

It has already been shown that pentosan colloids show maximum
hydration capacity in the presence and under the action of certain
amino-compounds, a subject to which the larger part of this chapter
will be given.

The actual acidity, or hydrogen-ion concentration of the sap, is
widely different from the total amount of acid, as some is always com-
bined with such bases as potassium, sodium, calcium, magnesium,
iron, and aluminium, making a "buffer" by which the degree of dis-
sociation is controlled within certain limits. This range of variation
as it appears in separate estimations is rather large as compared with
variations in animals. It is necessary to bear in mind, however, that
a cell-mass is not uniformly acid or that the entire mass of the cell-
colloids is saturated with a solution of the same concentration.

In any buffer situation, however, a lessening of the hydrogen-ion
concentration of the sap would be followed by increased dissociation
of the acid radicle of the salts, and increase of acidity beyond a certain
point would result in a reversal of the process. The actual acidity is
expressed by the negative common logarithm of the number of dis-
sociated hydrogen ions given as the value of Sorensen's symbol pH.
This may vary from 3.9 to 5.7 in various succulents examined by
Jenny Hempel, and may approach neutrality at pH-7 in some cases.
A singular instance of wide difference between actual and total acidity
is offered by lemon fruits, the sap of which has an actual acidity of
pH = 2.3, which is about one-tenth the total acidity, which may be
expressed as about 0.05 to 0.06 N.1

The variations in the hydrogen-ion concentration of the cell-sap
and the determination of the agencies which may cause such changes
offer a most inviting field for research.2 In a recent paper, R. B.
Harvey has described some extremely interesting changes in the
as determined by potentiometer methods, of cabbage leaves in acidity,

1 Hempel, Jenny. Buffer processes in the metabolism of succulent plants. Compt. Rend,
d. trav. d. Lab. Carlsberg, 13: No. 1. 1917.

2 Haas, A. R. The reaction of plant protoplasm. Bot. Gazette, 63 : 232. 1917.

56 Hydration and Growth.

freezing, and finds that the principal effect is an increase in the hydro-
gen-ion concentration followed by a general return to original values
on thawing, with changes in the proteins generally consisting in pre-
cipitations of some of the proteins.1

The results of Borowikow and those of Dachnowski show that the
growth of the higher green plants, does not depend upon the hydrogen-
ion concentration alone. Acids and bases both influence hydration
and growth. In addition the accelerating effects of ammo-acids and
amines on hydration of biocolloids and cell-masses, living and dead,
go far to support the conclusion that these substances facilitate or
increase total growth. These substances are built up from simpler
substances in the plant in a manner which is by no means clear, al-
though under investigation and discussion for a quarter of a century.
The evidence favors the assumption that they come together in the
field of photosynthetic activity. The structure of these amino-
groups may be no means be assumed to be identical with that of the
amino-acids of animal metabolism, in which they occur only as dis-
integration products of the proteins or albumins.

The total amount of ammo-compounds in a cell-mass of a plant
varies widely during the course of a day, and, as has been noted above,
the proportion of nitrogenous material in the organs of the cell or the
members of a shoot may be greatly different.

As the hydrogen-ion concentration of the sap is known to remain
fairly constant, as the salts or bases which affect growth also change
but slowly, attention naturally focuses on the amino-compounds
as a cause in modifying the rate, course, and total amount of growth.
As the acids and their salts may be assumed to act invariably in the
presence of amino-groups, a series of tests were planned which should
make possible a comparison of the action of some of the commoner
organic acids and then* amino-compounds.

Two groups were chosen for the tests — succinic acid and its amino-
compound, amino-succinic or aspartic acid, which are dibasic; and its
amide, as noted above, which is monobasic; and acetic acid and its
amino-compound, glycocoll, which are monobasic. Sections of plates
of agar, gelatine, agar-gelatine, agar-protein, and other mixtures were
used. Swellings were carried out in the equable chambers of the
Coastal Laboratory, at 15° to 16° C. The principal results are given
in table 45.

The two organic acids, succinic and acetic, are seen to exert the
classical effect on gelatine, the greatest hydration taking place in the
higher concentrations, the effect decreasing with dilution until at
0.0004 N the swelling in acetic acid was scarcely greater than in dis-
tilled water. At 0.0004 M, however, the dibasic succinic acid showed

1 Harvey, R. B. Hardening processes in plants and developments from frost injury. Jour.
Agric. Res., 15:83. 1918.

Effect of Certain Organic Acids and Amino Compounds. 57

a swelling less than that in distilled water, a result that suggests a
rapid solution or dispersion from the surfaces of the sections and alter-
ations of viscosity in the mass.

TABLE 45. — Hydration of agar, gelatine, agar-gelatine, and agar-oat protein in organic acids
and their amino-compounds at 16° to 17° C. Expansion in percentages of dried thickness.

Concen-
tration.
Mol.

Succinic
acid.

Aspartic
acid.

Aspar-
agine.

Acetic
acid.

Glyco-
coll.

p. ct.

p. ct.

p. ct.

p. ct.

p. ct.

0.3

1,950

.5

1 060

2 804

.1

1,000

2,260

1,333

AOAB.

.06

1,091

827

2,308

1,433

.01

1,273

1,270

2,365

1,560

2,965

.002

1,600

1,400

2,440

1,790

3,166

.0004

1,750

1,788

2,720

1,955

2,605

.00008

2,528

2,080

3,250

2,640

Water, aver. 2,600 per cent.

0.1

.05

1,200

1,500

320

952

370

GELATINE.

.01

700

1,033

480

714

.002

500

380

500

690

360

[

.0004

433

340

467

643

360

Water, aver. 600 per cent.

0.5

.1

850

AGAR 8 PARTS, GELATINE

.05
.01

716
850

910
1,017

1,485
1,574

850
900

1,233
1,960

2 PARTS.

.002

917

1,295

1,608

922

1,767

.0004

1,000

1,667

1,383

1,117

1,420

.00008

1,030

1,786

1,383

1,167

1,484

Water, aver. 1,684 per cent.

0 5

500

1

809

AGAR 8 PARTS, OAT-PROTEIN

.05
.01

700
864

855
900

1,867
2,455

1,090
1,255

1,983
2,340

PARTS.

.002

909

1,670

2,523

1,738

3,050

.0004

1,136

2,600

2,675

2,238

3,000

.00008

2,330

3,050

2,600

2,480

Water, aver. 2,365 per cent.

Mixtures of agar (8 parts) and gelatine (2 parts) were now tested,
and the hydration in succinic acid at 0.00008 M was but 1,030 per
cent, as compared with 1,684 per cent in water, while acetic acid was
slightly higher, 1,167 per cent. A similar statement would hold for
the action of these acids on agar and for agar-protein, the hydration
in water alone being reached more nearly* than in the agar-gelatine
sections.

When we now turn to amino-succinic or aspartic acid and amino-
acetic acid or glycocoll, some new relations are uncovered. The
aspartic acid appeared to exercise a notable influence on the hydration
of agar. The limit of its solubility appeared to be about 0.05 M
at 15° to 20° C. When more than this was added to the water

58 Hydration and Growth.

used for solution a swelling in excess of the expectancy resulted. It
was also seen that the surface of the liquid became covered with thin
crystals. In all probability the solution or dispersion of some agar
into the water resulted in the displacement of some of the acid, with
the result that the sections were actually hydrated from a solution less
concentrated, giving a swelling in excess of the expectancy.

Tests were now made at the same temperature and under the same
conditions with plates consisting of agar (8 parts) and gelatine (2
parts), in order to ascertain the results when the carbohydrate was in
colloidal combination with complex amino-compounds. The trios of
sections had shown swellings of about 1,700 per cent in distilled water
under the same conditions and had an average thickness of 0.28 mm.

TABLE 46.

p. ct.

Aspartic acid, 0.05 M 910

Aspartic acid, 0.0002 M 1 , 090

Aspartic acid, 0.00008 M 1,786

The effect of the acid is seen to vanish at a much greater concen-
tration than on the agar alone, the swelling at saturation being about
half that of distilled water.

After the experience noted above, new plates of agar (9 parts) and
aspartic acid (1 part) were made. The amino-acid was placed in the
water in which the agar was liquefied to a 2.5 per cent solution. The
usual translucency of the agar was modified to a pale milky appear-
ance, and its viscosity seemed to be decreased. Soon after setting,
cracks and fractures appeared in the plates. This of course allowed
shrinkage in the long axes of the plates and would make it impossible
for the sections to swell in thickness to the same proportion as the
coherent agar plates. These new plates came down to a thickness of
about 0.16 mm. and showed swellings of 220 per cent in distilled water
at 16° C. and of a slightly greater expansion in a solution of 0.05 M
asparagin, in which the swelling was 281 per cent. It is to be noted
that while the aspartic acid is present in a more concentrated condition
in these plates than is possible in water, yet the entire amount was
held in the colloidal mesh or plate and showed no formation of crystals
on the surface or in the sections, as in the case of the less-soluble tyrosin.
The hydration of the colloid with the acid incorporated in it is less
than that which may take place when the acid is dissolved to saturation
in the water in which the swellings are made. The influence of this
acid on agar was not widely different from that of succinic acid, but it
caused greater swelling in equimolecular concentrations hi gelatine,
agar-gelatine, and agar-protein.

The amine of this group was now tested both in solution and in-
corporated in agar sections. Plates of agar (9 parts) and asparagine
(1 part) were prepared and swelled in comparison with aspartic acid,
giving results shown in table 47.

Effect of Certain Organic Acids and Amino Compounds.
TABLE 47.— 16° to 18° C.

59

Citric

Sodium

Water.

acid,

hydroxid,

0.01 N.

0.01 M.

p. ct.

p. ct.

p. ct.

Agar 9, asparagine 1

640

300

402

Agar 9, aspartic acid 1 ....

296

250

625

The proportion of the acid and the asparagine being too high
to be of any physiological interest, new plates with half the
quantity of acid and amine were prepared, and these came down
to a thickness of 0.32 and 0.33 mm., which swelled 1,875 per cent
in distilled water as compared with agar, which showed a hydration
capacity of 2,700 per cent. The effect in this trial was not so
marked as in the first series, but it is evident that the incorpora-
tion of the asparagine in any proportion in the colloid affects
hydration to a greater extent than the perfusion of the asparagin
in the same concentration, which in this case gave swellings of 2,300
per cent. Even a 0.1 M solution with double the amount present in
the solution did not reduce the hydration to the limits shown by the
agar-asparagine plate used in this test. The asparagine was present in
such amount that if diffused out of the sections it would have made a
0.04 M solution in the 30 c.c. of water in the dish.

Asparagine was now applied in a series of concentrations to sections
of agar of the above swelling capacity in water and it was found that
hydration was actually increased or accelerated by the presence of the
amine. That this result did not simply appear by faulty comparisons
was shown by the following replacement test:

A trio of sections which had been swelled in distilled water to a total
of 2,630 per cent, and which had stood in the solution without any
perceptible change for a few hours after the close of the test, was now
treated with a 0.01 M asparagine solution. The mechanical disturbance
which might result from changing the liquid in the dishes was mini-
mized by fractionization. About one-third of the water was removed,
the level was raised by the addition of asparagine solution, and this
was repeated about a half-dozen tunes, the final result being a solution
which was diluted slightly below the hundredth normal. A slow
expansion began at once, which continued for about 20 hours, which
raised the total hydration of these sections to 2,890 per cent, an in-
crease of 230 per cent, due to the action of the asparagine on sections
which had undoubtedly been reduced in mass somewhat by solution
from the surfaces.

When asparagine is applied to mixtures in which the gelatine is
replaced by an albumin, the results included some special reactions.
Plates of agar and oat-protein were made up to contain 8 parts of the

60 Hydration and Growth.

first and 2 of the last, coming down to a thickness of 0.22 to 0.23 mm.
These swelled at 17° C. to the proportions shown in table 45, which in
some cases exceeded that in water. The swelling in concentrations as
high as 0.01 M were but little below that in water.

Glycocoll has been used in many cultural tests with plants and
various interpretations have been placed on its accelerative influence
on growth. The experiments with this material, therefore, included
the possibilities of the manner and extent to which this might accom-
pany or run parallel with hydration reactions.

The first trials were made with this reagent incorporated with
liquid agar in such proportion that the amount present in three sec-
tions would have been equivalent to that in 30 c.c. of 0.14 M solution.
Trios of such sections 0.15 mm. thick gave swellings of 1,133, 1,267
and 1,300 per cent hi water at 16° C., which is much less than that
shown in a solution at 0.3 M containing twice as much of the amino-
acid. (Table 45).

Thin sections of agar swelled in all glycocoll solutions less concen-
trated than 0.3 M to the amplitude attained in water and exceeded it in
some cases, a fact which for the first time gives a sound basis for cul-
tural tests in which growth was accelerated and the total increased by
this compound.

Another pentosan, gum tragacanth, was dried from solutions to form
sections 0.13 mm. thick on filter-paper. Swellings at 15° C. were ob-
tained, as shown in table 48.

TABLE 48. p. ct.

Distilled water 1 ,380

Glycocoll, 0.03M 1,382

Glycocoll, 0.05M •< 1,077

Glycocoll, 0.01 M 1,462

This gum liquefies irregularly, and hence the figures show the extent
of swelling before active dispersion of the mass begins.

A mixture of 9 parts gelatine and 1 part gum tragacanth was made
up at 25 per cent to correspond to a similar mixture of gelatine and
opuntia mucilage. Swellings as follows at 15°C. were obtained:

TABLE 49. p. ct.

Distilled water 1,320

Glycocoll, 3 M 1,520

Glycocoll, 0.05M 1,040

Glycocoll, 0.01 M 1,320

Nothing may be concluded on the basis of these figures, except that
the hydration of this material reaches a stage where it goes into dis-
persion unevenly and in a manner which makes auxographic readings,
as well as all mass or weight determination, of doubtful value.

The above tests were repeated with opuntia mucilage at 15° C.,
with results as shown in table 50.

Effect of Certain Organic Adds and Amino Compounds. 61

TABLE 50. p. d.

Distilled water 923

Glycocoll 3M 800

Glycocoll 0.05 M 664

Glycocoll 0.01 M 600

Here again the uneven dispersion of the mucilage results in auxo-
graphic records, the obvious meaning of which would be unsafe to
follow. It is highly probable that the high relative swelling in the
concentrated solution is due to coagulatory or aggregation effects,
especially on the surfaces of the sections, resulting in a sac-like con-
dition which would show considerable increase before dispersion
began, resulting in a final shrinkage. This dispersion began earlier
in the weaker solutions.

Swellings of gelatine in glycocoll ran uniformly low, the presence of
this substance apparently accelerating solution of the gel.

Sections consisting of 4 parts agar and 1 of gelatine which had an
average thickness of 0.3 mm. swelled as follows at 15° C. in glycocoll:

TABLE 51. p. a.

Glycocoll, 0.3M 1,550

Glycocoll, 0.05M 1,233

Glycocoll, 0.01 M 1,960

Glycocoll, 0.002 M 1,767

The average swelling of such sections in water was about 1,700 per
cent and the irregularity characteristic of auxographic measurements
of the action of this amino-acid is seen in the above results.

A preparation was now made in which 2 parts of the water-soluble
protein from oats was added to 8 parts of agar in a 2.5 per cent solution
of the latter. The plates dried to a thickness of 0.25 mm. When
sections of such biocolloids were swelled in the glycocoll series, the
results were as shown in table 45, the hydration in concentrations less
than 0.01 M approaching and surpassing those in distilled water.

A number of tests were made to determine the influence of glycocoll
on hydrations in acetic acid. The first was that of surface slices of
Opuntia, which had dried to a thickness of 0.8 mm. Trios swelled 163
per cent in 0.05 N acetic acid and 156 per cent in a 0.05 N solution
of acetic acid and glycocoll each. No especial significance can be
attached to the lesser swelling in the double solution, except that no
evidence as to acceleration of swelling by the addition of the amino-acid
was obtained.

Next, trios of sections of 8 parts agar and 2 parts gelatine 0.3 mm. in
thickness were swelled hi the acetic and amino-acetic solutions 0.01 N
at 18° C. The swelling in the acetic acid alone was 1,450 per cent,
while that in the combined solutions was but 1,300 per cent, which
agreed with the previous effects in being less than in the acid alone.
It is to be noted that the amount of the acetic acid in the combined
solution in the swelling-dish would be but half that when this acid was
used alone.

62 Hydration and Growth.

Trios of sections of agar swelled 1,875 per cent in a 0.01 N solution of
acetic acid at 18° C., while a combined solution of equivalent molecular
concentration showed a swelling of 1,750 per cent.

There now remained the test with living tissues. Some joints of
Opuntia blakeana of 1918, which had been brought from Tucson two
months earlier and had laid on the table, with the result that they had
lost much water but were still alive, were used for this test. A trio of
sections with an average thickness of 6 mm. swelled 60 per cent in the
hundredth-normal acetic acid, while a similar trio which measured
5.5 mm. on the average swelled but 45.5 per cent in the combined
acetic-glycocoll solution. A second feature distinguished the two
reactions, the swelling in the acetic acid being continuous and ap-
proaching zero during the 20 hours of measurement, while in the com-
bined solution full expansion was reached in 4 hours, after which a
shrinkage resulted in a loss of nearly 5 per cent, suggesting that the
H-ion concentration of the combined solution was greater than that
of the acid alone.

A return was made to the biocolloidal mixtures and trios of sections
of agar 8 parts and oat protein 2 parts, with a thickness of 0.22 mm.,
swelled at 18° C. The hydration in the hundredth-normal acetic acid
gave an increase of 1,318 per cent, while an equimolecular solution of
the acetic acid and glycocoll gave a swelling of 1,605 per cent. This
test is the only one of the series in which the addition of glycocoll to
the acetic acid enhances imbibition. In this last test the amount of
solution poured in each dish was such that the same quantity of the
acetic acid was present in both.

An additional test was made in which equal amounts of glycocoll and
acetic acid were brought together at a concentration of 0.001 M each
on agar-oat protein sections as above. The swelling in the acetic
acid was 2,681 per cent, or about the same of that possible in distilled
water (2,630 per cent), while the swelling in the combined solution was
slightly less, being 2,570 per cent.

Glycocoll and other amino-groups are present in the plant in com-
paratively great dilutions, and probably at no tune does the amount
present reach the concentration in which a retardation or limiting of
the hydration effect would be exerted. The experiments described
show that glycocoll and asparagin may actually increase the hydration
capacity of pentosan and of pentosan-protein colloids. The meager
results obtained from swelling plant-sections are not harmonious and
further experimentation is highly desirable. The accelerating effect
of glycocoll is a subject which has come up for notice many times.
Dakin connected its action with possible catalytic effects.1 The simi-
larity of the results obtained from agar and agar-protein mixtures

1 Dakin, H. D. The catalytic action of amino-acids, etc., in effecting certain syntheses. Jour.
Biol. Chem., 7: 49-55. 1909.

Effect of Certain Organic Adds and Amino Compounds. 63

and from the swelling of plants makes it fairly certain that the effect
is due primarily to the action of the pentosaus.

The most recent tests of the effects of glycocoll on plants are those
of Borowikow,1 completed in 1913 and published in the same year, and
those of Dachnowski, brought out in 1914.2 Borowikow took the
position that substances which facilitate hydration of the plasmatic
colloids accelerate growth, and that such hydration was one of pro-
teins, an assumption which is not sound. His trials consisted in com-
paring the growth of seedlings of Helianthus in distilled water as a
check or control with the substances to be tested added to water, the
measurements being taken during a few hours only. Glycocoll was
used in 0.01 N and 0.005 N concentration. Such concentrations are
relatively high for the plant, and only retardation effects were obtained.

Dachnowski's figures indicate that glycocoll added to hydrochloric
acid in concentrations of N/1,600 (50 c.c. N/800 of each substance)
causes an increase in the amount of water absorbed by bean seeds,
and a lesser increase of hydration in corn seeds.

Both absorption and transpiration by cuttings of tomato were less
in solutions of hydrochloric acid ranging from N/800 to N/6,400 than
in water, but this retarding effect was counteracted to some extent
when glycocoll was added to the solutions. This amino-acid also
caused an increased gain in weight in acid and alkaline solutions.

The hydration phenomena described in the preceding pages afford
some interesting parallelisms with the action of these compounds on
growth, absorption, and transpiration.

It is evident that we must definitely and finally cease to treat a plant
cell-mass as an amphoteric colloid with a dissociation expressed by the
actual acidity of the cell-sap. Such dissociation and resultant hy-
dration capacity may determine the action of protoplasts or of cell-
organs which are chiefly proteinaceous.

Vegetative cell-masses such as are responsible for growth, and the
activity of which constitutes growth in the larger sense, are composed
of colloids predominantly of a carbohydrate character. These pen-
tosans do not dissociate. Their swelling capacity in electrolytes is
less than in pure water. The hydration of agar and the pentosans in
acids is retarded or lessened by the action of H ions, so directly that
the proportionate swelling of agar in an acid such as acetic or succinic
might be used as a measure of the concentration of the acid solution
(see p. 57). This fact and the part played by the dissociation of
gelatine may be traced through all of the results on hydration of agar,
agar-gelatine, and agar-protein mixtures. Thus, for example, agar

1 Borowikow, G. A. Ueber die Ursachen des Wachstums der Pflanzen. Biochem. Zeitschrift,
50: 119. 1913.

2 Dachnowski, A. The effects of acid and alkaline solutions upon the water-relations and the
metabolism of plants. Amer. Jour, of Bot., 1: 412-439. 1914; also, Dachnowski and Gormley.
The physiological water requirement and the growth of plants in glycocoll solutions. Amer.
Jour, of Bot., 1 : 174-185. 1914.

64 Hydration and Growth.

alone gives an average swelling of about 2,600 per cent of plates 0.18
to 0.20 mm. in thickness at 13° C. When combined with gelatine in
proportions of 8 to 2, the swelling is less than 1,700 per cent.

The reactions of the pentosans and pentosan-protein colloids in
solutions of the ammo-compounds show some highly important depart-
ures, the chief of which is the fact that the hydration capacity is
greater than in distilled water in such monobasic acids, but not in the
dibasic aspartic acid. This last-named substance dissociates, so that
.01 M, pH = 3, in accordance with which it is found to lessen the
hydration capacity of agar, but, on the other hand, this action is not
shown by the pentosan-protein mixture. No explanation may be
offered for this behavior and for the excessive swelling of pentosans
and pentosan mixtures in amino- compounds, except that amino-com-
pounds may form salts with the carbohydrate, thus increasing the
hydration capacity of the latter. That this superior swelling is an
actuality is well demonstrated by the increase that resulted when agar-
albumin sections in a condition approaching complete hydration showed
a further marked increase when the water was replaced with an
asparagin solution. The positive action of the ammo-compounds is
also well demonstrated by the fact that the maximum effects were
produced at a concentration not coincident with the maximum con-
centration and at a point of great dilution.1

When these results are applied to the conditions in the cell, emphasis
is given to the fact that the total of amino-acids is always no more than
a fraction of the amount of organic acids present. It is highly probable
that these substances, originating constructively in the plant and
affecting growth in a profound manner, may do so partly by their
participation in the buffer processes.

1 MacDougal and Spoehr. The effect of organic acids and their ammo-compounds on the
hydration of agar and on a biocolloid. Proc. Soc. Exper. Biol. and Med., 16: 33. 1918.

VI. REACTIONS OF BIOCOLLOIDS AND CELL-MASSES TO

CULTURE SOLUTIONS, BOG, SWAMP, AND GROUND

WATER, AND OTHER SOLUTIONS.

The organism encounters a variety of substances in solution in the
substratum or medium to which, of course, the colloids of the cell re-
act in a manner determined by their own composition and that of the
impinging substances. The securest knowledge of the complex rela-
tions involved will in the end rest upon results obtained by analytical
experiments in which the effects of separate substances and graded
concentrations of the elements are first determined and then their
action in combination is measured. Meanwhile, a number of standard
or commonly accepted solutions are used for a variety of cultural and
experimental purposes and an effort was made to ascertain the reactions
of biocolloids and of sections of plants to them in terms of imbibitional
swelling. The idea was extended to include the "natural waters"
which are characteristic of some well-defined plant habitats, such as
bogs and swamps.

A large and important share of the knowledge of the physiology of
plants rests upon cultures made with " nutrient solutions." One of
these, after a formula devised by W. E. Tottingham, was chosen for
the test.1 Its composition was : potassium nitrate 4.048 g., dipotassic
phosphate 12.980 g., magnesium sulphate crystals 29.280 g., and cal-
cium nitrate 27.920 g., in 4,000 c.c. of water. A precipitate comes
down in the bottle on standing. This was filtered out and dissolved
in distilled water, which was used to dilute the solution to a concen-
tration of about 0.5 per cent total concentration.

The preliminary trial of the effect of the whole solution was made
with sections of a plate consisting of 95 parts agar and 5 parts of bean
protein, an old preparation which had been exposed to the damp air
for a month. The swelling measurements were as follows:

TABLE 52.

p. ct.

Water 617.6

Nutrient solution, 0.5 p. ct 500

Citric acid, O.Ql N 406.8

Sodium hydroxid, 0.01 M 431.4

The only feature of interest hi the results was the low imbibition in
water, the dried sheet being an old one. A fresh preparation was
made with the agar and bean protein hi the same proportion as before,
and a double series of instruments was used in the test.

In order to determine the possible interference or antagonism of the
constituents, tests were also made of the separate action of the four

1 Tottingham, W. E. A quantitative chemical and physiological study of nutrient solutions for
plant cultures. Physiol. Res., 1 : No. 4. May 1914.

65

66

Hydration and Growth.

salts in the concentrations in which they occur in the culture solution
(table 53).

The total amount of swelling in the culture solution is scarcely
more than half that in distilled water, that in the dipotassic phosphate
not falling much below that of water. Swelling in potassium nitrate
is much greater than that shown in the culture solution. The low
imbibition in calcium nitrate is in accordance with the expectancies.

TABLE 53.

Dist.
water.

Nutrient
soln.,
0.5 per ct.

Potass,
nitrate,
M 0.00285.

Magn.
sulphate,
M 0.00898.

Calc.
nitrate,
M 0.00844.

Di-potass.
phosphate,
M 0.00681.

Swelling in 10 hours

p. ct.
1,360
1,300

p. ct.
720
760

p. ct.
1,040
900

p. ct.
740
740

p. ct.
340
600

p. ct.
1,200
940

Average

1,330

750

970

740

470

1,070

Swelling in 24 hours ....

1,560

800

1,260

804
800

680
500

1,400
1,300

Average

1,560

800

1,260

802

590

1,350

The magnesium salt exercises an imbibitional action equivalent to
that of the complete solution. The potassium salts allow a notably
greater swelling. Apparently the calcium salt interferes, or exercises
an antagonism which results in the averaged total exemplified. The
actual relative action of these salts, however, can not be taken up at
this time. A consideration of the imbibitional action of the constit-
uent salts might yield some data which would be of value in deter-
mining the composition of culture solutions for special purposes.

A similar series of tests of the

effect of the nutrient solution TABLE 54.

and its components upon grow-
ing tissues were made with
sections of young stems of
Rudbeckia bearing young flower-
heads. Tangential slices were
removed from one side to allow
expansion and trios of pieces a
centimeter long and 3.5 mm. in
thickness were placed under the
auxographs in a dark room at

16° C. Air-dried sections of the same stems which had been exposed
to the air and light for a day and had shrunk to about half of their
original thickness were now placed in identical solutions. The swell-
ings of the fresh and of the dried sections are given in table 54.

Living
sections.

Dried
sections.

Water

p. ct.
5.8

p. ct.
28 6

Nutrient solution

4

15 7

Potassium nitrate

4.3

27.1

Magn. sulphate

2.8

20

Calcium nitrate

2

3.6

Di-potassium phosphate . .

2.9

12.8

Certain Reactions of Biocolloids and Cell-masses. 67

Several variations are apparent, but perhaps the relations of greatest
importance are those which may be expressed by saying that the
imbibition of dried specimens is five times that of living material in
distilled water, only four times in nutrient solution, over six times in
potassium nitrate, over seven times in magnesium sulphate, less than
twice in calcium nitrate, and over four times in dipotassium phos-
phate. It is to be noted that when a section of living tissue is dehy-
drated it is not possible to restore the cell-colloids to their original
condition simply by swelling. This is due chiefly to the fact that, as
desiccation proceeds, the salts, acids, sugars, etc., in the liquids are
concentrated until finally they are fixed by the solidifying proto-
plasmic gel in this condition. Rehydration must then take place as
in a salted colloid with the sugars in a concentration in which they
may modify imbibition.

Petioles of young leaves of Phytolacca, with a thickness of 3 mm.,
swelled 4.2 per cent in distilled water and an equal amount in potassium
phosphate, 5 per cent in magnesium sulphate, 3.3 per cent in calcium
nitrate, and -2.5 per cent in the nutrient solution. The equivalence
or uniformity of the material was in doubt, however, and the test was
not extended to dried sections.

The 4-angled stems of Mentha spicata offered certain mechanical
advantages, and the internodes near the apex of the stem which were
half-grown and with a thickness of 3 mm. were selected. Trios of
sections 3 or 4 mm. long were tested with distilled water, culture
solution, and its components. The swelling of fresh specimens was
very slight, varying from 0.05 to 0.1 mm., and no safe comparisons
could be made. Dried sections came down nearly half of their original
dimensions, and when a series was swelled in distilled water the in-
crease was but 12.9 per cent and in hundredth-normal citric acid 6.4
per cent, while in hundredth-molar sodium hydroxid the increase was
19.3 per cent. These results indicate that the plant colloids were in
an acidified condition, the swelling in water and hi acid being conse-
quently small, while that in alkali was a swelling in a neutralized or
nearly neutralized condition. The dried series in the culture solution
and its components gave swellings of 16.8 per cent in distilled water,
almost no swellings in nutrient solution (due to defective wetting
by the liquid), 19.3 per cent in potassium nitrate, 12.6 per cent in
magnesium sulphate, 9.6 per cent in calcium nitrate, and 4.8 per cent
in dipotassic phosphate. The swelling of dried sections presents some
possible sources of error in the limited surfaces presented for absorption
which may be "'waterproofed" in some cases, while in other instances
the sections collapse or do not swell toward their original form.

Direct effects of the waters of bogs and swamps in producing modi-
fications of growth, departures in structure and form, and in influenc-
ing general nutrition are well established and have long been known.

68 Hydration and Growth.

The numerous analyses of the water have failed to disclose physico-
chemical features which might be held responsible for the very direct
and positive action exercised in the determination of the plant form-
ations of such places. The history of such attempts is a long one.1
Bog water was furnished by Mr. E. R. Long, who procured a sample
from Ronalds, in the region of Seattle, Washington. Dr. J. M. McGee
reports the following constituents per liter:

TABLE 55. gm.

Organic matter 0 . 106

Ash (chiefly CaSO«) 048

Total soluble residue 154

Swamp water was procured by the kindness of Dr. S. A. Gortner, who
obtained a sample from near Anoka, Minnesota, concerning which he
says:

"This sample was taken about 20 miles north of Minneapolis, in Anoka
County, of which three-fourths of the area consists of peat lands. These
peat lands are of the grass and sedge formation, the peat being from 4 to 6
inches or more deep, fairly well decomposed, and one of the better grades of
peat for agricultural purposes in that it contains an appreciable amount of
lime. I believe that you will find this sample of water perfectly typical of
most of the large grass bogs of Minnesota."

The analysis of this water shows the following per liter:

TABLE 56. gm.

Soluble organic matter 0.094

Ash (CaSO4 with trace of NaCl) 128

Total soluble residue 222

The degree of acidity of the bog water was such that 1.1 c.c. of N/10
potassium hydrate was necessary to neutralize 100 c.c. of water. The
acidity of the grass-sedge water was scarcely a third of this, but 0.35
c.c. N/10 potassium hydrate being necessary to neutralize 100 c.c.

The first trial of the action of these waters and comparative solutions
was made with living material. Circular disks 12 mm. across and of
an average thickness of 11 to 13 mm. were cut from joints of Opuntia
discata which had matured at Carmel, California, in the summer of
1917. Tests were made with water, swamp water, bog water, and
various calcium solutions at 15° C. The measurements obtained were

as follows:

TABLE 57. p. ct.

Distilled water 18.2

Swamp water 13.6

Bog water 18.3

Calcium nitrate, 0.008M 15.5

Calcium nitrate, 0.008 M acidified with, nitric acid 16.8

Calcium nitrate, 2 M (shrinkage and subsequent swelling) ' 6.8

Calcium nitrate, 0.2 M (steady shrinkage)

Calcium nitrate, 0.02M 14.6

Calcium nitrate, 0.002M 15.5

Calcium nitrate, 0.0002 M 21

1 See Bigg, G. B., Summary of bog theories. Plant World, 19:310, 1916.

Certain Reactions of Biocolloids and Cell-masses. 69

The uppermost line shows a swelling in bog water equivalent to
that in distilled water, while the imbibition in swamp water is very
much less, sustaining about the same proportions as the measurements
of the swelling of biocolloids. The retarding action of the swamp
water, high in calcium, is greater than that of the solution 0.008 M,
in which this salt enters into nutrient solutions, and greater even than
that of a 0.02 M solution. The retarding action of swamp water may
be predicted to be about the same as a 0.03 M solution of calcium nitrate
acidified as in the solution used. Such acidification was made by adding

1 c.c. of hundredth-molar nitric acid to 25 c.c. of the calcium solution.
The final cessation of shrinkage and slight enlargement of sections

in the 2 M solution remains unexplained, since the shrinkage in a
solution containing but one-tenth of this amount of calcium was con-
stant during the entire 90 hours of the exposure. The final figures in

2 M to 0.0002 M are of swellings which were continued for 90 hours,
while the swellings in water, swamp water, bog water, calcium as in a
nutrient solution, and acidified calcium solution were taken at the close
of 40 hours. The total swelling of 21 per cent in calcium nitrate
0.0002 M is probably equivalent to that of distilled water. The swell-
ings of biocolloids in the same solutions should receive attention in this
connection.1

In addition to the above note on the swelling of the sections in the
2 M solution of calcium nitrate, the f ollowing facts are of interest :
Four sections with an aggregate thickness of 37 mm. were placed in a
dish and covered with such a solution at the time the auxograph
measurements were started. As the sections under the auxograph
were in an expanding stage when the measurements were closed, free
sections were allowed to remain in the dish after the records on the
instrument were ended. The free sections were measured 6 days after
being put in the concentrated solution, with the result that their total
thickness was found to be 42 mm., a gain of 5 mm. or 13.3 per cent.

Sections of a biocolloid consisting of agar 90 parts and oat protein
10 parts were swelled in a series of solutions of calcium nitrate parallel
to the above set. The increase in the 0.5 M solution was 975 per cent,
525 per cent in the 0.2 M solution, 650 per cent in the 0.02 M solution,
1,425 per cent in the 0.002 M solution, and 1,975 per cent in the
0.0002 M solution. The test was repeated with the following results
at the end of 24 hours: swelling in 2 M solution, 917 per cent; in 0.2 M
solution, 722 per cent; in 0.02 M solution, 777 per cent; in 0.002 M
solution, 1,555 per cent.

The minimum swelling in this series evidently lies between the con-
centrations of 0.2 M and a molar solution.

Another series was carried out in which sections of Opuntia were
swelled at temperatures of 18° to 20° C. in acidified and salt solutions,
as given in table 58.

1 MacDougal, D. T. The effect of bog and swamp waters on swelling in plants and in biocol-
loids. Plant World, 21:88. 1918.

70 . Hydration and Growth.

TABLE 58.

p. ct.

Swamp water 14

Swamp water, citric acid, 0.01 N 15

Potassium nitrate, 0.01 M 14

Citric acid, 0.01 N '.'.'.'.'. 7

Potassium nitrate, citric acid, 0.01 N 9

Potassium hydroxid, 0.01 M 12

The measurements in swamp water alone and with acid include the
full increase in 96 hours, while the others extended over from 20 to
40 hours.

No important effect can be ascribed to the acidification of swamp
water. The swelling of the sections in the hundredth-molar solution
of potassium nitrate was but little below that in the swamp water, but
when this solution was similarly and equally acidified, a decrease
ensued. The foregoing tests were made with sections in a living con-
dition, in which questions of permeability and osmotic action might
possibly play a part. Material was prepared to exclude the action of
the living cell. The chlorophyllous layers were removed from the two
sides of joints of Opuntia and slices 7 mm. in thickness were cut in the
plane of the joint and placed between two sheets of filter-paper, to
one of which they adhered. A third sheet was laid over them and
the preparation placed on a wire netting to dry without pressure. In
6 days the thickness had been reduced to about 0.5 mm. and enough
moisture still remained to give the slices a leathery consistency. Suit-
able sections free from visible fibrovascular tissue were prepared which

gave swellings as follows:

TABLE 59.

Living sections. Dried sections,

p. ct. p. ct.

Distilled water 660 47

Bog water 640 45

Swamp water 530 38

Culture solution, 0.5 per cent 625 44

The measurements were taken at the end of 24 hours, when a fair
rate of increase was still noticeable which would in the end have carried
the figures up to the next hundred in the dried sections. The swelling
of living material in bog water is but little less than in distilled water
and is also but little different from that in the culture solution, which
is of the concentration used in water cultures. Hydration is, however,
noticeably less in swamp water.

Attention was now turned to the biocolloids to ascertain whether
the action of plant material living and dried would find a parallel in
the action of mixtures of known composition. Sections of plates
composed of agar (90) and oat protein (10) were found to show the
following swellings at 15°C.

TABLE 60.

p. ct.

Distilled water 2, 188

Bog water 2,083

Swamp water 1,200

Certain Reactions of Biocolloids and Cell-masses. 71

The decreased swelling in swamp water and the high swelling in bog
water were marked and invariably shown.

However, the biocolloid approaches more nearly to the condition of
the protoplast when, in common with all living matter, it includes some
salts. The above mixture containing culture salts was not avail-
able, but some plates in which the oat protein was replaced by bean
protein to which had been added 0.8 per cent of culture salts were
swelled in a parallel series. The untreated mixture free from the
added salts does not show as high an imbibition capacity as that made
up with the oat protein. The measurements of the increase of the
agar-bean protein-salted sections at 15° C. were as shown herewith:

TABLE 61.

p. ct.

Distilled water 1 , 525

Bog water 1 , 525

Swamp water 1 , 100

Calcium nitrate, 0.008 M 825

These measurements were taken at the end of 40 hours, while some
increase was still in progress, but the final relations would not have
been materially altered by the use of the end-points for the compari-
sons. The retarding action of the swamp water and the equivalence
of swelling in pure water and bog water therefore runs plainly defined
through all of the experiments with living and dried sections of plants
and in tests with salted and unsalted biocolloids. Calcium nitrate in
the concentration used exercises a more marked effect on the salted
biocolloid than on the unsalted mixture and on the plant material.
The calcium content of the biocolloid is probably much higher than
that of the plant, so that the two sets of measurements are not strictly
comparable.

The calcium solution contained about eight times as much salt as
the bog water and nearly three times as much as the swamp water,
which also includes a trace of sodium chloride. A series was therefore
arranged to compare the action of the two with equivalent salt solu-
tions. Sections of agar and bean protein impregnated with 0.8 per
cent of culture salts were swelled at 15° C. with results as follows:

TABLE 62.

p. ct.

Distilled water 1,417

Bog water 1 , 444

Calcium sulphate 0.048 gram per liter 1,417

Swamp water 944

Calcium sulphate 0.128 gram per liter 1 , 083

Bog water and an equivalent calcium solution allow equal swelling,
but the increase in swamp water is much less and also less than in the
equivalent calcium solution, to which may be attributed most of the
retarding effect of the swamp water.

72 Hydration and Growth.

The imbibition capacity of the biocolloids varies with the propor-
tions between the carbohydrate and the proteins and protein deriva-
tives. A biocolloid was subsequently made up which included a high
nitrogen-content and a second carbohydrate and five albuminous com-
pounds. For this purpose, 70 parts agar, 5 parts each of dextrose,
peptone, gelatine, asparagin, nucleinic acid, and bean protein were
suitably liquefied and poured into plates which dried down to a
thickness of 0.2 mm. Swellings of sections of these plates at 15° C.
gave the following increases at the end of 24 hours :

TABLE 63. p. ct.

Distilled water 725

Bog water 592

Swamp water 550

Calcium chloride, 0.2 M 350

Calcium chloride, 0.1 M 400

The total swelling in distilled water for this biocolloid is low, al-
though it is to be noted that swellings as high as 1,200 per cent in dis-
tilled water have been measured at temperatures of 18° to 20° C.

Sections from plates made up as above, but to which had been
added 0.8 per cent of culture salts, gave increases as follows at tem-
peratures of 15° C. :

TABLE 64. p. ct.

Distilled water. 600

Bog water 525

Swamp water 625

This complex biocolloid, high in nitrogen and in the culture salts,
displays hydration capacity in swamp water superior to that shown in
bog water or pure water. The properties in question would enable a
plant so equipped to thrive in the waters of swamps, and it would be
interesting to determine whether such a condition actually prevails
in the plants of the sedgy swamps.1

The earlier attempts to interpret the swelling action of protoplasm
were founded on the assumption that such increase might be repre-
sented by the action of gelatine. The unsoundness of this assumption
and the inadequacy of the methods using this material have been
amply demonstrated by results previously published. At one end of
the scale stand some plants and some plant structures high in protein-
aceous compounds and low in pentosans, and these do show a behavior
approximating that of gelatine. This is illustrated by the following
series, in which sections of gelatine plates 0.18 mm. in thickness were
swelled at 15° C., giving measurements as follows:

TABLE 65. p. ct.

Distilled water 778

Bog water 889

Swamp water 939

Culture solution, 0.5 per cent 889

Potassium nitrate, 0.01 M 911

1 Schimper, A. F. W. Die Indo-Malayscher Strandflora, p. 142. 1891.

Certain Reactions of Biocolloids and Cell-masses. 73

The swelling of sections of agar plates 0.2 mm. in thickness at 15° C.
resulted in increases of:

TABLE 66.

p. ct.

Distilled water 700

Bog water 650

Swamp water 425

Nutrient solution, 0.5 per cent 375

It is to be seen that all of the solutions decrease the swelling capacity
of the agar below that displayed in distilled water, and that the greater
reduction in the nutrient solution is to be attributed to the higher salt-
content.

Plants of bogs and especially swamps are undoubtedly subjected to
great variations in the composition of the water by reason of inunda-
tions and floods. It was thought pertinent to extend experiments
in which alternations of solutions were made in such manner
as to test the effect of previous history on the behavior of a bio-
colloid. Sections of agar-oat protein 0.18 mm. in thickness swelled
972 per cent in 12 hours at 17° to 19° C. and reached a total of 1,233
per cent at the end-point in 108 hours, which are equivalent to results
previously attained and hence afford a fair basis of comparison with
the following, in which a trio of sections swelled 2,361 per cent in dis-
tilled water at the end-point in 72 hours. Replacement of distilled
water with swamp water was followed by a slow shrinkage, but this
amounted to only 36 per cent of the original volume. No swelling
agent yet tested has been found to reverse the action of another solu-
tion so fully as to bring the dimensions of the sections down to the
dimensions which might be attained in the second agent alone.

Sections of a biocolloid consisting of 90 parts agar and 10 parts of
bean protein to which 0.8 per cent of nutrient salts had been added,
0.18 mm. in thickness, were now swelled in swamp water at 18° to
20° C. The increase was measured at the end of 16 hours, at which
time the total swelling was 1,082 per cent. The swamp water was
now replaced with hundredth-normal citric acid-potassium nitrate for
36 hours, during which time no appreciable change was registered.
Replacement of this solution with swamp water was followed by a
resumption of the swelling, which carried the thickness of the sections
to 1,388 per cent of the original, which is greater than that attained
in the simple continuous swelling in swamp water.

Swamp water is high in salts, and it is probably this feature to which
its influence on swelling is due. A test parallel to the above was made
in which the sections were first swelled in a 0.5 per cent nutrient solu-
tion in which the salts are somewhat more concentrated than in the.
swamp water. A swelling of 888 per cent took place in 17 hours, at
which time the pen of the auxograph was tracing a horizontal line.
Replacement of the nutrient solution with the acidified potassium-

74 Hydration and Growth.

nitrate solution was followed by a very slight swelling. When this
solution was replaced by swamp water an increase of 333 per cent
followed in 40 hours. The two tests were parallel, except that the
initial treatment in one case was with nutrient solution in which the
salts were more concentrated than in the swamp water used in the
other. The final swelling in the second case is greater and may be
attributed to the initial salt action.

In a partial repetition of the above test, the sections placed in culture
solution swelled 861 per cent in 20 hours. Lengthening the immersion
in acidified potassium nitrate to 55 hours was accompanied by a
swelling of 55 per cent. Replacement with distilled water set up a slow
increase which resulted in a gain of 111 per cent in thickness in 40
hours. The total increase was 1,027 per cent, while the one finished
in swamp water swelled 1,388 per cent.

The relative effects of swamp and bog water on biocolloids were
tested in still another way. Plates of agar-oat protein were prepared
in which strips of webbing of cotton were embedded in the soft material
as it cooled for the purpose of testing the influence of certain purely
mechanical features on swelling. Portions of the plates dried down to
a thickness of 0.18 mm. in the clear portion of the plate and sections
from this swelled 2,111 per cent in bog water and 1,195 per cent in
swamp water at 15° C. Sections containing webbing were 0.58 mm.
in thickness and the actual increase of such sections was 491 per cent
in bog water and distilled water and 371 per cent in swamp water.
If the increase be calculated on the assumption that the webbed sec-
tions included as much biocolloid as the free sections, the proportions
would be 1,583 per cent in bog water and distilled water and 1,195
per cent in swamp water.

Swamp water has been found to affect absorption and swelling in the
same manner as an equivalent solution of calcium sulphate. Swelling
and absorption is retarded by swamp water in salted biocolloids and
in sections of plants containing a large proportion of pentosans and a
low protein-content. Biocolloids with a high protein and salt content,
on the other hand, show an enhanced absorption in swamp water.
Inferentially, plants of similar constitution would carry on absorption
readily and thrive in swamp waters.. Whether adaptation to swamp
habitats actually takes this course is not known.

An extension of these measurements was made to ascertain the
effects of water and soil solutions which were in common use at the
Desert Laboratory upon a biocolloid, a mixture consisting of 6 parts
of agar, 2 parts of mucilage of Opuntia, 1 part of gelatine, and 1 part
of bean protein. This had been poured in the usual manner and dried
to a thickness of 0.2 mm. Sections of the usual size were placed in
dishes under the auxograph. Distilled water of the grade used in
making up all of the solutions caused a swelling of 1,750 per cent; ram

Certain Reactions of Biocolloids and Cell-masses. 75

water which had fallen on a slate roof and collected in a closed cement
cistern gave a swelling of 1,500 per cent. The water from the system
supplying the Desert Laboratory, taken from a well 40 feet in depth
in the alluvium of the flats along the Santa Cruz River and pumped
through an iron pipe line 6,000 feet long to a cement tank, produced a
swelling of only 800 per cent. As a final test, a soil solution was used
which was obtained by shaking up 600 grams of surface soil with 1,200
c.c. of distilled water and then allowed to stand for 12 hours. The
filtered solution applied to sections in the same manner as the other
waters induced a hydration of 900 per cent. (Fig. 10).

These measurements afford a standard of desirability of the water
from these various sources for cultural work and for drinking purposes.
Since growth consists in the main of the hydration of plasmatic col-
loids, the nutritive solution most favorable to this process would be an
important factor in an environmental optimum. It was also possible
to make tests of these natural waters with a biocolloid which included
6 parts agar, 2 parts prosopis gum, 1 part gelatine, and 1 part bean
protein, to which had been added 0.2 per cent of culture solution.
Such a mixture, like one containing gum arabic, shows high swelling
in acids and less in salts, whether acid or alkaline. Swelling of plates
0.17 mm. in thickness were as shown herewith:

TABLE 67.

p. ct.

Distilled water (25° C.) 1,760

Rain water (27° C.) 1,794

Well water (27° C.) 1,706

Soil water (25° C.) 1,500

The higher temperatures at which the swellings in rain water and
well water were made prevents direct comparison, but it may be sup-
posed that a biocolloid already charged with salts to a point above the
average of land plants would be hydrated in the dilute solution
offered by the cistern water practically as readily as from distilled
water. The figure given expresses the increase at a temperature 2° C.
higher. The same would be true of the well water as compared with
the soil solution. It is to be noted that the difference between the
reaction in the solutions and in pure water is less than in the unsalted
colloid. Of course, the substitution of prosopis gum for opuntia
mucilage is also a factor. Relations to environmental conditions of
some importance are suggested. The reactions of the halophytes
should include some effects similar, in that there would be offered
the phenomena of the swelling of cell-masses high in salts (fig. 10).

Some experiments in the modification of germ-plasm in 1905 resulted
in the formation of embryos developing, into individuals not entirely
identical with the parental types. The essential feature of the experi-
ment consisted in the successful introduction of various substances
into the neighborhood of the embryo-sacs at the time that fertilization

76

Hydration and Growth.

was imminent, and when the first trials were made I had two main
purposes in mind : first, to ascertain whether or not foreign substances
could be introduced into ovaries in such manner as to affect the ovules
with a minimum of traumatic effects, so that the ovaries might reach
maturity; and secondly, to ascertain whether or not such changes
could be produced in an early stage of sexual specialization before the
development of the embryo-sac or after the union of the sexual ele-
ments in fertilization. The value of the results was much lessened by
the fact that the direct effects of the reagents could not be identified.
After some difficulty the actual diffusion of the liquids in the ovaries
was ascertained by substituting dyes for the salt solutions, but there
still remains to be determined the nature of the action of the reagents
on the cell colloids.

12 p.m.

FIG. 10. — Tracing of auxographic record of swelling of plates consisting of 6 parts agar, 2 parts
mucilage of Opuntia, 1 of gelatine, and 1 of bean protein, in distilled water, A ; rain water, B;
well water, C; and a soil solution, D.

The first step hi such an examination would naturally be the measure-
ment of the hydration effect. Plates of agar 90 parts and bean pro-
tein 10 parts, 0.25 mm. thick, were swelled to ascertain the possible
imbibition effects. A series in the dark room at 15° to 16° C. gave the
following measurements of expansion :

TABLE 68.

p. ct.

DistUled water 1,220

Methylene blue 860

Iodine, sat. solution in distilled water 1 , 080

A third reagent used in the later series of ovarial treatments, zinc
sulphate, was tested in the concentration of 1 part in 10,000 (0.00034 M)
in comparison with distilled water and hydroxid on sections of agar-
bean albumin. The swellings were as below :

TABLE 69.

p. ct.

Distilled water 1,388

Zinc sulphate, 0.00034 M 833

Sodium hydroxid, 0.01 M 194

The characteristic high rate of swelling of agar-albumin is exhibited,
the swelling in the hydroxid being but one-seventh that in water. The

Certain Reactions of Biocolloids and Cell-masses. 77

zinc salt, although very dilute, retards swelling noticeably, and exerts
a greater effect on the imbibition capacity than does either of the
other reagents named.1

Whatever value may attach to this procedure, it seems reasonable
to assume that salts less toxic in effect than those of zinc and identical
with those already present in the embryo-sac offer the greatest promise,
and their most intense effect might be secured when acidified.

The presence of free amino-groups in the cell and their rapid penetra-
tion of plasmatic structures makes it highly probable that some of
these substances singly or in combination might diffuse throughout the
cytoplasmic structure of the embryo-sac and also reach the chromo-
somes with a high degree of possibility of affecting then* genetic con-
tent or potentiality.

1 MacDougal and Spoehr. The effect of organic acids and their amino-compounds on the hydra-
tion of agar and on a biocolloid. Proc. Soc. Exper. Biol. and Med., 16:33. 1918.

MacDougal, D. T. The experimental modification of germ-plasm. Annals Mo. Bot. Garden,
2:253-274, Feb.-Apr., 1915.

VII. FLUCTUATING OR ALTERNATING HYDRATION EFFECTS.
BASIS OF XEROPHILY AND SUCCULENCE.

The experiments described in the previous chapters have dealt
chiefly with sections of colloidal material artificially compounded to
represent the materials and conditions which affect hydration in plants.
Measurements of the swelling of dried sections of this material have
been used as a basis for comparison with the action of slices or sections
of plant cell-masses similarly dehydrated or in living condition.

No estimate of results of this kind will be valid and no perspective
of their bearing will be correct which does not take into account
the fact that the growing parts of the higher plants contain 90 per
cent or more water and that the colloids of the protoplast, the action
of which makes for the distension or enlargement of growth, are even
more highly hydrated. These gels also invariably contain the culture
salts, in combination or simply adsorbed, and are inevitably in a con-
dition of acidity resulting from their carbohydrate metabolism.

The elementary facts obtained by the experiments described in the
foregoing pages made it possible to plan a series of treatments of
hydrating material in which the previous experience of biocolloids
would be apprehended in swellings in salts, acids, etc., in a sequence of
interest to the physiologist, and to obtain additive, alternating, or
superposed effects. Variations in the carbohydrate and proteinaceous
substances of a colloidal mixture may not be readily produced to
simulate the changes which form the basis of some of the most funda-
mentally important phenomena of the cell. The worker must approach
this phase of the subject by studying the reactions of separate masses
or lots of material compounded to represent various stages in the
condition of the protoplasm. Something of this kind has been done
in the measurement of the reactions of biocolloids of several kinds. It
is of course impossible to imitate any of the important metabolic
processes which make cell-colloids continuously acting machines, al-
though no hint has been found of any source of energy or directive
action outside of surface tension and chemical action.

In the subjection of colloidal masses to the action of hydrating
solutions, as described in the following pages, it is obvious that the
soluble constituents of the sections would be partially and unequally
removed with every change in the solutions in which they were sub-
merged, and while the fact that the colloidal mass changed its com-
position continuously during the immersion in the various reagents,
yet it can not be said that these alterations were identical with those
of the growing cell.1 Some highly suggestive results or situations were
produced, however, by the replacements of hydrating solutions, as
described in following pages.

NOTE. — All measurements of swelling and shrinkage are given in terms of original or dried
thickness of sections. — AUTHOR.

1 MacDougal and Spoehr. The effects of acids and salts on biocolloids. Science, 46 : 269. 1917.
78

Fluctuating or Alternating Hydration Effects.

79

An experiment of this kind was carried out July 30 to August 9,
1917, in the equable-temperature chambers of the Coastal Laboratory
at 15° to 16° C., in which sections composed of 9 parts agar and 1 part
bean protein were subjected to alternating action of acids and hydro-
xid after they had first been swelled in water for 5 hours. Citric, malic,
and formic acids were used in separate sets at hundredth-normal con-
centration, but no determination was made of the hydrogen-ion con-
centration, and as the initial swellings in water were widely divergent,
the final totals have no especial significance, entire interest lying in the
changes in volume resulting from the replacements. (See fig. 11.)

I2pm. m. 12p.m. m. 12p.m. m. 12p.m. m. 12p.m. m. 12p.m. m. 12p.m.
~r 7 f r r 1 r ? 7 r T / '•

FIG. 11. — Record of variations in thickness of sections of agar and bean protein subjected to the
action of water, acids, and alkalies, as described in text pages 79 and 80.

Trios of sections which were 0.25 mm. in thickness were placed in
dishes into which distilled water was poured in the usual manner.
At the end of 5 hours, designated as A on the tracing, the water was
drawn off by a pipette and a solution of hundredth-normal sodium
hydroxid substituted, which was followed by an expansion which
reached almost to the possible total of 580 to 860 per cent of the
original dried sections in 8 hours. At the end of this time the sections
were in an advanced stage of hydration and were also probably
impregnated with salts formed by potassium with the carbohydrates
and proteins in the gel, and the possible presence of these compounds
as modified by subsequent experiences must be kept in mind.

The three acids were now added to the separate sets of sections,
and from this point their experiences diverge. All agreed in under-
going a retraction during the next 9 hours which was most pro-
nounced in the citric acid. Shrinkage at a slow rate was still in prog-
ress at the end of this period, B, when the acid solutions were removed
and distilled water substituted. No effort was made to wash the
sections which were saturated with the acid and salt, and the operation
resulted simply in a reduced acidity in which some slight swelling took
place during the 5 hours ending at C, at which time the reaction was
practically at an end.

The hydroxid was now used for the second time, replacing the acid,
for 2 hours, in which time a further slight swelling ensued. Acids

80 Hydration and Growth.

were again used at D and a slow shrinkage again set in which fol-
lowed on during the 18 hours until the change was made at E to
hydroxid, which, as before, simply neutralized the acid and increased
the salt-content, with the result that the volume of the colloid swelled
to the dimensions occupied before the last treatment with acid. At
the end of the 5 hours, F, the hydroxid was removed and water, re-
newed once, poured into the dishes. The effect was very marked,
as a rapid swelling ensued during the next 4 hours, at the end
of which time it was in progress at an undiminished rate, being greatest
in the formic and least in the citric acid. The sections now contained
a large proportion of water, sufficient to bring them into the condition
of hydration of active protoplasm, and the addition of the acids at G
arrested the swelling abruptly and caused a shrinkage which was di-
minishing at the end of 4 hours. The shrinkage was checked by replace-
ment with water at H and a slight swelling took place in the ensuing
11 hours.

The water now being replaced by hydroxid, a sudden slight ex-
pansion occurred in all of the sections, followed by a shrinkage which
had not ceased entirely at the end of 9 hours, during which period
additional salts were being formed in the colloidal structure. Now,
when the hydroxid was partially washed out by water at J, a swelling
ensued which in 15 hours brought the sections very nearly to their
final thickness and consequently near their maximum imbibition
capacity. It is in this condition, of course, that growing protoplasts
are normally active. The greatest expansion in this phase was in the
material treated previously with malic acid.

The addition of hydroxid (K) was now followed by a marked
shrinkage which had not ceased at the end of 8 hours. Replace-
ment of the hydroxid with acid at L caused a further abrupt
retraction which soon ceased, and after 5 hours the acid was re-
placed with water once renewed (M), only a slight swelling resulting
during the next 10 hours. Again hydroxid (N) caused a sudden
expansion to be followed by a slow shrinkage which had not reached
its end in 13 hours. Water now following hydroxid at 0, a much
greater expansion took place than when water followed acid. Alka-
losis at P now came after an hydroxid-water period and was not
followed by the abrupt enlargement consequent upon adding hy-
droxid to the colloid after an acid-water period. The shrinkage,
however, was as marked as in previous experiences, and had not ended
in 8 hours.

Replacement with water at S was followed by an expansion which
had not ceased at the end of 6 hours. Finally, acids caused a diminu-
tion the most marked of any produced by these substances. Both
acids and hydroxids caused the most marked changes ni the highly
hydrated and salted sections near the end of the week over which the

Fluctuating or Alternating Hydration Effects.

81

experiments had been extended. It is to be noted that, in addition to
hydration and possible salt formation, the colloid was also undergoing
some alteration by the unequal solution out of the solution of pro-
tein and agar.

Fio. 12.

Continuation of record
of variations of sec-
tions of agar and bean
protein in fig. 11. For
description see text,
pages 81 and 82.

12p.m. m. I2p.m

12p.m.

12p.m.

New sheets were fitted to the recorder of the auxograph and arrange-
ments made to follow further changes (see fig. 12). During the next
4 days an additional swelling of 280 per cent in the malic series, 320
per cent in the citric, and 300 per cent in the formic were recorded.
Replacement of the acid by hydroxid (1) resulted first in an expan-
sion which was partially lost in 7 hours, so that the net gain was very-
light. When the hydroxid was washed off (2) hydration in distilled
water was followed by expansion of lesser amplitude than in the
previous procedure of this kind, but it had not ceased at the end
of 14 hours. A diminution in each repetition was found. Hydroxid
(3) failed to bring the sections back to the dimensions preceding the
last hydration. Replacement of the hydroxid by acid (4) caused a
further slight contraction, but not to the last pre-hydration dimen-
sions. In fact, every hydration included an irreversible element.
Hydroxid (5) again produced shrinkage, and then contraction which
soon ceased. After 13 hours in hydroxid, water applied and re-
newed (6) produced a swelling which was in progress at the end of
12 hours. Replacement with acids (7) was followed by very abrupt
shrinkages which were more gradual in formic acid. Substitution
of hydroxid after 11 hours at 8 was followed by the expected initial
expansion and subsequent shrinkage. The final hydration (9) on the
tenth day of the test gave swellings with net expansions of 7, 9, and
8, as compared with 21, 18, and 17 on the sixth day. The biocolloid
is thus seen to progress through a period of reactions of increasing
amplitude to a climax, followed by one of diminishing alterations in

82

Hydration and Growth.

water. Such progress is probably accompanied by, or consequent
upon, changes in the proteins and in the pentosans. On the other
hand, as the sections come nearer to their total imbibitional or
hydration capacity, they are more sensitive to acidity and not only
the speed but the amount of retraction increases.

Another series was based on the behavior of an agar (90 parts) bean
albumen (10 parts) mixture in plates 0.18 mm. in thickness. Three
sets of sections were given treatment as nearly identical as possible,
a special feature being made of the action of salts.

An initial swelling of 1,055 to 1,222 per cent, averaging 1,137 per
cent, was first produced in 7 hours in distilled water, at which tune
it is evident the plates had increased to a thickness of .about 2 mm., or
one-half of the total displayed in the test. (See fig. 13.) The replace-

m. l?p.m. m. 12p.m. m. I? p.m

12p.m.

12p.m. m 12 p. m

12p.m. m.

Fio. 13. — Variations in thickness in sections of agar and bean albumen subjected to the action

of acids, salts, and hydroxid.

ment of the water by a hundredth-molar solution of potassium nitrate
(A) for 14 hours was characterized by a slight initial shrinkage, followed
by very slow swelling, which was probably accompanied by a solution
of the albumin from the sections. Replacement of the salt solution
.by another one acidified by a hundredth-normal citric-acid solution (B),
produced a shrinkage which brought the dimensions of the sections
to about that at the end of the initial swelling in water. The material
might now be considered as a biocolloid containing some salt and
acidified to a point probably equivalent to conditions in living material.
Replacement of the acidified salt with alkaline salt of the same con-
centration resulted in a swelling averaging 200 per cent of the original
thickness of the dried plate.

The sections were now washed (D) and allowed to swell for 9 hours in
distilled water, making an increase of over 1,100 per cent and, as will
be obvious, bringing the water-content of the biocolloid to a point
nearing the maximum capacity.

Replacement of water by an alkaline salt solution (E) now pro-
duced a greater shrinkage than when the biocolloid had a water-con-
tent about a half less, the loss being 200 per cent of the original thick-

Fluctuating or Alternating Hydration Effects. 83

ness. Replacement with acid (F) resulted in a further slight shrink-
age, no noticeable loss ensuing when the acid was replaced by acidified
salt (<?). Replacement with alkaline salt (H) was followed by an in-
crease which had ceased at the end of 4 hours. Water now caused a
slow continued swelling (/), at which time the maximum size of the
sections was reached and they had swelled 2,400 to 2,800 per cent of
the diameter of the dried plate.

Replacement of the water by an acidified solution (J) resulted in a
very rapid shrinkage, which was nearly complete in an hour and re-
duced the sections nearly 200 per cent of the original. Replacement
of the acidified salt by water (K), which was renewed three tunes,
regained only about half of the thickness lost in the acidified salt. It
is to be remembered that the sections were in a condition of acidosis,
and this would not be reduced below a certain point until alkali was
introduced. This was done in an alkaline salt (L), which produced a
very slight shrinkage. The swelling in water following this treatment
was of such reduced amplitude that the measurements were discon-
tinued. The successive treatments had resulted in the incorporation
of water to the full imbibition capacity of the colloid, and the reagents
had produced such changes that mere variations in acidity, alkalinity,
etc., caused but little variation in this amount and in the volume of
the sections, a condition in general analogous to that of a mature organ.

A series of tests were now planned to induce alterations in sections
in which imbibition was first carried to a degree near the saturation-
point. Sections of agar 90 parts, bean protein 10 parts, 0.16 mm. in
thickness, swelled 2,156 per cent in 18.5 hours at 16° to 18° C., and
the rate of expansion had fallen very low. Substitution of a hun-
dredth-normal potassium-nitrate solution checked further increase and
induced a very slight shrinkage in 6.5 hours, and no perceptible alter-
ation took place by the substitution of citric acid (0.01 M) for 15.5
hours. Replacement with potassium hydroxid + potassium nitrate
(0.01 M) resulted in a swelling of 156 per cent of the original dimen-
sions in 8 hours, at the end of which time this action had entirely
terminated. The sections now being in an alkaline condition, their
immersion in water was followed by an increase of 1,250 per cent of
the original dimensions, the total now being 3,406 per cent.

Replacement of the water by a hundredth-normal solution of potas-
sium nitrate and citric acid resulted in a greater change than at a
lower water-content, the shrinkage now being 190 per cent of the origi-
nal diameter of the air-dry plate. When the sections had swelled but
2,156 per cent, the action of a similar solution did but little more than
check swelling.

The biocolloid at this last point was equivalent to a protoplasm in
its water-relations which contained over 95 per cent water and less
than 5 per cent dry matter. In a later stage the biocolloid was made

84 Hydration and Growth.

up of less than 3 per cent dry matter and more than 97 per cent
water, and hence approximated the dispersion of active protoplasm.

Other sections from the same plate swelled 2,094 per cent in 18.5
hours at 16° to 18° C. The use of potassium nitrate and citric acid
(0.01 N) resulted in a shrinkage of less than a hundred per cent within
an hour, after which the volume was stationary. As the experiences
of the two sets of sections had been identical hitherto, it is interesting
to note that in this stage of imbibition (95 per cent water) acidified
salt produced more shrinkage than the salt alone, a result in harmony
with those obtained by swelling biocolloids. Imbibition in the salt
being generally greater than in the acidified salt, replacement of the
acidified salt with the alkaline salt was followed by a swelling amount-
ing to over 300 per cent of the original thickness of the sections in 9
hours, at the end of which time the swelling had not reached its limit.
Replacement with water speeded the imbibition to a rate which made
an increase of over 800 per cent in 9 hours. The sections now con-
tained nearly 97 per cent water, having swelled 3,156 per cent, and
when the water in the dish was replaced with acidified potassium
nitrate (0.01 M), the shrinkage was greater than in the previous experi-
ence, now amounting to 300 per cent of the original thickness of the
sections. A further shrinkage followed the substitution of alkaline
salt solution.

Tests were arranged to ascertain the effect of the initial hydration
medium, using salts and acidified salts. Trios of sections of agar 90
parts and oat protein 10 parts, 0.18 mm. in thickness, were put under
the auxograph in a darkened chamber which remained steadily at 18°
C. during the week in which the measurements were made.

The sections which were immersed in hundredth-molar potassium
nitrate reached a size within 100 per cent of the possible total in
16.5 hours, at which time the expansion was about 1,200 per cent.
The replacement of the salt by distilled water which was renewed
twice induced an additional expansion of 1,750 per cent in 22.5 hours,
which was the practical limit of the sections. Replacement with
hundredth-normal acidified potassium nitrate resulted in a shrink-
age of about 100 per cent, the movement being complete in 6 hours.
Replacement of the acidified salt with water (renewed three times)
induced a slow, long-continued swelling, which, at the end of 18.5 hours,
had resulted in an increase of about 100 per cent, and which carried
the sections back to the maximum thickness attained before the acidi-
fied salt was added. The water was now replaced by a simple hun-
dredth-normal potassium-nitrate solution, which produced a shrinkage
slightly less than that displayed 24 hours before in acidified salt. This
was followed by a long-continued swelling, which again, in 21 hours,
brought the sections to the maximum thickness. The change to an
acidified salt solution resulted in a shrinkage about equivalent to the

Fluctuating or Alternating Hydration Effects. 85

previous one, and the experiment was discontinued after a total period
of 100 hours.

The use of hundredth-normal potassium nitrate-citric acid as the first
solution caused a swelling of 708 per cent in 7 hours, at which time
the rate of increase was very slow and would not have carried the
thickness to more than 800 per cent of the original. Replacement of
the acidified salt by the salt alone in the same concentration and its
renewal accelerated imbibition, which proceeded at a rate which les-
sened very slowly for 63 hours. The swelling was at first at the rate
of 100 per cent in 4 hours and about 25 per cent during the last 4 hours.
The increase during this period of 87 hours was about 700 per cent,
making the total increase over 1,400 per cent. The behavior of the
colloid during this period was that of a plant with diminishing acidity.

The salt was now pipetted off and replaced with distilled water which
was renewed. The acceleration following was so abrupt that the rate
jumped from 25 per cent in 4 hours to 750 per cent in 2 hours. The
swelling at the end of 24 hours was 1,778 per cent, at which time in-
crease was still in progress. The total increase now amounted to 3,195
per cent. The unsatisfied capacity would have doubtless carried the
swelling to a thickness as great as any yet observed in any of the bio-
colloids and in excess of the sections which were swelled initially in the
salt alone.

The mass was now nearly 97 per cent water. Replacement of the
water by acidified salt solution was followed by a shrinkage of about
200 per cent of the original thickness of the sections in 8 hours. When
this was washed out, the loss was regained in a few hours. These
sections were now set aside for desiccation. The final period of 16
hours in the swelling was in distilled water, so that the acidified potas-
sium nitrate in which the sections had been immersed for the preceding
8 hours must have been reduced to a very small amount.

The last desiccation had left the sections warped to some extent,
which interfered with accurate remeasurements, but the thickness had
been reduced from 0.18 to 0.16 mm. and even thinner. The irregulari-
ties of course operated to introduce some error in the swelling, which
would make the total appear to be 100 to 200 per cent less than it
should be.

The swelling in distilled water at 18° to 20° C. now progressed over
a period of about 96 hours, reaching a total of 1,625 per cent. Both
the amount of the total and the length of time necessary to reach full
capacity would indicate that the sections still contained some of the
protein or its derivatives and perhaps some of the salts. Agar alone
would attain full capacity at a lesser volume and hi a shorter time.

A second set of sections of agar 90 and oat protein 10 parts were
first treated with acidified potassium nitrate, in which the swelling was
about 500 per cent (the solution being freshly made) hi 16 hours. This

86 Hydration and Growth.

was now washed out with water and the swelling, which had been
interrupted in the previous test, was allowed to go on for 147 hours.
Swelling was still in progress after 6 days of continuous imbibition, the
increase being 1,833 per cent. The total increase up to this time was
2,388 per cent. The shrinkage caused by a solution of acidified potas-
sium nitrate was very slight, being less than 100 per cent of the origi-
nal, and was scarcely more than the effect of the potassium nitrate
which was first applied. The original thickness of 0.18 mm. was now
reduced to 0.16 mm., much of this loss being attributable to the solu-
tion out of some of the water-soluble oat protein and some of the agar
(see p. 107). The sections also probably contained some salt and
probably a trace of acid.

The trio of sections were placed under the auxograph in a chamber
where the temperature of 'the distilled water in which they were im-
mersed varied between 17° to 20° C. in the 6 days during which the test
was carried on. The initial swelling, hi contrast with the action of
fresh sections, was very slow. Furthermore, the increase was contin-
ued over a long period, and came to a total of 938 per cent, or less than
one-half of the original expansion.

Sections of plates 0.18 mm. in thickness of agar 90 parts and bean
protein 10 parts, to which 0.85 per cent of nutrient salts had been
added, were tested in the chamber at 16° to 19°C. The initial treat-
ment with distilled water induced a swelling 1,861 per cent in 20
hours, during which time the nutrient salts must have been partially
dissolved out, so that at the end of this period the liquid was a salt solu-
tion in which none of the various components was as concentrated as
0.001 M, since about 30 c.c. of water was poured in the dish. The
capacity for increase in this solution having been approached, the water
(or saline solution) was replaced with a hundredth-normal solution of
malic acid, which caused a shrinkage of about 100 per cent hi 2 or 3
hours. After 7 hours the acid was replaced with distilled water, and a
slow swelling ensued. The effects of the alternations here are not sep-
arable from those in which the salt is incorporated in the colloid with
the first swelling solution.

A similar set of sections containing nutrient salts were prepared by
cutting a trio of strips 7 mm. in length to be placed under the auxo-
graph. A free strip 30 mm. long was also placed in the dish.

The initial immersion in 0.5 per cent nutrient solution was practi-
cally complete as to its swelling effects in 18 hours, with an increase of
850 per cent. Substitution of hundredth-normal acidified potassium
nitrate caused a total additional increase of 75 per cent in 22.5 hours.
Replacement of this solution with an alkaline solution of potassium
nitrate of the same concentration was followed by a further swelling
of 350 per cent in 44 hours. The total swelling of the sections during
a period of 110 hours was 1,250 per cent. The concentration of the

Fluctuating or Alternating Hydration Effects, 87

salts was reduced during one period of 23.5 hours and during the re-
mainder of the time the concentration ^was about a hundredth molar
at a temperature of 18° to 21° C. No appreciable change in length of
the tested trio of sections or of the free long strip occurred (see p. 19).

The effects of swamp water, which has already been shown to retard
hydration, were tested in this connection. The first lot of water of this
kind was procured from the sedge-grass swamps near Anoka, Minnesota.
Sections of agar 90 and oat protein 10 parts, which were 0.18 mm.
in thickness, were swelled in this at 17° to 19° C. Imbibition was
rapid during the first 12 hours, during which time a total enlargement
of 972 per cent took place. At the end of this time the rate had
decreased notably, but was maintained in such manner that in 4 days
an additional swelling of 361 per cent was recorded. The volume
was now practically stationary and the test was closed at the end of
108 hours. This long-continued swelling at a low rate and low total
was in contrast with swellings of similar sections in distilled water and
in nutritive salts.

The trio of sections placed in distilled water at the same time as the
above swelled 2,361 per cent in 3 days, and as increase had ceased, the
distilled water was replaced with swamp water, with the result that a
slow shrinkage ensued, which, however, amounted to only 36 per cent
of the original volume in 2 days.

It was next thought important to test the swelling of sections con-
taining salts in swamp water, and a mixture of agar 90 parts, bean
protein 10 parts, and nutrient salts 0.85, 0.18 mm. in thickness, was
swelled at the temperatures ofl7°tol9°C. The initial swelling was prac-
tically complete in 16 hours, at which tune an increase of 1,082 per
cent had taken place. Replacement with acidified potassium nitrate,
hundredth normal, for 36 hours caused no appreciable change in vol-
ume, but when this solution was washed away in swamp water, swell-
ing was resumed and a further increase occurred, most of which took
place in the first 4 hours, but which was still in progress at a very slow
rate at the end of 38 hours. The enlargement in the swamp water
during this final period was 306 per cent, the total swelling being
1,388 per cent.

Sections identical with the above were first placed in a 0.5 per cent
nutrient solution, in which a swelling of 888 per cent occurred in
17 hours, at which time the pen of the auxograph was tracing a hori-
zontal line. Replacement with acidified potassium nitrate produced
no effect beyond a very slight swelling. When the acid solution was
washed away with the swamp water swelling began and an increase
amounting to 333 per cent followed in 40 hours. This final action was
fairly equivalent to that of the preceding series, except that the sub-
stitution of swamp water for the acidified salt was followed by a much
slower but more extended rate of initial swelling.

88

Hydration and Growth.

Another test was made for the purpose of comparison of the effects
of the swamp water with those of a solution of nutrient salts in which
the initial swelling in the nutrient solution was 861 per cent in 20
hours, which was less than in swamp water. A solution of acidified
potassium nitrate was substituted and a swelling of 55 per cent ensued
in 55 hours. Replacement of the acidified salt solution by distilled
water was followed by a long-continued swelling which increased the
volume 111 per cent in 40 hours. The total increase of this section
was thus 1,027 per cent, as compared with 1,333 per cent in bog
water alone.

The obvious importance of these reactions is amply illustrated by
the changes in environmental conditions to which many aquatic plants
are subjected in the course of a season or even in a day.

Growth in all probability implies the incorporation of new material
in the colloid which adds to the hydration total of the mass. It is not
easy to arrange the introduction of unhydrated particles in a swelling
mass, but it was deemed worth while to bring a desiccated trio of sec-
tions into swelling under a similar trio which had already undergone
some expansion.

Three sections of an agar 90, peptone 10 parts, 0.18 mm. in thick-
ness, were swelled in distilled water until an expansion of 1,195 per
cent had been reached in 4 hours; a second series of sections were gently
slid under the first lot, the pen was readjusted to register continuously,
and a fresh lot of water was added (fig. 14).

I2p.m

7T

/ /

1 i

\ i

1

— . i

\ j

V

\

rzs \

\

\

\ \

FIG. 14.

Course of swelling of a
double trio of sections
of agar and peptone,
the second being added
after the first was par-
tially hydrated. See
text, page 88.

A swelling of the two trios now followed which reached satisfaction
in about 14 hours, which amounted to 708 per cent, calculated on the
thickness of both. Most of this swelling, however, was in the freshly
added sections, in which the swelling was 1,083 per cent in 4 hours,
and a lesser amount was due of course to the original trio. The final
total of the pair of trios swelled only 1,153 per cent, while the first set
had increased 1,195 per cent before the second pair was added and had
not yet completed its swelling. It seems fair to infer that increase
of thickness diminishes the proportionate increase.

A second test was made in which the swelling of the first trio reached
about 800 per cent in 2 hours and then a fresh section was thrust under

Fluctuating or Alternating Hydration Effects.

89

one of the old ones. The swelling during the next two and a half hours
was equivalent to 500 per cent on the basis of the average thickness
of the four sections in action.

A second fresh section was placed under another old one, making
five sections in action. The swelling during the next 3 hours was
something over 300 per cent, calculated on the basis of the five sections
engaged. The third fresh section was now added, giving a preparation
in which sections were included in four different stages of swelling, with
an initial average thickness of 0.36 mm. The swelling during the next
three and a half hours was about 225 per cent of the initial thickness
and reached an end-point 20 hours after the experiment was begun
at 1,333 per cent of the total, which was in excess of that reached in the
previous test. The temperature underwent a range of from 15° to
22° C. (fig. 15).

12 p.m.

5
15
25
35
45
55
65
75
85
95
105

FIG. 15. — Courses of swelling of sections of agar and peptone, a fourth, fifth, and sixth fresh (dry)

section being added as indicated.

The water was now removed from the dishes and the double trio of
sections was subjected to the action of a 2 M solution of calcium
chloride. A shrinkage followed which terminated with some abrupt-
ness at the end of an hour and reduced the thickness of the swelled
sections 1.05 mm. or nearly 300 per cent of the original. Calculated
on the basis of the swelled sections, which had reached a total average

55
65
75

8am in 12 pm.

"1-1 ' "

/ '

X- _\ \

FIG. 16. — Variations in volume of double trio of sections of agar and peptone which had reached
full hydration in distilled water and were then immersed in calcium chloride, 2M.

thickness of 4.8 mm., the reduction was nearly 22 per cent. A swell-
ing now followed and practically half of the thickness lost was regained.
Most of this was completed within 2 hours, after which a very slow
rate of increase followed, which was not at an^end at the close of the
third day. This test also was carried on at room temperature, which
underwent a wide variation, as noted above (fig. 16).

Hydration and Growth.

These elementary experiments open a field of possibilities as to the
incorporation of new material in masses of swelling colloids, in exempli-
fication of some phases of cell-mechanics, inclusive of the action of
embryonic cells in growing regions. The author, in collaboration with
Spoehr and Richards, has recently been able to outline the manner
in which the xerophilous and the succulent types of shoots or organs
are due to the action of such superposed effects in the cell colloids.
A series of detailed analyses of the carbohydrate-content of the opun-
tias, which was arranged to determine the principal sugars not only
during developmental stages, but also to follow the changes through-
out the seasons, established the fact that when these plants were sub-
subjected to long periods of drought, resulting in partial desiccation,
polysaccharids were dehydrated, with the result that the sugars, which
have a low water capacity, became converted into pentosans or muci-
lages, which have a large imbibition capacity. This fact was amply
confirmed by the coefficients of swelling which were obtained in my
own tests, which ran through several years.

Following this, the fortunate discovery was made that Castilleia
latifolia, which is native to the region about the Coastal Laboratory,
has thin, highly acid leaves when growing under mesophytic conditions,
but has less-acid succulent leaves in arid locations, the increased size
of the leaves being due to the hypertrophy of the thin-walled paren-
chymatous tissues. The hydration reactions of the two types of
leaves in a fresh and dried condition are shown in table 70, which gives
the swellings in 0.01 normal citric acid at 15° C.

TABLE 70.

Thin.

Succulent.

Thickness.

Swelling.

Thickness.

Swelling.

Fresh leaves

mm.
/ 0.4
I -41

p. ct.
125
184

mm.
1.4
1.4

p. ct.
21
25

Above leaves dried and re-hydrated

/ .23
\ .25

42
20

0.5-0.6
0.63

95
91

(Expansion in terms of dried thickness.)
Fresh-dried leaves

{ .38
•2
| .38
I -2

1.2
.5
1.1

.38

25

120

(Expansion in terms of dried thickness.)

62

92

In the interpretation of these results, it is to be noted that drying,
both from the fresh state and from the hydrated condition, reduces the
hydration capacity of the thin leaves, but not of the succulent ones.
The principal changes in hydration include the extraction of acids and
salts as well as the hexoses, while the mucilaginous pentosans diffuse

Fluctuating or Alternating Hydration Effects. 91

not at all or very slowly. The swelling of the succulent leaves is
therefore high after extraction by reason of the presence of these sub-
stances, which have been partially freed from the retarding action of
the organic acids and salts.1

The conversion of the diffusible sugars to the mucilaginous pentosans
is one of the alterations which may result in the cell as a result of partial
desiccation, with a striking morphogenetic result as indicated. Under
certain conditions of desiccation, the lessening of the water-content of
the cell accelerates the formation of the anhydrides of which wall
material is composed, with the result that the hydration capacity of
the product in this case is less than that of the polysaccharids, and in
addition to this direct diminution of the water-absorbing material of
the cell, the production of the heavy wall at an early stage of the
development of the cell checks growth, resulting in the restricted
shoots or organs with indurated membranes which are characteristic
of xerophytic plants.2 Two striking and highly important vegetational
types are thus seen to result more or less directly from the action of the
environment upon the cell-colloids, hi accordance with a view expressed
by the author in a previous publication.

1 MacDougal, Richards, and Spoehr. The basis of succulence in plants. Bot. Gaz., 67: 405
1919.

2 MacDougal, D. T. The Salton Sea, etc. Carnegie Inst. Wash. Pub. No. 193. 1914. See
p. 179. Also MacDougal and Spoehr. The origination of xerophytism. Plant World, 21: 245.
1918.

VIII. WATER DEFICIT, OR UNSATISFIED HYDRATION

CAPACITY.

Much of the present confusion as to the nature, mechanism, and
course of growth is due to the fact that premature attempts have been
made to institute comparisons in measurements derived from organ-
isms fundamentally different.

Thus, the growth of bacteria consists in the enlargement to a unit
size of cells high in proteins which become independent when this
volume is reached, and not being attached to other cells, their presence
does not directly affect the rate of growth calculated upon their num-
ber, except in so far as their excretions hi the medium may do so.
Furthermore, these, as well as organs which are submerged hi the
liquid nutrient media, are in a condition approaching complete hydra-
tion in the complex of conditions in which they live, and these may
vary only within very narrow limits in many cases.

Growth in the higher complex plants implies the multiplication of
embryonic cells and the development of the greater number of them
into special static tissues to which the growing cells are inseparably
attached. Thus an internode, or a leaf, barely makes its appearance
before some of its cells have passed beyond the growing-stage and into
a condition of lessening change to a nearly static condition of maturity.
Enormous numbers of senescent and highly specialized cells are formed
through which the water-supply of the growing cells must pass and
which compete for the supply. The water-deficit, or the amount which
a cell-mass hi the plant may take up, may therefore vary widely, as,
in addition to the internal changes, water is constantly being lost from
the surfaces by transpiration. An instrument attached to the terminal
portion of a plant records the changes hi volume of cell-masses in all
of the possible stages between the apex and the base of the internode
or the point at which the stem may be fixed in the experimental pre-
parations.

The tips of roots offer a generalized type of growth, which has been
the subject of more experimentation than any other part of the higher
plants. Even here the measurements invariably include the enlarge-
ments of masses of embryonic cells by imbibition alone, the inter-
mediate stage in which the increase in the size of the vacuole doubtless
plays an important r61e, to a final stage in which imbibition again
may be the only force of distension. Only in individual unicellular
organisms and hi such structures as pollen-tubes may growth-enlarge-
ments be dealt with to the exclusion of variations of mature tissues.

Again, growth is a resultant of the play of molecular forces in sur-
face tensions and of a series of metabolic transformations, hi which
features notable differentiations are found in the groups of organisms.

92

Water Deficit or Unsatisfied Hydration Capacity. 93

The main features of imbibition are determined by the carbohydrate-
protein ratio and the presence of salts. Animals and some vegetal
organisms are high in proteins. The vacuole and the external layer
of the protoplasmic units in plants give a play of osmotic activities
not duplicated in the animal; and lastly, as has been so clearly estab-
lished by the work of Dr. H. A. Spoehr, the plant has a characteristic
carbohydrate metabolism with the capacity for the synthesis and com-
bination of the amino-acids, while in animal processes metabolism is
more largely concerned with the proteins, and amino-acids result
chiefly or entirely by disintegration of albumins.

The ordinary conception of growth implying the changes in dimen-
sions of the developing parts of organs, shoots, roots, etc., is, however,
a well-rounded one of sound value and worthy of attention as a uni-
fied procedure. The ultimate physical forces concerned are those
which find play in elastic gels, and the interaction of these forces may
be modified by the self-altered composition of the living colloids and
by the action of external agencies under the influence of which these
colloids operate.

The course of growth in the succulents will come in for a large share
of attention in the following chapters. The form of the organs of
such plants facilitates the making of observations in which the actual
temperature of the living mass may be found with some exactness,
and the large bulk which characterizes them makes it comparatively
easy to obtain analyses showing the varying proportions of the con-
stituents of importance in growth. Some attention has been given
to plants with thin stems and slender leaves, which in reality constitute
the dominant vegetative type of plants.

The arrangement of imbibition tests of the growing terminal inter-
nodes of stems, and other organs, and the interpretation of the results
was made upon the basis of the supposition that such material was in
effect a complicated salted colloid, with an altering and unsatisfied
hydration capacity. The manner in which saturation might be
reached by immersion in various solutions might well offer some
profitable comparisons with the behavior of biocolloids of known
composition and structure. The earlier trials were made with the
growing parts of stems of Phytolacca decandra, Micrampelis calif arnica,
and Rudbeckia laciniata. The young internodes furnished sections
about 1 cm. long and of a thickness from 4 to 8 mm. A tangential
slice was cut from one side to remove a segment of the fibrovascular
tissue, and the plane surface exposed served to seat the sections firmly
in the glass testing-dishes of the auxographs. The first series was
made with distilled water, sodium or potassium hydroxide, and citric
and formic acids, all in hundredth-normal concentration.

The series which were run in a day-lighted laboratory at tempera-
tures of 18° to 22° C. agreed in swelling most in distilled water, less in

94

Hydration and Growth.

41.9

the hydroxid, and least in the organic acids mentioned, in propor-
tions of 40, 20, and 15 in Rudbeckia, and 30, 25, and 15 in Micrampelis.
Phytolacca gave proportions of 30, 25, and 12 under the same circum-
stances. It was noticeable that distension due to swelling by these
acids was relaxed after a few hours, probably due to the solution out
of some of the colloidal contents of the cells.

Further tests were made with the stalks of Rudbeckia bearing heads
of flowers about ready to open. The greater part of the total growth
had been accomplished, although they were still in rapid action and
the material for the developing flowers was being drawn from them.
Tangential slices less than 1 mm. in thickness were removed, leav-
ing the stalks 4 mm. in thickness at
the larger end and about 3.4 mm. at
the smaller. Sections about 1 cm. hi
length were cut from four such stalks
and the whole number was divided into
two lots. One lot of sections was taken
into the dark room and swelled at
18° C. and the others were set on a
window-ledge to become air-dry. The
average thickness was taken to be 3.7
mm. Twenty hours later swellings of
6.8 per cent in distilled water, 4.8 per
cent in alkali, and 1.4 per cent in acid
had been made (fig. 17). Actual en-
largement by imbibition in the acid had
lasted only about an hour, after which
a shrinkage ensued that reduced the
thickness 5.4 per cent from the original
volume and 6.8 per cent from the max-
imum. The decrease here and the in-
crease in water and in hydroxid were
all in progress at the close of the test.
The dried sections came down to about
half of their original diameter, but, on account of the irregular
shapes assumed, could not be measured with accuracy. Trios of these
under the auxograph gave swellings of 41.9 per cent in distilled water,
21.6 per cent in hundredth-normal citric acid, and 37.8 per cent in
hundredth-normal sodium hydroxid. The sections were thus seen
to return to about their original size in the living condition in dis-
tilled water — nearly this size in hydroxid, but far short of it hi
the acid solution, after the manner of a salted biocolloid consisting
of a large proportion of carbohydrate and a smaller one of protein.
Here, as in all comparisons between the hydration of living and of
dried sections, it must be taken into account that in the desiccation

Dried

Fresh

6.8

37.8

sections

material
4.8

21.6

1.4

Water Alkali Acid

FIG. 17. — Swelling reactions of fresh
and dried flower-stalks of Rud-
beckia, in percentages of original
thickness. The proportionate in-
crease of dried sections is denoted
by the heights of the vertical lines,
and that of fresh sections by the
marks near base.

Water Deficit or Unsatisfied Hydration Capacity.

Young.

Mature.

Dried.

Water.

p. ct.
40

p. ct.
68

p. ct.
419

Acid

68

14

48

Hydroxid . . .

419

216

378

of cell-masses, the acids, salts, sugars, etc., in the vacuoles and
syncretic cavities of the protoplasm increase in concentration as the
dehydration proceeds, until as the end is neared these substances are
taken up by the protoplasmic colloids from solutions which may be
saturated.

The swelling of a living section continues until hydration capacity
is satisfied or balanced against the mechanical restraint of the cell-
walls, and includes the possibilities of osmotic action by reason of their
differential action. The colloids are subjected to the action of the
salts and acids only to attenuations in which these substances are
usually present. When desiccated sections are swelled, the cell col-
loids are now in the condition of having adsorbed substances previously
hi great dispersion and the external layers of the cells now exercise
only the effect of a dense filter-
paper; consequently any osmotic TABLE H-*"** «/ Rudbeckia.
pressure which might be set up
is soon equalized, and hence con-
tributes but little to the ex-
pansion of the cells.

Returning to the matter of the
fresh sections of Rudbeckia, the
nature of the increase in hydra-
tion capacity of the material with the march of development may be
set forth more clearly by the figures in table 71 , in which the percent-
ages of swelling are multiplied by 10.

Swelling was greatest in water in both young and old stalks and the
increase of the hydrogen-ion concentration or acidity of the medium
resulted in reducing the final hydration capacity of the tissues, more
water being taken up when hydroxid was used, but the total
capacity being less than in water. The desiccation of the stems,
with attendant possible coagulatory effects, was accompanied by an
increase in hydration capacity in the acid solutions. The greater
swelling shown by old tissues suggests increased pentosan or mucil-
age content rather than a deficit due to loss of water.

No opportunity was lost to make measurements of other material
which might possibly show the diversity of action of different plants.
The terminal internodes of Verbena ciliata were available at the Desert
Laboratory in March 1918, and sections of these with a tangential
slice removed were about 1.8 mm. in thickness. Swellings at 18° to
20° C. were as shown in table 72.

TABLE 72.

p. ct.

Distilled water 7

Citric acid, 0.01 N 2.8

Sodium hydroxid, 0.01 M 7

Potassium chloride, hydrochloric acid, 0.01 M 4

96 Hydraiion and Growth.

The familiar relation much seen in young organs, by which the
imbibition is least in the acid and most in water and alkaline solutions,
is exhibited. Some interest attaches to this plant from the fact that
its expressed and centrifuged juice has been found by Dr. H. A. Spoehr
to aggregate and form a fairly firm jelly without concentration, an
action probably due to the large proportion of pentosans present.

Forms of Brodicea native to Tumamoc Hill start into activity late
hi February and by the end of the first week in March have formed new
conns on the crowns of the older ones nearly 1 cm. in diameter, from
the apices of which the long, slender leaves extend at a rapid rate.
Halves of these corms arranged in the dishes under the auxographs
gave swellings at ,20° C. as shown in table 73.

TABLE 73.

p. ct.

Distilled water 5

Citric acid, 0.01 N 2.7

Sodium hydroxid, 0.01 M (not completed) 2.2

Potassium chloride, hydrochloric acid, 0.01 M 2.5

The carbohydrate material in these organs is starch, and this and
the products of its hydrolysis constitute the main components of the
cells. The increases noted above are low, that in the alkaline solution
being still in progress. The old corms form tapering extensions from
the lower surf ace/ which hi the earlier stages are made up only of thin-
walled cells, filled with the products of starch hydrolysis. These
show swellings of 17 per cent hi distilled water at the above tem-
perature. Two other variations were tried to ascertain whether the
young corm just formed, having swelling capacities as above, differed
in hydration capacity from the older basal corms being emptied of their
contents. This was accomplished by setting up a preparation of three
plants in which the young corms were seated in place on the older
conns. The average height of the preparation was 12 mm. and the
increase in distilled water was 9.6 per cent at 20° C. Another set of prep-
arations was made, in which older corms from which the young portion
had been removed were immersed in water at similar temperatures.
The increase in this case was 24 per cent, showing that these structures,
hi which the accumulated starch was hi an advanced stage of hydrolysis,
had an imbibition capacity much greater than that of the young corms.
The fact must be taken into account that the young corms were firm
and solid to the touch and were to be regarded as in a high state of
imbibition, while the old corms had been partially emptied, but were
capable of returning to the dimensions of their original turgid con-
dition.

Tangential slices from the terminal internodes of asparagus tips,
bought hi the market in Tucson in March, were made to have a thick-
ness of about 3 mm., and these gave swellings as shown in table 74.

Water Deficit or Unsatisfied Hydration Capacity.

97

TABLE 74.

p.ct.

Distilled water 7

Citric acid, 0.01 N 4

Sodium hydroxid, 0.01 M 6.2

Potassium chloride, hydrochloric acid, 0.01 M. Immediate and marked shrinkage.

The rapid and immediate shrinkage of these thin sections hi the
acidified salt solution was so striking that a second measurement was
made, in which thicker sections swelled 4.4 per cent hi the acidified
salt solution within a few minutes and then began to shrink to the
original dimensions and to smaller volume. Imbibition hi the salt solu-
tion alone showed two phases : first, a very rapid swelling to a volume
near the maximum capacity, then a slow increase which was still hi
progress 24 hours later and which at that time had brought the section
to a thickness 10 per cent greater than the original. Such material hi
acids swells quickly and more gradually than in the combined solu-
tion, then slowly shrinks to the original dimensions and below. The
presence of the salt accentuates the action of the acid, as it did also in
the swelling of stems of Verbena hi similar solutions, the increase hi the
acid being but 2.8 per cent, while it was 4 per cent hi the combined solu-
tion, the explanation of which is probably to be sought hi the com-
bined effects of acids and bases (fig. 18).

8a.m 9 10 II m. lp.m.2 345 6p.m.7 8 9 10 II I2p.mlain.2 3.

,7 A— A — A—-/- -/— -1— /- -/ / -V --A -A— A -^--V A -/— V -A— /-

*2 111 il r T T 1 irrilfllri

//////////// _--/- / / / / / / / i

\

/— /— 4— I—

I-H-

ri

1 1

1

1

r,

d

.-'"

FIG. 18. — Swelling of tangential slices from tips of asparagus shoots at 14° to 17° C., X 20, on
scale ruled to 5 mm. and 1-hour intervals, a, swelling of trio 3.8 mm. in thickness 7 per
cent in water; b, swelling of trio 3.7 mm. in thickness 4 per cent in hundredth-normal citric
acid; complete within a few minutes, followed by gradual shrinkage; c, swelling of trio of
sections 6.2 per cent in sodium hydroxid with final shrinkage; d, slight swelling of trio of
sections in hundredth-normal potassium chloride and hydrochloric acid and then rapid
shrinkage.

Other aspects of the matter of permeability and swelling are offered
by the reactions of young bean seeds which had not attained more than
an eighth of their final volume and had a diameter of 2.8 mm. These
were tested hi a series parallel to the above and swelled as follows:

TABLE 75.

p.ct.

Distilled water 8.2

Potassium nitrate, 0.01 M 9.9

Potassium nitrate, citric acid, 0.01 N 8.2

Citric acid, 0.01 N 5.4

Potassium hydroxid, 0.01 M 12.5

The proportions here were also those of an agar-albumin mixture,
but the high swelling hi the acidified salt was not shown. The beans

98 Hydration and Growth.

were intact, with an unbroken coat. It was probable, therefore, that
osmosis may have played some part in the absorption in water and the
solutions.

A second lot of the growing beans, which had an average diameter
of 2.4 mm., was allowed to desiccate for 3 days, and when they had
become hard and dry and were dead, the swelling tests were made
with them, yielding the f olio wing results :

TABLE 76.

p. cL

Distilled water 29.2

Potassium nitrate, 0.01 M 22.9

Potassium nitrate, citric acid, 0.01 N 32 . 5

Citric acid, 0.01 N 29.2

Potassium nitrate, potassium hydroxid, 0.01 M 31 .3

Potassium hydroxid, 0.01 M 15.8

It is evident that the activity of the vacuole is not the determining
or dominating factor in the water-deficit or unsatisfied hydration
capacity; if it were, the greatest swelling would have taken place in
water. The action of the salt was not increased by the addition of
acid in the swelling of the living cells, but such an effect was produced
in the dried beans. The interferences were much less in the driejl
material, the swelling in acid being but little short of that in water,
while it was relatively much less in the living material.

Young leaves of Abronia latifolia which had been allowed to wilt for
two days while attached to stems in a ventilated room were cut into
suitable strips free from the midrib and the larger veins in preparation
for swelling tests. The spongy texture made it impossible to measure
thickness with accuracy, but this was estimated as 1 mm. Increases
in thickness were measured, as follows:

TABLE 77.

p. ct.

Distilled water 40

Potassium nitrate, 0.01 M 80

Potassium nitrate, citric acid, 0.01 M 55

Citric acid, 0.01 N 50

Potassium nitrate, potassium hydroxid, 0.01 M 50

Potassium hydroxid, 0.01 M 60

Potassium nitrate gave a maximum swelling, a lesser one when acid
was added with the salt, and still less swelling ensued when acid alone
was used. The swelling in distilled water probably represents a nor-
mal imbibition total under the undisturbed conditions in the leaf.
The partial neutralization of the acid by the addition of hydroxid
gave a swelling determined by the acidified salts formed.

Half-grown succulent leaves of Cakile sp. were taken from the beach
at Carmel, August 23, 1917. The total acidity, as determined by Pro-
fessor H. M. Richards, was found to be equivalent to 0.30 c.c. hun-
dredth-normal sodium hydroxid per gram of fresh material. The
strips of leaf-blades, which were cut in such manner as not to include
any of the main veins, had an average thickness of 1.2 mm. One lot

Water Deficit or Unsatisfied Hydration Capacity.

99

was immersed in solutions in a fresh condition, and others were allowed
to lie on a window ledge for 24 hours, at the end of which time they
were dead, shrunken, but limp, so that they remained in any shape
into which they were bent or twisted. The swellings were as follows,
on the basis of the original thickness :

TABLE 78.

Living.

Dried.

Distilled water

p. ct.
12.5

p. ct.
20 8

Potassium nitrate, 0.01 M

16.6

25

Potassium nitrate, citric acid, 0.01 N

16.6

4.2

Citric acid, 0.01 N

8.3

16 6

Potassium nitrate, potassium hydroxid, 0.01 M
Potassium hydroxid, 0.01 M

12.5
16.6

37.5
33.3

The swelling of the living tissues was greatest and was equal in salt,
acidified salt, and hydroxid, while it was low in acid. The dried
material swelled most in the salt, while it was least in acidified
salt. Increases occurred in all the other solutions, the swelling in
alkaline salt being three times as great as hi the living material.
It was evidently desirable to measure the water-relations of some
simple plant structure which could be brought into the tests with-
out anatomical injury and which would not suffer material and
immediate injury by submersion. The small tubers of the potato
seemed to meet these requirements.

A number of tubers of the second generation of a hybrid between a
domesticated potato and Solarium fendleri of Arizona were available,
and as these bodies were in a condition in which they were ready for
planting and sprouting, tests were arranged to obtain the swelling
measurements in various solutions. Trios of tubers 5 to 8 mm. hi
diameter were placed in the dishes after the total and average diameters
had been ascertained. The first set swelled 7.5 per cent in 5 days hi
distilled water at 14° to 21° C., and the second increased the same
proportion in hundredth-normal citric acid in 4 days. A series ar-
ranged to obtain the auxographic record as complete as possible was
allowed to run for 11 days, at the end of which time the swelling in
water amounted to 13 per cent, in hundredth-normal citric and
hydrochloric acids 14 per cent, and less than 7 per cent in hydroxid,
at temperatures between 14° and 20° C. identical for the lot. Actual
shrinkage had not begun in any of the solutions and all solutions were
renewed three or four times during the period.

A set of three with an average diameter of 8.3 mm. was placed in a
solution of calcium chloride 3 N acidified to 0.01 N with hydrochloric
acid. A steady shrinkage amounting to 3.4 per cent in 4 days began
at once.

100 Hydration and Growth.

Later, when the tubers had begun to sprout, a set was arranged to
obtain another series of tests, extended over a period of 22 days, with
many renewals of the solutions. The swellings were as follows at
20° C:

TABLE 79.

P. ct.

Distilled water 34 . 4

Citric acid, 0.01 N 24.4

Sodium hydroxid, 0.01 M 13 . 8

Potassium chloride, hydrochloric acid, 0.01 M 20

All of the tubers, with two exceptions, were turgid and firm at the
end of the tests, even when extended over 22 days. Immersion in
any of the solutions for a day or two usually prevented sprouting by
killing the buds, although the remainder of the tuber kept alive. One
of the three tubers in water in the last test sprouted in the dish at the
end of a week and the excessive increase shown in this lot may be
attributed in part to the action of the continued hydrolysis of starch
as the growth of the etiolated stem proceeded.

Stiles and J0rgensen have made an extensive series of tests of the
swelling of the potato, in which a wholly different technique was used.
Plugs were cut from tubers, from which slices 2 mm. in thickness were
taken, and these were immersed in solutions in bottles. Variations
were taken by weight and the unavoidable difficulties of the method
were dealt with in an exact manner. The interpretations of the action
of the sections in the different solutions by Stiles and J0rgensen are
all based on the assumption that the hydration is one based entirely
on osmosis and that swelling ceases when the parenchymatou? walls
become permeable.1

The conditions presented by my experiments included the action of
the external coat of the tuber, which is composed of non-living elements
the permeability of which to water has been tested in a careful manner
in certain structures by Denny. The fact that the swelling was
greater in water than hi any of the solutions might lead to the con-
clusion that absorption was largely by osmosis. Such an explanation
can not be accepted as an adequate one, however. The cell colloids
of the potato, being high in carbohydrates, would show the greatest
swelling hi water, and hydration would be retarded and limited by
any contained acid or salt, except the ammo compounds. In addi-
tion to this action of the living cells, the retarding action of the outer
coat, with its possible differential action with respect to the acid, salt,
and hydroxid, are to be taken into account.2

1 Stiles, Walter, and Ingvar J0rgensen. Studies in permeability. The swelling of plant tissue
in water and its relation to temperature and various dissolved substances. Annals of Bot., 31 :
415. 1917. See also Stiles and J0rgensen. Quantitative measurement of permeability. Bot.
Gazette, 65:526. 1918.

2 Denny, F. E. Permeability of certain plant membranes to water. Bot. Gazette, 63 : 373-397.
1917. Permeability of membranes as related to their composition. Bot. Gazette, 63: 468-
485. 1917.

Water Deficit or Unsatisfied Hydration Capacity, 101

It is to be added that when the dried slices of the commercial prep-
aration of potatoes known as "Anhydrous" were tested at 22° to 23°
C., the sections, which were 1 to 1.1 mm. in thickness, showed a
maximum swelling of 265 per cent in potassium hydroxid 0.01 M,
250 per cent in citric acid (0.01 N), and 200 per cent in acidified
potassium chloride, at 0.01 N concentration, while the swelling in dis-
tilled water was 222 per cent. The relatively lessened swelling in
distilled water, as compared with salts and acids, might be attributed to
the elimination of the osmotic action of the semi-permeable membrane.
The exact history of the preparation of the material is not available,
however, and here, as in other dried material, the coagulatory effects
of contained salts and acids in desiccation must be taken into account.
This will become apparent from the results obtained from the swelling
of dried apples made from machine-cut strips. Sections cut from
these strips had an average thickness of 2.6 to 3.4 mm. and gave
swelling increases as follows at 16° to 18° C.:

TABLE 80.

p. ct.

Distilled water 72

Citric acid, 0.01 N 69

Sodium hydroxid 0.01 M 105

Calcium chloride, 3 M 123

The expected proportionate swellings in water, acids, and alkaline
solutions are noted, but the great imbibition from the solution of
calcium chloride which would afford an osmotic pressure of 68 atmos-
pheres is a fact of extraordinary interest. An extension of the test
was made in different concentrations of this salt, in which it was found
that at 19° to 20° C., swellings of 82 and 95 per cent only were obtained
in a solution of 0.01 M and in other tests of the original concentrated
solution; 3 M gave an increase of 230 per cent on one trial and 106
per cent on another at the above temperatures. This maximum was
obtained from sections 1 mm. in thickness, and it is suggested that
they were in a state of undue compression. Similar sections swelled
180 per cent in a 2.7 M solution of potassium nitrate capable of exert-
ing an osmotic pull of 84 atmospheres.1 Thicker sections in the potas-
sium solutions of this concentration swelled only 62 per cent and gave
an expected greater swelling of 94 per cent in a 0.01 N solution. The
maximum swelling in the concentrated calcium solution suggests that
compounds of the calcium with the pectin may be formed of higher
hydration value, which makes possible these unexpected imbibition
reactions. Such swellings, however, have been found hi the case of
colloidal mixtures which simulate the action of the plant in concen-
trated solutions of potassium nitrate.

1 MacDougal and Spoehr. The behavior of certain gels useful in the interpretation of the ac-
tion of plants. Science, 45:484-488. 1916.

102

Hydration and Growth.

The cell-sap of Echinocactus grown at the Desert Laboratory has a
low content of solid matter, and shows osmotic pressures of 3 to 5
atmospheres, calculated by cryoscopic methods. The acidity of the
massive body is greatest in the external layers and decreases toward the
center, and also shows daily variation which is greatest in the exter-
nal layer. This is illustrated by the data in table 81, obtained by
Mr. E. R. Long, in which A designates the external layer and D
the innermost parenchyma, B and C being intermediate.1

TABLE 81.

Plant.

Weight.

Dates and
hours.

Sample.

Afternoon.

Morning.

Dry wt.

Acidity.

Dry wt.

Acidity.

kg.

No. 27.

20

June 30-

A

11.9

0.376

10.0

0.342

July 1. . .

B

10.7

.269

9.0

.278

4 p. m.,

C

7.7

.154

7.5

.166

8 a. m. . .

D

6.7

.143

7.1

.166

The freshly expressed juice of regions C and D of such a plant at
midday caused sections of a biocolloid consisting of 5 parts agar, 3
parts mucilage of Opuntia, and 1 each of gelatine and bean protein to
swell about 840 per cent at 20° C., while similar sections increased
about 2,500 pe^ cent in distilled water. Dried slices of the paren-
chymatous tissue similar to that from which the juice was expressed
swelled 115 per cent in the juice, while they increased but 42 per cent
in distilled water at the same temperature.

Some of the sap of Echinocactus caused dried slices of Opuntia, the
swelling of which has been described in detail in Chapter VII, to swell
372 per cent at 20° C., which is to be compared with 550 per cent in
distilled water. It is notable, however, that such dried sections of
Opuntia increased 325 per cent in the sap expressed from living joints
of the same kind, thus swelling less in its own sap than in that of
Echinocactus. It seems probable that the possibilities of parasitism
might be more profitably sought in these hydration relations rather
than in the simpler osmotic coefficients to which the author attributed
great importance in his original study of this matter.2

It is highly probable that any cell-mass would absorb water and
swell in fresh sap from other cell-masses of the same kind in an equiva-
lent condition. Kunkel placed spores of Monilia sitophila (Mont) in
the sap of equivalent spores and obtained no plasmolytic effects and
did not measure for swelling. When the sap was reduced to one-tenth

1 Long, E. R. Acid accumulation and destruction in large succulents. The Plant World, 18:
No. 10, 261. 1918.

2 MacDougal and Cannon. The conditions of parasitism in plants. Carnegie Inst. Wash.
Pub. No. 129, 1910. See also MacDougal, The beginnings and physical basis of parasitism. The
Plant World, 20: p. 238. 1917.

Water Deficit or Unsatisfied Hydration Capacity. 103

of its volume by boiling, spores immersed in the concentrate plasmo-
lyzed, as might be expected. The temperature implied would of course
cause many changes in the constitution of the sap. The fresh juice
was seen to cause plasmolysis in vegetative cells of Spirogyra setiformis.1

In any consideration of the general facts which are brought into a
discussion of permeability in the experimental laboratory, one of the
most disturbing features is the frequency with which results are en-
countered which can not be duplicated. The effects of phlorizin on
the diffusion of sugar is an example of such a matter. Preparations
of the fresh and turgid inner layers of onions prepared by Dr. H. A.
Spoehr were found to contain an amount of sugar suitable for experi-
mentation, and when such sections were placed in distilled water for
a short period the amount of sugar extracted was fairly equivalent
to that coming out of other sections placed in the solution of potas-
sium carbonate and phlorizin (0.02 M) customarily employed in the
experiments with this glucoside, and also to the amount of sugar
which was found to diffuse out of sections placed in the potassium-
carbonate solution alone. Wachter believed he had proved that the
addition of a trace of potassium salt to water would prevent the
diffusion of sugar from- tissue of onions.2

Sections of the layers of onion bulbs were placed in distilled water
and in solutions of phlorizin and potassium carbonate, to ascertain
to what relative extent they might influence hydration. Trios of sec-
tions having an average thickness of about 2.3 mm. were prepared
for measurement under the auxograph and a series of three prepara-
tions were swelled at 20° C. The set in water increased 1.5 per cent,
that in the potassium carbonate 4.3 per cent, and the one in potas-
sium carbonate and phlorizin 9 per cent, the maximum amount.

In the repetition of the above with other material the results were
somewhat different. It was found that trios at 20° C. swelled 4 to 8
per cent in water, 7 to 11 per cent in the potassium-carbonate solutions,
and between 5 and 6 per cent in the solution of phlorizin and potas-
sium carbonate. Such results are indicative of an inequality of the
material, but it is evident that not only does potassium carbonate
not prevent the diffusion of sugar from the onion, but that a hydra-
tion of greater amount may occur in its presence than in water alone.
Nothing could be determined as to the action of the phlorizin on plant
colloids with relation to sugar.3

It has also been found impossible to bring the results of theoretical
antagonisms of substances taken up by protoplasm into harmony
with the results of hydrations made in the present connection, a matter

1 Kunkel, Louis Otto. A study of the problem of water absorption. 23d Ann. Rep. Missouri
Botanical Garden, pp. 26-40. 1912.

* Wachter, W. Untersuchungen ileber den Austritt von Zucker aus den Zellen der Speicher-
organe von Allium cepa und Beta vulgaris. Jahrb. f. wiss. Bot., 41: 165. 1905.

3 See Brooks, S. C. Permeability of cell-walls of Allium. Bot. Gaz., 64:509. 1917.

104 Hydration and Growth.

which for the present may be ascribed in part to the fact that generaliza-
tions on permeability rest upon results obtained with a narrow range
of material or under highly specialized conditions. It would seem
allowable to assume, if antagonistic effects are to be attributed to
the opposed action of two salts, that sections of living plants would
show differentiating swelling capacities in balanced solutions and in
their two components. The first trial of this matter was made with
terminal internodes of Mentha spicata, which had attained about half
of their final length and had an average diameter of 3 mm. The
imbibition capacity of this plant in living and dried conditions has
been described elsewhere in this paper. An estimation by Professor
H. M. Richards made the approximate acidity of the "pure juice"
such that 1 c.c. = 0.45 c.c. N/20 KOH, and that the total acidity of a
gram of fresh material was equivalent to 0.64 c.c. N/20 KOH.

A few simple tests were planned which might furnish results having
a bearing upon the present question. The neutralizing or balancing
action of sodium and calcium being one of the commonest conjunctions,
the action of these two elements in the form of chlorides were tried.1

The balanced solution, consisting of 100 parts of 0.375 M sodium
chloride and 10 parts 0.195 M calcium chloride was used. Such a
solution plasmolyzes cells of Spirogyra, but according to Osterhout's
findings, the presence of two salts in the proportions given prevents
either from passing the membrane. It would appear that Osterhout
in later papers takes the position that the essential feature of antagon-
ism between two substances consists in the fact that they produce
opposed effects upon it.2

Sections of stems of Mentha, as described above, swelled about
0.075 mm. in distilled water, shrunk 1 mm. in the balanced solution,
but came back to a volume slightly greater than the initial size when
distilled water was run into the dish. The shrinkage in the solution
of sodium chloride, 0.375 M, was very marked, dilution being fol-
lowed by a resumption of the original volume. Similar effects were
obtained with the solution calcium chloride.

The terminal parts of growing stems of an Erigeron, which were
higher in acid than Mentha, were next tested. The sections had a
diameter of about 3.7 mm. and included a length of 5 to 7 mm. of the
stem. The balanced solution and its constituents were diluted to
one-thirtieth of the above concentration to avoid shrinkage by plas-
molysis. The swelling in distilled water (average of 3 sections by
auxographic method) was 0.2 mm.; in the balanced solution, 0.15
mm.; in sodium chloride (6 sections), 0.2 mm.; and in calcium chlo-
ride (6 sections), 0.15 mm. These minute swellings progressed for a

1 Osterhout, W. J. V. The permeability of living cells to salts in pure and balanced solutions.
Science, 34: 187-189. 1911.

2 Osterhout, W. J. V. Nature of antagonism. Science, 41 : 255-256. 1915.

Water Deficit or Unsatisfied Hydration Capacity.

105

period of 9 or 10 hours (inclusive of the action of distilled water).
The sections then slowly shrunk at rates which would carry them back
to original volume in a similar period. The swelling in the balanced
solution was equivalent to the effect of its calcium, while the sodium
alone gave a greater swelling than the balanced solution. The swell-
ing in this salt was as great as in distilled water.

Sections of young joints of Opuntia have been subjected to a wide
variety of tests, and a set of these was placed under conditions similar
to those noted for Erigeron. The swelling in distilled water was 7.1
per cent, dilute balanced solution 8.7 per cent, sodium chloride 7.2
and 8.2 per cent, and in calcium chloride 7.7 and 8.8 per cent. The
small differences in the proportionate swelling do not appear to have
any bearing on possible antagonisms.

The masses of tissue used in the above tests were complex as to
composition and doubtless already contained some of the salts of the
balanced pair. It seemed important to test the effect of antago-
nistic salts on a biocolloid inclusive of bean protein, and a simpler one
in which agar was combined with glycocoll. The results of the meas-
urements were as shown in table 82.

TABLE 82.

Agar 90 parts,
bean protein 10 parts,
0.23 mm. in thickness.

Agar 90 parts,
glycocoll 10 parts,
15 mm. in thickness.

Distilled water

p. ct.
569.6
195.6
195.6
228.3
195.6

p. ct.

p. ct.
1,133 3

p. ct.

Balanced solution

239.1

466.6
400
533.3
333.3

533.3

Calcium chloride, 0.195 M. .
Sodium chloride, 0.375 M. . .
Sodium hydroxid, 0.01 M . .

The amount of imbibition hi the balanced solution in both colloids
was less than half that in distilled water, and was not much different
from that in the sodium chloride. The amount of imbibition hi the
calcium component used separately was less than that in the sodium
chloride alone. No aspect of the above experimentation seems to
promise anything of importance concerning the nature of the external
layer of the cell, differential action of which to solutions of various
kinds is so largely a matter of assumption. In fact, but little of the
evidence obtained by the extended experiments described in this
volume requires such a conception for its explanation, a conclusion
previously set forth very clearly by Kunkel after an experimental
examination of some features of the reactions of cells supposedly
associated with semi-permeable membranes.1

1 Kunkel, Louis Otto. A study of the problem of water absorption. 23d Ann. Report,
Missouri Botanical Garden, pp. 26-40. 1912.

106 Hydration and Growth,

The swelling of dried sections is clearly under conditions exclusive
of the action of a semi-permeable membrane, except in so far as
the cell-walls may show such properties. If desiccation resulted in
simple loss of water such as that which ensues in an unsalted plate
of biocolloids, the action of living and dried material might be expected
to be identical. The presence of salts and acids, however, causes some
irreversible changes, and the relative swelling of dried sections in various
solutions is different from that of a series obtained from the use of liv-
ing cell-masses. That the cell does act as an osmotic machine is estab-
lished beyond all question. That it is an enormously complex system
of osmotic sacs is well established. That the differential action of the
specialized layers formed at all phase boundaries can be made to
account for the entire relations of the protoplast is more than doubtful.
While all hydration in the broadest sense, including osmotic action,
ultimately depends upon molecular affinity, it is evident that the con-
ception of the semi-permeable sac does not offer a suite of possibilities
which may account for the range of action of the protoplast.

A second proposal is that which has been most clearly outlined by
Dr. E. E. Free, based upon the general acceptance of specialization of
colloidal conditions in the external layer of the protoplast, but explains
its action upon changes in the relations of the colloidal phases in it.1

Shrinkage of such a cell would follow a change in the dispersion or
phase relations of the external layer, and might be accompanied by the
solvation or passage out of the cell of substances which would reduce
its water-holding capacity still further. The explanation based on
osmosis might account for the escape of the free liquid of the vacuoles,
or from syncretic cavities, but some mechanism such as that suggested
by Free is necessary to account for the lessening of the amount of
water held by the cell colloids.

Some further evidence on the matter of extraction of substances
from colloidal masses and the action of contained substances on desic-
cating colloids remains to be considered. The facts which seem to
warrant the prolongation of the discussion were obtained by a com-
parison of the results of swelling of living and dried sections, with
determinations of their acidity, measurements of substances extracted,
and measurements of repeated swellings of the same material.

These treatments as applied to median slices of maturing joints of
Opuntia discata grown at Carmel gave measurements at 18° C. as
shown in table 83.

The second swelling of dried sections which had been once swelled
(extracted) indicate that some material of high hydration capacity had
been dissolved out, as the sections which had been simply dried swelled
25 times as much in potassium nitrate. Material which was dried
directly without extraction did not undergo any lessened hydration

1Free, E. E. A colloidal hypothesis of protoplasmic permeability. Plant World, 21:141. 1918.

Water Deficit or Unsatisfied Hydration Capacity.

107

capacity by swelling, as it swelled a second time to the same amplitude,
except in the acid solution.

TABLE 83.

Opuntia discata.

Living
median
slices.

Same sections dried.
Swelling calculated
on basis of
original thickness.

Dried median slices not
previously treated.

First swelling.

Second swelling
after drying.

Distilled water

p. ct.
11.4
6.4
6.6
9.2

p. ct.
17.5
26.4
24.5
22.4

p. ct.
430
357
315
541

p. ct.
430
250
352
541

Citric acid, 0.01 N

Potassium hydroxid, 0.01 M.
Potassium nitrate, 0.01 M . .

TABLE 84.

Opuntia discata,
median slices.

First
swelling.

Second
swelling.

Distilled water

p. ct.
225

p. ct.
100

Citric acid, 0.01 N

225

107

Sodium hydroxid, 0.01 M . . .
Potassium hydroxid, 0.01 M .

281

287.5

87.5
137.5

TABLE 85.

Swelling of fresh material implies bursting of cells by combined
imbibition and absorption and the consequent escape of the mucilages
which would not occur hi the hydration of dried sections, and these
would consequently, in reswelling, regain their original dimensions, or
repeat the first swelling.1

That this action depends
on the condition of the cell-
masses was demonstrated by
the fact that another series
of sections of the same plant
which included the chloro-
phyllose layer and the epi-
dermis did not show such du-
plication of results on the
second swelling. The in-
creases at 16° to 18° C. were
as shown in table 84.

It would be unsafe to as-
sume that such a result is
associated only with a chloro-
phyll-bearing tissue,as median
slices of a second opuntia
with a smaller proportion of
mucilage gave similar swell-
ings at 16° C., as shown in table 85.

The lessened swelling in this case can not be attributed to the escape
of pentosans from bursting cells, and attention naturally is directed
to the readily diffusible amino-acids, the presence of which facilitates
hydration in a remarkable way.

The swelling of the pentosan agar, which has been used so widely
in the imbibition measurements in connection with growth, would be

1 MacDougal and Spoehr. The solution and fixation accompanying swelling and drying of
biocolloids and plant tissues. Plant World, 22: June 1919.

Opuntia discata,
median slices.

After
first
drying.

After
second
drying.

p. ct.

p. ct.

Distilled water

361

42

Citric acid, 0.01 N

306

56

Potassium hydroxid, 0.01 M .

250

100

Potassium nitrate, 0.01 M . .

325

75

108 Hydration and Growth.

accompanied by the solution or dispersion of material from the exter-
nal part of the sections and by the diffusion of whatever salts or acids
might be present in the interior of the mass. Sections 0.25 mm. in
thickness swelled 2,420 per cent in water, and material equivalent to
200 similar sections, weighing 1.6731 g., placed in water for the same
length of time, lost 0.2570 g., or 15 per cent of the total. A similar
test of sections composed of 8 parts agar and 2 parts gelatine showed a
swelling of 1,684 per cent, with a loss of 18 per cent of the original
weight by dispersion or solution in the water.

Sections of Opuntia swelled in water for 24 hours at 16° to 18° C.
lost 7 per cent of the average total dry weight of similar sections. As
much of the dry weight is insoluble cell-wall, it is to be seen that
the actual percentage of soluble or diffusible material extracted was
large, a fact which would readily account for the lessened reswelling of
sections.

A single effort was made to ascertain to what extent the acids are
extracted in the hydration of living cell-masses of Opuntia and in dried
sections of the same. Determinations by Professor H. M. Richards of
the acidity of the water in which fresh slices of the Opuntia were swelled
showed that this might be expressed as follows: 10 c. c. solution from
dish in which set of fresh sections were swelled in water = 0.44 N/20
KOH.

Dried slices of the above material, when swelled in water 24 hours,
gave a solution the acidity of which might be expressed as 10 c. c. of
solution = 0.10 N/20 KOH.

When such sections were immersed in citric acid 0.01 N, the strength
of the solution was increased so that at the end of 24 hours the acidity
was expressible as 10 c. c. of solution = 2.25 N/20 KOH.1

The extraction of acid from the fresh sections in water is marked
and is much greater in the acid solution. This action in setting
free the amino-acids would cause a loss in hydration capacity which
would become apparent on reswelling.

The chief interest in the present work is centered in the hydration
of protoplasm associated with growth The development of embryonic
cells from the stage of a highly granular, dense colloidal mass with a
large nucleus to maturity is characterized by the migration of pro-
teinaceous material from the nucleus into the remainder of the mass;
by the formation of syncretic cavities, including those designated as
vacuoles; and by a constantly varying metabolism which results in
continuous alterations in the composition of the vacuolar fluids, and
in the composition and hydration capacity of the protoplasm.2

The peripheral part of the colloidal mass is probably of greater
density than the interior, and it is the behavior of this layer which

1 MacDougal, Richards, and Spoehr. The basis of succulence in plants. Bot. Gaz., 67:405.
1919.

2 See Thoday, D. On turgescence and the absorption of water by the cells of plants. The
New Phytologist, 17: 108. 1918.

Water Deficit or Unsatisfied Hydration Capacity. 109

gives rise to most of the phenomena known as permeability and osmosis.
The external layer, as well as the entire colloidal mass, reacts to solu-
tions in a manner determined by its composition and its history,
especially with respect to the salts which have come into the cell since
its formation, and particularly to the salts which may be dissolved in
the water in which the cell is immersed. The absorption of such salts
alters the capacity of the colloids in various ways. In the case of the
alkaline solutions, it has already been pointed out that nearly all plant
tissues show a long-continued swelling which may be reasonably
ascribed to the formation of a salt between the sugar and the hydroxide,
which salt has a greater capacity for hydration than the carbohy-
drates alone.

Acids decrease the hydration capacity of agar and of the pentosans
which enter into the composition of certain types of plant cells, of
which the tissues of the potato would be a good example.

The death of cell-masses by desiccation probably produces less dis-
arrangement of the material of the cell than any other method of
killing, and not only the colloidal mass, but its external layer, probably
retains its condition with respect to phases with but little serious
disturbance. The only positive movement which might be recog-
nized as of importance in connection with the point now being
discussed would be that as drying-out proceeds the salts and acids
of the mass would pass toward the periphery and might accumulate
at that place, although it could by no means be assumed that such
action would result in a uniform deposition in the membrane. The
hydration of such dried material would take place as in other dead
material, being largely by means of imbibition and adsorption, with
osmosis by the action of vacuolar material almost eliminated. The
records of many measurements of dried plants, of which fresh and
living portions have been tested, as described in this chapter, are
meant to be explanatory and illustrative, but they show conclusively
that any serious development of our conceptions of the water-rela-
tions of cell-masses must be based upon and take seriously into con-
sideration the hydration processes of the protoplastic colloids.

Colloids are never at rest, yet it is possible to secure combinations
of conditions in mixtures in which hydration, for example, is all but
complete. Protoplasm, however, is the seat of complex transforma-
tions and is the medium of such diverse diffusion movements that
ideally it is never in a condition of satisfied hydration. The amounts
which it may take up from different solutions and under various con-
ditions previously described may, on proper analysis, serve to show
the nature of the protoplastic colloid with respect to its principal
components. The determination of the index of unsatisfied hydra-
tion, or the water deficit, is an indispensable feature of any serious
effort to analyze growth into its physical components.

IX. TEMPERATURE AND THE HYDRATION AND GROWTH
OF COLLOIDS AND OF CELL-MASSES.

Living material is a colloidal mass consisting of a mixture of
colloids, the most important components of which are carbohydrates
and proteins or protein derivatives. Salts of sodium, potassium, and
magnesium in various combinations are dissolved in the system. The
denser portions of the protoplasm, including all of the continuing
structures, or those of morphological rank, have the properties of an
elastic gel with the general structure of a fine sponge or a honeycomb
with irregularly broken or incomplete walls. The materials which
make up this fairly continuous structure are also in a disperse or liquid
condition in the cavities and may even fill large syncretic spaces in the
general structure.

The essential feature of growth consists in the accretion of material
entering into this colloidal structure, its hydration, and its arrangement
into additional structures or portions of honeycomb. This may be
pictured as taking place by an initial increased dispersion or enlarge-
ment of the colloidal network to a point where new masses of gel would
be formed in the liquid phase of the existing mesh. Considering living
material as an intimate mixture of minute particles of its main colloidal
components (and the scanty evidence on this matter is to the effect
that the carbohydrates and proteins do not diffuse into each other), it
is on this basis to be assumed that the new material would accrue to
these separately and in a characteristic and differentiated manner.

Aggregations of the introduced material might also take place in
syncretic cavities, with opportunity for the development of specialized
structures. The absorption and diffusion of material in liquid form
and its diffusion into the colloidal mass would in all cases be the initial
step in growth. The consequent swelling with all of its accompani-
ments and consequences in cell-masses of plants and in my biocolloidal
mixtures has been found to depend upon the character and the pro-
portion of the proteins or protein derivatives in the colloid, the pro-
portion of the pentosans, and the amount of salts present. In addition
to these features, which change but slowly, the active metabolism of
the growing cell includes respiration in which substances such as sugars
may be adsorbed on surfaces of aggregates of enzymes which catalyze
them, acids occurring at certain stages of the resultant reactions.
Acidity in a growing cactus, for example, may vary between a value of
0.1 N malic acid and one-twentieth of this amount during the course
of a daylight period, causing very marked changes in the imbibition
and absorption of the cell-colloids.

The exposure of a growing or swelling colloid to different tempera-
tures has several effects: First, the rate of absorption and diffusion of

110

Hydralion and Growth of Colloids and Cell-masses.

Ill

water is modified, being generally accelerated by a rise within the range
of ordinary temperatures at which plants grow. Next, adsorption is
affected in a contrary manner, but the complex series of reactions
associated with respiration are speeded up, with consequences far too
complex to be characterized here. The first step, that of absorption
and diffusion, would be far simpler, as the rate of acceleration here
would be nearly identical with that of the diffusion of the same material
in water.

A determination of the effect which temperature might have on
growth necessarily takes into account, first, the fundamental increase
which might accrue from simple absorption under various conditions.
In recognition of this fact, it was arranged to carry on swellings of
some of the biocolloids in order to gain some appreciation of the im-
bibition factor in growth. Sections 0.2 mm. in thickness of plates of
agar 90 parts, bean protein 10 parts, and culture salts 0.85 per cent
were swelled at 15° C. and at 22° C. The rate of swelling may be
appreciated by a consideration of the total amounts at the end of 4
hours, 8 hours, and at the end of the test (fig. 19).

A p.m.

8p.m

12p.m. A a.m.

8a.m.

1

T

. I5°C.

22°C.

30°C.

39'C.

[\

FIG. 19. — Tracing of auxographic record of swelling of dried plates 0.2 mm. in thickness of a
salted "biocolloid" consisting of 9 parts of agar, 1 part bean protein, and 0.015 part
nutritive salts. Increase is denoted by downward movement of the pen, amplified 20 times.
The initial rate and continuance of the swelling at various temperatures may be compared.

TABLE 86. — Swelling of mixture of agar, bean protein, and cultures in distilled water.

Swellings, per cent.

Averages.

4 hours (15° C.) .

800

875

838

8 hours (15° C.) .

1,150

1,075

1,113

22 hours (15° C.) .

1,325

1,325

1,325

4 hours (22° C.) .

1,325

1,450

1,388

8 hours (22° C.) .

1,400

1,700

1,550

22 hours (22° C.) .

1,900

1,900

1,900

The hydration during the first 4 hours includes any process of
chemical union of water with the colloidal material by which water
in definite proportions enters into the molecular aggregates. The
greater part of this action probably takes place within the first half

112

Hydration and Growth.

hour of immersion, with consequent great rapidity of swelling. It is
not possible to separate this action from adsorption, which follows more
slowly, but if the first 4-hour period be considered, it is to be seen that
the total rises from about 9 to 14, with a change from 15° to 22° C. at
which the immersion was made. If the first 8 hours is considered, the
hydration as expressed by the total swelling is as 11 to 16 at the two
temperatures and as 13 to 19 for the entire period of hydration, a ratio
which includes the accelerating effects of a rise in temperature both on
the initial chemical action and the continued absorption.1

A biocolloid without salts consisting of 9 parts agar and 1 part oat
protein was now tested for comparison, with results shown in table 87.

TABLE 87.

Swellings, per cent.

Averages.

4 hours (15° C.) . . .

1,111 1,222

1,167

8 hours (15° C.) . . .

1,417 1,500

1,459

23 hours, final(15°C. .

1,722 1,866

1,794

4 houra (23° C.) . . .

1,278 1,333

1,306

8 hours (23° C.) . . .

1,944 1,861

1,903

23 hours (23° C.) . . .

2,528 2,444

2,486

The swelling during the initial period of this salt-free mixture is
actually greater than that of the first mixture, but is affected in far
less degree by the rise in temperature, the ratio at 15° C. and at 23° C.
being as 12 to 13. The difference, if the 8-hour period is considered,

FIG. 20. — Tracing of auxographic records of swelling of plates 0.18 mm. in thickness, consisting
of 9 parts agar and 1 part oat protein, a " biocolloid " with very high hydration capacity.
The 5 mm. and 4-hour intervals of the record sheet are shown. Increase is denoted by the
downward movement of the pen, which amplifies the actual swelling 20 times. The varia-
tion in initial and following rates and time necessary for hydration are shown.

was as 16 to 10, and for the 23-hour period, as 18 to 25. It is suggested
that the chemical action during the beginning stages of swelling is far
less important than in the salted mixture and that the entire set of
reactions is one in which absorption and hydrolysis play the determin-

1 MacDougal. The relation of growth and swelling of plants and biocolloids to temperature.
Proc. Soc. Exper. Biol. and Med., 15:48. 1917.

Hydration and Growth of Colloids and Cell-masses.

113

ing r61e. A much more pronounced effect, however, was secured at the
temperature of 31° C. (fig. 20).

Plates of agar 90 and oat protein 10 parts which were 0.16 mm. in
thickness were swelled at a temperature of 31° C. at intervals of 2
hours or more, with the results given in table 88.

TABLE 88.

Swellings, per cent.

Averages.

4 hours

2,031
2,556
2,906

2,312
2,968
3,250

2,172
2,763
3,078

8 hours

20 hours

The acceleration, as expressed by the total during the first 4 hours,
was as 13 to 22 in the rise from 23° C. to 31° C., as 19 to 28 when the
first 8 hours is considered, and as 25 to 38 when the entire swelling is
taken into account. Raising the temperature to 39° C. gave swellings
as shown in table 89.

TABLE 89.

TABLE 90.

Agar 90, oat protein 10.

4 hours

Swellings.
p. ct. p. ct.
2,361 2,722
2,555 3,166
2,611 3,222

Average,
p. ct.
2,542
2,861
2,917

8 hours

12 hours

Agar 90, oat protein 10.

Swellings.

p. ct.

4 hours

2,555

8 hours

2,833

10 hours

2,888

The acceleration accompanying this rise of 8° C. was followed by
an increase which was as 22 to 25 during the first 4 hours, 28 to 29
during the second 8 hours, and the hydration for the entire period was
actually less at this higher temperature. That the hydration passed
beyond the limits of acceleration and of total water-holding capacity
in this temperature region was denoted by the fact that swellings at
46° to 47° C. were as given in table 90.

TABLE 91.

Swellings.

Averages.

4 hours

1,444
1,666
2,083

1,500
1,805
1,972

1,472
1,735
2,022

8 hours

20 hours

It is to be seen that while the final capacity for swelling at 15° C.
had not been reached in 20 to 22 hours, it was practically complete
at 39° C. in 12 hours, as the continuance of the measurement would

114

Hydration and Growth.

have detected a very small expansion, while satisfaction was nearly
complete in 8 hours at 46° to 47° C.

We may now profitably turn to an extension of the reactions of the
salted colloids (see p. 111). Sections of agar, bean protein, and nutrient
salts 0.18 mm. in thickness gave the expansions shown in table 91 at
31° C.

The temperature series was taken another step by raising the air
in the chamber to 42° C., at which point the liquid in the dishes showed
38° to 39° C., being cooled by evaporation to this point. The measure-
ments are given in table 92.

TABLE 92.

Agar 90, bean protein 10, nutrients salts 0.85.

4 hours

Swellings
p. ct. p. ct.
2,381 2,611
2,638 2,833
2,693 2,888

Averages,
p. ct.
2,486
2,736
2,791

8 hours

12 hours

The increase of the temperature of the air in the chamber to 52° C.
gave a temperature of 46° to 47° C. to water in the dishes. The swell-
ings at 46° to 47° C. are shown in table 93.

TABLE 93.

Agar 90, bean protein 10, nutrient salts 0.85.

4 hours

Swellings,
p. ct. p. ct.
2,000 2,305
2,194 2,500
1,111 2,500

Averages,
p. ct.
2,153
2,347
2,361

8 hours

10 hours

A review of the action of the salted mixture shows a very slight
acceleration by the rise in temperature from 22° C. to 31° C. and also
a total but slightly increased above that of the lower temperature.
The next step, from 31° to 39° C., however, was one marked by a
distinct acceleration, the rates during the first 4 hours being as 15 to
25, during the first 8 hours as 17 to 27, and the final total at the lower
temperature at 20 hours was as 20 to 28 at the higher temperature at
the end of 12 hours.

The absorption of water fell off at temperatures above 39° C., which
may be regarded as in the region of the optimum or maximum water-
holding capacity, which is somewhat lower than that of the salt-free
mixture. Many repetitions of the tests would be necessary before the
matter of the failure to show expected increase of hydration between
23° and 31° C. was accepted as a reality. The measurements in ques-

Hydration and Growth of Colloids and Cell-masses.

115

tion, however, suggest that even in hydrating colloids, in which meta-
bolism is not in progress, abrupt modifications may occur by the con-
junction or disjunction of two important reactions of the constellation
present.

The chamber in which these tests were made was under ground,
with earthen and board walls, and was reached through an entrance
chamber. Electric heaters were used. The chamber was 2.7 by 2
by 2.2 meters and it was necessary to enter and work in it when making
the tests. The air was stirred by a fan and a mercurial thermometer
suspended from the ceiling showed air temperatures several degrees
above that of the water in the dishes. Thus, in the preceding test,
the observer was in a humid atmosphere at 55° C. (131° F.) when
the readings of the swellings were 48° to 49° C.

The compilation of averages (table 94) affords a ready means of
comprehension of the essential features of the entire series.

TABLE 94.

4 hours.

8 hours.

20 to 22 hours.

Agar 90, bean protein 10,

culture salts 0.85 :

p. ct.

p. ct.

p. ct.

15 to 16° C, 0.20 mm.

888

1,113

1,325

21 to 23° C, 0.20 mm. . .

1,388

1,550

1,900

30 to 31° C., 0.18mm.

1,472

1,735

2,022

39° C., 0.18mm

2,486

2,736

2,791 (12 hours).

46 to 37° C., 0.18 mm..

2,163

2,347

2,361 (10 hours).

48 to 49° C., 0.18 mm..

2,361

2,514

No swelling after 8

hours.

Agar 90, oat protein 10:

15 to 16° C., 0.18 mm.

1,167

1,459

1,794

22 to 23° C., 0.16mm.

1,388

1,550

1,900

30 to 31° C., 0.16 mm.

2,172

2,763

3,078

38 to 39° C., 0.18 mm.

2,541

2,861

2,917 (12 hours).

46 to 47° C., 0.18 mm.

2,555

2,833

2,889 (10 hours).

48 to 49° C., 0.16 mm.

1,906

2,031

No swelling after 8

hours.

A series of swellings of agar sections 0.18mm. in thickness, made
at the same time, affords valuable data for comparison with the in-
creases of the complex biocolloids (table 95).

The hydration reactions of the agar are not so positive and uniform
as those of the more complete systems. Increase by absorption had
not reached a positive final in 24 hours at the lowest temperature. A
similar stage of satisfaction was evident in half this time at about
40° C. and in 8 hours at 49° C. The rate of swelling is graphically
illustrated in figure 21.

The maximum or " optimum" of swelling of such agar plates occurs
at some temperature near 40° C. Initial rate and total increase are
greatest at this point. The maximum swelling of the agar, bean pro-

116

Hydration and Growth.

tein, and salt mixture lies below 46° C. and is probably above 40° C.,
as both rate and total capacity become uncertain at 46° C. and above.

The agar-oat protein mixture has a higher initial capacity at 46°
to 47° C., but does not appear to absorb as much water as it did at

TABLE 95.

Swelling of agar.

4 hours.

8 hours.

10 hours.

12 hours.

16 hours.

20 hours.

24 hours.

18 to 21° C .

875
806
944
1,000
1,056
1,292
1,472
1,750
1,611
1,306
1,333

961
889
1,083
1,083
1,167
1,389
1,556
1,778
1,667
1,389
1,417

1,069
1,000
1,139
1,278
1,208
1,417
1,583
1,833

1,111
1,054
1,168
1,333
1,250
1,444

1,153
1,083
1,222

27° C

29 to 31° C

39 to 41° C

40 to 41° C

1,708

46 to 47° C

1,416

48 to 49° C

Averages.
18 to 21° C ...

875
1,028
1,611
1,320

978
1,125
1,667
1,403

1,069
1,244
1,708

1,111
1,222

1,153

27° C

39 to 41° C

46 to 49° C

1,416

a temperature a few degrees lower. The probable error at the high-
est temperatures is great, however, and conclusions as to a separation
of initial rate and final capacity should be made with caution.1

The relation of temperature to swelling of agar and of these colloidal
mixtures is of interest because of the fact that they lie within the range
of activities of plants. The greater number of seed-forming plants

6p.m.

12 p.m.

8a.m.

FIG. 21. — Tracing of auxographic records showing swelling of plates of agar 0.18 mm. in thickness
at various temperatures. The 5 mm. and 4-hour intervals of the record-slip are shown.
Increase is denoted by the downward movement of the pen, which amplifies the actual
increase 20 times. The initial and following rates are well illustrated.

do not endure air-temperatures of above 45° or 46° C. The tempera-
ture of the stems or growing-zones in such exposures can not be cal-
culated from such data unless sunlight exposure is known. Actual
temperatures of the tissues have been taken by a few observers, from

1 See Freundlich, H. Kapillarchemie, 1909, pp. 504-511, and references given also Ostwald,
W., Transl. by M. H. Fischer. An introduction to theoretical and applied colloid chemistry,
pp. 84-92. 1909.

Hydration and Growth of Colloids and Cell-masses.

117

which it is seen that the cells may at times have a temperature 20° C.1
higher than the air. The previous maximum record is that of 51.5 °C.
for Euphorbia virosa, obtained by Pearson in South Africa in 1913.2

40

30

20

10

\

Neon

12 I Z3456789

5a.m. 6 7 6 9 10

Ss.m. 6

FIQ. 22. — Temperatures of joints of Opuntia in the open at Desert Laboratory on a day in March
and a day in June 1916. The solid line denotes air temperatures, the dotted line the temper-
ature of a joint in an east-and-west position with the faces north and south. The broken
line gives the course of the temperature of a joint with its faces east and west, and showing
a drop in temperature when the margin is toward the sun at midday. The upper part of the
figure is compiled from the records of March 9, 1916. The lower set of lines is compiled
from the records for June 2, 1916. Redrawn after diagram furnished by Dr. J. M. McGee.

A proper interpretation of the reactions of the plants used as experi-
mental material in the present work might be made only when the ordi-
nary exposures were measured, and this was undertaken at the request

1Askenasy, E. Ueber die Temperature, welche Pflanzen im Sonnenlicht annehmen. Bot.
Zeitung, 33:441-442. 1875. See also U 'sprung, A. Die physikalischen Eigenschaften der
Laubblatter. Bibliotheca Botanica, 12: (Heft 60), 68. 1903-4.

2 Pearson, H. H. W. Observations on the internal temperatures of Euphorbia virosa and Aloe
dichotoma. Annals of the Bolus Herbarium, vol. i, part 2. Nov. 1914.

118 Hydration and Growth.

of the author by Dr. J. M. McGee at the Desert Laboratory in 1916.
Mature joints of Opuntia were arranged vertically in two sets, one with
all in a meridional position and the other in an east-and-west position,
so that the edges only were exposed to the rising and setting sun. Sets
of two each were observed for short periods, in which the joints were
kept moving at a rate which kept the edges or the full surface exposed
to the direct rays of the sun during the entire day.1

The course of the temperature on two days is shown in figure 22.
The joints which were in a fixed north-and-south position showed a
dry weight slightly hi excels of those which were in an east-and-west
position. The temperature of the plants facing north and south rose
steadily until about 2 p. m., then began to decline, falling to a point
below that of the air soon after sunset. Joints facing east and west
rose in temperature until about 11 a. m., then a slight drop took
place until the sun shone on the western face of the joint, at which
time it began to rise, reaching a maximum at 4 p. m. It is evident
that the position of these members may through the temperature-
relation modify the rates of growth or of hydration. During midsum-
mer the mature joints may attain daily temperatures of 53° to 55° C.
The data obtained by the author show that such delicate members as
etiolated shoots, as well as green joints, may endure temperature of
51.5° C. and to show enlargement a degree or two below that point.
(See page 135.)

The newly developed method for determining temperatures of leaves
by a thermo-electrical method developed by Mrs. E. B. Shreve at the
Desert Laboratory promises to be of great use in the accurate deter-
mination of this important condition in plants.2

A series of measurements were made with joints of Opuntia grown
at the Coastal Laboratory, in which the degree of hydration was high,
so that the total possible swelling in fresh material was small, although,
as may be seen by reference to page 132, the increase of dried sections
was indicative of the presence of a carbohydrate-protein complex of a
high hydration coefficient.

Two recently matured joints grown in the open, and subjected to
air-temperatures of 16° to 22° C., were taken for each test. The sections
at the highest temperature had an average thickness of 9.4 mm. Those
used in the tests at 24° to 25° C. were about 12 mm. in thickness, and
the third lot had an average thickness of about the same.

Trios were placed hi glass dishes containing about 25 c.c. of liquid,
the total volume of the sections being about 3 c.c. Temperatures were
taken by mercurial thermometers, the bulbs of which were thrust into
the dishes from time to tune. The testing-chamber was not illumi-

1 McGee, J. M. The effect of position on the temperature and dry weight of joints of Opuntia.
Rep. Dept. Bot. Research, Year Book No. 15 for 1916, Carnegie Inst. Wash. 1917.

z Shreve, E. B. A thermo-electrical method for the determination of leaf temperature.
Plant World, 22: 100. 1919.

Hydration and Growth of Colloids and Cell-masses.

119

nated, except during the few minutes when examinations were being
made. The swelling measurements are given in table 96.

TABLE 96.

18 to 20° C.

24 to 26° C.

44 to 45° C.

Distilled water

p. ct.
11.8
14

8.8
7.3
12.2

hours.
42
42

32
20

44

p. ct.
13
14.4

13.2
7.8
13.2

hours.
40
42

42
14
42

p. ct.

7
8.4

4.9
4.9

5.8

hours.
5
7

25
2
4

Potassium nitrate, 0.01 M.
Potassium nitrate, citric
acid, 0.01 N

Citric acid, 0.01 N

Potassium hydroxid, 0.01M

The data given in table 95 yield a number of exceedingly interesting
suggestions as to the absorption and incorporation of water by living
sections in the presence of various substances. The time in which
saturation takes place varies widely in the range of temperatures
which is a normal one for this plant. Satisfaction of the water capacity
of the sections requires 42 hours at the lowest temperature and but 5
hours at the highest in distilled water. The change is from 42 hours
to 7 in potassium nitrate and from 32 to 25 in the acidified salt and from
20 to 2 in the acid. The living material already contains a normal
supply of both substances and immersion in them involves an increase
in their action. The action of the sodium hydroxid is, as has been
found in many instances, long-continued. The maximum swelling is
seen to be accomplished at about 25° C. The general optimum for all
of the solutions is, however, probably above this point.

The initial rate of increase at the highest temperature is great-
est, but it is soon checked. Etiolated shoots of the same species of
Opuntia were available and the results of the swelling measurements
obtained from them afford some interesting comparisons, although
it was not possible to run them as nearly parallel as might be wished.
These shoots were 18 to 25 cm. long and 1.5 to 2 cm. in width, some of
which were hi a chamber at 17° to 19° C. and others in a chamber in
which their temperature ranged from 30° to 31° C. Swellings were
made at both temperatures, with the results given in table 97.

The most striking feature of the results is the fact that the material
etiolated at the higher temperature shows a more extended and
greater swelling at the lower temperature. Unfortunately, the fact
that the limiting effects of acidity on imbibition increase with rise hi
temperature was not known at the time these tests were carried out.

If the case of the low-temperature material be taken up, it is seen
that its increase is greatest at the high temperature hi water, potas-
sium nitrate, acidified potassium nitrate, and acid, while the swelling
in alkaline salt and in hydroxid is greatest at the low temperature.

120

Hydration and Growth.

This fact suggests that the general optima of the plants grown at the
different temperatures differ in such manner that the one grown at the
higher temperature has the lower optimum, and conversely, that the
one grown in the cool chamber has its optimum at a higher temperature,
which is not necessarily at the point at which the tests were made.
The exceptions in the last case in the alkaline solutions may afford a
clue to the actual physical conditions upon which such differential

TABLE 97.

Opuntia etiolated at
17° to 19° C.

Opuntia etiolated at
30° to 31° C.

Swelled at
17° to 16° C.

Swelled at
30° to 31° C.

Swelled at
17° to 19° C.

Swelled at
30° to 31° C.

Distilled water

p. ct.
4.2
4.8
4.9
3.2

13.7
10.6

hra.
16
30
3
3

36
36

p. ct.
7.5
6.5
9.5
4

9.5

8

hrs.
1.5
6
1
1

9
1.3

p. ct.
13.2
12.7
7.3
4.4

hrs.
40
48
4
4

p. ct.
3.7
7.3
3.7
3.3

hra.
8
8
1
1

Potassium nitrate

Potassium nitrate, citric acid, 0.01 N
Citric acid 0.01 N

Potassium nitrate, potassium hy-
droxid, 0.01 M

Potassium hydroxid, 0.01 N

action rests. According to Jenny Hempel, the hydrogen-ion concen-
tration of etiolated lupine shoots was not much different from that of
normal green plants, although the actual quantity of acid was much
greater. The amount of acid in the plant grown at the higher temper-
ature would, hi accordance with general experience, be less than in
those grown at the lower temperature.1 The only suggestions available
for an explanation of the behavior in question would rest upon the as-
sumption that the amount of acid was the critical feature, and also
that the plants grown at higher temperature had experienced the con-
version of the polysaccharids into the pentosans, which have a rela-
tively high coefficient for swelling (see p. 91).

It is to be noted, in any consideration of the action of the colloidal
systems, that fresh or living sections of plants are already hi the con-
dition of the colloidal sections which have been immersed 4 to 8 hours,
and that it is the behavior of the sections after they have taken up
90 per cent of their total capacity for water which comes into the
realm of the living plant. Dried sections of plants come down to a
water-content not much above that of colloids. It also is to be remem-
bered that in the advanced stages of the desiccation of cell-masses the
protoplasmic colloids are subjected to the action of the dissolved elec-
trolytes in a concentrated condition in the final stages of drying. The
fixation of the salts and acids which takes place under these circum-

1 Hempel, J. Buffer processes in the metabolism of succulent plants.
Lab. d Carlsberg, 13: No. 1. 1917.

Compt. Rend. d. Trav. d.

Hydration and Growth of Colloids and Cell-masses. 121

stances would have such effect that the hydration of the sections would
not result in the exact resumption of the former condition of the cell-
colloids. The field of action of the colloid within the limits mentioned
offers a most promising group of phenomena, the study of which may
be expected to contribute in an important manner to knowledge of the
mechanics of protoplasm.

Adequate parallel measurements of the effects of temperature on
growth of Opuntia were not made, but some records are available
which show the variation caused by the rise upon material grown at
the lower temperature hi the above etiolated series. Elongation by
growth of the stems in question was at the rate of 5.2 mm. daily at
16° to 18° C. and 11 to 17 mm. daily at 30° to 32° C. The increase
amounted practically to a doubling for a rise of 10° C. The swelling
in transverse sections of similar material was 4.9 per cent at 17° to
19° C. and 7.5 per cent at 30° to 31° C. in distilled water; and 4.9 per
cent at the lower temperature in acidified potassium nitrate and 9.5
per cent at the higher temperature. The increase by swelling trans-
versely was therefore slightly less than double, with a fair inference that
it would have been greater in the axis of elongation or growth. It is
to be seen, therefore, that in the elongation of the vegetative axes of
plants the temperature effect is a complex one, and that the accel-
erating effect of rising temperature may be primarily an increase
in absorption capacity by altered metabolism, including lessened
accumulations of acids.

An illustration of the failure of rising temperatures to increase
hydration and swelling under some conditions is furnished by the
behavior of sections of joints of Opuntia taken in a condition of acidosis
hi the morning. These increased no more at 27° C. than at 20° C.
(see p. 119). The experiences with biocolloids must be drawn upon
with care when a parallel is sought. The sections of a biocolloid
containing the mucilage from Opuntia and bean protein were seen to
swell 2,400 per cent at 22° C., while the swelling was less than 1,800
per cent at a temperature 5 degrees higher, but the lower figure in this
case was probably due to the fact that the plates of colloidal material
began to break up and dissolve out at a lower stage of hydration,
thus ending the record.

Various tests of material were made for the purpose of ascertaining
the conditions prevailing among plants of different types. In mid-
April, sections were taken from the terminal elongation internodes
of Phoradendron growing near the Desert Laboratory, parasitic on
Parkinsonia microphylla. Such sections were about 3 mm. in length
and half that thickness. Swelling was 3 and 5 per cent in distilled
water at 20° and 30° C., respectively, but no appreciable difference
could be detected between the increases in hundredth-no mal citric acid
at the two temperatures.

122

Hydration and Growth.

The petioles of some young plants of a Solanum hybrid in the glass-
house at Tucson were available on April 21, 1918. Two series of sec-
tions were placed in distilled water and acid at 18° and 38° C., with
results shown in table 98.

TABLE 98. TABLE 99.

18° C.

38° C.

p. ct.

p. ct.

Distilled water

4.2

11.8

Citric acid, 0.01 N. .

4.2 .

2.6

18° C.

38° C.

a.

b.

a.

6.

p. ct.

p. ct.

p. ct.

p. ct.

Distilled water

14

8

9.6

11.7

Citric acid, 0.01 N.

11

9.7

6.6

4.4

The swelling in distilled water was nearly three times as great at the
higher temperature, while in the acid solution a retardation took place
which limited the total at the higher temperature to something over
a half that possible at the lower point. The total swelling in acid at
the lower temperature occupied an hour and at the higher temperature
it was a matter of 10 or 15 minutes. A similar speeding-up of imbi-
bition in water was observed. The total capacity at the lower tem-
perature was not reached for 8 or 10 hours, while at the higher it was
something under 2 hours.

Plants of Phaseolus which formed the experimental material for
measuring the growth of pods and seeds bore some pods in which the
beans were nearly mature. Pods of the same stage of development as
one which was under the auxograph for recording daily changes (see
p. 156) were opened and the unripe beans removed. The ends were cut
away and the outer coat removed. The remainder of each cotyledon
made one section, of which three were taken from separate pods for
swelling. The average thickness was 3.2 to 3.4 mm. and the swellings
of duplicate series were as given in table 99.

The higher temperature to which series a was subjected appears to
be above the point at which maximum absorption or imbibition takes
place in distilled water, as the swelling was 30 per cent less than at the
lower temperature. The retarding effect is much more marked in the
acid solution, however, as the reduction of the total capacity below
that shown at 18° C. amounted to 40 per cent.

The material in series b, taken at a later date and with seeds which
seemed to be more nearly mature, showed an increase in swelling in
distilled water of about 45 per cent over the total at the lower tempera-
ture, while the swelling in acid was less than half that at 18° C. The
average of the two series is such that the swelling in distilled water is
nearly the same at both temperatures, while in acid the average at
18° C. is 10.4 per cent, which is nearly double that at 38° C., at which
point the hydration capacity seems to be invariably lower than at the
lower temperature. These averages represent a total of 6 cotyledons
each.

Hydration and Growth of Colloids and Cell-masses.

123

A final test of variations in temperature upon material in an acidi-
fied condition was made with dried sections of Opuntia. These sec-
tions were made by slicing away the chlorophyllous layer from one
side of the flat joint and drying the remainder in the desiccator and in
sheets of blotting-paper in such manner that buckling and crumpling
were prevented. After all of these precautions were taken, however,
the measurement of the sections was subject to some error, due to the
fact that the fibrovascular strands remaining would increase the thick-
ness under the calipers without reacting in due proportion to the action
of the swelling agent. A wide range of figures was obtained, but it
was apparent that a rise in temperature did not have an effect on
material in acid equivalent to that in distilled water, as will be apparent
from the measurements obtained from sections which were 0.43 to
0.46 mm. in thickness (table 100).

TABLE 100.

Swelling at
18° C.

Aver-
ages.

Swelling at
28° C.

Swelling at
38° C.

Aver-
ages.

Distilled water

p. ct. p. ct. p. ct.
315 385 486

p. ct.
395

p. ct.
453

p. ct. p. ct.
500 413

p. ct.

457

Citric acid, 0.01 N

360 430 460

417

460

477 400

439

The increase in swelling in distilled water is seen to be about twice
that in the acid in the rise from 18° C. to 38° C. The influence which
the condition in question may exert on the rate of growth is obvious.
Thus the course of enlargement of an organ or of a cell-mass, in so
far as this consists in hydration, may vary widely in the first instance,
because of the residual acids in the colloids, and the balance or accumu-
lation of this will in turn depend upon the effect of the enzymic or
respiratory processes in metabolism. It is obvious that a rise of 10
degrees from the customary morning temperature of 15° C., which has
accompanied so many of these experiments, might result in an ac-
celeration of growth determined by the reduced acidosis of the plant.
A rise from the same temperature later in the day or under other con-
ditions of illumination would necessarily have a different result. An
extension of the attempts to bring rates of growth into a figure or
formula, therefore, would be a forced application of knowledge of one
process to a complicated procedure which results in no positive advance.
Variation of temperature results in modification of the rate of enzymic
processes and of the forms of metabolism included under and associated
with respiration, in modification of the rate of absorption of water by the
organism from its medium or substratum, and modification of the water-
holding capacity of the cell-colloids after a mode determined by their
carbohydrate-protein ratio and by their state of acidosis. The actual
increase in volume, will also be influenced to some extent by the continual
water-loss from the surface.

Hydration and Growth.

The conception of a temperature coefficient of growth must be taken
as an integration of the action of a constellation of forces acting upon
colloidal material of varying constitution.1 The agencies in question
do not run parallel in their effects and interlock hi the most intricate
manner.2 It is not surprising, therefore, to find that the coefficient of
temperature as applied to growth, which is usually calculated in terms
of relative effect for each variation of 10° C., has but little usefulness,
except between 10° and 30° C.2 The value of Q10 as it is usually written
varies between 1.12 and 5 or 6, and the variation in any given organism
is usually very great above 30° or 35° C. Thus, in my own experi-
ments with Opuntia, the actual range of growth was found to extend
from as low as 7° C. under some circumstances to 51.5° C. under others,
but no single individual was seen to grow throughout this range. The
combination of conditions which would enable it to do so are not likely
to occur in a state of nature and would be difficult to bring about
experimentally.

In addition to the complexities of interplay of molecular forces to be
reckoned with — and they appear most formidable to the chemist
familiar with their nature — the application of ratios or formulae to
variations in growth produced by temperature in the higher plants
encounters still other difficulties. Chief among these is the fact that
the growing region of a plant may vary in actual and relative amounts
of embryonic cell-masses and of fixed non-expanding tissues. External
measurements of elongation, even when applied to root-tips, may have,
in consequence, but doubtful value.

A brief description has been given on page 96 of the unsatisfied
water capacity of the conns of Brodicea, from the apices of which two
or three leaves 20 to 30 cm. long arise and elongate by the action of a
mass of embryonic cells, which maintain a basal position during the
entire development of the leaf. The corms habitually lie 5 to 10 cm.
below the surface of the soil and the growing region operates under the
influence of the soil, not the air, temperature. It was therefore ar-
ranged to grow some of these plants hi pots, taking the temperatures
by thermometers thrust into the soil, hi the vicinity of the bulbs. Such
cultures in small chambers with thermostatic control yielded some
data of interest. An attempt was made to ascertain the relative rates
of increase or amount of water which might be absorbed by the corms
and by the growing regions of the leaves at different temperatures.
A trio of corms, each of which consisted of the older basal corm and
the recently formed younger one, having an average height of 12 mm.,
were at first swelled to saturation at 19° C. and after two days, when
quiescence had been reached, the preparation was placed in a warmer

1 Osterhout, W. J. V. Some aspects of the temperature coefficients of life-processes. Jour.
Biol. Chem., 32: No. 1, p. 23. 1917.

2 See Barry, F. The influence of temperature upon chemical reactions in general. Amer.
Jour. Bot., 1: 203-225. 1914.

Hydration and Growth of Colloids and Cell-masses.

125

chamber, where the water in which they were immersed was raised
to 28° C. and kept at that point for 40 hours. Increased imbibition
ensued, which resulted in an elongation of 1 mm. or 8 per cent of their
total height. Measurements of the growth of leaves arising from the
apices of such corms showed that they maintained a rate of about 0.25
mm. per hour at air-temperatures of 19° C., which was increased to 0.49
mm. per hour at 27° to 28° C.; the temperature coefficient of such
elongation would thus be nearly 2.5.

A second pair of preparations with young leaves about 10 to 12 cm.
long was placed in a small, well-lighted thermostat, and the apices of
the second or younger leaf were attached to the auxographs. The
action of these plants was followed for a period of about 20 days,
during which time the leaves reached a length of 25 to 30 cm. The
growing region remains basal to the leaf and the rate of growth during
the course of development is much flatter than in stems, presenting
some of the features of root-tips.

Three objects were in view in the measurements of the rates of
elongation: (1) estimation of the rate in alterations from a low to a
high temperature and vice versa; (2) accelerations due to changes from
one temperature to another; (3) the effects of small and of wide vari-
ations in temperature.

The principal data concerning the experiment are given in table 101.

TABLE 101.

No. 1

No. 2

Rates
per hour
for—

At tempera-
tures of —

Rates
per hour
for—

At temera-
tures of —

mm.

hrs.

°C.

mm.

hrs.

•C.

0.24

24

31-33

1.7

12

25-27

0.1

24

9.5-11

0.11

24

9.5-11

1.1

18

20.5-21.5

0.8

15

19.5-21

1.4

7

20-21

1.1

7

19-20

0.3

11

9-10

0.4

11

9-10

1.2

8

19.5-20

0.9

8

19-20

0.6

12

8-9

0.27

12

8-9

1.4

4.5

27-28.5

1.22

4.5

26-27.5

0.3

10

7

0.3

10

7

1.25

6

20-21

0.8

6

19-20

2.25

8

28

0.9

5

30-31

0.7

5

33

0.7

9

20-21

0.7

9

23

0.3

5

17

0.15

5

17

0.8

6

27-28

0.27

15

30-31

0.2

15

17

0.2

15

17

0.5

28

17

0.5

28

17

1.1

15

26-28

1.7

7

26-28

0.7

12

17-18

0.6

16

16-17

The maximum rate displayed was at a temperature slightly under
30° C., varying, of course, with the preceding experience. Rising
temperatures are seen to accelerate growth with a coefficient slightly

126 Hydration and Growth.

above 2, except in cases in which the rise carried the temperature to
30° C. or above and also when the change was as much as 20° C. In
the earlier stage of development the rate rose from 0.1 mm. per hour at
11° C. to 0.8 mm. per hour at 21° C., and in another leaf from 0.1 mm.
to 1.1 mm. per hour in passing from 9° to 20° C. A change from 9°
to 27° C. resulted in a decreased rate in one case, while in others rais-
ing the temperature from 27° or 28° to 33° C. had no effect.

Falling temperatures were accompanied by reductions, with a usually
lower coefficient, it being noticeable that, as exceptions, the rate in
one case decreased from 0.7 mm. per hour at 23° C. to 0.15 mm. per
hour at 17° C., while in another case the rate decreased from 0.8 mm.
per hour at 27° C. to 0.2 mm. at 17° C. This unusual reduction is
ascribed to the fact that the door of the thermostat was opened to
facilitate the cooling, which resulted in the exposure of the leaves to
low relative humidity during the greater part of this period.

Closing the chamber and continuing the temperature of 16° to 17° C.
for 28 hours longer gave a record in which the rate gradually rose from
the low figure mentioned to 0.5 and 0.6 mm. per hour, which was nearest
the expectancy in comparison with the rate of 0.7mm. per hour at
23° C. which had been displayed in the previous period.

In the following period a rise of temperature from 17° to 26° or
28° C. was accompanied by accelerations of 0.5 to 1.7mm. and from
0.5 to 1.1 mm. per hour. The reversal of the temperatures brought the
rates down. to 0.6 mm. from 1.7 mm. in a change from 28° to 16° or 17°
C. and from 1.1 mm. to 0.7 mm. in a change from 26°-28° to 17°-18°
C. No other variations were observed in the three weeks in which
these organs were under observation which could be ascribed simply
to the change of temperature. Usually the change in the thermostatic
temperature would be followed within an hour or two by that of the
soil around the bulbs and the result would be a gradual adjustment of
the rate, increasing or decreasing, with no breaks or erratic features,
in the tracings which gave a continuous records of the lengths.

In conclusion, it is to be pointed out that the foregoing observations
show that the hydration capacity of cell-masses may bear some re-
lation to the temperature at which they are formed and under which
they have functioned for some time, and also that the unsatisfied water
capacity of a tissue will be affected by the relation between absorption
and water-loss by transpiration. Some gross measurements, which
have been made with an accuracy quite adequate for the determination
of the point in question, show that plants in the tropics show a rate of
growth which may be directly correlated with relative humidity and
the transpiration to be inferred.

Retardation of growth of an organ of a higher plant may be the
result of such direct water-loss, or, on the other hand, it may be directly
connected with the lessening of the water capacity of colloidal masses

Hydration and Growth of Colloids and Cell-masses. 127

under acid conditions, as has been demonstrated both in sections of
pentosan-protein biocolloids and in living and dried sections of plants.
The application of a temperature coefficient derived from a simple
equation to the rates of growth 6f organs of green plants has a certain
practical value when temperatures between the minimum and the
maximum rate are under consideration. As changes in temperature
affect a number of constituent processes in growth, including absorp-
tion and diffusion, transpiration, adsorption, action of acidity or
hydrogen-ion concentration, formation of the amino-acids, enzymic
action and oxidations, and all transformations of the carbohydrates,
the empirical character of such indices must be kept well in mind.
The character of temperature coefficients is sufficiently indicated by
the fact that they are not found to apply through the range of practi-
cable or habitual conditions of the organism and that the values change
within the range of 8° to 30° C. when falling and rising temperatures
are contrasted.

X. IMBIBITION AND GROWTH OF OPUNTIA.

Living cells and colloidal masses are never in a state of perfect
equilibrium with the environment, and the possible adjustments in
growing cells in which the colloidal substances are increasing, changing
in constitution, and varying in condition, may be great both as to
velocity and amplitude. Artificially compounded colloids, such as
the agar-protein mixtures which have been so freely used in this work,
show a similar delicate series of adjustment and correlations as they
pass from the dry state to one of almost completely satisfied hydration;
but when they reach this stage they are in the general condition of
mature cells or tissues and then show only the minute adjustments
which follow the modifications of the environment very closely.
Living cell-masses are to be considered as masses of colloid the inti-
mate substance of which is being constantly altered by metabolism
and by the incorporation of new material. The actual capacity of a
gel for water and its consequent state of swelling may be practically
satisfied at any stationary temperature. Such equable temperatures
do not occur in natural environments and may be observed only in
control chambers. The pen of the instrument employed in methods
of accurate measurement of a hydrated mass of colloid indicates con-
stant variations in volume, due to the solvation or dispersion of some
of the mass in the water in which it may be immersed.

The swelling of a cell-mass is to be considered as determined by the
hydration capacity of its colloids at the beginning of immersion plus
whatever additional capacity or variation may be developed by the
rearrangement of its material, reformation of its compounds, and
migrations of its molecular aggregates. Of the increased volume char-
acterized as growth, 98 per cent results from hydration. The grow-
ing cell-mass, however, in addition to the initial hydration capacity
of its mass, is continually adding material which has hydration ca-
pacity, and metabolic activities result in the accumulation of acids
and other substances which affect the coefficient of swelling. The
major procedure in growth would theoretically be imitated if minute
particles of powdered colloid could be continuously introduced into a
swelling mass.

Measurements of hydration in plants were made with disks about
12 mm. across, cut from the flattened joints of an opuntia, which
ranged from 5 to 20 mm. in thickness (fig. 23). Such sections
consisted of the indurated epidermal layers, between which was a
cylindrical mass of parenchymatous cells, the outer ones being chlo-
rophyllose. An anastomosed network of thin, fibrovascular strands
was included in the parenchymatous mass, and this mechanical tis-
sue checked expansion, so that care was necessary not to include the
larger, firmer strands in the section. Three of such disks about 12 mm.

128

Imbibition and Growth of Opuntia.

129

across the epidermal surface and from 6 to 11 mm. in thickness were
arranged in a triangle in the bottom of a Stender dish and a triangle of
thin sheet-glass arranged to rest its apices on the three disks (see fig. 1)
The vertical swinging arm of an auxograph was now adjusted to a
shallow socket in the center of the glass- triangle, while the pen was set
at zero on the recording sheet. Water or a solution being poured into
the dish, the bulb of a thermometer was adjusted in it and the course
of the swelling was traced, the record showing the average result of
the action of the trio of specimens.1

FIG. 23. — Calipers used in obtaining thickness of trio of sections.
Swelling is calculated on average.

Extensive records of the growth of the flattened joints of certain
platyopuntias having been made at the Desert Laboratory, and as a
series of analyses of the carbohydrate constituents of these plants was
being carried out, parallel measurements were planned which might
show possible connection between the composition of these members
and their imbibition or swelling reactions. The first series began with
the young joints which are formed in April and May, reaching maturity
in about 40 or 50 days and extending through the seasons, including
the dry foresummer, the summer rainy season, then the dry after-
summer, merging into the winter with its final rainy season. The im-
bibition capacity of sections taken from the joints ran a course shown
in table 102.

1 MacDougal, D. T. Imbibitional swelling of plants and colloidal mixtures. Science, 44: 502-
505. 1916. Also MacDougal. Mechanism and conditions of growth. Mem. N. Y. Bot. Gar-
den, 6: 5-26. 1916.

130

Bydration and Growth.
TABLE 102. — Swelling of joints formed in 1916.

Water.

HC1, 0.01M.

NaOH,
0.01M.

May 18, 1916

p. ct.
24.3

p. ct.
30 0

p. ct.
40 0

June 2, 1916

23.6

16 4

22 9

Nov. 2, 1916

20.5

21 0

22 2

Nov. 23, 1916

48 0

45 4

35 3

Jan. 24-25, 1917 (12 sections). . .
Feb. 20-21, 1917 (6 sections)
Mar. 23-24, 1917 (6 sections) . . .
Apr. 24, 1917

25.7
10.7
9.4
21.8

27.9
11.7
12.0
20.4

25.0
10.8
10.9
13 9

No record was made of the temperatures of the swelling sections,
but these in general were determined by the seasonal conditions.
Thus, sections were swelled in November at 16° to 18° C. and at 25°
to 28° C. in April and May.

The measurements given in table 102 were made primarily for the
purpose of following the changes in the flattened stems of Opuntia as
they go toward maturity. The joints, having reached the mature con-
dition in November, the changes for the next 4 or 5 months are not
directly connected with growth, although the action of living cells is
concerned. The period of late summer is one of progressive desic-
cation, in which the water-loss is much greater than the supply
obtained from the soil, and this drying out continues until some time
in January and February, when the winter rains saturate the soil.
It would be expected that if sections of the partially desiccated
plants were taken during the dry period, the swellings which they
would show when placed in solutions would be a direct function of
the net water-loss which they had sustained, rather than of changes
in composition. It was therefore necessary to devise a method by
which the material could be reduced to a standard in which the
seasonal water-condition would be eliminated. This was accom-
plished by the use of dried slices from the median portion of the
joints in such manner as to exclude nodes and spines.

The joints were laid flat on a table and against a strip of wood 5
or 6 mm. in thickness, which served as a guide for sliding a knife to
cut away one epidermal region. This being done, the piece was turned
over and a horizontal movement of the knife along the lowered guide
would cut away the other epidermal region, leaving a slice 3 to 5 mm.
in thickness, which was dried in the air at temperatures of 15° to 18° C.,
using filter-paper or blotting-paper to keep the sections plane during a
part of the process. Sections cut from the dried pieces were calibrated
by scales of the type used commercially for measuring the thickness
of paper (fig. 8), and then swelled under the auxograph in the usual
manner. The calculation of the percentage of swelling on the final
thickness of the dried material eliminated the factors attendant upon

Imbibition and Growth of Opuntia.

131

the condition of the fresh material. It is important that the thick-
ness of the slices before drying be approximately equivalent.

The measurements obtained from living sections inclusive of the
entire thickness of joints and from dried slices, given in table 103,
illustrate the seasonal variations in mature stems.

TABLE 103. — Swelling of mature joints at different seasons.

Date.

Solutions.

Water.

Citric
acid,
0.01 N.

Sodium
hydroxid,
0.01 M.

Potassium
chloride,
hydro-
chloric
acid,
0.01 M.

Oct. 1917 (living sections) (18-25° C.)

p. ct.
98
277
214
194
55
143
20
238
4.8
550
37
304
54
195

p. ct.
116
300
238
317
80
143
14
210
5.5
463
40
322
51.6
166

p. ct.
100
177

p. ct.

Dec. 1917 (dried slices) (16-18° C.)

Jan. 3, 1918 (living sections) (16-18° C.)

(Dried slices) (12-14° C.)

282
85
143
14
210
5.7
600
36.5
310
50.3
208

Jan. 24, 1918 (living sections)

(Dried slices) (16-18° C.)

March 4, 1918 (living sections)

14.3
210
3.5
388
37
274
42.7
230

(Dried slices) (18-20° C.)

March 30, 1918 (living sections)

(Dried slices) (20-22° C.)

Apr. 25, 1918 (living sections)

(Dried slices) (23° C.)

May 28, 1918 (living sections)

(Dried slices) (20-25° C.)

The continued dry condition is seen to result in desiccation, which,
of course, is followed by increased hydration when such living sections
are swelled, the maximum effect being reached in January, before the
beginning of the winter rains.

The tests of the dried sections which show the swelling after the
colloids have been subjected to the action of concentrating salts have
a value derived from the fact that all are reduced to an equivalent
water-content, accompanied by irreversible coagulations of the more
complex proteins. Although we here introduce a new set of conditions,
the effects of which are not easily to be evaluated, yet it is none the
less significant that the entire series of preparations reach their
maximum water capacity at the end of March. Since some changes
may have been brought about in the proteins, it is important to
follow the course of the carbohydrates.

The variation in the sugar-content is best illustrated by the data
given in table 104, as determined by Dr. Spoehr.1

It is clear that the colloidal material of the cell-mass of this plant
does not come to a condition of highest imbibition capacity at the end
of the period of desiccation in the season of low temperature, but the
maximum is found after the beginning of a season of rising temperatures
and of accumulating sugars, coupled with an inadequate water-supply.

Spoehr, H. A. The pentoae sugars in plant metabolism. The Plant World, 121 : 365-379. 1917.

132

Hydration and Growth.

The extended measurements of the swelling of biocolloids containing
sugars, dextrose and sucrose, and of such mixtures in solutions of
sugars, show that these have but little influence upon imbibition capac-
ity. These results suggest that it is to the variations in the pentoses
as represented by the mucilages of the opuntias that we must look for
a part of the varying imbibition capacity of these and perhaps other
plants as well.

TABLE 104. — Seasonal variations in sugar-content of Opuntia sp.

Date.

July
5.

July
31.

Sept.
20.

Oct.
27.

Nov.
15.

Dec.
20.

Jan.
11.

Feb.
16.

Mar.
17.

April
25.

May
22.

Dry weight

36.38

16.45

19.66

20.30

23.05

30.10

22.20

22.33

19.50

24.30

25 25

Total sugars, p. ct

20.03

13.24

18.44

20.90

18.75

28.95

19.10

21.32

28.05

32.40

30 15

Total hexose sugars, p. ct. . .
Total pentose sugars, p. ct . .
Pentosans, p. ct

10.45
9.26
9.04

8.60
4.39

8.83
9.08
8.86

9.32
10.95
10.47

5.50
12.50
11.35

7.90
10.45
10.10

14.95
4.73
4.40

14.90
6.07
5.51

22.16
5.55
4.75

22.70
9.15

8 68

17.08
12.34
12 17

Pentoses, p. ct

0.20

0.24

0.48

0.82

0.35

0.43

0.65

0.82

0 48

0 16

Ratio of total pentose sug-
ars to total sugars

0.462

0.332

0.492

0.624

0.667

0.551

0.248

0.283

0.198

0 283

0 409

Ratio of total hexose sug-
ars to total sugars

0.522

0.650

0.479

0.446

0.293

0.417

0.783

0.698

0 791

0 702

0 567

The vegetative conditions at the Coastal Laboratory are widely
different from those at the Desert Laboratory, at which the above
results were obtained. Sections of fresh material at the end of August
at the first place showed swellings of 7 to 10 per cent at tempera-
tures of 15° to 16° C. This was characteristic of the end of the sum-
mer cool and foggy season. Higher temperatures and greater average
daily illumination resulted in increased desiccation during August and
September, with the result that living sections showed an unsatisfied
imbibition capacity of 18 per cent at 15° to 16° C. The method of
preparing dried median slices was developed at this time, and these
showed swellings of 500 per cent at 15° C. and 570 per cent at 20° C.
The total proportion of pentose sugars at this time was 19.10 per cent
as compared with 4.39 per cent at Tucson, calculated on dry weight.
Dried sections at the Coastal Laboratory taken at the end of Janu-
ary, after 2 months of the cool season but with a fair supply of
moisture, were found to swell 300 per cent at 14° to 16° C. A test
was also made of sections comprising only the epidermal and chloro-
phyllose layers of the same material, and these were found to show
an increase of 262 per cent at the same temperature.

Decrease in swelling capacity is seen to occur in the cool season at
Tucson and at Carmel. The composition of the joints in September,
representing approximately the condition of the material at both
places in October, as determined by Dr. H. A. Spoehr, is shown by
table 105.1

1 MacDougal and Spoehr. Growth and imbibition. Proo. Am. Phil. So*., 66: 289. 1917.

Imbibition and Growth of Opuntia.

133

TABLE 105.

Cannel.

Tucson.

Water

p. ct.
91.15

p. ct.
80.34

Total sugars

2.61

4 30

Total polysaccharides
Hexose polysaccharides. . .
Disaccharides

1.94
.09
.07

3.50
1.65
04

Hexoses

.52

06

Pentoses

.14

05

Pentosans

1 70

1 74

The material to which the high hydration capacity of Opuntia is due
is the mucilage which exudes from the damaged cells of cut surfaces
of the parenchymatous tissues. This is included among the pentosans
together with agar and various gums, including tragacanth, acacia,
cherry gum, and prosopis gum, many of which have a high hydra-
tion capacity and infinite dispersion in water.

The manner of the formation
of this material is a matter of
no little importance in connec-
tion with varying growth and
hydration capacity. Briefly
stated, the depletion of the
water-content of a cell accel-
erates the conversion of poly-
saccharids with low hydration
capacity into pentosans (muci-
lages and gums) which have
an extremely high water capac-
ity, and it is this change which is followed by the large swelling of
tissues in certain stages or in dry seasons. The transformation in
question is not reversible. While some decrease in these pentosans
may occur, it is not known by what steps it takes place.1

The composition of etiolated plants presents proportions of the
principal constituents different from those of the green plant, and if
one of the main conclusions of the present work is valid, they might
be expected to exhibit some water-relations different from those of
green plants.

Some shoots of Opuntia which had developed in a dark room at
Carmel in equable temperatures below 20° C. were available in July

1917, and these displayed some singular temperature effects. The
stems were from 20 to 30 cm. long and compressed ovate in cross-
section. Short sections were cut out and immersed in dishes in the
usual manner. Most of the development had been at 16° to 18° C.,
and some of the shoots were placed in a second chamber at 30° to
31.1° C. for 2 days before being swelled. The illumination of a
small incandescent bulb used in obtaining these measurements had
given the stems a slight greenish tinge, but the energy derived from
this source was not sufficient to have caused any serious change in
the composition of the shoot. Sections were about 5.5 mm. in dia-
meter, and sets of three were selected for testing under the auxo-
graph, so that the older basal parts of the stem and the newest
apical portions were represented in the separate measurements. One

1 See MacDougal and Spoehr. The origination of xerophytism. The Plant World, 21 : 245.

1918. Also MacDougal, Richards, and Spoehr. The basis of succulence in plants. Bot. Gaz.,
67: 405. 1919.

134 Hydration and Growth.

series was swelled at the temperature of 30° to 31° C., at which the
shoot had stood for several days. The second series was swelled at
17° to 19° C., in which equivalent shoots stood, which were to be
used as a comparison.1

The most remarkable feature of these measurements is that in
which the material grown at 30° to 31° C. for two days showed a greater
increase when swelled at a lower temperature (see p. 119). The entire
set of reactions is indicative of a colloidal complex of different con-
stitution from that of green plants.

Analyses necessary to establish the nature of such divergent con-
stitution of etiolated opuntias could not be made, but data obtained
from other species grown in darkness are available. The results of
Palladin show that stems of such plants have less nitrogen than nor-
mal green plants, and all workers who have dealt with this subject
agree that the ash-content of etiolated organs is relatively greater than
in green stems.2

There is not general agreement as to the distribution of nitrogen in
etiolated plants, but there seems to be unanimity in the conclusion
that the total nitrogen-content is less than in normal plants. Accord-
ing to Karsten, all parts of the plant have less sugar and "gums" when
grown in darkness.3 As the gums include the pentosans, which with the
nitrogenous compounds make up the hydration machine, it is to be
seen that the etiolated plant presents the features of lessened protein,
decreased pentosans, and increased salts, all of which would tend to a
lessened imbibition capacity in both fresh and dried material. How
much the lessened nitrogen-content would contribute to this general
decrease might only be known by a determination of the character of
the compounds in the two instances.

All of the facts concerning imbibition by living plants and by bio-
colloids tend to show that the proposal of Palladin to ascribe the vari-
ous departures of growth in plants in darkness to transpiration effects
is not tenable. There is much foundation for the belief that form may
be largely affected by the water-relation, but with respect chiefly to
imbibition capacity, as a resultant of the protein-pentosan-salt com-
plex with varying acidity. It would seem, therefore, that for Palladin's
contention that the ash constituents of the etiolated plant exercise a de-
termining effect on the growth and development in darkness by in-
fluencing transpiration, we may safely substitute an assertion that
the important effect of the salts is that which they have on the imbibi-

1 MacDougal. The influence of etiolation upon chemical composition, in "The influence of
light and darkness upon growth and development." Mem. N. Y. Bot. Garden, 2: 300-305.
1891.

2 Palladin, W. Eiweissgehalt der griinen und der etiolirter Blatter. Ber. d. deut. hot. Ges.,
9:191. 1903.

3 Karsten, H. Die Einwirkung dea Lichtes auf das Wachstums der Pflanzen. Jena. 1870.

Imbibition and Growth of Opuntia.

135

tion or absorption action of the plasmatic colloids, which in the case
of Opuntia are probably low in proteins.1

The growth of etiolated shoots of Opuntia is of an indefinite char-
acter, as the length which such members may reach depends to
a large extent upon the amount of available material and other
features. Shoots which were already a few weeks old, and which had
developed in a dark chamber kept at 16° to 18° C. were found to be
growing at the rate of 5.2 mm. daily. These were removed to a
second chamber, hi which the temperature was kept steadily at 16° C.
for 3 days, during which time the rate varied from 3.1 mm. to 3.4 mm.
daily. The temperature was brought up to 21° to 23° C. in 3 hours,
and the rate was 5 mm. for the first day. During the second and
third days at this temperature the rate rose to 7 mm. per day. The
rate was 7.6 mm. daily during the next 2 days and about 8 mm. daily
for a final period of 16 hours. The temperature now being raised to
30° C. in 2 hours, and after that varying from 30° to 32.5° C., the rate
was 11 mm. daily during the first 16 hours, then at the rate of 16.8 mm.
during the succeeding 12 hours, during which time the elongation pro-
gressed in a remarkably uniform manner.

I2p.m.2 4 6 8 10 m. 24 6 6 10p.m.

FIG. 24. — Auxographic record of varia-
tions of length of etiolated shoot of
Opuntia X26, at temperatures as be-
low, sheet ruled to 10 mm. intervals: (a)
downward movement of pen 7h30m a. m.
to 9h40m a. m. denoting growth at tem-
peratures of the stem of 45° to 49° C.;
(b) growth checked for 20 minutes at
49° C. ; (c) growth resumed at temper-
ature of 49° C. ; (d) shortening at 48.5°
to 52° C. ; (e) stationary at 50.5° C.;
(/) growing at temperatures of 48° to
49° C.; (0) shortening at 49° C.; (h)
growing at 38° to 41° C. ; (t) shorten-
ing at 49° C.

The rate itself was one which might have been identified with that
of a green plant, in which, however, the length of the cell-mass might
not be equivalent. The chief point of interest in the present con-
nection is that which comes from a comparison of the imbibition capac-
ity and growth. Sections grown at 17° to 19° C. showed an imbibi-
tion capacity at 30° to 31° C., nearly double that displayed at the lower
temperature, and it was also to be seen that shoots growing at the rate

x Palladia, W. Transpiration als Ursache der Formanderung etiolirter Pflanzen. Ber d. deut.
bot.Ges., 8:364. 1890.

136

Hydration and Growth.

of 5.2 mm. daily at 16° to 18° C. showed a rate of 11 to 17 mm. daily
at 30° to 32° C. The rate of growth would be one which would be
accepted as being in general conformity with the van't Hoff formula of
chemical reaction, while as a matter of fact it is not widely different from
the capacity for imbibition under the influence of equivalent tempera-
tures. Experimental tests have already been described in which the
upper limits of growth and the behavior of etiolated and green stems of
Opuntia between 46° and 51.5° C. are not widely different. (Fig. 24.)1
The rapid and wide variations at these high critical temperatures
are to be contrasted with the steady rate of a growing green joint main-
tained at 30° C. for 38 hours. The illumination in the daylight period
apparently did not affect the rate of 0.07 mm. per hour.

12p.m. m. 12p.m. m 12p.m. m. 12p.m. m. 12 p.m.

1

1

r —

\

1

1

I

1
1

1
I

v L

\

•

\

\ — \ — -

t '<

1

\ \

\ \

\ \

r^

\x*5\

\ \

\ \

\

\

\\

\ \

\

\

\

\

\

75

FIG. 25. — Tracing of auxographic record of variations in thickness of joint of Opuntia approach-
ing maturity. Upward movement of pen denotes increase in thickness, X45. Measurements
made from place between areoles near base of joint. Scale ruled to 5 mm. and 12-hour
intervals. It is to be noted that the hour is set forward on the summer schedule.

In addition to the great number of records of the variation of joints
of Opuntia as to length, a few days' measurements of the thickness of a
maturing joint were made in April 1918 hi order to ascertain whether
or not increases and shrinkages took place in all dimensions at the
same time. The auxographic tracing in figure 25 gives the daily
variation in thickness near the base of a maturing joint, which are seen
to be correspondent to those in length. It is to be noted that the
instrument was necessarily adjusted so that increase was denoted by
the upward movement of the pen, in a manner opposite to that in
nearly all the other records. Later the bearing-lever of the instru-
ment was adjusted to a place near the apex of the joint and the shrink-
age, which was now pronounced, was seen to set hi about 9 or 10 a. m.
and continue until sunset, at which time thickening began and lasted
for 3 or 4 hours, after which but little change took place until the rising
temperature of the following day was encountered.

The singular retardations and variations in growth which are highly
characteristic of Opuntia, together with its unusual features of trans-
piration and its readily measurable imbibition, offer unusual oppor-
tunities for the examination of certain agencies affecting growth,
especially as the action of some of these factors is so plainly discernible
in the variations in volume of mature organs.

1 MacDougal and Spoehr. Growth and imbibition. Proc. Amer. Phil. Soc., 56: p. 308. 1917.

Imbibition and Growth of Opuntia.

137

The size or swelling of any colloidal mass, such as a growing organ,
or particularly a joint of Opuntia, will depend upon the integrated
effects of several agencies, including transpiration, absorption, the
hydration coefficient (as determined by the acid, salt, and protein
content of the protoplasm), and the temperature.

Several series of measurements of the separate processes involved
have been made at the Desert Laboratory. The earliest results of
importance with reference to transpiration as affecting growth were
obtained by Mrs. E. B. Shreve, who found that the actual amount of
water which an excised section of one of the cylindropuntias, Opuntia
versicolor (fig. 26), might contain begins to decrease some time after
midday and continues to do so until about daybreak of the fol-
lowing morning, then increases during the forenoon.1 If the plant

B

Apr.ZO

Apr.ZZ Apr.Z3 Apr.t4

Fio. 26.

A, tracing of an auxo-
graphic record of the
variations in length of
a stem of Opuntia ver-
sicolor. Below is a line
showing the daily vari-
ations in temperature.
Thegraph is composed
of sections of straight
lines representing con-
ditions of illumina-
tion. Interruptions
of the sunlight by
cloudiness are shown
on April 21, 22, 23, and
29. B, auxographic
record of variations
in thickness of stem
of Opuntia versicolor,
with temperature and
illumination indicat-
ed. (After Mrs. E. B.
Shreve.)

was under conditions of satisfied imbibition capacity, the plotted line
showing such capacity would also be that of growth. Such a condition
does not exist, however. Also, if the simple capacity for imbibition
determined volume, and this capacity were always satisfied, the plotted
line of water capacity might be identical with that of the daily varia-
tion in volume of the mature organ. Neither does this condition
exist (fig. 27).

The record of variations in volume of a joint from the time of its
beginning to maturity and through the following season, and selected
portions of this graph, are reproduced in figures 28 and 29.

The aspect of the daily variations in volume shows seasonal altera-
tions and depends to some extent upon the age of the mature joint,
but it is evident that the joint begins to increase in volume in the

1 See Shreve, E. B. An analysis of the causes of variations in the transpiring power of cacti.
Phyaiol. Researches, 2: No. 13. August 1916.

138

Hydration and Growth.

morning or at some time in the forenoon and continues to do so until
some time late in the afternoon, at which point a shrinkage ensues which
continues until the next morning. The rate of water-loss was not paral-
lel with the changing volume, as transpiration was most rapid between
midnight and morning, and was in excess of the amount which might

Var.

Tr.

Ab.

12p.m. 6a.m. m. 6p.m 12p.m. 6a.m. m. 6p.m.

FIG. 27. — Absorption, transpiration, and variation in length of growing shoot of Opuntia. Ab-
sorption by roots denoted by dotted line is least about midnight and is greatest about midday,
exceeding transpiration during the greater part of the daylight period. Transpiration is
greatest about midnight and least about midday, according to data by Mrs. E. B. Shreve.
The wavy line is an auxographic tracing of actual changes in length of a joint of Opuntia
in a mature condition, X 50.

be absorbed by the roots during this period. It is obvious without
further discussion that the variation in volume of a member like that
of a joint of Opuntia must be the resultant of the transpiration, absorp-
tion, and water capacity of the cells modified by the action of the new
colloidal material which may

be added to the cell-masses. TABLE 106.

It is notable, however, that
at some time approaching
midday the pen recording
the variations in volume
traces a line not far from the
horizontal for a brief period,
perhaps half an hour, at
which time absorption and

transpiration balance, and the absolute water capacity of the cell-masses
is greatest (see figs. 28 and 29). An equally marked confluence of fac-

Time.

Weight

Increase

Percentage

in grams.

in grams.

increase.

5h30m a. m. . .

23.62

6 30 a. m. . .

25.25

1.63

6.9

4 30 p. m. . .

22.25

5 30 p. m. . .

25.00

2.75

12.4

Imbibition and Growth of Opuntia.

139

tors is to be seen late in the afternoon, with lessening water capacity,
minimum acidity, and the beginning of decreased absorption by the
root-system.

Some measurements of growth and hydration of the Platyopuntia,
the growth of which has been observed so extensively, were made by
Mr. E. R. Long in 1915. Imbibition was tested by noting the increases

12p.m

I? p.m.

12p.m.

FIG. 28. — Portions of auxographic record of variations in length of a joint of Opuntia from October,
1915 to October 1916. The joint had formed roots which depended in a glass jar of tap- water
and the joint was held firmly in a natural erect position. The preparation stood in the south
end of a glass house and was exposed to the alternations of sunlight and darkness with accom-
panying temperatures as low as 8° C. in the morning and as high as 45° C. in midsummer
afternoons. The actual variations are amplified 30 and elongation is denoted by the down-
ward movement of the pen. Divisions of scale =1 cm. The joint was in a stage of com-
pleted growth during the week beginning October 18, 1915, at which time elongation occurred
from evening until the middle of the forenoon, and shortening during the remainder of the
day, with the length less at the end of the week than at the beginning. The records for March
and April show a similar daily periodicity, but with an increase in length which may be
ascribed to imbibition under the advancing temperatures. The daily losses and gains are
more nearly equal in May and June and the variations of narrow amplitude. Some loss is
shown in July and the reactions of the joint a year from the beginning of the record show a
daily variation different in many particulars from those of the previous year. See figure 29
for a continuation of the record.

140

Hydration and Growth.

TABLE 107. — Growth of flower-buds of
Opuntia, March 25 to April 24.

in weight of disk-shaped sections taken from the joints at sunrise and
sunset, with the results shown hi table 106. (Fig. 30.)

These results afford a comparison only between conditions at the
beginning and end of the daylight period and the tune of the maximum
and minimum imbibition capacity was not determined as in Mesem-
bryanthemum. (See p. 145). Mr. Long
also tested the effects of acids, hy-
droxids, and salts upon the growth of
Opuntia. Preparations for this purpose
consisted of mature joints bearing
young flower-buds. The bases of the
joints were suspended with their
freshly cut surfaces in solutions in
glass jars, and the lengths of the buds
were taken at intervals of 3 or 4 days.
The final amount of growth in each
case is given in table 107. (Fig. 31. )*

Total

growth

Medium.

increment

Time.

before

flowering.

mm.

days.

Distilled water

42.0

27

N/50 NaOH

40 5

30

N/50 malic acid. . . .

36.0

28

N/50HC1

31.0

28

12p.m. m. 12p.m.

12p.m.

12p.m. m. 12p.m. m. 12p.m. m. 12p.m.

i

/

/ '

j

1

1

' j

1

i ___

i ___

1

1

|

-> r~

NOV. 6 1916

1

i

1

|

12 p.m. m. 12 p.m.

12 p.m. m. 12p.m. m. 12p.m. m 12p.m.

12 p.m.

1

1

l

i

-^- — 1 — • —

"— — i

v-^ " ' — •-;

DEC. 2&, 1916

1

|

m. 12 p.m m. 12 p.m. m. 12 p.m. m. 12p.m. m. 12p.m. m. 12 p.m. m. 12 p.m.

i

f

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1

1

• 1

i

i

1

'*~ |

JAN. id, 1917

iCIoudy

and r4iny all

week ',

"

1

m. 12p.m. m. 12p.m. m. 12p.m. m. 12p.m. m. 12p.m. m. 12p.m. m. 12p.m.

__!__! '

1 i

i

v

I ~± ',

1

\ i. r^ i \\/—

1 \

\ i \ \ \ ~ \ \ v \ \ X^1 — ~\ — **Nl ( /^ \ \

\ > \ \ \

4 \ * \ * K ^_. ~-^ ~\ , — i— •*"

\FEB.\B\\9\7 \ \ \

\ \ \ \ \^ \ W \

m. 12p.m. m. 12p.m. m. 12 p.m. m. 12p.m. m. 12p.m. m. 12p.m. m.

12 p.m.

>_ I

1

i 1 ;

i I

'

!

1 1

i

L — ' S^ ^~~i" — '"x.

^^ ^_

t

'ill*1

\-~~^"^\ \^-S~ N

, ^S^^

\APR.2J, 1917

; \lncrea^in£ incize all\ week \ \

m. 12p.m. m. V2p.m, m. 12p.m. m. 12p.m. m. 12p.m. m. 12p.m. m. 12p.m.

1

1

1

!

APR. 2!?, 1917

1

! 1 '. _

i

•^ \ ^-V «v

i ^-* — ~v\ s~i — N! ^s~ \

"^ » ~>^__x^"

* ^

r-' \ '

^\~^ \

\^~S \ \^s \ \' \ \ \ \

', \ \

FIG. 29. — Continuation of record of variations in length of mature joint of Opuntia (see fig. 28)
amplified 50 times. The amplitude of the daily variations has been reduced to a minimum
November to January, with a general shrinkage in length. Equable conditions accompanying
clouds and rain are illustrated by the record of the week beginning January 15, 1917. The
influence of the advancing temperatures in inducing increased imbibition is illustrated by
the records of February to April, and a balance of daily losses and gains occurred about the
end of April.

1 Long, E. R. Growth and colloid hydratation in cacti. Bot. Gaz., 59: No. 6. 1915.

Imbibition and Growth of Opuntia.

141

50

o30

£20

10

%o Sodium hydro/id
Nutrient solution
Distilled water
%w Hydrochloric acid
%o Malic acid

%o Hydrochloric acid

12
Time in hours

18

Fia. 30. — The course and amount of swelling of sections of Opuntia in solutions as indicated dur-
ing a period of 24 hours. Fresh sections 12 mm. across were cut from green joints and placed
in glass dishes in a dark room at 18° C. The lines are traced from the results of measure-
ments made at 6-hour intervals. Compare with fig. 31. (Redrawn after E. R. Long.)

50

40

30

20

10

Nutrient solution
Distilled water

N/oo Sodium hydroxid
N/so Malic acid

%>o Hydrochloric acid

April 10 13 16 20 23 27 30

FIG. 31. — Record of growth of flower-buds of Opuntia, on joints the bases of which were immersed
in solutions, the imbibition effects of which are shown in fig. 30. The record covers a period
of three weeks, or the entire measurable period of extension of the buds to the time of open-
ing. The preparations stood in a glass house and were exposed to alternating conditions of
daylight and darkness and to temperatlres of about 15° to 30° C. (See fig. 30.) (Redrawn
after E. R. Long.)

142

Hydration and Growth.

Mr. Long's results were confirmed by the author in April 1918, at
which time auxographic methods and technique of measuring the
swelling of such sections had been brought to an advanced stage of
efficacy. Sections were cut at sunrise, noon, and sunset, from young
joints 8 to 10 cm. long growing hi the open under natural conditions.
Such sections had an average thickness of about 4 mm. and when
swelled at 20° C. gave the increases shown in table 108.

TABLE 108.

Opuntia,
median
sections.

Time taken.

Dried
slice
taken
7 a. m.

7 a. m.

8 a. m.

Noon.

5 p. m.

Distilled water. . . .
Citric acid

p. ct.
7
6

p. ct.
4.6
6
8.2

p. ct.
8.2
8.2
12.0

p. ct.
4.6
5.2
11.8

392
400
440

Sodium hydroxid. .

The great increase of dried slices is indicative of high water capacity
of living material. The relative swelling of the sections in the different
solutions is identical with that of the fresh sections, demonstrating that
the dominant process is imbibition rather than osmosis.

The time required for satisfaction varied widely with the time at
which material was taken and the character of the solution. The
sections taken at the end of the day were fully hydrated in distilled
water and began to shrink in 6 hours. The sections taken at sunrise,
which were most highly acid, as those taken in the evening are least
acidified, were satisfied in 2 hours and began to shrink in 3 hours. The
material taken in the morning was saturated in the acid solution in less
than an hour and was shrinking rapidly an hour and a half after

8a.m.

8 a.m.

I I I I I

FIG. 32. — The tracing on the left shows the variation in volume of a trio of sections of a young
joint of Opuntia 4.6 mm. in thickness which swelled 6.5 per cent in citric acid, 0.01 N, less
than three hours at 20° C., then began to shrink. The tracing on the right shows the
reaction of a similar trio which had an average thickness of but 3.9 mm., and which
swelled 6.4 per cent in an hour at 28° C. and then began to shrink very rapidly.

immersion (see fig. 32). Swelling in the alkaline solution was charac-
teristically slow and long-continued. Material taken in the morning
continued to increase for 4 hours, and that taken hi the evening was
still showing some increase 12 hours later.

Another feature in conformity with the condition of the joint at differ-
ent times of the day was the fact, confirmed by repeated tests, that the

Imbibition and Growth of Opuntia. 143

difference between the amount of swelling of sections in distilled water
taken at sunrise and swelled at 20° and 28° C. was so small as to be
negligible. The same condition prevailed in dried slices taken in the
morning and dried rapidly in the desiccator. Slices which came down
to a thickness of about 0.6 mm. swelled 392 per cent at 20° C. and a
second set increased an equivalent proportion at 27° to 28° C. The
sets of living sections used for these tests gave an increase of about 6.5
per cent in water. Sections taken at noon swelled 8.2 per cent at 20° C.
and 9.8 per cent at 28° C. Sections taken at the end of the day
swelled 4.6 per cent at 20° C. and 6.5 per cent at 28° C. The material
used for these paired tests was selected to be equivalent as far as pos-
sible, and comparisons transverse to those stated are not allowable.

The acidosis produced by the residual acids of the morning condition
may be taken to be accentuated by the rising temperature and to
cancel or mask any additional absorption which might accrue as a
direct effect of temperature on a neutral solution. The increases at
other times would be due to the direct action of rising temperatures
on absorption.

It became evident in the earliest work done with the opuntias that
the young joints showed a swelling in hydroxid solutions greater than
in water or acids, a fact probably attributable to the formation of
compounds of the sodium hydroxid with the carbohydrates present,
in conjunction with possible sodium albuminates. The carbohydrates
are in greatest proportion in young joints. The swelling in hydroxid
in later stages comes down to a level near that in acid and in water.
An additional fact of interest was the action of the juice of the joints
taken in the condition in which it is found at midday. Duplicates of
the dried slices, which have already been noted as showing an increase
of 390 per cent in distilled water, swelled 325 per cent at 20° C. in the
expressed juice. Similar dried slices swelled 372 per cent in the freshly
expressed juice of Echinocactus wislizeni. Its hydrating effect on thin
plates of a biocolloid consisting of agar 6 parts, opuntia mucilage 2
parts, bean protein 1 part, and gelatine 1 part was much marked.
Such sections swelled 2,450 per cent at 20° C. in distilled water, and
only 700 per cent in the fresh juice of Opuntia taken at midday.

The principal factors which influence the rate and course of growth
of Opuntia have been described in the preceding pages. The joints
when in the youngest stage have a mode of growth which does not
differ materially in the record which it makes from that of many other
green plants. As soon as it reaches medium size its innate peculiarities
of transpiration and metabolism and certain morphological features
operate to give it a highly characteristic daily chart of elongation and
retardation or shrinkage. The respiration of these plants is of a
character which results hi the accumulation of acids to an amount
equivalent to as much as 0.1 N malic acid at daybreak, which is suffi-

144 Hydration and Growth.

cient to have a distinct effect on the hydration capacity of the cell-
colloids. Late in the daylight period the acid may be reduced to a
point below the hundredth normal which has been used so exten-
sively in this work as a standard solution. The effects of acidosis
would, of course, vary as the composition of the pentosan-protein-salt
colloid of the joint passed from its embryonic aspect to that of the
mature member. The actual amount of water lost by the greater
number of plants, especially those with thin stems and broad leaves,
is greater for the daylight period than for the night. Opuntia is a
notable exception to this generalization, and its rate of transpiration
is greatest during the night, usually between midnight and morning.
All of these agencies affecting imbibition likewise have a determining
influence oh growth, and the resultants in Opuntia and presumably in
other massive succulents are such as to constitute a characteristic
type of growth.

Another feature about which but little has been said is that of the
morphology of the compound members of the stems of Opuntia. At
first, when a young joint is but 2 or 3 cm. in length, it is wholly in an
embryonic condition and its colloids show the reactions of such mix-
tures. As development progresses much permanent tissue is formed,
hi which finally the embryonic tracts lie as a network or reticulum.
Any given section of a growing joint contains growing and mature
tissue after a certain stage is reached, but when the mature tissue
reaches a proportion something greater than that of the growing
masses its characteristic variations in hydration overshadow those of
the growing cells and give rise to the retardations and shrinkages
which are so marked a feature of these plants. It is probable that
many features of the rate and course of growth may be ascribed to
anatomical relations, of which the above is an illustration.

XL THE HYDRATION REACTIONS AND GROWTH OF MESEM-
BRYANTHEMUM, HELIANTHUS, AND PHASEOLUS.

The results obtained by a study of the hydration of the cacti are
especially valuable because of the possibility of their correlation with
features of varying composition which could be determined by chemical
analyses. Measurements of a second type of succulent were sought
for the purpose of bringing into relief the possibilities of rapid changes
in the water-content of growing and mature organs. A mesembryan-
themum (Mesembryanthemum edule) which flourishes in the open
at the Coastal Laboratory and in the glass-house at the Desert Labora-
tory furnished material suitable for such studies. The leaves attain a
length when mature of about 6 to 10 cm. and are triangular in cross-
section, the three faces being about 10 to 12 mm. across. Metabolism
runs a course in these organs similar to that of the opuntias, as a result
of which acid accumulates during the night, and decreases with the
disintegrating action of light during the daytime, as illustrated by the
data given in table 109. The total daily range in the concentration of
the acids is much less than that displayed by the cacti.

TABLE 108. — Acidity of juice of leaves of Mesembryanthemum in cubic centimeters

of N/100 KOH.

8 a. m.

Noon.

4h30m p. m.

Sample A.
Fresh juice, per c.c

p. ct.
0.0280

p. ct.
0.0279

p. ct.
0.0232

Total acidity per gm. dry material . .
Total acidity per gm. fresh material .

Sample B.
Pure juice, per c.c

1.584
.0356

.0273

1.509
.0351

.0225

1.191
.0264

.0205

Total acidity per gm. dry material . .
Total acidity per gm. fresh material .

1.072
.029

1.091
.0241

1.056
.0275

Measurements of the varying diameter of young and of mature
leaves indicate that the direct water-loss from the surfaces are so im-
portant as to mask the imbibition capacity as affected by acidity and
other factors, as will be apparent from auxographic measurements.

Mention has been made previously of the general similarity of the
course of growth of Mesembryanthemum to that of Opuntia. It was
possible to go into this matter in greater detail in the experiments
described in this volume.1

A series of tests was arranged to ascertain the alterations in volume
of these leaves both in a mature condition and when in the course of

1 MacDougal and Spoehr. Growth and imbibition. Proc. Amer. Phil. Soc., 56: 314. 1917.

145

146

Hydration and Growth.

growth. Their regular surfaces made it possible to place one side
snugly on a small wooden block, and to bring the cork-tipped vertical
auxograph lever in bearing with the uppermost angle of the leaf. The
stem was held firmly a few centimeters from the base of the leaf, which
was exposed to no disturbing conditions (fig. 33). First, the diurnal
variations of a mature leaf were
followed without any attempt being
made to equalize temperatures,
which were taken by a thermometer
with a thin bulb thrust into a
second leaf and allowed to remain
there. The readings were as low
as 10° C. at daybreak and as high
as 31° C. at midday. The varia-
tions in volume amplified 45 times
shown hi figure 34. The record
beginning at noon on February 14,
with the temperature of the leaves
at 25° C., was at a juncture when
shrinkage set in, lessening the
thickness of the leaf about 1 mm.,
or 10 per cent of its turgid thick-
ness before 5h30m p. m., at which
time, with the temperature still at a
high point (27° C.), the shrinkage
came to an end and enlargement
began, which continued through the
night, so that at 8 o'clock the follow-
ing morning the- leaf was actually
thicker than on the preceding day.
The temperature on this day rose
to 31° C. and the shrinkage exceeded
that of the preceding day, amount-
ing to about 13 per cent of the
turgid thickness of the leaf. Swell-
ing began again in the evening,
which restored the leaf to about its
original dimensions 48 hours after
the beginning of the record.

The variations appeared to run parallel in part only to those already
described in detail f or Opuntia, and to the extent that the daily variations
take the form of alternate shrinking and enlargement with the increase
in excess of the loss. The chief features of growth may be illustrated by
the record of one of a pair of leaves which had attained about two-thirds
of the full size. This was put in bearing with the auxograph in the

FIG. 33. — Detail of arrangement of auxo-
graph to record variations in thickness of
leaf of Mesembryanthemum. Leaf in a
horizontal position resting on a wooden
support cut away below to make place for
the younger terminal leaves.

Some Hydration Reactions and Growth.

147

I2p.m. m. J2p.m.

T

X45

12 p.m.

manner described, and a portion of the record is given in figure 35. As
may be seen, the period of shrinkage, which begins about the same time
in the older leaf, continues for a shorter period, on some days not more
than 2 hours, and enlargement sets in in mid-
afternoon. The thickness on each successive
morning was greater than at the same tune on
the preceding day, demonstrating that actual
growth was in progress.

Two series of measurements were now un-
dertaken to secure new records of the elonga-
tion of leaves which had reached about half
the final length and of others still younger.
Such a pair of young leaves, with their surf aces
still appressed in an erect position, were
brought into bearing on an auxograph lever in
a sunny place in a glass-house. The length of
the exposed portion was about 25 mm. and
their thickness was not over 0.5 mm. at the
beginning of the tests. Here, as in previous
preparations, it was found that whatever the
causes of the stoppage of growth and of
shrinkage might be, they were not effective in
producing an actual cessation of elongation,
which hi these young leaves continued through-
out the 24 hours of the day variously respon-
sive to alterations in temperature (fig. 36). l

Another pair which were about to spread by the growth of the bud
ensheathed between their bases were attached to the auxograph and
set in a place where they would be shielded from the direct rays of the
sun. Preparations of all three stages increased in length and thickness

FIG. 34. — Upper part of fig-
ure is an auxographic rec-
ord of variations in thick-
ness of leaf of Mesembry-
anthemum on a cloudy
day with but little change
in temperature, as taken
by a mercurial thermom-
eter from a similar leaf.
Upward course of line de-
notes increase. X45.

Lower figure is an auxo-
graphic tracing of same
leaf on sunny day with
temperatures of 16° to 25°
C. Shrinkage occurred
during the entire day-
light period. X45.

5
15
25

35
45
55
65

I2p.m m.

12p.m. m. 12p.m. m. 12 p.m. m.

T

/

_T

I

24"C,

,27'C.

\

\

\

±

FIG. 35 — Auxographic record of thickness of pair of leaves about half mature size. X45. A
daily shrinkage between 8 a. m. and midday occurred, which amounted to an increasing pro-
portion of the increase taking place during the remainder of the day.

during the entire night, the increase in thickness being very rapid
during the first half of the night and slowing down to a very low rate

1MacDougal and Spoehr. Growth and imbibition. Proc. Amer. Phil. Soc., 56: 310. 1917.

148

Hydration and Growth.

afterward, beginning actual shrinkage by 9 a. m. Growth of the leaf
of middle age slows down with the low temperature of daybreak, but
accelerates at 9 a. m., at the time the older leaf begins shrinking, at
17° C. The youngest pair of leaves grows with a high rate all night,
with a perceptible slowing at daybreak, but it also accelerates at 9 a. m.
with the rising temperature, at the time the oldest leaves are shrinking
(fig. 37).

12p.m. m.

12p.m.

15
25
35
45
55
rr

V / /

\

I4*C.\

zz'o.

\

\

X20

\ v°

f-

12 p.m.

12p.m.

5
15
25
35
45
55

' / /

J / /

[\I6°C.

J

[

I8°CV

otfr

X20

^

"X

i

i

\

B

I2p.m.

3l°C.

FIG. 36. — A, auxographic record of elongation of pair of leaves 2 cm. long during a 24-hour
period; temperatures of 14° to 25° C. Retardation during period of highest temperature is
illustrated. X20. B, action of same leaves in shade.

The general features of growth under the usual varying conditions
of alternating daylight-high temperature and night-low temperature
complexes being determined, it became necessary to test the swelling
of the leaves, as had been done with the joints of Opuntia to ascertain
their unsatisfied hydration capacity.

Preparations for testing the swell-
ing of living material of leaves were s
made by placing these triangular !5
organs on a flat surface alongside a 25
guide of 5 mm. in thickness. A
razor slid along this slices away the
uppermost angle, leaving a trun-
cated section 5 mm. in thickness, in
which the central fibrovascular
tissue could be seen through the
translucent parenchymatous tissue.
Segments about 1 cm. long were
taken, to the exclusion of the basal and apical parts of the leaf. It is
to be noted that active enlargement is usually in progress morning
and evening in young leaves, while mature leaves are enlarging in the
morning but shrinking in the afternoon and night. The results of the
hydrations are shown in table 110.

In the above tests the amount of swelling was most in distilled water,
less in hydroxid, and least in acid, in mature leaves taken in the even-

35

45

55

FIG. 37. — Variations in thickness of a mature
leaf of Mesembryanthemum. X45. Tem-
peratures taken from a similar leaf with
a mercurial thermometer.

Some Hydration Reactions and Growth.

149

ing while in a shrinking stage. These differences hold for the morning,
except that the effects of acid and hydroxid are about equal.

TABLE 110.

Mesembryanthemum,
median slices of
leaves.

Taken at 6 p. m.,
swelled at
15 to 17° C.

Taken at 8 a. m.,
swelled at
13 to 15° C.

Mature
leaves.

Young
leaves.

Mature
leaves.

Young
leaves.

Distilled water

p. ct.
18
16
15

p. ct.
12
16
12

p. ct.
13

8
8

p. ct.
22
13
47

Sodium hydroxid, 0.01 M
Citric acid, 0.01 N

An additional set of measurements with young leaves decreased the
differential between the more acid condition of the morning and the
less acid condition of the evening, as is shown in table 111.

TABLE 111.

Young leaves,

Young leaves,

taken at 6

taken at 8

Mesembryanthemum.

p. m., and
swelled in

a. m., and
swelled in

darkness at

darkness at

12 to 15° C.

12 to 15° C.

p. ct.

p. ct.

Distilled water

13

12

Sodium hydroxid, 0.01 M . .

11

10

Citric acid, 0.01 N

11

10

Young leaves are generally in a state of enlargement both morning
and evening, and should show but little difference in swelling capacity
in the two stages, while mature leaves are enlarging in the morning
and shrinking hi the afternoon, in expression of a condition which is
reflected in the swelling reactions. Thus, for example, sections of
mature leaves taken in the morning, which had a thickness of about
9.3 mm., swelled 6 per cent at 14° to 19° C. in the dark room, which was
about the temperature at which they were taken. An equivalent set
taken at 1 p. m. swelled 15 per cent in water at 17° to 19° C. From
which it may be seen that the measurements of variations in thickness
of entire leaves (see pp. 147, 148) are in entire conformity with other
known facts.

The foregoing measurements were obtained from segments cut
from leaves about 1 cm. in length and with the epidermis on the three
faces intact. The measurements made for the purpose of determining
the possible effects of acidity were obtained by cutting slices which
removed one angle of the leaf and the epidermal face parallel to the
excised surface. If the water-loss is a factor, its effects would be most

150 Hydration and Growth.

pronounced in the layers which had been removed. The mature
leaves were distinctly flabby to the touch and the external layers were
in a state of partial collapse during the midday period.

Segments of young leaves in the stage in which these organs con-
tinued elongation and increase during the entire day were now taken
for comparison. Their thickness was a little more than half that of
the mature leaves. The trio of sections taken from leaves at 8 a. m.,
when the highest turgidity prevailed, showed a swelling of 13.6 per cent,
while those taken at midday swelled 16.7 per cent at 14° to 20° C.
The difference in the two cases is much less proportionately than that
which is set up in mature leaves and fully accounts for the shrinkage
of the older organs. Beyond this the auxographic tracings made obvi-
ous another difference between the young and mature leaves. The
total swelling in the mature leaves was reached in 6 or 8 hours, most
of which developed within 2 hours of immersion. The swelling of
young leaves was much more gradual, and the rate was less rapid at
first and then decreased much more gradually and had not actually
ceased at the end of 20 hours. The material in the young leaves was
not only alive, but in a growing condition; consequently new colloidal
material in the process of aggregation would provide a continuing
source of hydration capacity.

Confirmatory evidence consists in the fact that when two pairs of
leaves are put in bearing with auxographs, the one exposed to the sun
soon reaches the stage where the water-loss at midday is equivalent
to growth and neutralizes it on the record, while the pair of leaves
of the same age shaded from the sun continues elongation scarcely
checked or retarded during the same period. Now, if the preparation
in the sunny location experiences a cloudy day or is shaded, it too
continues growth during the entire day in a manner which shows con-
clusively that the daily retardation is in the main due to excessive
water-loss in the case of the Mesembryanthemum. A similar action by
the cacti has already been discussed in Chapter X.

The practice of testing the swelling of dried sections for comparison
with that of living material was followed as in Opuntia. Sections of
living leaves about 5 mm. in thickness were prepared as above, and
these were placed between folds of filter-paper and weighted only
sufficiently to prevent warping and curling during desiccation, which
continued for a week. Their final thickness was about 0.25 mm. and
their swelling, unlike that of the segments of Opuntia, did not come
back to the approximate size of the fresh material. The final measure-
ments in percentages of the dry thickness were as follows:

TABLE 112.

p. ct.

Distilled water 100

Citric acid, 0.01 N 120

Sodium hydroxid, 0.01 M 100

Some Hydration Reactions and Growth. 151

It had been previously concluded that the dominant factor in the
varying rate, and in producing shrinkage, was that of modified hydra-
tion capacity as dependent chiefly upon the acidity of the sap and the
balance between absorption and water-loss by transpiration. The
effects of the last-named feature were marked, as the experiments were
carried out under conditions of drought approaching the limit of
endurance of this plant. Sudden changes in hydrogen-ion concen-
tration of the sap of another species of this genus, with other unusual
features of the sap as noted by J. Hempel, may be responsible for some
of the aberrances shown by this plant.1 While this author gives nitro-
gen determinations showing much greater uniformity than in other
succulents, this uniformity of total nitrogen may include many changes
in amino-groups which might affect the water capacity of the colloids.
One species was found especially high, in nitrogen.

The sunflower has been used extensively in studies on growth-
rates, and the behavior of young and older internodes of the same stem
of Helianthus furnishes some homologies with the alterations exhibited
by the succulents. A stock of Helianthus annuus was grown in the
glass-house of the Desert Laboratory in February and March 1918,
at which time they showed vigorous and normal development. Dur-
ing most of the time in which these observations were made the tem-
perature of the air rose to 25° to 31° C. in the glass-house and some
slight wilting effects were noticeable in the leaves at midday, a fact
included in the records given below, to which are also attached the tem-
peratures taken by thermometers thrust into the stems. The young
parts in which growing cells constitute the greater part of the mass con-
tinue to increase during a period in which the older parts are shrinking.
The older parts consist of cylinders of tissue, almost mature, fully
saturated, with no continuing increase in hydration capacity; and the
growing cells are in the form of an irregular cylindrical shell under-
neath the epidermis, the thickness of which is no more than a small
fraction of the entire diameter. Consequently the external measure-
ments of the stem are chiefly determined by the changes in the mature
cell-masses.

The preliminary swelling tests were made with sections of the ter-
minal internodes from which tangential slices had been removed, leav-
ing them with a thickness of 2.7 mm. Such sections at 14° to 16° C.
swelled 7.4 per cent in distilled water, 9.2 per cent in hundredth-normal
sodium hydrate, 5.5 per cent in a similar solution of citric acid, and
7.4 per cent in a similar solution of potassium nitrate. These results
are fairly representative of this type of plants.

The preceding series was made up before the daily shortage of water
in the terminal parts of the stems had been detected. A second set of

1 Hempel, J. Buffer processes in the metabolism of succulent plants. Compt. Rend. d. Trav.
d. Lab. d. Carlsberg, 13. 1917. See pp. 45-51.

152 Hydration and Growth.

sections were therefore taken at Ih30m p. m., when the plant stood at a
temperature of 22° C., and these were swelled hi the dark room at
once with solutions which stood at 18° to 20° C. during the time of the
swelling. The increases were as follows:

TABLE 113.

p. ct.

Distilled water 70

Citric acid, 0.01 N 19

Sodium hydroxid, 0.01 M 46

Potassium chloride, hydrochloric acid, 0.01 M 14

The examination of the sections after the records were complete
showed that they were variously twisted and curled, due to the fact
that the internal parenchymatous tissues had swelled more than the
external layers. So far as could be estimated by simple observation
without measurement, the error did not double the measurement, how-
ever. Consequently it is to be seen that the imbibition capacity of
these stems, due to a depletion of the water-balance, is much greater at
noon than in early morning.

A repetition of the first test with whole sections that could not so
readily twist showed that another series of sections taken at 8 a. m.
swelled as follows, at 18° to 20° C.:

TABLE 114.

p. ct.

Distilled water 4

Citric acid, 0.01 N 4

Sodium hydroxid, 0.01 M 6.5

Potassium chloride, hydrochloric acid, 0.01 M 6

This series was characterized by the satisfaction of the full hydration
capacity within an hour or two, except in the case of the alkaline solu-
tion, in which the increase was very gradual. The material had re-
turned to its original dimensions within 6 hours in the other liquids
and continued to shrink. This action, coupled with decoloration, was
especially marked in the acidified saline solution. As a still further
verification of the above results, a trio of sections exactly like those of
the above series were taken at midday on the following day, and these
swelled 14 per cent in water, while another trio increased 10 per cent in
the acidified saline solution before shrinking.

The earlier measurements of the swelling of the sunflower having
been made with the terminal internodes of growing stems, a final series
was made in which were used the cotyledonary stalks from which the
plumules had been cut a day or two earlier. Measurements as follows
were obtained at 17° to 19° C.

TABLE 115.

p. ct.

Distilled water 6

Citric acid, 0.01 N 4

Sodium hydroxid, 0.01 M 7

Potassium chloride, hydrochloric acid, 0.01 M 7

Some Hydration Reactions and Growth. 153

Another trio of sections from an older stem measuring 5 mm. in
diameter was taken at midday, and when immersed in distilled water
at 21° C. increased 7.5 per cent. The entire lot of observations con-
firms and supports the conclusion that the stems of Helianthus have
their hydration capacity more nearly satisfied in the morning than at
noon, when the leaves may be in a wilting condition. This fact would
inevitably have an important influence on the rate at which such stems
might elongate.

The growth of stems of Helianthus was measured on stems growing
in the soil of a large bed. Heavy wooden bases were placed on the
surface of the soil of the greenhouse bench and the stems were brought
closely against this and fastened at the base of the growing internodes
in such manner that only the elongation above this point would be
registered by the auxograph, and the movements of the base due to
softness of soil or other features would have no effect. The growing
part consisted of one internode approaching maturity and a terminal
one less than 3 cm. in length. A fine wire loop was passed around this
and carried up over the arm of the auxograph lever.

Temperatures were taken by thermometers with thin bulbs thrust
into the stems of similar plants within a few centimeters of the one
being measured. As an example of the rate, the older and the younger
internodes, together having a length of about 15 cm., increased 2.7 mm.
during an hour at midday at a temperature of 30° C., while in the 2
hours immediately afterwards, when, as will be seen, the stem of another
plant was showing shrinkage in thickness, the rate was but 1.2 mm.
per hour at 29° C. During the next 4 hours the temperature slowly
fell to 19° C., but the rate of elongation came up to 2.1 mm. per hour,
a fact plainly due to decreased water-loss.

A similar behavior ensued on the following day, when the rate was
2.4 mm. per hour at midday at 29° to 30° C., then fell off to 1.4 mm.
per hour during the next two hours at 26° C., and then to 0.6 mm. per
hour during the following 2 hours. Such diminished growth might
be attributed to the falling temperature if it had not been observed
that a higher rate was shown at temperatures as low as 14° to 16° C.
This lowered rate in the afternoon was accompanied by a distinct
wilting of the leaves.

An auxograph was now provided with a cork bearing hollowed to
fit against a stem about 15 cm. from the apex, and the stem was held
firmly in place, so that any variation in thickness would be expressed
by the free arm of the auxograph and traced on the revolving cylinder
by the pen. The daily action may be exemplified by the following
transcript from the notebook :

" Feb. 8. The temperature had risen from about 16° C. in the morning to
23° C. at 10 a. m., at which time an increase in the thickness of the stem at a
point 15 cm. from the tip had been in progress for 4 hours. The pen was

154

Hydration and Growth.

stationary at midday, with a stem temperature of 26° to 29° C. Actual
shrinkage now began and continued through the afternoon, but all action
ceased at night. On the following day swelling or increase in thickness began
at 8 a. m. at a temperature of 12.5 °C., but continued for an hour only. The
leaves were beginning to flag at 10h30m a. m., as the plants had not been
watered, and shrinkage was in progress before noon at a temperature of
23.5° C." (See fig. 38.)

FIG. 38. — Detail of arrangement for recording variations in thickness of stem of Helianthus.
A, stem; B and C, parts of clamp holding stem rigidly in place; D, support; E, cork bearing
of short lever of auxograph; F, pen arm of auxograph, and G, rack-and- pinion column of
auxograph.

A second preparation was set up on February 10, in which the
variations in thickness were taken at a place but 6 cm. from the tip of
the stem. The adjustment had been completed by night and an in-
crease in thickness of 0.38 mm. took place in 12 hours at temperatures
between 24° and 18° C. Constant readjustment was necessary to
obtain reliable measurements, and on February 12 another record was
obtained, at which time an enlargement of 0.4 mm. was recorded
between 11 a. m. and 2h30m p. m. at temperatures of 22° to 25° C.
After this time a slight shrinkage occurred, although the plant was so
well supplied with water as to show no indications of wilting. Here,
as in the leaves of Mesenibryanthemum, elongations may be taking
place at a lessened rate in the extreme terminal part of a stem in which
the hydration capacity is kept continuously higher than in older inter-
nodes, while at the same moment an actual decrease in thickness may
be taking place within a few centimeters of the elongating active zone.
This shrinkage may ensue in a section of the stem which has not lost
the capacity for elongation altogether, so that its daily record shows a
period of elongation at a moderate rate during a part of the day, then
a cessation due to the depletion of the water-balance.1 It is obvious
that the action in question is one which may be responsible for many
mistaken generalizations bearing upon cessation of growth and effect
of temperature on growth (fig. 39). Attempts at interpretation of the

*See Brown and Trelease. Alternate shrinkage and elongation of growing stems of Cestrum
nocturnum. Philipp. Jour, of Science, 13: No. 6, 333. 1918.

Some Hydration Reactions and Growth.

155

rate and course of growth of any plant with differentiated tissues which
does not take into account the mechanical composition of the organs,
and especially the arrangement of the growing cell-masses with respect
to mature parts, may encounter many pitfalls and can hardly fail to
be inadequate.

5

15
25
35
45
55
65
75
85
95

Feb.22

8a.m.

I5°C.

X5

I2p.m. m. 12p.m. m. l?p.m. m. 12p.m. m. 12p.m. m.

FIG. 39. — The upper part of the figure shows the course of elongation of a stem of Helianthus
annuus for 24 hours beginning at 5 p. m., with temperatures of the plant as indicated, X 5.
Increase in length denoted by downward movement of the pen. Shown on a scale of milli-
meters as indicated, the total elongation during the peiiod being 19 mm. The lower tracing
shows variations in thickness of a stem of Helianthus 15 cm. from apex, the increase being
denoted by the upward movement of the pen, with temperatures of the plant as indicated.
Shrinkage or cessation of enlargement began after midday, but increase was again mani-
fested by evening. The variation is amplified 30 times and is shown on a millimeter scale,
the actual increase during six days being about 0.6 mm.

Opportunity for the measurement of growth in another type of
structure was presented by the legumes of Phaseolus cultivated in the
glass-house of the Desert Laboratory in April 1918. These pods are
first measurable when they have attained a length of about 3 cm. and
a thickness of 2 mm., and as they attain a final length of 10 to 12 cm.
in a week, the rate is rapid enough to afford ready means of detecting
variations and connecting them with possible modifying agencies.
The thickness of a mature pod through a full-sized bean may be as
much as 6 to 8 mm. The imbibition or swelling capacity of the entire
structure and its contents was tested at two different stages. The
measurements of this capacity in the earlier stage was made upon
sections of the pod less than a centimeter in length, which, by their
bulging contour, showed the presence of an embryo bean inside, al-
though this was much less than a millimeter in diameter and probably
played a very small part in the swelling. The increases of such sec-
tions at two different temperatures were as noted in table 116, the
average thickness of the trios of sections being 2.7 to 2.8 mm.

156

Hydration and Growth.

It will be seen by reference to the records of growth cited in table 116
that the higher temperature lies above the point at which the most
rapid elongation or thickening takes place, a matter which might be
due to excessive water-loss or to the action of residual acids at high
temperatures.

TABLE 116. TABLE 117.

Swelling of bean
pods.

18° C.

38° C.

p. ct.

p. ct.

Distilled water

2

2.7

Citric acid, 0.01 N. .

2

Shrinkage.

Swelling of beans.

18° C.

38° C.

p. ct.

p. ct.

Distilled water

11

10.6

Citric acid, 0.01 N. .

10.4

5.5

Beans nearly mature but still in the process of enlargement were
removed from green pods, the ends of the cotyledons cut away, and
then a slice removing the hypocotyl; the remainder of the cotyledons
came away free from the outer coating or mem-
brane. The average diameter of trios of such sec-
tions was 3 to 3.2 mm., and their swelling was as
given in table 117.

The amount of hydration was less at the higher
temperature in distilled water, suggesting that the
point of maximum imbibition or swelling lies be-
low 38° C. In the presence of acid the amount of
water absorbed is distinctly less than at 18° C.
These data being available, attention may now be
profitably turned to the features of enlargement of
the pods.

Preparations were made by which delicately
weighted auxographs recorded the variations in
thickness of pods in the stage when about half the
final length had been reached. The end of the
bearing-lever rested over an enlarging bean, and
variation during a week is shown in figure 40.

The localization of growth had been previously
determined by the well-known expedient of mark-
ing a young pod which was in the stage of initial
growth of the young beans into four 1-centimeter
intervals (fig. 41). Swelling tests had been made
of the pods in this stage (table 116). Ten days
later the basal and apical intervals had increased
to 2.5 cm., while the other two had each elongated
to 4 cm. All measurements of variations in thick-
ness were made in this median region of maximum elongation (fig. 42).

Growth both in length and thickness of the young pods was at the
lowest rate at night, during which period the temperature was at 15° C.

FIG. 40. — Arrangement of
vertical lever of auxo-
graph making record
of variations in thick-
ness of growing bean
and pod.

Some Hydration Reactions and Growth.

157

or below. As the temperature of the air rises above this point in the
morning, acceleration ensues and a high rate prevails until the ther-
mometer shows 25° C. or above, at which time retardation takes place,
which may continue to complete
cessation or even shrinkage. It was
noted that the behavior with respect
to the temperature in this region
might be modified by varying the
conditions of water-loss, and that
growth might be maintained at a
higher rate if high humidity around
the growing member is maintained.
As the temperature falls at the

FIG. 41. — Diagram showing the elonga-
tion of four 1-cm. intervals into which
a young pod was divided. Maximum
increase ensued in third centimeter
from base.

FIG. 42. — Arrangement of auxograph to
take variations in length of pod of
Phaseolus. The mature tip of pod is
fastened to cork buffer on end of lever of
instrument. ,

close of the day the rate accelerates again and growth is rapid until
checked by the falling temperature. (Fig. 43.)

These records were all made of plants not subjected to the direct'
action of the sun. One was placed in such position that the sunlight
fell directly upon the pod for about 15 minutes before 6 p. m., with the

m. tfpnti. m. 12p.m. m. 12p.m. m. 12p.m. m. 12p.m. m. I2.p.m. m. 12p.m. m.

FIG. 43. — Tracing of auxographic record of growth in length of bean pod, X 45. The features
of active elongation are similar to those described for variations in thickness in fig. 44.
Retardation of growth is seen at temperatures above 30° C. Downward course of pen
denotes increase.

result that a sudden enlargement followed, which was quickly retracted
and quiescence or slow shrinkage followed. Such sudden variations
have been seen in other types of organs, such as the joints of Opuntia,
and seem to be enlargements due to expansion of gases in the organ,
the cavity of the pods in this case being of such size that a marked
response might be expected (fig. 44). The features of variation of

158

Hydration and Growth.

thickness are recognizable in changes in length, although the action
of 120 mm. of tissue is involved as against 2 or 3 mm. in the measure-
ments of thickness. The structural arrangement of the cell-masses
and the shape of the cavity of the pod would operate to minimize the
shrinkages so apparent when thickness is measured.

i2p.m. m. 12p.m. m. 12p.m. m.

15

/

/

/ ;

/ •' '

/

">c.

/ I6°C
1~ \

/ 7

/ ;
(

|

•3K

1

^ f~i?£sJ

t, r

X45

i

28°C. \

i

I

FIG. 44. — Tracing of an auxographic record of growth in thickness of pod of Phaseolus. Down-
ward movement of the pen denotes increase in thickness, X 45. Temperatures given are
of the air near plants. The sudden shrinkage between 5 and 6 p. m. took place during
a brief daily illumination by direct rays of sun. Scale ruled to 5 mm. and 12-hour inter-
vals. Summer-time schedule. (See fig. 40 for illustration of the arrangement of auxo-
graphic levers.)

A pair of tests was now arranged in which one pod was placed in-
side a cell consisting of a short section of a glass T-tube of about 1 cm.
internal diameter. The pod was placed in a horizontal position in
the main section of this tube, which rested solidly on a concrete

f WHMW \VVW\V

FIG. 45. — Glass chamber for controlling humidity in making auxographic record of pod of Pha-
seoliis. Ends of horizontal part of chamber closed by cork stoppers fitted to stem of pod
and thermometer. Tube to be closed around vertical arm of instrument with cotton wool.

block. The end around its stem was closed loosely, and the opposite
end of the tube held a cork and small thermometer. The vertical
arm of the auxograph reached its bearing on the pod through the
upright arm of the T-tube and opportunity was given to keep record

Some Hydration Reactions and Growth.

159

of the temperature of the air, which in this setting must have been
practically identical with that of the inclosed pod (fig. 45). The air
inside this chamber was at a high degree of humidity, and it was
found that the afternoon cessation or retardation of growth was not
so marked in actively growing pods and that it did not come on so
early in the development of the pod as in those exposed to the
evaporating influence of the freely circulating air. The influence of
high humidity approaching saturation was shown more directly by the
application of wet slips of filter-paper in such manner that the pod
was completely swathed and evaporation reduced to a minimum.
This treatment was also successfully applied to pods resting upon a
cork base and not inclosed in the chamber. When a slowly growing
young pod was thus given an atmosphere of high humidity at tem-
peratures from 20° to 27° C. no alteration in the rate would be visible
for nearly an hour, but at the end of this time an abrupt acceleration
would ensue which would continue for as much as 2 hours, and then,
if the supply of moisture were not renewed, a slackening would ensue
which would bring the rate back to the point at which it was growing
previous to the treatment (see figs. 46 and 47).

•n. 12 p.m. i

n. 12 p.m.

7i. 12 p.m. r

n. 12p.m.

n. 12p.m.

TI. 12p.m. . m.

i

1

1

^5

»- 1
I

^\ ,'

1
j

i

i
I

l"

1

. i

i

1
1

i
i

H^

__^ jir'C.

30°C.l |nor

^

\ \ X45

\

\

r30°ai~ — -

— •*•-, "-* •-"
Y 27^^

f 3°"C< 1

\ \

\ \

\ \

\ V

\ \

\ '\ \

FIG. 46. — Tracing of auxographic record of pod of Phaaeolua which was 2 mm. in thickness at
beginning of record. Downward movement of pen denotes increase in thickness, X 45.
The range of active growth lies between 15° and 30° C. and consequently acceleration ensued
in the morning, retardation occurred as temperatures about 30° C. were reached in the after-
noon, and the rate increased again at sunset, when the temperature fell to a point below 30°
C., but slowing down followed in the cooler night temperatures.

12p.m. m. I2p.m. m. 12p.m. m. 12p.m. m.

5
15

//'/,' /'////

/ ' T — ^^— /— (/ 21 !' 3-l^

1 I 1 1 1 111!

FIG. 47. — Tracing of auxographic record of variations in thickness of young pod of bean. The
enlargements caused by humidity are seen at 1, 2, and 3.

The greater part of the enlargement registered in the above measure-
ments was due to the growth of the beans, and the imbibition capacity
of such seeds has been measured separately. A. Dachnowski (see
reference, p. 63) found that mature seeds absorbed and held more
water in acids and hydroxids than in distilled water at temperatures
not given, and that the amounts taken up in alkaline solutions was
greater than that in acids. The general imbibition reactions, in my
own experiments, of young seeds which had reached a thickness of

160 Hydration and Growth.

2.8 mm. to 3 mm., is shown by the measurements of swellings, made
at 15° to 16° C., shown in table 118.

TABLE 118.

p. ct.

Distilled water 8.2

Citric acid, 0.01 N 5.4

Potassium hydroxid, 0.01 M 12.5

Potassium nitrate, 0.01 M ; citric acid, 0.01 N 8.2

Actually lessened imbibition took place in acid as compared with that
in water; the addition of equimolecular solution of potassium nitrate
to acid brought the swelling up to that hi water. The total absorbed
in alkali was markedly greater than in any solution tested.

The measurements of variations in length and thickness of the suc-
culent leaves of Mesembryanthemum, stems of Helianthus, and of the
pods of Phaseolus, and the flattened stems of Opuntia, yield ample
evidence that the fluctuations hi growth show a direct relation to the
hydration capacity of the growing cell-masses, and that as a morpho-
logically complex member or organ approaches maturity, the fully
developed tissues show a varying water capacity different in many
respects from that of the embryonic cell-masses. Some of the irreg-
ularities hi the course of growth of internodes are due to the fact
that these members include regions of embryonic tissue and tracts hi
all stages of differentiation approaching maturity.

XII. WATER-CONTENT, DRY WEIGHT, AND OTHER
GENERAL CONSIDERATIONS.-

Two different types of organs or shoots with respect to the variations
in the water-content and dry weight are recognizable in the material
which has served for studies in growth as described in this volume and
in the work of other writers. The commoner types of woody stems,
of thin leaves, and of the organs of the greater number of the higher
plants undergo a development which terminates in a mature stage in
which the proportion of solid material is very much higher than that
found in younger material. A parallel procedure is the prevalent one
in the tissues of the higher animals. Thus, by way of illustration,
Donaldson found that the proportion of water in the bodies of mammals
diminishes with age, and Hatai has shown that the percentage of water
is an indicator of phases of chemical alteration in the composition of
the body.1

Growth and differentiation of cell-masses into specialized tissues is
not inseparably connected with increases in dry weight, however, as
has been demonstrated by studies of the growth of frog larvae2 in the
earlier stages, and it is highly probable that similar phenomena are
prevalent in the fleshy fungi and other lower forms of plants.

The distinction between the two kinds of growth has not been made
previously in studies of plants, and the matter was finally taken into
consideration in the experiments late in 1918. Stems of Helianthus
and pods of Phaseolus illustrate the kind of material in which dry
weight increases with age, upon which the greater part of all studies
in growth have been carried out.

Etiolated plants furnish examples of growth with a diminished
increase in dry weight. Chief interest attaches to plants which nor-
mally show such action, and the most striking illustrations are
furnished by the organs of succulent plants and by fruits. The
relative amount of solid material in the flattened joints of Opuntia does
not increase with the course of development toward maturity, and
joints which have reached full size may contain over 91 per cent of
water. Secondary thickening, especially that which results from
branching and the development of additional fibrovascular tissue,
may cause an added amount to be formed. The proportion of dried
material and water hi the leaves of Mesembryanthemum does not vary
greatly with age. These and probably all succulent forms are char-
acterized by an exaggerated production of mucilages or pentosans,
and have certain implied cycles of metabolism, including an incomplete

1 Donaldson, H. The relation of myelin to the loss of water in the mammalian nervous system
with advancing age. Proc. Nat. Acad. Sc., 2: 350. 1916. Hatai, S. Changes in the composi-
tion of the entire body of the albino rat during the life span. Amer. Jour. Anat., 1 : 23. 1917.

1 Ostwald, W. Ueber zeitlichen Eigenschaften der Entwickelungsvorgange, p. 49. 1908.

161

162 Hydration. and Growth.

type of respiration which leaves large acid residues. These, con-
stituting the total acidity of the cell-masses, may vary greatly during
development and during the course of a day, and the actual acidity or
hydrogen-ion concentration of the sap resulting from the buffer situa-
tion may also show a marked variation, but within narrower limits.

Although the development and maturation of fruits such as berries
obviously includes a growth in which the total effect is one of practical
maintenance or increase hi the water-content, studies of their growth
seem to be lacking. It was therefore planned to arrange a final series
of experiments in which the enlargement of fruits with increasing dry
weights and with small and more nearly constant dry weights should
be measured. The walnut was taken to represent a structure with
accumulating solid matter and the tomato for the other type.

The walnut consists of a thick, fleshy exocarp and a heavy endocarp
which finally becomes hard and bony with the deposition of anhydrous
wall material. The inclosed embryo also accumulates a large amount
of condensed food-material. The tomato is a large globose berry hi
which deposition and thickening is confined to the small, hard seeds.
The greater part of the fruit is a fleshy, watery pulp, which becomes
more highly hydrated as progress is made toward maturity.

Nuts of Juglans californica var. querdna Babcock, of various sizes
from 3 mm. in diameter to that approaching maturity, were borne on two
trees in the garden at Carmel, California, in June 1918. Suitable supports
being provided, the bearing lever of an auxograph was rested as lightly on
the young nuts as was consistent with a clear record, and temperatures
were taken by thin thermometers thrust into similar nuts or into young
stems near the preparation. 15 nuts were measured for periods of 2
or 3 days, or for as long as 2 months in the case of No. 10.

Coincidently with the measurements, an effort was made to determine
the degree of saturation or hydration of the stems on which the nuts
were borne. A well-defined "negative" pressure was detected hi the
basal branches of Juglans major, which was growing near the experi-
mental tree. A basal branch 1.2 meters from the trunk gave a dry-
looking surface when it was cut off.

A section of a similar branch about 8 mm. in thickness and 42 cm.
long was cut away from another basal branch of the tree, the end of the
detached portion quickly sealed with vaseline, and when all was in readi-
ness the tip was excised and the cut thrust into water to ascertain the
actual deficiency hi this portion; 14 hours later a total of 6 c. c. of
water had been absorbed and 24 hours later 8.5 c. c., which was a practi-
cal saturation, at a temperature of 18° to 20° C. The volume of the
branch proved to be 35 c. c., so that the amount of water absorbed was
24 per cent of the total.

Sections of young internodes of Juglans californica querdna which
had an average diameter of about 2.5 mm. were swelled in solutions as

Imbibition and Growth in Fruits.

163

below, then dried, and swelled again, with results as shown hi table 119
at 16° C.:

TABLE 119.

Swelling of sections of stems of
Juglans.

Fresh
living.

After
drying.

Distilled water

p. ct.
10

p. ct.
34

Citric acid, 0.01 N

14

34

Potassium hydroxid, 0.01 M

13.2

34

Potassium nitrate, 0.01 M

12

32

(On basis of original thickness.)

The unsatisfied water capacity of these sections taken from young
terminal internodes was comparatively great, doubtless due hi part to
the constant drain of the active leaves they bore. The older wood, in-
cluding that formed the previous year, showed an absorptive capacity
of 22 per cent hi water. It is from these older internodes that the nuts
arise.

The nuts were highly turgid, exuded sap when cut into, and hence
must have had a colloidal composition which acted to withdraw water
from the stems, which were less highly hydrated. The soil was low in
moisture-content at this tune, as it had been 4 or 5 months without
rain.

Tests of nuts 8 to 10mm., from which tangential slices had been removed
to give a uniform thickness of 7.5 mm., were made in July, and these
swelled at temperatures of 17° to 20° C. in solutions as follows:

TABLE 120.

Distilled water

Citric acid, 0.01 M

Potassium hydroxid, 0.01 M .
Potassium nitrate, 0.01 M . . ,

p. ct.
1.4
1.8
1.4
2

A useful conception of the hydration conditions in the stems and
fruits may be formed, if due weight is given to the measurements cited
above. The woody branches of the previous year, on which both the
leafy green twigs and those bearing the nuts are borne, had a relatively
large deficiency hi water, so that sections a few centimeters long absorbed
about 20 to 25 per cent of their volume of distilled water hi 24 hours at
20° C. No swelling test was made, but it is obvious that an enlarge-
ment of only a small fraction might be shown by this or any branch
with a mature woody cylinder. The active green twigs still hi a state
of elongation arising from these branches had a swelling capacity of 10
per cent. The growing nuts arising from the drier stems exuded water
from cut surfaces, the cotyledons being sacs of watery fluid, hi contrast
to the dry appearance of sections of the youngest internodes, and showed
a swelling of less than 2 per cent and soon shrunk when placed in a cylin-

164 Hydration and Growth.

der of distilled water after being cut in halves. In a system of this kind
any alteration of the conditions which would facilitate transpiration
would have a differential effect on the older stems, the green leafy twigs,
and the fruits. The loss from the stems would be affected least, since
the bark would effectually prevent any notable increase hi evaporation
from the relatively dry woody tissues. The loss from the leafy twigs
would of course tend to become greater and the deficit in both leaves
and twigs would be increased and their absorbing power correspondingly
increased. The outer integument of the nuts being still hi an embryonic
condition and being highly hydrated, the loss would reach a maximum
rate, with the daily effect of causing a cancellation of enlargement begin-
ning mid-forenoon at 20° to 22° C. and continuing until mid-afternoon,
when a fall in temperature brought transpiration to a rate below that of
accession from the stem.

A large percentage of the nuts which were placed under the auxo-
graph lever were cast off at various stages of development by abscission
of the stalk. The inciting causes of the anatomical change which
results in abscission lie outside the scope of this article. It was
noted, however, that it was preceded by a period in which the nut
showed a shrinkage by day in the higher temperatures and lessened
humidity, alternating with equalizing enlargements, at nights. Fin-
ally, an abrupt, rapid, and continuous shrinkage resulted in the separ-
ation of the stalk.

The general features of growth of these nuts may be illustrated by a
re'sume' of history of No. 10, which was under continuous observation
from July 15 to September 9, 1918, during which period of 56 days its
diameter increased from 16 mm. to 26.5 mm. Of this, 2.25 mm. was
gained in the first 5 days of cool, foggy weather. This effect was con-
firmed by the fact that a cessation or retardation occurred at midday
and was most pronounced on hot, sunny days, suggesting a direct
water-loss. In the week ending July 29 the total growth was an in-
crease of 1.7 mm. This period was characterized by heavy fogs and
mists in the forenoon, both the amount of shrinkage and rate of in-
crease being lessened — an equalization to be ascribed in part to ap-
proaching maturity. The temperature taken from a thermometer
thrust in a young branch of the thickness of the nut ranged from 13°
to 22° C. The completion of the record of No. 10 was followed by cut-
ting of the branch bearing it at a distance of 30 cm., placing the excised
end in water, and arranging the entire preparation in the dark room at
17° C., with the nut under the bearing lever of the auxograph. Swell-
ing continued for about 20 hours, after which shrinkage began, which
rapidly accelerated (see fig. 48).

The general features of growth are also well illustrated by the
following notes on No. 15, which was brought under observation when
it was about 15 mm. in diameter and put under an auxograph ampli-

Imbibition and Growth in Fruits.

165

fying 45 on August 3. Great daily variations in size, with a net total
increase, were displayed every day. Usually enlargement could be
detected between noon and 2 o'clock, which continued until 8 or 10
the following morning. If the sun rese clear, shrinkage began imme-
diately. If the morning was foggy it would be delayed. Minor vari-
ations might be brought about by the shade of clouds, especially notice-
able at noonday August 6 and to be seen at other times.

12p.m.

I2o.m. m. 12 p.m. m.

I5-22°C. during this beriod

25 I-

35

12p.m.

5

15
25

/

SEPT. 9, 1918 Or/e day in dark room at I7°C.
/ X 20 Swelling for 20 hours

Branch 30 cm
loni in vessel

-. / then slight shrinkage

of/water

-t— ^— — ; —

FIG. 48. — Variations in volume of nut of Juglans during 56 days. Enlargement is denoted by
downward course of pen tracing, X 10. The lowermost section of the figure gives auxographic
record of swelling of a nut in a dark room on a branch 30 cm. in length with the cut end in a
vessel of water. Swelling for 20 hours occurred after shrinkage began as denoted by upward
course of pen tracing.

After these facts were noted, experimental modifications were
arranged. Temperatures were taken from a branch 16 mm. in thick-
ness which were probably within a degree of that of the nut at all
times. A screen was arranged to cut off the direct rays of the sun at
midday, the nut being exposed for about 4 hours in the forenoon to di-
rect illumination. The temperatures ranged from 14° to 25° C. The
occurrence of fogs and of rain added to the variations in the conditions
affecting transpiration. The shrinkage in the forenoon was abrupt
and marked, being lessened on foggy days, and reaching an extreme of
4 mm. when the temperature rose from 14° to 25° C. in the 4 hours,
while it was on no day less than one-fourth this amount. The increase
varied from a minimum growth of less than 0.1 mm. on a cool, foggy

166

Hydration and Growth.

day to 0.7 mm. when shaded on August 6, and to a similar amount in
a rain on September 11, at which time it was in an advanced state of
development (fig. 49).

It is to be seen from the above that the fruit of the walnut in an
environment favorable to its development exhibits daily variations in
growth clearly attributable to the balance between transpiration and
absorption. The nut in a growing condition has a high water-content
and a small unsatisfied capacity, but its supply from the relatively
dry stems must come slowly — so slowly that any marked increase in
transpiration would overbalance the absorption by the nut and result
in cessation of enlargement or even shrinkage.

IZp.m. m. Ifp.m. m 12p.m. m. 12p.m. m. 12p.m. m. 12p.m.

/ NtylS i Jug|ans Calif. quqlrcina /

FIG. 49. — -Variations in volume of a growing nut of Juglana 15 mm. in diameter at beginning
for a period of 45 days. The marked acceleration under the conditions of high humidity
and abundant water-supply are illustrated in the record beginning September 9. Retarding
or shrinking effects of noonday temperature and low humidity and masking effects of fog
are also illustrated.

The fruit of the tomato (Lycopersicori) presents features of water-
content unlike any other organ the growth of which had been under
observation in present studies. The most striking feature of this
phase of the matter is that the proportion of solid material is higher in
young fruits than in mature ones. In the determination of the pro-
portions, first young fruits less than a week old were taken and 4
tomatoes with radial diameters of 14, 16, 17, and 18 mm. were found to
weigh 14.650 grams. These were fragmented and placed in a beaker
on a water-bath at about 100° C. for 48 hours, at which time the dry
material remaining was 1.90 grams. From this it is to be seen that the
young fruit contained 87 per cent of water and 13 per cent of dry
material. A mature fruit of the same kind as those measured was
46 mm. in axial diameter and 58 mm. in radial diameter and weighed
93.050 grams. This was dried over water-bath for 2 days, at which time

Imbibition and Growth in Fruits. 167

8.400 grams remained. From this it is to be seen that the ripe fruit con-
tained 91 per cent of water and 9 per cent of dry material. In fact,
these fruits show a better parallel to the hydration reactions of the
prepared biocolloids than any living material which has hitherto been
examined for the purpose of estimating the value of the physical fac-
tors in growth.

A number of plants of the tomato were grown in suitable boxes of
soil at a ranch in the Carmel Valley, and were in such a stage of de-
velopment that young fruits were available at the Coastal Laboratory
early in August 1918. Six plants in all were used and continuous rec-
ords from fruits of an axial diameter of 3 to 4 mm. to maturity at 50 to 55
mm. were obtained. The fruits were obla,te-spheroid in form and the
auxograph was arranged to register increase in diameter nearly par-
allel to the axis in some cases and radially or at right angles to it in
others. In addition to the other advantageous features of this mate-
rial, the regular form and mode of growth made it possible to use the
Variations in diameter as a basis for calculating the changes in volume
of the fruits taken as spheres.

Temperatures were taken by thrusting the thin bulbs of small
thermometers into fruits near the one tinder measurement. The
development of such fruits was but little affected by this wounding
and the thermometers remained firmly in place, as in the fleshy joints
of Opuntia, in the measurement of which this method was first practiced.
The preparations stood in a well-ventilated glass-house and the soil
around the roots was kept moist in accordance with the cultural re-
quirements of these plants. The results may be best set forth by the
description of the action of the several fruits measured.

No. 1 was placed in the greenhouse and a fruit 29 mm. in diameter
was fixed on a block of hard cork in such position that it gave a radial
bearing to the auxograph, which was set to amplify changes in volume
by 5, on August 9. The record was kept continuously until September
18, at which time the radial diameter of the fruit was 51.5 mm. The
fruit was turning yellow on September 16 and was showing fluctuations
in volume comparable to those in No. 2, with which it was run in close
comparison and under almost exactly the same conditions of moisture
and temperature as recorded.

No. 2 was adjusted to the auxograph in the greenhouse on August 9,
in such manner as to give modifications of the axial diameter, which
at this tune was about 27 mm. The record was continuous until
September 18, at which time the diameter was 50.5 mm. This fruit,
like No. 1, was beginning to turn yellow on September 16.

No. 3, 10 mm. in diameter, was adjusted to the auxograph to record
variations in radial diameter on August 21, and a record was kept
continuously with frequent notations of temperature and sunshine,
etc. It is to be noted that 1, 2, and 3 were under equable temperatures,

168

Hydration and Growth.

19° to 20° C., and high relative humidity during the rainfall of Sep-
tember 11 and 12.

The fact that the greatest increase in growth occurs in fruits at
diameters between 16 and 25 mm. hi diameter, before half the final
size is reached, is a point to which we shall recur in the discussion of
growth in terms of volume. Thus, in No. 3 the increases in thickness
weekly were as follows: 6 mm., 6.3 mm., 2.5 mm., 3.5 mm. (fig. 50).

I2p.m. m. 12p.m. m. 12p.m. m. 12p.m. m. 12p.m. m. 12p.m. m. 12p.m.

FIG. 50. — Variations in radial or transverse diameter of a tomato during development in 28 days.
Increase is denoted by downward course of the auxographic tracing, and the direct effects of
temperatures and relative humidity are illustrated by the record and the accompanying
notations in the figure. Amplified 5 times on scale ruled to 5 mm. intervals.

Imbibition and Growth in Fruits.

169

If this method be followed it would at once be obvious that while the
rate of increase in diameter would be a direct measurement, yet as the
fruit increases as a globe the actual material added could be regarded
as a shell on this globe. The rate in terms of volume would therefore
be the amount of this shell to be calculated by finding the difference
between the initial volume and the volume at the end of each period.
The rate by direct measurement of diameter and by volume increases
may be compared as hi table 121, for periods of one week beginning
on the date given.

TABLE 121. — Average daily rate of growth. TABLE 122.

Date.

Diameter,
millimeters.

Volume,
cubic
millimeters.

Aug. 9.

1.7

2,604

16.

1.1

2,513

21.

0.7

2,064

28.

0.4

1,373

Sept. 4 .

0.28

976

11.

0.17

695

Date.

Diameter,
millimeters.

Volume,
cubic
millimeters.

Aug. 9.

0.95

2,072

16.

0.7

1,852

21.

0.56

1,800

28.

0.3

660

Sept. 4.

0.2

508

11.

0.2

560

The rate on September 11 by direct measurement would appear to
be one-tenth that of a month earlier, yet actually water and new mate-
rial was being added at a rate equivalent to one-fourth of the earlier
rate. The radial proportions would make the rate on August 21 not
much more than 40 per cent of the rate on August 9, while the increase
in volume was over 96 per cent. The rate hi the week beginning
August 28 would appear to be less than a fourth that by direct measure-
ment on August 9, yet actually the increment of water and material is
more than half that in the younger stage and smaller size.

A second plant with the auxograph arranged to take axial varia-
tions in the fruits which measured 33 mm. was arranged to run con-
currently with No. 1 and under identical temperature and conditions
of moisture. The daily rates of increase in diameter were as shown
in table 122 for weeks beginning on the dates given.

Here again the actual course of growth as calculated in terms of
volume shows that simple measurements of the thickness do not express
the real values in growth of such organs.

The third test was made on a fruit taken at a much earlier stage at a
diameter of 16 mm. with a transverse or radial bearing, the tempera-
ture and moisture conditions being similar to those of 1 and 2. The
daily rate of increase was as shown in table 123 for the weeks begin-
ning on the given dates.

The actual volume of this fruit at the close of the experiment was
approximately 2,900 c. mm. and its growth had been followed for a
period of 40 days. It is notable that in the earlier stage in the advance
of the fruit from 20 to 26 mm. in diameter (August 21 to August 31),
while the increase of the diameter seems constant, yet the actual

170

Hydration and Growth.

accession of material is very much greater. Then, in further develop-
ment, the average increment to the diameter was smaller, yet the actual
accession of material was greater (see September 4). Following this,
the rate falling from 0.8 to 0.3 mm. daily, the accession decreases less
than half. (See figs. 51 and 52.)

TABLE 124.

TABLE 123.

Date.

Diameter,
millimeters.

Volume,
cubic
millimeters.

Aug. 21.

0.85

537

28.

0.85

851

Sept. 4.

0.64

885

11.

0.8

1,643

18.

0.3

594

25.

0.37

662

20°

C.

30°

C.

Diam.

Volume.

Diam.

Volume.

Sept. 1.

mm.
1

c. mm.
72

mm.

c. mm.

Sept. 7. ...
Sept. 14. ...
Sept. 21....

2
1.4
0.08

33
33
9

0.8
.3

.085

128
91
27

Attention was now directed to temperature effects as measured in
this manner. Two plants were placed in chambers subjected to equiv-
alent diffuse illumination and humidity. The fruits similar to those

\

i 1 1 • . ,

FIG. 51. — Diagram illustrating the course
of growth of a tomato during the six
weeks of its development. The broken
line is plotted from the average daily
rate of growth during each week, and
the solid line from the calculated in-
creases in volume.

Fro. 52. — Similar to fig. 51, but begin-
ning at an earlier stage. The average
daily rate is seen to form a graph which
presents notable differences from the
one plotted from variations in volume.

measured in one showed thermometer readings of 19° to 21° C. and
in the other 29° to 31° C. The daily rates of axial increase were as
shown in table 124 for the weeks beginning on the given dates.

The conditions under which both plants were grown were unfavor-
able to development, but it is to be noted that the rates of increase

Imbibition and Growth in Fruits. 171

sustained a changing relation as growth slackened. The enlargement
of such highly watery fruits must be so largely a matter of diffusion
and hydration that any formula expressive of the temperature re-
lations of chemical transformation must be wide of the facts in many
stages of development.

The record of growth of No. 3, which is given in full in figure 50,
shows beyond question the effect of transpiration and water-loss on
growth. As the daily temperatures of the fruits rose from 12° C. and
14° C. to 26° C. and 28° C., acceleration ensued up to a point where
the rise caused a water-loss overbalancing the gain by hydration.
Higher temperatures, therefore, did not facilitate or accelerate growth
unless accompanied by high relative humidity. Thus the highest
growth rates are those of midday and afternoon, with fog or showers.
This is especially marked on the records of September 10, 11, 12, and
13, in which a 50-hour rainy period was anticipated and followed by
high humidity. (See fig. 50.) It was not possible to increase the
water-supply by watering the soil around the roots in such manner as
to cancel the midday shrinkage or slackening in growth. One espe-
cially striking effect is that in which the rise in temperature conse-
quent upon the cessation of the rain, from 20° to 25° C. at 3 p. m. on
September 13, was followed by a lessened rate of growth. On the
cloudy days growth was uniformly high. Similar effects were exhib-
ited by a small fruit of a potato in a greenhouse at Tucson in May
1918.

The two types of fruits are seen to show a concordant behavior with
respect to the balance between the water-supply and transpiration.
A rise in temperature with accompanying lessened relative humidity
had the effect of retarding or stopping growth or of producing an actual
shrinkage in volume. The nut and the berry are both more highly
hydrated or more watery than the stem through which their water-
supply must be drawn. This was established by measurement in the
walnut and is obvious with respect to the tomato and its stems.

A distinction must be made between the water-relations of a fruit
and its stem and that which prevails between a parasite and its host,
or between a swelling colloid and the solution in which it may be im-
mersed. The water deficit of the stems as measured by swelling in-
cludes that of the entire structure. The fruits, however, receive their
supply through special conduits which sustain only a mechanical rela-
tion to the other parts of the stem which may be active in its swelling.
Such non-cctaducting tissues of course draw their supply from this
system of conduits also, but it is highly probable that the dispropor-
tion between the water-content of the fruit and of the tracts in the
stem from which it receives its supply is not so great as might be
indicated by the measurements given. The hydration capacity of the
fruits would be the resultant of many factors, including the pentosan-

172 Hydration and Growth.

protein ratio, the hydrogen-ion concentration, the action of salts, and
the effect of the amino compounds.

The delicate balance between water-loss and absorption as revealed
by measurements of growing organs of all kinds is very striking. The
rate at which water is received is generally so little hi excess of the
transpiration that a rise of 10 to 15 degrees centigrade may extinguish
the balance. At the same time, such rise in temperature may also
result in a lessened hydration capacity, so that by the action of the
acids at the higher temperature, water may theoretically be forced out
of the colloidal complex.

It is plainly evident that growth consists of two fundamental
features — hydration of the colloidal material of the plasma and the
arrangement of additional material hi colloidal structures with the
entailed additional capacity for adsorbing water. The first may
occur without the second, and increase in volume might occur
in a pentosan-protein colloid at any time by the action of its
own metabolic products, such as the hydrogen-ion concentration
or the proportion of ammo-compounds formed. Growth by acces-
sion of solid material without a corresponding absorption of water
is characteristic of cell organs or walls, and such deposition of
material can only result in changes hi volume which would not be
measurable by auxographic methods.

Hydration consists, in the first instance, of the union of molecules of
water with the molecules of solid material in the colloidal masses, and
it is this action which is entailed in the initial and almost instantaneous
enlargement of dried sections when water is poured on them. No serious
reason has yet been advanced, however, against the extension of the
term to apply to the accompanying and subsequent adsorption of an
indefinite number of molecules on the surfaces of the molecular aggre-
gates. Cell-masses are already in an advanced stage of hydration, and
all of the tests with living material are simply modifications of such a
condition. The swelling of dried sections of plant tissue may include
some chemical action, or some union of water with the solid material
in definite proportions.

The manner in which hydration ensues, or rather the character of the
process, will naturally depend upon the character of the cell colloids.
If these are albuminous, swelling will be largely determined by
the hydrogen-ion concentration of the solution. It also follows that
any cell-organ or cell-mass which is dominantly proteinaceous will
show such increases of hydration capacity with acidity, modified by
other facts, including the presence of salts or bases.

These effects are modified or reversed in colloidal material which
consists more largely of carbohydrate material. The pentosans
represented by various gums and mucilages are abundant in plant
cells, and these present some variety of composition and differences

Imbibition and Growth in Fruits. 173

in solubility or dispersibility. One group which may. be illustrated
by agar has a definitely limited swelling capacity under temperatures
below 50° C. and other conditions, and of course is not soluble. Others,
like the mucilages of Opuntia or acacia or tragacanth, are soluble, and
when placed in water pass from a dry solid state to a complete solution.
The solubility of protoplasm will depend upon the presence of these
substances, as well as upon the albumins which may be present.

The ideal capacity for hydration and growth of any mass of proto-
plasm would be a resultant of the composition and proportions of its
organic material and of the relation of the phases in which they occur.
The theoretical maximum hydration of a carbohydrate-protein system
is invariably modified by the nutrient salts adsorbed in its structure and
by the products of unceasing metabolic changes, especially the trans-
formations which are comprehended in respiration and which carry
compounds through a stage in which acids are formed. These features,
as influenced by temperature, determine the rate, daily course, and total
expansion in growth. In addition, a certain amount of material is lost
from the plant in the form of carbon dioxid, and, as has been emphasized
on the preceding pages, the surface loss of water may on occasion be
greater than the amount passing into the growing cell-masses. The
above-mentioned processes and agencies affect the rate, course, and
amount of growth.

LITERATURE CITED.

ASKENASY, E. Ueber die Temperature, welche Pflanzen im Sonnenlicht annehmen. Bot.

Zeitung., 33:441-2. 1875.
BARRY, F. The influence of temperature upon chemical reactions in general. Amer.

Jour. Bot., 1:203. 1914.

BEIJERINCK, M. W. Ueber Emulsoidenbildung bei der Vermischung wasseriger Losungen
gewisser gelatinierendern Kolloide. Zeitschrift f. Kolloid-Chem., 7: 16.
1910.

BERMAN, N., and L. F. RETTGER. The influence of carbohydrates on the nitrogen meta-
bolism of bacteria. Jour. Bacter., 3:389. 1918.
BOROWIKOW, G. A. Ueber die Ursachen des Wachstums der Pflanzen. Biochem. Zeitschr.,

48:230, and 50: 119. 1913.

BROOKS, S. C. Permeability of cell-walls of Allium. Botan. Gaz., 64:509. 1917.
BROWN and TRELEASE. Alternate shrinkage and elongation of growing stems of Oestrum
nocturnum. Philip. Jour, of Science 13: No. 6. Bot., p. 353. Nov. 1918.
CLARK, Lois. Acidity of marine algae. Puget Sound Marine Sta. Publ. 1, No. 22. 1917.
CONKLIN, E. G. Effects of centrifugal force on the structure and development of the eggs

of Crepidida. Jour. Exper. Zool., 22: 356-364. Feb. 1917.

DACHNOWSKI, A. The effects of acid and alkaline solutions upon the water-relations and
the metabolism of plants. Amer. Jour. Bot., 1:412-439. 1914.

and GORMLEY. The physiological water requirement and the growth of plants

in glycocoll solutions. Amer. Jour. Bot., 1: 174-185. 1914.
DAKIN, H. D. The catalytic action of amino-acids, etc., in effecting certain syntheses.

Jour. Biol. Chem., 7:49-55. 1909.

DENNY, F. E. Permeability of certain plant membranes to water. Bot. Gazette, 63: 373-
397. 1917.

. Permeability of membranes as related to their composition. Bot. Gazette, 63:

468-485. 1917.
DONALDSON, H. The relation of myelin to the loss of water in the mammalian nervous

system with advancing age. Proc. Nat. Acad. Sci., 2:350. 1916.
FENN, W. 0. Similarity in the behavior of protoplasm and gelatine. Proc. Nat. Acad.

Sci., 2: 539. 1916.

FREE, E. E. Note on the swelling of gelatine and agar gels in solutions of sucrose and
dextrose. Science, n. s., 46: 142. 1917.

. A colloidal hypothesis of protoplasmic permeability. The Plant World, 21: 141.

1918.
FREUNDLICH, H. Kapillarchemie; eine Darstellung der Chemie der Kolloide und verwandte

Gebiete, pages 504-511. 1909.
GRAHAM, E. A., and H. T. GRAHAM. Retardation by sugars of diffusion by acids in gels.

Jour. Amer. Chem. Soc., 40: 1900. 1918.

HAAS, A. R. The reaction of plant protoplasm. Bot. Gazette, 63 : 232. 1917.
HARDY, W. B. On the structure of cell protoplasm. Jour. Physiol., 24: 158. 1899.
HARVEY, R. B. Hardening processes in plants and developments from frost injury. Jour.

Agric. Res., 15:83. 1918.
HATAI, S. Changes in the composition of the entire body of the albino rat during the life

span. Amer. Jour. Anat., 1:23. 1917.
HEMPEL, JENNY. The buffer processes in the metabolism of succulent plants. Compt.

Rend. d. trav. d. Lab. Carlsberg., 13: No. 1. 1917.
HOFMEISTER, F. Die Betheiligung geloster Stoffe an Quellungsvorgange. Archiv f.

Exper. Pathol. u. Pharm., 27: 395, 1890, and 28: 210, 189 V
KARSTEN, H. Die Einwirkung des Lichtes auf das Wachstums der Pflanzen. Jena.

1870.
KITE, G. L. The physical properties of protoplasm of certain plant and animal cells.

Amer. Jour. Physiol., 32: 146 (especially pp. 161 and 162). 1913.

KUNKEL, Lotus OTTO. A study of the problem of water absorption. Twenty-third Annual
Report, Missouri Botanical Garden, pp. 26-40. 1912.

174

Literature Cited. 175

LLOYD, FRANCIS E. Colloidal phenomena in the protoplasm of pollen tubes. Report

Dept. Bot. Research, Carnegie Inst. Wash, for 1917, Year Book No. 16.

1918.
. The origin and nature of the mucilage in the cacti and in certain other plants.

Amer. Jour, of Bot. 6: 156. 1919.
LOEB, J. The similarity of the action of salts upon the swelling of animal membranes and

of powdered colloids. Jour. Biol. Chem., 31: 343. 1917.
. Amphoteric colloids. I: Chemical influence of the hydrogen-ion concentration.

Jour. Gen. Physiol., 1:39. 1918.
LONG, E. R... Growth and colloid hydratation in cacti. Botan. Gazette, 59: No. 6. June

1915.
. Acid accumulation and destruction in large succulents. The Plant World, 18:

No. 10, 261. 1918.
MACCALLUM, A. M. The distribution of potassium in animal and vegetable cells. Jour.

Physiol., 32: 95. 1905.

. Presidential address, Brit. Assoc. for Adv. of Sci., Report for 1910, 744.

MAcDouGAL, D. T. The influence of etiolation upon chemical composition, in "The

influence of light and darkness upon growth and development." Mem.

N. Y. Bot. Garden, 2: 300-305. 1903.
. The Salton Sea: A study of the geography, the geology, the floristics, and ecology

of a desert basin. Carnegie Inst. Wash. Pub. No. 193. 1914.
. The experimental modification of germ-plasm. Annals Mo. Bot. Garden, 2: 253-

274., Feb.-Apr. 1915.

. Mechanism and conditions of growth. Mem. N. Y. Bot. Garden, 6: 5-26. 1916.

. Imbibitional swelling of plants and colloidal mixtures. Science, n. s., 44: 502-

505. 1916.

. The beginnings and physical basis of parasitism. The Plant World, 20 : 238. 1917.

. The relation of growth and swelling of plants and biocolloids to temperature.

Proc. Soc. Exper. Biol. and Med., 15: 48. 1917.
. The effect of bog and swamp waters on swelling in plants and in biocolloids. The

Plant World, 21 : 88-99. 1918.
, and W. A. CANNON. The conditions of parasitism in plants. Carnegie Inst.

Wash. Pub. No. 129. 1910.
, H. M. RICHARDS, and H. A. SPOEHR. The basis of succulence in plants. Botan.

Gazette, 67:405. 1919.
, and H. A. SPOEHR. The behavior of certain gels useful in the interpretation of the

action of plants. Science, n. s., 45:484-488. 1916.

. Growth and imbibition. Proc. Amer. Phil. Soc., 56: 289. 1917.

. The effects of acids and salts on biocolloids. Science, n. s., 46 : 269. 1917.

. The effect of organic acids and their amino-compounds on the hydration

of agar and on a biocolloid. Proc. Soc. Exper. Biol. and Med., 16 : 33. 1918.

. The origination of xerophytism. The Plant World, 21: 245. 1919.

. The solution and fixation accompanying swelling and drying of bio-
colloids and plant tissues. The Plant World, 22:—. 1919.
MATHEWS, A. P. Physiological chemistry, 2d ed. p. 211. 1916.
McCLENDON, J. F. The physical chemistry of vital phenomena, page 95. 1917.
McGsE, J. M. Theeffectof position on the temperature and dry weight of joints of Opuntia.

Rep. Dept. Bot. Research, Carnegie Inst. Wash, for 1916, Year Book No.

15. Feb. 15, 1917.

. The imbibitional swelling of marine algae. The Plant World, 21: 13. 1918.

OSTERHOUT, W. J. V. The permeability of living cells to salts in pure and balanced solu-
tions. Science, n. s., 34: 187-189. Aug. 11, 1911.

. Nature of antagonism. Science, n. s., 41: 255-256. 1915.

. Some aspects of the temperature coefficients of life processes. Jour. Biol. Chem.,

32: No. 1. 1917.

OSTWALD, W. Ueber zeitlichen Eigenschaften der Entwickelungs-vorgange, page 49. 1908.
, translated by M. FISCHER. An introduction to theoretical and applied colloid

chemistry, pages 84-92. 1909.
, and M. FISCHER. Theoretical and applied colloid chemistry. 1917.

176 Literature Cited.

PALLADIN, W. Eiweissgehalt der griiaen und der etiolirter Blatter. Ber. d. deut. Bot.

Ges., 9: 191. 1891.
. Transpiration als Ursache der Formanderung etiolirter Pflanzen. Ber. d.

deut. Bot. Ges., 8:364. 1890.
PEARSON, H. H. W. Observations on the internal temperatures of Euphorbia virosa and

Aloe dichotoma. Annals Bolus Herbarium, vol. i, part 2. Nov. 1914.
RIGG, G. B. Summary of bog theories. The Plant World, 19: 310. 1916.
ROBERTSON, T. B \The physical chemistry of proteins. 1918.
SCHIMPER, A. F. W. Die Indo-Malayscher Strandflora, page 142. 1891.
SHREVE, E. B. An analysis of the causes of variations in the transpiring power of cacti.

Physiol. Researches, 2: No. 13. Aug. 1916.
. A thermo-electrical method for the determination of leaf temperature. Plant

World, 22:100. 1919. P. 118.
SPOEHR, H. A. The pentose sugars in plant metabolism. The Plant World, 21 : 365-379.

1917.

. Carbohydrate economy of the cacti. Carnegie Inst. Wash. Pub. No. 287. 1919.

STEWART, E. G. Mucilage or slime formation in the cacti. Bull. Torr. Bot. Club, 46:

157. 1919.
STILES, WALTER, INGVAR J0RGENSEN. Studies in permeability. The swelling of plant

tissue in water and its relation to temperature and various dissolved sub-
stances. Annals of Botany, 31: 415. 1917.
, . Quantitative measurement of permeability. Botan. Gazette, 65: 526.

1918.
SWARTZ, M. D. Nutrition investigations on the carbohydrates of lichens, algae, and

related substances. Tran. Conn. Acad. Arts and Sciences, 16:247. 1911.

P. 29.
THODAY, D. On turgescence and the absorption of water by the cells of plants. New

Phytologist, 17: 108. 1918.
THOMPSON, D. A. W. Growth and form. 1917.
TOTTINGHAM, W. E. A quantitative chemical and physiological study of nutrient solutions

for plant cultures. Physiol. Res., l:No. 4. May 1914.
URSPRUNG, A. Die physikalischen Eigenschaften der Laubblatter. Bibliotheca Botanica,

12 (Heft 60), 68. 1903-4.
WACHTER, W. Untersuchungen tiber den Austritt von Zucker aus den Zellen der Speicher-

organe von Allium cepa und Beta mdgaris. Jahrb. f . wiss. Bot., 41 : 165.

1905.
ZSIGMONDT, R., translated by E. P. SPEAR. Chemistry of colloids, p. 222. 1917.

SCHOOL U8RW