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MONOGRAPHS ON EXPERIMENTAL BIOLOGY

EDITED BY
JACQUES LOEB, Rockefeller Institute
T. H. MORGAN, Columbia University
W. J. V. OSTERHOUT, Harvard University

INJURY, RECOVERY, AND DEATH, IN
RELATION TO CONDUCTIVITY AND
PERMEABILITY
BY
W. J. V. OSTERHOUT.

MONOGRAPHS ON EXPERIMENTAL
BIOLOGY

PUBLISHED
FORCED MOVEMENTS, TROPISMS, AND ANIMAL
CONDUCT

By JACQUES LOEB, Rockefeller Institute

THE ELEMENTARY NERVOUS SYSTEM
By G. H. PARKER, Harvard University

THE PHYSICAL BASIS OF HEREDITY
By T. H. MORGAN, Columbia University

INBREEDING AND OUTBREEDING: THEIR GENETIC
AND SOCIOLOGICAL SIGNIFICANCE
By E. M. EAST and D. F. JONES, Bussey Institution, Harvard University

THE NATURE OF ANIMAL LIGHT
By E. N. HARVEY, Princeton University

SMELL, TASTE AND ALLIED SENSES IN THE
VERTEBRATES
By G. H. PARKER, Harvard University

BIOLOGY OF DEATH
By R. PEARL, Johns Hopkins University

INJURY, RECOVERY, AND DEATH IN RELATION TO
CONDUCTIVITY AND PERMEABILITY
By W. J. V. OSTERHOUT, Harvard University

IN PREPARATION
PURE LINE INHERITANCE
By H.S. JENNINGS, Johns Hopkins University
LOCALIZATION OF MORPHOGENETIC SUBSTANCES
IN THE EGG
By E. G. CONKLIN, Princeton University

TISSUE CULTURE
By R. G. HARRISON, Yale University

THE EQUILIBRIUM BETWEEN ACIDS AND BASES IN
ORGANISM AND ENVIRONMENT
By L. J. HENDERSON, Harvard University

CHEMICAL BASIS OF GROWTH
By T. B. ROBERTSON, University of Toronto

COORDINATION IN LOCOMOTION
By A. R. MOORE, Rutgers College

OTHERS WILL FOLLOW

—- ~
MONOGRAPHS ON EXPERIMENTAL BIOLOGY

INJURY, RECOVERY, AND DEATH, IN
RELATION TO CONDUCTIVITY AND
PERMEABILITY

BY
W. J. V. OSTERHOUT.

Mt
PROFESSOR OF BOTANY.

S533
9 Le meme Re TT
WR ee os

PHILADELPHIA AND LONDON
J. B. LIPPINCOTT COMPANY

C)\J
i, A “

PRINTED BY J. B. LIPPINCOTT COMPANY
AT THE WASHINGTON SQUARE PRESS
PHILADELPHIA, U.S.A.

EDITOR’S ANNOUNCEMENT

THE rapidly increasing specialization makes it im-
possible for one author to cover satisfactorily the whole
field of modern Biology. This situation, which exists in
all the sciences, has induced English authors to issue
series of monographs in Biochemistry, Physiology, and
Physics. A number of American biologists have decided
to provide the same opportunity for the study of
Experimental Biology.

Biology, which not long ago was purely descriptive
and speculative, has begun to adopt the methods of the
exact sciences, recognizing that for permanent progress
not only experiments are required but that the experi-
ments should be of a quantitative character. It will be
the purpose of this series of monographs to emphasize
and further as much as possible this development of
Biology.

Experimental Biology and General Physiology are one
and the same science, by method as well as by contents,
since both aim at explaining life from the physico-chemical
constitution of living matter. The series of monographs
on Experimental Biology will therefore include the field
of traditional General Physiology.

- Jacques Logs,
T. H. Morean,
W. J. V. OstEeRHOUvT.

AUTHOR’S PREFACE

THis volume endeavors to treat certain aspects of
biology according to the spirit and methods of the exact
sciences. The treatment is confined to certain funda-
mental problems which have been studied quantitatively.
These studies lead to a theory of some aspects of injury,
recovery, and death, as well as of antagonism and per-
meability. The behavior of the organism in these
respects may be predicted with a satisfactory degree of
accuracy by means of the equations which express the
theory in mathematical form.

The author is under great obligation to the Marine
Biological Laboratory at Woods Hole for the facilities
generously placed at his disposal. He desires to make
grateful acknowledgement to Mr. F. 8S. Mathews and Mr.
G. B. Ray for the preparation of drawings and to Mr.
Lee Morrison for technical assistance in conducting

the experiments.

Tue AuTHOR.
Cambridge, Mass.

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CONTENTS

EE ace ek hana aera kid edu s Marviicdk ak kie Bilalae G bem eave 7
I Sia Tee i roe Be an Be gare Os eer eee Ue ele 15
CHAPTER I. METHODS oF MEASURING ELECTRICAL CONDUCTIVITY. ...... 21
CHAPTER IJ. THe MECHANISM OF THE PROCESS OF DEATH............. 40
See 511, INJURY AND RBCOVERY .. o.oo 6 cic cee cccecwcuctaace 79
Cis MMP MUMMMEEM GS S00. es rs eds wate gd Gave eae 124
I a MSE A Pe a ec oe 184
CHAPTER VI. CONDUCTIVITY AND PERMEABILITY..............cceceecs 195
NR edd ah a st fe | OE ee cata d ato echt eh Se me ee 237

re La

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CW ies, ak an ia

ve
U

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i a et f
¥, hy’ ay iv’ r eer p , 4 re oy ast mn U Arey fi
: t Pry co ae ’

it ree
aes Pa yh hh) af

by 4 thy aif ie ha he

ILLUSTRATIONS

FIG. PAGE

1. Apparatus for Measuring the Electrical Conductivity of Muscle
ET Soest s Kal tects peed Te Kote ais aaieh ee be aid Canineans wicaes bile 22

2. Apparatus for Measuring the Electrical Conductivity of Sea
CERNE Da oS asian at aed eau METS ay eR EE Pap eRe S 23

3. Apparatus for Determining the Electrical Conductivity of Living
NN ae oh eae Nave F's sn bn & oan ENE LTD Meo 4) Spe Lea ae 24
EN Ni a ss baa dia Mas pe wei eeawld opin ow S leak LRU cae 25
5. Hard Rubber Disks, Alternating with Disks of Tissue............ 29
6. Diagram to Show Bridge and Connections..................+++-. 30
rn CORNER ok isc hitless wiles a MGea ee Nahads pa tethe aes 31
EAM EMP Las oe Ce haw bebe tee pak Bh ee See Ue ee ale hvac 32

9. Two Glass Cells each Provided with an Electrode with Strip ot
EMINENT wots Gs kG Gir tik os OE eNO ete + Fai en sane hora aE 34

10. A Disk of Hard Rubber, One of Tissue and One of Celluloid,
faa Toth With ubber PANGS... 25. se. os ea een ae aaes 34

11. Disk of Rubber with a Mass of Tissue Wedged in the Central
ET auld 0 ge ae aa MU a pe eh GURL AC Re Re ee 34
12. Apparatus for Measuring the Conductivity of Nitella............ 36
13. Curves of Net Electrical Resistance of Laminaria Agardhii....... 41
14. Curves of Net Electrical Resistance of Laminaria Agardhii...... 42
15. Curve of Net Electrical Resistance of Laminaria Agardhit........ 43

16. Curve Showing Changes in the Hydrogen Ion Concentration of
NINE a oe ce ane ale Oe cia ty aki nah Fonte need 44
17. Curves of Net Electrical Resistance of Laminaria Agardhii....... 45
18. Curve Showing the Net Electrical Resistance of Laminaria Agardhii 46

. Curve Showing the Net Electrical Resistance of Laminaria Agardhii 47
. Curves Showing the Net Electrical Resistance of Laminaria Agardhii 48
. Curves Showing the Effect of CaCl, 0. 278 M...............-... 50
. Curves Showing Net Electrical Resistance of Laminaria Agardhit 51
. Curves of Net Electrical Resistance of Laminaria Agardhit....... 52

12 ILLUSTRATIONS
FIG. PAGE
24. Curves Showing Net Electrical Resistance of Laminaria Agardhii. 53
25. Curves of Net Electrical Resistance of Laminaria Agardhii... ae
26. Curve Showing Rise in Net Electrical Resistance of Laminaria
Agar hts. oo o0is bv dais daxd Gad een ae ne a cats ee 56
27. Diagrams Illustrating Consecutive Reactions..................+++- 58
28. Death Curve of Laminaria Agardhit.......cccccceccecceccccccces —~=59
29. Curve Showing the Net Electrical Resistance of Laminaria Agardhii 64
30. Curve Representing Velocity of Processes..............eeeeeeeees 69
31. Curves Showing Changes in the Net Electrical Resistance of Tissues 71

32. Curve Showing Value of M under Various Velocity Constants..... 76
33. Graph Showing the Fall of Net Electrical Resistance of Laminaria
Agardhit... 02005 fb nt doe inet eealeeebe ee Sinica mee ee ee 81
34. Graph Showing Loss of Net Electrical Resistance of Laminaria
Agar. Cobo on iiseite. hv eeee tenia. Wie eee a8 eka Pe we eae ee ee 82
35. Rise of Net Electrical Resistance of Laminaria Agardhit............ 84
36. Extreme Alterations of Net Electrical Resistance of Laminaria
AGOPAWAL o.oo vs pi eve eo ain nas bo oo) ean aielele eed 86
37. Curves Showing Net Electrical Resistance of Laminaria Agardhii.... 92
38. Curves Showing Net Electrical Resistance of Laminaria Agardhii.... 93
39. Curves Showing Rate of Respiration of Laminaria Agardhit........ 96
40. Curves Showing Rate of Respiration of Laminaria Agardhii........ 97
41. Curves Showing Fall of Net Electrical Resistanceof Laminaria
AGUA oo o.oo vin seinin. ss hovdua ugne tne kn oe mee pase en ae eke 102
42. Curves Showing the Rise and Fall of Net Electrical Resistance in
Laminaria Agardhit.. ... 5. 0cvevenceccceneusnaiee ve =e cane 107
43. Curves Showing the Value of 0+10 in Various Solutions......... 109
44, Curves Showing the Net Electrical Resistance of Laminaria Agardhii 111

. Curves Showing the Net Electrical Resistance of Laminaria Agardhit 116
. Curves Showing the Net Electrical Resistance of Laminaria Agardhii 117
. Curves Showing the Net Electrical Resistance of Laminaria Agardhii 118
. Curves Showing the Net Electrical Resistance of Laminaria Agardhii 120

. Curves Showing the Growth of Roots in Toxic Solutions.......... 125
. Curve Showing the Antagonism Between Two Salts.............- 128
. Curves Showing Growth in Mixtures of Unequally Toxic Solutions.. 129
. Types of Antagonism Curves.......cesecececcccserscceecccececs 131

. Diagram Representing the Composition of Various Mixtures...... 132

ILLUSTRATIONS 13

FIG. PAGE
54. Solid Model Showing the Forms of the Antagonism Curves...... 134
mm mecthod Of Expressing Antagonism. ...+....sccsenscosccccessesase 135
56. Effect of Dilutions on the Forms of Antagonism Curves.......... 136
57. Diagram Representing the Composition of Solutions.............. 138
58. Solid Model Showing Antagonism Affected by Altered Solutions.. 139
59. Curves Showing the Net Electrical Resistance of Laminaria Agardhii 140
60. Curves of Net Electrical Resistance of Laminaria Agardhii......... 143
61. Curve of Net Electrical Resistance of Laminaria Agardhii......... 144
62. Curve of Net Electrical Resistance of Laminaria Agardhii......... 145
63. Curve of Net Electrical Resistance of Laminaria Agardhii......... 146
64. Increase of a Hypothetical Salt Compound....................... 149
65. Graph Showing Increase of the Velocity Constant................ 151
66. Curves Showing the Rise of Resistance After Exposure to Toxic
SRM reins CUS Lio kie etl > So iaas baa tis Kel Ek Obtains coca es 154
67. Curves Showing Calculated Value of s in Various Solutions........ 155
68. and 69. Curves Showing the Net Electrical Resistance of Laminaria
DERE OOT Peete Lue cn oti vn eee ekg Shwe kee ee Row gb Knee Oe 156
70. Curves Showing the Net Electrical Resistance of Laminaria Agardhii 157
71. Antagonism Curve of Laminaria Agardhit.............. cece eeeees 165
72. Curves Showing Antagonism Between NaCl and Na-taurocholate.. 169
73. Curves Showing Antagonism Between NaCl and Nicotine.......... 170
74. Curves Showing Antagonism Between NaCl and Caffeine.......... 171
75. Curves Showing Antagonism Between NaCl and Cevadine Sulfate.. 172
76. Curves Showing Antagonism Between NaCl and CaCle........... 174
77. Curves Showing the Resistance of Laminaria Agardhii............ 176
78. Electrical Resistance of Laminaria Agardhii in Sodium Acetate 177
79. Increased Toxicity of Laminaria Agardhit............0e.ceeeeeeee 178
80. Net Electrical Resistance of Laminaria Agardhii...... ......-... 185
81. Net Electrical Resistance of Laminaria Agardhit................. 186
82. Net Electrical Resistance of Laminaria Agardhii................. 189
83. Curves Showing the Net Electrical Resistance of Laminaria Agardhii 190
84. Cross Section of Monostroma Latissima..............eeeeeeeeeeee 196
ER UTERINE GOK 2) OE FADO soca ciate on oe bie bins der aaciew see 196
86. Cross Section of Rhodymenia Palmata....... ..........2-0000-00- 197
87. Cross Section of Laminaria Agardhii.................-...00 seco 198

FIG,

88.
89.

90.
91.

92.

93.
94.
95.
96.

ILLUSTRATIONS

PAGE
A Vegetable Cell Showing Plasmolysis.................cceeeeecees 201
Apparatus for Testing Rate of Diffusion of Salts through Living Tissue 206
Diffusion of Various Solutions through Laminaria Agardhii....... 207
Exosmosis into Distilled Water from Taraxacum Officinale........ 208

Recovery of Taraxacum Officinale from Effect of Various Hypertonic
Solutions.) «nag. o/c. Pee eee ee Cees ean ee ele on ee 209
Diagram Showing Principle of Resistance....................002 218
Increase in Value of Velocity Constants ..........:.....eeeeeeee 222
Decrease in Value of Velocity Constants. .......0. 6500... eee 223
A Cell of Grifithsia Bornetiana::. 000025 cc2 ss ons ee eee ee 231

INJURY, RECOVERY, AND DEATH, IN
RELATION TO CONDUCTIVITY AND
PERMEABILITY

INTRODUCTION

Some of the fundamental ideas of biology are most
difficult to define with precision. This is especially true
of such conceptions as life, vitality, injury, recovery, and
death. To put these conceptions on a more definite
basis it is necessary to investigate them by quantita-
tive methods.

To illustrate this we may consider some researches
on the electrical conductivity of organisms. These ex-
periments show that the electrical resistance of a plant
or animal is an excellent indicator of what may be called
its normal condition of vitality. Injurious agents in-
variably change its electrical resistance. For example,
if the marine plant, Laminaria, is taken out of its normal
environment of sea water and placed in a solution
of pure NaCl it is at once injured, and if the exposure
be sufficiently prolonged it is killed. During the
whole time of exposure to the solution of NaCl its
electrical resistance falls steadily until the death-
point is eventually reached; after this there is no
further change. A study of the time curve of this
process shows that it corresponds to a monomolecular
reaction (slightly inhibited at the start). This may
be expressed in the form of an equation whigh ean be
utilized to predict the curve of death under various con-

16 INJURY, RECOVERY, AND DEATH

ditions. We find that in testing these predictions we
must ascertain when the death process reaches a definite
stage, (2. e., when it is one-fourth or one-half completed).
This can be determined experimentally with a satisfac-
tory degree of accuracy.

We can therefore follow the process of death in the
same manner that we follow the progress of a chemical
reaction in vitro; in both cases we obtain curves which
may be subjected to mathematical analysis, from which
we may draw conclusions regarding the nature of the
process. ‘T'his method has been fruitful in chemistry
and it is possible that it may prove equally so in biology.

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

The process of death which occurs in a solution of
NaCl may be checked by adding a little CaCl, to the
solution. In this case we speak of antagonism between
sodium and calcium. When the calcium is added in the
proper proportion the fall of resistance is very slow and
the tissue lives for a long time. Any deviation from
this optimum proportion hastens death.

In order to explain these results we may assume that

1The general eae 4 ef the de ony process goes on n ‘contin
is in harmony with the ideas expressed by many physiologists from
Claude Bernard (1879; I, 28), down to the present day. Cf. Lipschiitz,
A. (1915).

INTRODUCTION 17

both sodium and calcium combine with a constituent of
the protoplasm, forming a compound which inhibits the
death process. This enables us to formulate an equa-
tion by means of which the death curve in any mixture
of sodium and calcium can be predicted with consider-
able accuracy. )

The changes in electrical conductivity which occur
under the influence of reagents run parallel to changes
in the permeability of the protoplasm. This is to be
expected, since it is evident that when a current passes
from a salt solution into living protoplasm, ions must
enter the protoplasm, and if there is an increase in the
permeability of the protoplasm to these ions its electri-
cal conductivity must increase, and vice versa. The
electrical conductivity of the protoplasm may therefore
be regarded as a measure of its permeability to ions.

The resistance of the tissue does not depend upon
the protoplasm alone, but also upon the cell wall and the
cell sap. But we find, as a matter of fact, that the re-
sistance of the protoplasm rises and falls with that of
the tissue as a whole. Hence when we observe that the
conductivity of the tissue increases in a solution of
NaCl and decreases in a solution of CaCl,, we may con-
clude that the permeability is increased by NaCl and
decreased by CaCl,. This is in harmony with experiments
in which permeability is measured by other methods
(such as plasmolysis, specific gravity, exosmosis, tissue
tension, and the diffusion of salts through living tissue).
It is likewise confirmed by direct determinations, in which
the penetration of various substances is ascertained by
testing for their presence in the cell sap.

It has been observed in the course of these investi-

gations that plants which have developed in a normal
2

18 INJURY, RECOVERY, AND DEATH

environment are fairly uniform in their electrical resis-
tance, so that we may speak of a normal degree of
resistance as indicating a normal condition. If the plant
is injured and the resistance falls, we may consider
that the loss of resistance gives a measure of the amount
of injury. This enables us to place the study of injury
upon a quantitative basis. As the result of this we are
able to formulate a definite conception of the mechanism
of recovery. We find that if injury in a solution of NaCl
amounts to 5% the tissue recovers its normal resistance
when replaced in sea water. But if the injury amounts
to 257% recovery is incomplete: instead of returning to
the normal the resistance rises to only 90% of the
normal. ‘T'he greater the injury the less complete
the recovery. When injury amounts to 90% there is
no recovery.

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

The significance of such methods is further shown
by the fact that they have led to the development of
equations which enable us to predict with a satisfactory
degree of accuracy the recovery curves which are ob-
served under a great variety of conditions.

As the result of these investigations we are led to
look upon recovery in a somewhat different fashion from
that which is customary. While recovery is usually

INTRODUCTION 19

regarded as due to the reversal of the reaction which
produces injury, the conception here developed is funda-
mentally different. It assumes that the reactions in-
volved are irreversible (or practically so) and that
injury and recovery differ only in the relative speed at
which certain processes take place. The reasons for
this are fully explained in the following pages.

The experiments of the writer lead to the view that
life depends upon a series of reactions which normally
proceed at rates bearing a definite relation to each other.
If this is true it is clear that a disturbance of these rate-
relations may have a profound effect upon the organism,
and may produce such diverse phenomena as stimulation,
development, injury, and death. Such a disturbance
might be produced by changes of temperature (if the
temperature coefficients of the reactions differ) or by
chemical agents. The same result might be brought
about by physical means, especially where structural
changes occur which alter the permeability of the plasma
membrane or of internal structures (such as the nucleus
and plastids) in such a way as to bring together sub-
stances which do not normally react.

Throughout these investigations the aim has been to
apply to the study of living matter the methods which
have proved useful in physics and chemistry. In this
attempt no serious difficulty was encountered after ac-
curate methods of measurement had been devised: nor
does there seem to be any real obstacle to an extensive
use of methods which lead biology in the direction of the
exact sciences.

It is evident from what has been said that we may
investigate such fundamental conceptions as vitality, in-

20 INJURY, RECOVERY, AND DEATH

jury, recovery, and death by quantitative methods anc
obtain a set of equations by which they can be predicted
It may be added that the predictive value of these equa.
tions is quite independent of the assumptions upon whicl
they were originally based. The importance of suck
equations is fully as great in biology as in physic:
or chemistry.

The measurements described in this volume, and the
accompanying mathematical analysis, lead to a quantita.
tive theory of the mechanism which underlies certair
important phenomena. The theory can be tested by
exact methods and, as far as experiments have gone
appears to be sound. This investigation of certain fun.
damental life processes seems to show that they obey}
the laws of chemical dynamics: it likewise illustrates
a method which promises to throw light upon the under.
lying mechanism of these processes and to assist in the
analysis and control of life-phenomena.

CHAPTER I.

METHODS OF MEASURING ELECTRICAL
CONDUCTIVITY

Srnce the conclusions set forth in this work depend
largely upon researches on the electrical conductivity of
organisms it seems desirable to give an account of the
methods of conducting such investigations.

In the experiments of some investigators! platinum
electrodes have been applied directly to the tissue. It
is difficult to obtain good contact by this method and
there is danger that some of the platinum black may be
rubbed off. Kodis (1901) states that it is impossible
to obtain trustworthy results in this manner. He there-
fore placed the tissue in a U-tube in each arm of which
was a funnel plugged at the bottom with plaster of Paris.
Kach funnel was filled with a solution of NaCl into which
an electrode dipped (Fig. 1).

In measuring the conductivity of red blood corpuscles
or of unicellular organisms the electrodes are placed
directly in a suspension of the cells, either with or with-
out previous centrifugation.’(Fig. 2.)

In experiments of the writer on unicellular plants
(such as Euglena and Chlorella) the organisms were

* Regarding methods see Galeotti (1803), Alcock (1905, 1906), Polacci
(1907), Mamelli (1909), Stone and Chapman (1912), Henri et Calugareanu
(1902, A, B), McClendon (1912), Stiles and Jérgensen (1914), Héber
(1914) pp. 381, 440, Small (1918).

*See R6th (1897), Bugarsky und Tangl (1897), Stewart (1897, 1899,
1909-10), Woelfel (1908), McClendon (1910), Gray (1913, 1916),
Shearer (1919, A, B, C).

22 INJURY, RECOVERY, AND DEATH

placed in a centrifuge tube near the bottom of which
platinum electrodes were inserted (through the walls of
the tube) a short distance apart. The material was then
centrifugated and the resistance was measured. The
supernatant liquid was then poured off and replaced by
a different solution. The material was agitated in order

Fic. 1.—Apparatus for measuring
the electrical conductivity of muscle
(Kodis): E, electrode, contained in a
funnel (filled with a solution of
NaCl) plugged at the bottom with
plaster of Paris (P); M, muscle: “_
whole is placed in a water bath,

to disperse it through the solution and the process of
centrifugation and washing was repeated until the first
solution was removed. This must be done frequently
since otherwise the organism may change the conduc-
tivity of the external solution (by absorbing or giving
out electrolytes) and this may be confused with a change
in the conductivity of the cells.

A method of measuring the electrical conductivity
of bacteria has recently been proposed by Thornton
(1912), who states that it depends upon the principle
that in an electric field bacteria orient themselves in a

MEASURING ELECTRICAL CONDUCTIVITY 23

solution having a lower conductivity than the bacteria,
but not in a solution of the same conductivity.
The method therefore consists in placing the bacteria

in an electric field and increasing the
strength of the salt solution until they
cease to orient. The results indicate
that the conductivity of living bac-
teria is usually greater than that of
the medium in which they grow. This
is opposed to the results of Shearer
(1919, A) in which the conductivity
Was measured in the usual manner.
On theoretical grounds there are ser-
ious objections to Thornton’s tech-
nique as well as to his conclusions.

The method* used by the writer
gives under the most favorable condi-
tions, measurements which are
accurate to within 1%. This degree
of accuracy may be regarded as satis-
factory for biological purposes.

In the original method‘ aquatic
plants with leaf-like fronds were
employed, particularly one of the
common kelps of the Atlantic coast
(Laminaria agardhi): disks were cut

from this by means of a cork borer ¢

and packed together, like a roll of coins,

S S

Fig. 2.— Apparatus for
measuring the _ electrical
conductivity of sea urchin
eggs (Gray): S, silver wires,
,G, glass tubes: E, plat-
inum_ electrodes.

in, an apparatus which is shown in Fig. 3. It consists
of two platinum electrodes (covered with platinum
black), A, sealed into glass tubes, B, which are filled

*This was developed without reference to the methods previously

used and differs somewhat from them.
*Osterhout (1918, #),

24 INJURY, RECOVERY, AND DEATH

with mereury and into which dip copper wires, C,
which go to the Wheatstone bridge. These tubes are
contained in electrode holders of hard rubber, D,
through which pass a rod, H#, and a long screw, F,
by means of which the electrode holders may be drawn
toward each other and held firmly in any desired posi-

Fic. 3.—Apparatus for determining the electrical conductivity of living tissue. The disks

of tissue, Z, are packed together like a roll of coins. At each end is a platinum electrode, A,

fastened in an electrode carrier, D. By means of the screw, F, the electrode carriers can be
drawn together, compressing the tissue and holding it firmly in place.

tion. This screw engages an internal screw contained —
in the electrode holder at the right. This is not the
case with the electrode holder at the left in which the screw
passes through a sleeve, and in consequence this electrode
holder is drawn toward the other only when the block,
M, is fastened in place by the set screw, N, and the
screw, F’, is turned in the proper direction.

An end view of an electrode holder, D, is shown in |
Fig. 4. Its lower portion (which contains the platinum
electrode) is shown inserted in a hard rubber support,
G. The support is pierced by a series of seven holes

MEASURING ELECTRICAL CONDUCTIVITY 25

(arranged in a circle as shown in the figure) each of
which receives the end of a glass rod about 9 inches long,
the other end of each rod being fastened in a similar
support. The cirele (dotted line) just inside the seven
small circles represents a disk of tissue inserted between
the glass rods with its surface at right angles to them.
The smaller circle, H, in the centre
represents an opening in the electrode
holder through which the current
passes from the platinum electrode to
the disks of Laminaria. The arrange-
ment is shown in Fig. 3, where H
represents the opening and JL repre-
sents the disks. Before reaching the
disks the current passes through K,
(Fig. 3), a hard rubber disk (with an
opening in the centre) which provides
mechanical support for the tissue.

The disks are cut from the fronds
by means of a cork borer and have Fics from the ond. rest:
about the diameter and thickness of a Which Gre cet lass rods
silver quarter. They are packed to- of circles) which. hold
gether like a roll of coins (about 100 sue (seen in section arf
in all). They are firmly held in
place by the glass rods which surround them’ and by
the electrode holders which press against them at either
end. At the same time the spaces between the glass
rods allow free circulation of liquid.

Each disk is placed in sea water as soon as it is cut’;

*It was at first thought that cutting might injure the tissues at the
edge of the disk sufficiently to interfere with the results, but experiments
proved that this is not the case. Not only do the cells adjoining the cut
surface live as long as those in the centre of the disk, but it is found that
experiments (made by another method) on intact fronds give the same
results as experiments on the cut disks,

26 INJURY, RECOVERY, AND DEATH

from this the disks are transferred to the support G,
which is submerged in sea water. They are arranged
inside the glass rods by means of forceps, and eare is
taken to see that no bubbles of air are caught in the
space around the electrode or in the opening at H.

When the effect of a number of different solutions is
to be compared the following procedure is adopted.
If there are seven solutions seven disks are cut from
the same part of a frond: each disk is placed in a
separate tumbler of sea water. A second lot of seven
disks is cut, as close to each other as possible, and
placed in the tumblers, so that each tumbler contains
two disks. This is continued until each tumbler con-
tains one hundred disks. By this means the material
in the different tumblers is made as similar as possible.
The disks in each tumbler are then packed together
(like a roll of coins) to form a cylinder whose resistance
is measured. Throughout the experiments the differ-
ent lots are kept side by side and treated as nearly
alike as possible, except that they are placed in dif-
ferent solutions.

The electrode holders are now pressed against the
ends of the roll of disks, the block, M, is firmly fastened
by means of the set screw, N, and the screw, F’, is turned
until the electrode holders are tightly clamped against
the roll of disks. The pressure used in this operation
should be fairly uniform.® 7

The apparatus is now lifted out of the sea water’

°It was at first thought necessary to use a dynamometer, but it was
found that the operator soon becomes so proficient as to make it un-
necessary. The resistance is very little affected by variations in pressure.

"In the earlier experiments the resistance was taken with the cylinder
submerged in sea water, and this may be preferable in special cases,

MEASURING ELECTRICAL CONDUCTIVITY 27

and allowed to drain’ for a definite time (not over
one minute) after which the resistance becomes practi-
eally constant.

The current passes for a short distance through sea
water before reaching the disks. There is a film of sea
water between each pair of disks and likewise a film
around the cut edges. Otherwise the current passes only
through the tissue.

As soon as the resistance has been measured the
apparatus is replaced in sea water; the set screw, N, is
loosened so that the electrode holders can be moved
apart and the disks separated from each other by means
of forceps. After standing for a few minutes in sea
water the resistance is again determined. The disks
are then separated as before and allowed to stand in sea
water. This procedure is continued until it becomes
evident that the resistance is practically stationary.®

The apparatus is then transferred to another solu-
tion (e. g., NaCl 0.52 M) having the same conductivity
(and temperature’’) as the sea water. There should
be at least 1,500 ec. of solution, contained in a shallow
dish of glass or enameled ware. The disks are at once
separated by means of forceps and thoroughly rinsed

* Each support rests on a block of paraffin. Care must be taken that
there is no conduction between the blocks; e.g., along the wet surface of
the table.

* Unless this is the case the material is rejected. With good material
the resistance remains stationary for a long time; in one experiment it
remained so for 10 days at about 20° ©. In this case the tissue was
kept in running sea water and was only half-submerged, thus ensuring
an abundant supply of oxygen. See Osterhout (1915, B). When placed
on ice Laminaria can be kept in good condition for a much longer time.

* All readings should be made at the same temperature or, if this is
not practicable, should be corrected to the standard temperature. For the
temperature coefficient, see p. 37.

28 INJURY, RECOVERY, AND DEATH

in the new solution, the whole apparatus being moved
about in the dish to secure thorough mixing. By means
of a medicine dropper the sea water around the platinum
electrodes is thoroughly washed out. In some cases
it is desirable to transfer to a second dish to ensure
against contamination by sea water.

By this means a very rapid change is effected and,
as the disks are thin, diffusion is soon completed (this is
often the case in 5 minutes and should not in any event
require more than 10 minutes). Since the outward diffu-
sion of salts may take place at a different rate from the
inward diffusion there may be an apparent rise or fall
of resistance in consequence. This effect lasts but a
short time and is found in dead as well as in living tissue.
It is therefore easy to guard against error due to
such causes.7?

The resistance of the disks at the ends is much
greater than that of those in the middle since the current
spreads out after issuing from the small opening, H,
in the rubber disk (Fig. 1). For this reason the best
disks of tissue should be placed at the ends and their
positions should not be changed. Care should be taken
that they are not cut or injured by contact with the
edges of the opening in the rubber disk.** The inequal-
ity between the disks at the end and in the center may
be minimized by introducing at intervals rubber disks

“Cf. Osterhout (1918, D).

4 It results from this that the resistance does not increasg in direct
proportion to the number of disks. If we plot the resistance as ordinates
and the number of disks as abscisse, we obtain a curve which is concave
toward the base line. The curve is approximately logarithmic.

™ These edges may be rounded by filing. A soft rubber disk may be
placed between the hard rubber disk and the tissue.

MEASURING ELECTRICAL CONDUCTIVITY 29

provided with openings in the center (Fig. 5). This is
desirable in many cases and makes it possible to get a
high resistance with less tissue.

Care must be taken to see that liquid does not leak
out of the space around the electrodes while the appa-
ratus is out of the liquid. If a leak should occur fresh
liquid may be added by means of a
medicine dropper. With a proper ad-
justment of the rubber disks and suf-
ficient tissue to give elasticity no
leakage should occur.

In regard to the accuracy of the
readings it may be said at the outset sic. 5 Hararubberdiske
that under favorable conditions suc- ¢ tissue "B “Gall ‘seen
cessive readings on the same material etsy
do not vary more than 1% from the average. This is as
great accuracy as can ordinarily be hoped for in biological
work and there is no object in striving to get greater
accuracy than this in the apparatus itself.

It is usually desirable to introduce a variable capaci-
- tance or an arrangement such as is suggested by Taylor
and Curtis (1915), by Taylor and Acree (1916) or by
McClendon (1920). In the writer’s experiments the
capacity of the apparatus, filled with living Laminaria
and lifted out of the sea water, was about one thousandth
of a microfarad.

An advantageous arrangement suggested by Profes-
sor G. W. Pierce is shown in Fig. 6.

The frequency is of some importance. The writer
has found a thousand cycles convenient; this may be
obtained by means of an ‘‘audio oscillator’’ (such as
is used in wireless telegraphy) as furnished by the
General Radio Co., or by means of a toothed iron wheel re-

‘i

J”

30 INJURY, RECOVERY, AND DEATH

volving in a suitably arranged magnetic circuit such as
is furnished by Leeds and Northrup. The results so
obtained did not differ from those secured with a
Vreeland oscillator.

Mou
S
\\. G
5000 Q 5000.2
A B
0
io”
C

v &

Fic. 6.—Diagram to show bridge and connections. S is an alternating source (1000 cycles

or more), A and B are the ratio arms of the bridge, C is the variable resistance of the bridge,

X is the unknown resistance (tissue and holder), 7’, telephone, V, variable condenser, G, a

ground wire from the centre of a high resistance (in case the ratio arms of the bridge are
unequal the two parts of the high resistamce should also be unequal).

The use of the ordinary lighting cireuit (60 cycles)
with a vibration galvanometer is recommended by Green
(1917). The use of an alternating current galvanom-
eter in connection with a recording device is suggested
by Weibel and Thuras (1918).

MEASURING ELECTRICAL CONDUCTIVITY 31

For details regarding apparatus the reader is re-
ferred to the papers of Hibbard and Chapman (1915),
Washburn", Taylor and Acree’®, Rivers-Moore (1919),
Schlesinger and Reed (1919), Newberry (1919), Hall
(1919), and Stiles and Jorgensen (1914).

A P| Ehanaea C

Fie. 7. Electrode carrier, A, consisting of a glass tube provided with a series of side tubes

to hold an electrode tube, D, ‘and a thermometer, EZ, also an inlet tube and an outlet tube.

To the right two glass cells, B, C, each with an inlet hing and an outlet tube, with disks of
tissue, F an

We may now turn to another form of apparatus
which may for convenience be called Type B. Fig. 7
shows one end of the apparatus, which consists of an
electrode holder, A, and a series of glass cells, B, C,
ete. The electrode holder consists of a glass tube pro-
vided with side arms for the admission of the electrode
tube, D, (which is similar to the tube used in Type A)
as well as of a thermometer, H. In addition there is an
~ ™ See Washburn, E. W. and Bell, J. E. (1913), Washburn, E. W. (1916),
Washburn, E. W. and Parker, K. (1917).

* See Taylor, W. A. and Acree, S. F. (1916), and previous papers in the
same journal.

32 INJURY, RECOVERY, AND DEATH

inlet tube and an outlet tube by means of which the
solution may be changed. Each of the glass cells, B, C,
etc., has a similar inlet and outlet tube. Each outlet
tube has a rubber connection through which liquid can
be discharged without wetting the outside of the cells.
All of the inlet tubes are connected (by rubber tubing and

NYS

weal

[ WIN

Fig, 8.—Disk of tissue, M, the edges surrounded by vaseline, VV, with an electrode carrier
on each side.

a system of Y-tubes) to the same funnel, so that all the
cells can be filled simultaneously.

The edges of the glass cells are ground in a plane
exactly at right angles to the long axis of the cell. When
pieces of Lamimaria are placed between them (as at Ff
and G) and they are pressed together, a tight joint is
formed. The series of glass cells (with pieces of mate-

MEASURING ELECTRICAL CONDUCTIVITY 33

rial) and an electrode carrier at each end are placed in
a V-shaped trough with rigid ends; at one end is a screw
by means of which they can be forced together and held
with any desired degree of pressure. At the places
where the pieces of material are located, the trough is
eut away so that they do not come in contact with it.
Care is taken to keep the current from leaking along the
trough (its surface is covered with paraffin).

The current therefore flows through the glass cells
and through the pieces of material placed between them.

The advantages of this type of apparatus are: (1)
the end pieces do not have more resistance than those
in the middle; (2) the solutions may be changed without
disturbing the material.

Types A and B may be combined by substituting
disks of Laminaria for the glass cells.

Type C is shown in Fig. 8. It consists of two elec-
trode carriers similar to those in Type A. The material
is shown at M, its edges being completely surrounded
by vaseline, V, V, so that the current cannot leak out.
In many cases it is preferable to use chicle, grafting wax,
’ or art gum in place of vaseline. The apparatus remains
partly submerged (the water line being indicated at W,
W), thus keeping the temperature more nearly constant.
The solutions are changed by siphoning through the
openings which admit the electrode tubes. This makes
it unnecessary to unscrew and separate the electrode
carriers during the experiment.

Type D is shown in Fig. 9. It permits the use of in-
tact plants. One end of the plant is inserted in each
of the cells 4 and B and held in place by a split rubber
stopper. The cells 4A and B are filled with solution.
The free portion of the plant is bathed in any desired

34 INJURY, RECOVERY, AND DEATH

+
tty

solution until a reading is to be taken, when the solution
is allowed to drain off and the reading is made. Care
should be taken to prevent the current from leaking
through or around the stopper.

The part of the frond which is contained in the stop-

Fig. 10.—A disk of hard rub-

ber, A, one of tissue, B, and

one of celluloid, C, tied to-

gether with rubber bands, D

(all seen in section). Surface
view at the left.

Fia. 11.—Disk of hard

rubber, D, with a mass

of tissue, M, wedged in

the central opening (seen
in section).

Fia. 9.—Two glass cells,
Aand B, each provided with
an electrode with a strip of
tissue stretched between.

per and in the cell may be killed to lessen its resistance.

Material which is too soft to be handled in the man-
ner recommended for Laminaria may be treated as
follows: If it forms sheets or membranes it may be fas-

MEASURING ELECTRICAL CONDUCTIVITY 35

tened to thin disks of hard rubber’* provided with a
central opening as shown in Fig. 10, in which A repre-
sents the rubber disk (seen in section), B the material,
and C another disk of thin rubber or celluloid. These
are fastened together by rubber bands, D. For this
purpose three projecting knobs are provided as shown
in the surface view at the left of Fig. 10. The disk is
placed in the frame described under Type ‘A, and the
knobs fit in between the glass rods in the manner shown
in Fig. 10 (where the rods appear in section). Every
other disk is turned upside down so that the knobs of
adjacent disks do not touch and interfere with the close
packing of the disks. The disks are treated precisely
like the disks of Laminaria as described under Type A.

Most of the experiments on frog skin and on Ulva
were made with this type of apparatus.

Material which cannot be handled in this way may be
treated as shown in Fig. 11, where D represents a hard
rubber disk with a central opening into which the mate-
rial is tightly wedged. The disks are then handled like
so many disks of Laminaria. A special type of appa-
ratus has been used in experiments on Zostera".

Experiments were also made with large cells of
Nitella, some of which reach a length of 5 or 6 inches
and a diameter of a thirty-second of an inch or more.
They were packed (Fig. 12) in a trough cut in a block of
paraffin (this was then covered with a plate of glass). The
trough was previously filled with a solution: this could
readily be changed after the cells were in place. The cur-
rent could be sent lengthwise or across the cells: usually
both methods were employed.

* The edges of each piece of tissue are protected by vaseline.
"Of. Osterhout (1919, A).

36 INJURY, RECOVERY, AND DEATH

In order to ascertain the conductivity of the cell sap
of Nitella small amounts were expressed (see page 212)
and allowed to fill a capillary tube. Platinum electrodes
were then inserted into the opposite ends of the tube,
eare being taken to exclude air bubbles.

By means of these methods a variety of plant and
animal material has been studied by the writer.'® Cer-

Fia. 12.—Apparatus for measuring the conductivity of Nitella. The cells, N, are placed in

a trough in a block of paraffin, P, and covered with plate glass, G. The solution is

poured in through the funnel, 7, and runs out through the opening, O. At EF and £E are
platinum electrodes.

tain precautions have been observed in the choice of
material. It is desirable that the intercellular space
or substance shall be constant in amount. This is the
case in tissues, such as those of Laminaria, where the
cell walls are of a firm consistency and do not change
during the experiment.4® On the other hand many
flowering plants present difficulties, since the spaces
between the cells are largely filled with gas, which is

cy Cf. Osterhout (1919, A, iON
” Plasmolysis must be avoided since this increasea the space between
the protoplasmic masses.

MEASURING ELECTRICAL CONDUCTIVITY 37

displaced to a varying extent when the tissue is placed
in a solution, with the result that the conductivity is
altered. In such cases we must select material in which
the displacement is very slow or else we must get rid
of the gas at the start by submerging the tissue and evacu-
ating by means of an air pump.

As the writer’s investigations were largely concerned
with alterations in permeability it was necessary to
provide for quick changes of reagents and for rapid
penetration. This was accomplished by the use of thin
sheets of tissue. For example it was found that when
Laminaria was transferred from sea water to sea water
diluted with an equal volume of distilled water,
diffusion was practically completed in 5 to 10 min-
utes; this was also the case with the other material used
in his investigations.

It is desirable that the thin sheets of tissue should be
stiff enough to be handled easily and that they should
not adhere to each other, but should tend to separate
spontaneously when the pressure is removed so as
to allow a free circulation of liquid between them (this is
assisted by choosing pieces with a slight curvature).

The material should be able to stand laboratory condi-
tions and the manipulation required by the experiments.
It is desirable that it should be available throughout the
year. All these requirements are so admirably fulfilled
by the marine alga Laminaria agardhi (a common
kelp of the Atlantic coast) that it has been largely used in
the investigations of the writer. It forms fronds several
feet in length, 3 to 6 inches wide (having somewhat the
consistency and thickness of a thin leather belt). It
remains in normal condition in the laboratory for several
weeks if kept in sea water (near O°C.) and is not injured

38 INJURY, RECOVERY, AND DEATH

by the pressure and the weak electric currents to which
it is subjected during the experiments.

The solutions were made with all possible precautions.
The salts used were the purest obtainable. The distilled
water was, as a rule, twice distilled from quartz or
glass,2° using cotton plugs in place of cork or rubber
stoppers in the distilling apparatus. The first and last
parts of the distillate were discarded.

The reaction of the solutions is of great impor-
tance. Unless otherwise stated it was close to neutrality.

It may be desirable to add a word of explanation
regarding the treatment of results. Most of the curves
here presented are time curves in which each point
represents the average of several experiments. In such
curves it is desirable (as indicated on page 68) to average
times (abscisse) rather than resistances (ordinates).

The probable error of the mean has been calculated
in all cases by Peter’s formula and expressed as per
cent. of the mean.?!_ Since, however, space is lacking to
present all the data, a general idea of the accuracy of
the results may be given by saying that there is no point
on the curve whose probable error of the mean exceeds
a certain per cent. of the mean.

The temperature was controlled in short experiments
so that the fluctuations did not amount to more than
+2°C. In longer experiments (lasting several days)
greater fluctuations were unavoidable, but the effect of
these was minimized by starting all the experiments of
a series at the same time so that the fluctuations affected

* Water distilled from a copper still should never be used.

"Thus, if the observations are 99, 104, 102, 97, 100, 96, 103, 101, 98,
the mean is 100, the sum of the deviations 20 and the probable error of
the mean 20 (.0332) = .664 which is .664% of the mean.

MEASURING ELECTRICAL CONDUCTIVITY 39

all of them equally. This answers very well as long as
we are comparing experiments which last about the
same length of time, but it may happen that one of the
series lasts but a short time and after its completion the
others proceed at a different temperature. In this case
the whole series should be rejected unless the difference in
temperature is small.

The temperature coefficient of the electrical conduc-
tivity of living Laminaria”? is about 1.331; this is higher
than that of dead tissue (1.26) which is very close
to that of sea water. This coefficient may be employed to
eorrect readings which are not made at the standard
temperature, provided the deviation in temperature does
not exceed two or three degrees.

2 Of. Osterhout (1914, JZ).

CHAPTER II
THE MECHANISM OF THE PROCESS OF DEATH.

In studying Laminaria it is found that toxic sub-
stances may be divided into two classes according to
their effects upon the conductivity of the tissue. The
first class includes those which cause a progressive loss
of resistance, ending in death;! the second class produces
a rise in resistance,” followed by a fall which continues
until the death point is reached.

The first group includes salts of monovalent metals.
The investigations of Raber (1920) have shown that the
higher the valency of the anion (which is combined with
the monovalent kation) the more rapid is the fall
in resistance.*

In subsequent studies® it was found that the trivalent
arsenate anion is more efficient than the bivalent molyb-
date and sulphate and these in turn are more efficient
than the univalent formate and chlorate® Further

* Effects of this aort are also produced by hypo- and hypertonic solu-
tions, by drying, by moderate heat (e.g. 35° OC), by lack of oxygen, or by
exposure to ordinary laboratory conditions.

7In some cases a temporary rise is observed, due to the fact that ions
diffuse out of the tissue faster than they diffuse in. This is easily recog-
nized because it is as pronounced with dead tissue as it is with living.
See Osterhout (1918, D).

7 Of. Osterhout (1912, A).

*This does not apply to OH, which is exceptional,

5 Raber, O. L. (1921, A).

*In these studies the solutions were not of the same concentration, but
all had the conductivity of sea water, except the molybdate, which had
the conductivity of 75% sea water plus 25% distilled water. Hence
the conclusions stated above should be taken in a qualitative rather
than a quantitative sense.

MECHANISM OF PROCESS OF DEATH = 41

studies by Raber? indicate that the rise in resistance
which is produced by bivalent and trivalent kations is

100 PER CENT OF CONTROL

1 ae 5 HOURS

Fie. 13.—Curves of net electrical resistance of Laminaria agardhii in 1793 c.c. NaCl 0.52
M +207 c.c. CaCl: 0.279 M with the addition of 0.01, 0.02, and 0.03 M NaOH. The per-
centages were calculated on the basis of the net resistance of the control. All readings were
taken at 18° C. or corrected to this temperature. Each curve represents a single experiment

less when they are combined with trivalent anions than
when combined with univalent.

The writer’ has found that OH is more effective than
any other anion in producing a fall in resistance. Since

Raber, O. L. (1921, B, 0).
8 Of. Osterhout (1914, F).

42 INJURY, RECOVERY, AND DEATH

the addition of alkali to sea water produces a precipitate
of Mg(OH), the experiments were made by adding vari-

100 PER CENT OF CONTROL

30 MI
me 80 MINUTES

pram

id ~1Hour
a

60
= ee
£0
LH,
m
O1 O2 038 M N.OH

Fig. 14.—Curves of net electrical resistance of Laminaria agardhii in 1793 c.c. NaCl 0.52

M +207 c.c. 0.279 M containing various amounts of NaOH. The abscisse represent the

concentration of NaOH in the solution: the ordinates represent the percentage of electrical

net resistance calculated on the basis of the net resistance of the control. All readings

were taken at 18° C. or corrected to this temperature. Each curve represents a single
typical experiment.

ous amounts of NaOH to a mixture of 1793 ec. NaCl
0.52 M + 207 ec. CaCl, 0.279 M. The results are shown
in Figs. 13 and 14.

Experiments were also made by adding alkali to the

-
:
; [

MECHANISM OF PROCESS OF DEATH = 438

sea water until a slight precipitate of Mg(OH)., was
formed. When tissue was placed in this its resistance

100 PER CENT
90
80

1 2. 4 HOURS

Fig. 15.—Curve of net electrical resistance of Laminaria agardhii in 1975 c.c. sea water plus

48c.c. NaOH 0.22 M (pH about 10), unbroken line, and of a control in sea water, broken

line. The percentages were calculated on the basis of the net resistance in sea water at the

beginning of the experiment. The readings were taken at 18°C. or corrected to this figure.
Each curve represents a single typical experiment.

steadily decreased, falling to 68% in about six hours.
(Fig. 15). Haas® has shown that in this case the pH

* Haas, A. R. C. (1916, A). From the table given by Haas it is evident
that when the concentration of added NaOH is about 0.005 M (the writer
used 0.0052 M) the burette reading has risen from 7.28 to about 7.33,
giving a pH of about 10.

44 INJURY, RECOVERY, AND DEATH

value is about 10. This is evident from Fig. 16, which
shows the increase of pH value as alkali is added to sea

water.’® It is therefore evident that small amounts of
alkali affect the resistance.

aki LL LL PUR AA Lia
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BOGUGLGUDL LOOGAGTUOLSQAODEOOA) OEDU DONE UOOHDSROEREODUDE AnOUEADONLODUOLD VONOL VOGUE OOD TEGO ad MAAEUHATGL OODLVDEOUAORELUOUU

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iit
SCT
WON 0000) CTT HATH OOGRSOU CORDA SUDAL ARGOS IEROD SORURDONDG ODORS DER

iin TTT
suas

RONORDGD ARDOUOODANIDROUSODRGDOGNO ODORS DE i

Boascbanesseacssases sensssssaaseses cance sasescucessscegsasesssanasesss snanessses cones sess

ii iit MG A Pe UH MH HN fn sity
Hu TS OT I PE Pf WITTE El TMI 1) HE

TR AIIM
Pea pent tas oa stQGHD0Do LAMAN HAANLVOGACAAUAU LL a AT Lee LOU HUGNTUOOGU PVGUOLAUESLTUEU TROY ATL TOA
Pech SU CGOONAGARUUOEGHOGSG4 SUDU00006 0G00QR04EU0RN0L50000000U0ODEOG0bAU0GuED0QuEROEOA! One SaUAN SUDRUOOAGE UODOUIEL H

Fria. 16.—Curve showing changes in the hydrogen ion concentration of sea water upon the
addition of alkali at 21° C. Ordinates show the hydrogen ion concentration. In passing
from 1X10-* to 1X10-* the successive divisions are read as follows: 9,8, 7,6, 5, 4,3, 2,1,5,1,

each multiplied by 10-*. Abscisse show burette readings beginning at 7. 28 ce. "and "ending
at 9c.c. The curve shows that on adding alkali to sea water the hydrogen ion concentration
at first falls rapidly and then very slowly until the magnesium hydrate has all been precipi-
tated. After this further additions of alkali cause a more rapid fall in the concentration of the
hydrogen ion, but this is soon checked by the precipitation of the calcium hydroxide. After
the calcium hydroxide is all precipitated further additions of alkali will cause a corresponding

decrease in the concentration of the hydrogen ion.

The efficiency of OH in decreasing resistance is strik- —
ingly shown in Fig. 17, which illustrates the rapid fall
of resistance in NaCl+ Ca(OH), as compared with
NaCl + CaCl.,.

Since the alkaline solution contains fewer calcium
ions (thoug oh the concentration of caleium molecules i is the

2 The sea watat was Bioletul fvaut Wobda Hole and was the’ same as
that used in the writer’s experiments.

MECHANISM OF PROCESS OF DEATH

same) an experiment was made in
which the concentration of cal-
cium ions was kept undiminished.
For this purpose there was added
to a saturated solution of
Ca(OH), (in distilled water) suffi-
cient CaCl, 1.42 M to make the
conductivity equal to that of sea
water. Tissue was placed in this
and also in CaCl, 0.278 M. In
spite of the fact that the concen-
tration of calcium ions was practi-
cally the same in the two solutions
the behavior of the tissue was
markedly different. In pure
CaCl, the net resistance rose to
171% of the original net resis-
tanee while in CaCl, + Ca(OH),
it rose to only 118%. At the end
of forty-five minutes the resis-
tance in CaCl, was 146%, while m
CaCl,+Ca(OH), it was only 23%.
These experiments make it
evident that small amounts of
NaOH are able to produce a con-
siderable increase in permea-
bility.1}
Let us now consider those sub-
stances which increase the resis-
tance of the tissue. In general we

100 PER CENT

1 HOUR

Fic. 17.—Curves of net electrical
resistance of Laminaria agardhti
in a solution containing NaCl 97.2
mols of NaCl to 2.8 mols of CaCle
(uppermost curve) ; a solution con-

taining 97.2 mols NaCl to 2.8 mols
Ca(OH): (middle curve) and in
NaCl 0.52 M (lowest curve). All
the solutions had the conductivity
of sea water. All readings were
taken at 18° C. or corrected to this
temperature. Each curve repre-
sents a single typical experiment.

find that bivalentkations are very effective in this respect?”

“Of. Osterhout (1914, F).
“Of. Osterhout (1915, D).

46 INJURY, RECOVERY, AND DEATH

and trivalent still more so.*? In most cases the effect is so
striking that the addition of the solid salt to the sea
water, although decreasing the resistance of the solution,
nevertheless increases the resistance of the living tissue
so greatly that the net result is an increase in the resist-
ance of the tissue plus solution. As this does not happen

100

= —
ee — — ——
——

0 20 HOURS 40

Fig. 18.—Curve showing the net electrical resistance of Laminaria agardhii in sea water to
which was added sufficient cobalt chloride (in the form of dry salt) to make the concentration
0.005 M. Solution neutral to litmus. All readings were taken at 18° C. or corrected to this
temperature. The curve represents a single experiment. Dead tissue showed no rise.

with dead tissue the increase must be due to an alteration
in the living protoplasm.** It is therefore evident that
the current must flow in part through the living proto-
plasm, as well as through the cell walls.

In the case of certain bivalent kations (Mn, Co, Cd, Ni,

*% Of. Osterhout (1915, #).

“It might be suggested that the increase in resistance is due to a de-
crease of the spaces between the protoplasmic masses brought about by an
expansion of the protoplasm or by a shrinkage of the cell wall. Micro-
scopic and macroscopic examination shows that this does not occur. The
tendency is, on the other hand, to increase the spaces between the cells,
as the result of incipient plasmolysis.

When dead Laminaria is transferred from sea water to CaCl, 0.278 M
there is a very slight shrinkage which, however, is entirely inadequate to
cause a noticeable rise in resistance if it occurs in the cell walls ofr
living tissue.

MECHANISM OF PROCESS OF DEATH 47

Zn) the addition to sea water of sufficient dry salt to
make the concentration 0.005 M kept the resistance above
that of the control for several hours (Fig. 18). In other
eases (FeSO, and SnCl,) the resistance rose, but soon
fell below that of the control.

In similar experiments with trivalent and tetravalent

110
% LIVING
1
0)
{
1
{
15
DEAD
re)
0 5 10 HOURS

Fic. 19.—Curve showing the net electrical resistance of Laminaria agardhii in 1000 c.c. sea
water plus 10 c.c. CaCle 5.0 M. Upper curve, living tissue; lower curve, dead tissue. All
readings were taken at 18° C. or corrected to this temperature. Each curve represents a
single experiment.
a

kations it was found that while the resistance remained
above that of the control for ten hours or more in the
ease of La (NO,), and Y (NO;), this was not the case’®
with Fe,(SO,),; and Th (NO,),.

These experiments were varied by adding strong
solutions to the sea water in place of the dry salt. The

*In the case of SnCl, this may be due to the acidity of the solution.
The concentration is .005 M in the case of each of these salts.

* The solutions containing Fe, (SO,), and Al, (SO,),; were acid. The dry
salts were added to the sea water in sufficient amounts to make the
concentrations as follows: 0.042 M La(NO,);, 0.006 M Ce(NO,),, 0.007
M, Y(NO;)s3, 0.0025 M Fe,(SO,);, 0.01 M Al, (SO,)3, 0.006 M Th (NO,),.

48 INJURY, RECOVERY, AND DEATH

result of such an experiment with CaCl, is shown in Fig.
19. It will be observed that while the resistance of

La CNO,),
AN

|
fogs TER AER oe oleh ee
V/
\7 1X
50 MgCle
\A
VA
NaCl -
fo)
fe) 250 500

MINUTES

Fia. 20.—Curves showing the net electrical resistance of Laminaria agardhii in solutions of

the same conductivity as sea water, t.e., in La(NOs)s about 0.126 M, CaCl 0.278 M., MnCle

about 0.317 M, MgCl: about 0.28 M and in NaCl0.52 M. The curves for La(NOs:)3, MnCle

and MgCl represent single typical experiments (all readings were taken at 18° C. or cor-

rected to this temperature); the curves for CaCle and NaCl represent the averages of six or

more experiments; probable error of the mean less than 10% of the mean (all readings were
taken at 18° C. or corrected to this temperature). _

&
the living tissue increases’ to 109.2% that of the dead

the solution contained in the apparatus and in the cell walls.

MECHANISM OF PROCESS OF DEATH = 49

tissue falls from 15.2% to 13.53%, thereby losing 11% of
the resistance it had in sea water. This corresponds
quite closely to the loss of resistance of the sea water
itself upon the addition of this amount of CaCl,.

Experiments were also performed by placing tissue in
solutions (having the same conductivity as sea water)
which contained only one salt. It was found (Fig. 20)
that the bivalent Ca, Ba, Sr, and Mn raised the resistance
(increasing it to 160% or more) while the trivalent La
and Ce gave a much greater rise (increasing it to 220%
or more). On the other hand, Mg gave very little rise
(increasing the resistance as a rule to not more than
115%). In this respect its behavior is not unexpected,
since it is usually less effective than other bivalent kations
(as for example in antagonizing the effects of Na).'8

It was found that Ulva (sea lettuce) and Zostera (eel
grass) resemble Laminaria in showing a rise with MgCl,
and a much greater rise with CaCl,.° Rhodymenia
palmata (dulse) agrees with Laminaria in showing a
rise in resistance in 7°CaCl,, BaCl., SrCl., MnCl., and
NiCl,, and a greater rise in alum, Ce(NO,),;, and
La(NO,),. The rise with MgCl, is less than with
CaCl,, but the latter is less than that found with Lami-
naria (Fig. 21).

Experiments on frog skin®! showed a rapid rise in
CaCl, (resistance increased to 140%, or less) followed
by a fall. In La(NO,), the rise is greater (resistance
increased to 190% or less) and in MgCl, it is less
(resistance increased to 110%, or less). It is evident

*See Chapter IV.

*® Osterhout (1919, A).

*® Osterhout (1919, A).
“Of. Osterhout (1919, C.).

30 INJURY, RECOVERY, AND DEATH

that the behavior of frog skin in these solutions resem-
bles that of Laminaria. On the other hand, such
substances as NaCl and KCl produce in frog skin and
in fhodymenia no rise, but only a fall of resistance,
just as in Laminaria.

Gray (1915) found that a rise in resistance is pro-

)404x

60-
Hrs. O | 5 10
Fia. 21.—Curves showing the effect of CaChk 0.278 M on the net electrical resistance of
Laminaria agardhii (upper curve) and of Rhodymenia palmata (lower curve). The ordinates

denote net electrical resistance. Temperature 17 + 2° C. Average of six experiments.
Probable error of the mean less than 3% of the mean.

duced in echinoderm eggs by La and Ce. Shearer (1919,
B) was unable to find such an effect in bacteria.

It was of especial interest to investigate the effect
of the hydrogen ion, which in many respects behaves
unlike other monovalent kations. The first experiments
were made by adding to sea water a solution of HCl of
the same conductivity as the sea water (about 0.119 M).
The results are shown in Fig. 22.

It will be seen that a rise occurred and that in higher

MECHANISM OF PROCESS OF DEATH 51

concentrations the maximum was reached earlier, while
in the lower concentrations it occurred later. It is evident
that as the concentration increases, the rise in resistance
is more rapid and the maximum point is passed more
quickly. If the concentration be sufficiently increased,

SEA WATER

eee

©)
20 og

50 100

MINUTES

Fig. 22.—Curves showing net electrical resistance of Laminaria agardhii in sea water con-

taining various amounts of HCl, as shown by the figures attached to the curves. Each

curve represents a single experiment. All readings were taken at 18°C. or corrected to this
temperature. Each curve represents a single typical experiment.

the period of increased resistance becomes less and less,
until it becomes difficult to detect it. The relation between
concentration and changes in resistance is better shown
in Fig. 23.

Experiments with acetic acid also showed a rise
in resistance.

o2 INJURY, RECOVERY, AND DEATH

erste, 120_PER CENT

100 |

20
001 01 .015 = .02 .03 M

Fic. 23.—Curves of net electrical resistance of Laminaria agardhii in sca water containing
various amounts of HCl. ‘The ordinates represent net resistance. The abscisse represent

concentrations of HCl. All readings are taken at 18° C. or corrected to this temperature.
Each curve represents a single typical experiment.

In the previous experiments with alkali? it was found
Of, Osterhout (1914, F). naa

MECHANISM OF PROCESS OF DEATH — 53

necessary to employ a solution containing NaCl+-CaCl,,
since the addition of alkali to sea water causes a precipi-
tate of Mg(OH),. In order to compare the effect of acid
with that of alkali a solution of HCl having the same

7 eS
120 \\
apa

“~
100 <2) Ge —~— yer er Ker Kr mr Om rm rmr rr nrreanr as ssn —->X
SEA WATER

20
OQ 50 100 150
MINUTES

Fic. 24.—Curves showing net electrical resistance of Laminaria agardhii in 1793 oa.c. NaCl
9.52 M + 207 ¢.c. CaCk 0.279 M containing various amounts of HCl, as shown by the
figures attached to the curves. Each curve represents a single experiment. All readings

were taken at 18° C. or corrected to this temperature.

conductivity as the sea water was added to a solution
containing 1793 ec. NaCl 0.52 M + 207 ee. CaCl, 0.278 M
(this solution had the same conductivity as sea water).
The results are shown in Fig. 24; it will be noted that
they are in good agreement with those obtained by adding

o4 INJURY, RECOVERY, AND DEATH

130 PER CENT

001 01 N15 O02. ‘03M
Fia. 25.—Curves of net electrical resistance of Laminaria agardhii in (1793 c.c. NaCl
0.52M +207 c.c. CaCh 0.278 M) containing various amounts of HCl. The ordinates repre-
sent net resistance; the abscisse represent concentrations of HCl. All readings were taken
at 18° GC. or corrected to this temperature. Each curve represents a single typical experiment.

acid to sea water. These relations are still more clearly
shown when the results are plotted as shown in Fig. 25.

MECHANISM OF PROCESS OF DEATH — 55

These results present a marked contrast to those
obtained by the use of alkali; with the latter there is no
rise in resistance, but, on the contrary, a fairly rapid
fall which continues until the death point is reached.

In view of the great importance of acid and alkali in
life processes these results deserve especial consideration
since it would seem that slight changes in the reaction of
the medium affect conductivity and permeability.

It may be added that experiments with frog skin*
showed that in this case also HCl produces a rapid rise
in resistance followed by a fall. Shearer (1919, A) found
a rise in the case of bacteria.

The writer has also found* that high concentrations
of KCN (0.01 to 0.381 M) produce a slight temporary
rise in the resistance of Laminaria.”

It is of considerable interest to find that certain
organic substances are able to increase resistance. As
an example of this we may consider experiments with
bile salts.2° In these investigations Na-taurocholate was
added to the sea water, which was then restored to its
normal conductivity and made approximately neutral
to litmus.?7_ All concentrations employed produced an
immediate increase in resistance followed by a fall,
as illustrated in Fig. 26. Under the conditions of the
experiment, the rise lasted about an hour. An increase in
resistance was also observed with Ulva rigida (sea

*Osterhout (1917, A).

* Possibly this might have been greater had not the solution
been alkaline.

* Osterhout (1919, B).

* The amounts varied from 0.8 to 1.5 gm. added to 1000 c.c. of sea

water. If the Na-taurocholate were pure 1 gm. in 1000 c.c. would make
the concentration about 0.002 M, but its purity is doubtful.

56 INJURY, RECOVERY, AND DEATH

lettuce) and Rhodymenia palmata (dulse) ; in the latter
case it was much less than in Laminaria.

An increase has also been observed in experiments
with ether, chloroform, chloral hydrate, and alcohol.*®

100%

%-
alas 100 200
MINUTES

Fig. 26.—Curve showing rise in net electrical resistance of Laminaria agardhii produced by

adding 1 gm. of Na-taurocholate to 1000 o.c. of sea water (solid line). Control in sea water,

dotted line. Average of two experiments; probable error of the mean less than 2.3% of the
mean. Temperature 19+ 2° C.

The question arises whether the rise due to these
organic substances differs from that produced by inor-
ganic salts and by acids. The writer is inclined to believe
that this may be the case, but prefers to await the
results of additional experimentation before reaching a
definite decision.

MECHANISM OF PROCESS OF DEATH — 57

The fact that in all these cases there is a rise in
resistance followed by a fall suggests that there are
two processes at work, one producing an increase and the
other a decrease. In order to picture a mechanism which
would account for this the writer has assumed that at
the surface of the cell there is a substance, M, forming
a continuous layer®® whose thickness determines the
amount of resistance. It is assumed that the thickness
of this layer is increased by the breaking down of a
substance, A, to form M, according to the monomolecular
reaction A—> M. At the same time M breaks down to
form a substance, B, according to the monomolecular
reaction, M—>B. The two reactions go on simultan-
eously according to the scheme*®” A—> M—> B.

It is obvious that if the rates of the reactions are
such that 17 is formed as rapidly as it is decomposed, it
will remain constant in amount; and that an increase in
the velocity of the first reaction will cause M to increase,
while an increase in the velocity of the second reaction
will cause M to decrease.

The nature of this process is evident from a considera-
tion of Fig. 27. If the reservoir A be filled with water
while M and B are empty, and if water be allowed to
flow from, 4 into M, the amount of water in M (for
convenience this amount is called y) will first increase
and then decrease. The rate of increase and decrease
and the maximum attained will depend on the relation
between the two outlets K, and K,. We may suppose
that if K, is equal in diameter to K., we get the upper

*It is recognized that the hypothesis will apply if the layer is not
continuous and also if the change in properties of the layer is other than
that of thickness.

* These reactions are regarded as reversible or practically so.

08 INJURY, RECOVERY, AND ae ne

curve aay in Bie figure, while if K, is less than K, we
get the lower curve (in the latter case both outlets are
supposed to be smaller than in the former). This is
analogous to what occurs in the reaction 4 —M—>B
if K, is the velocity constant of 4—>WM and K, is the
velocity constant of M—>B.

We assume that in sea water A is renewed as fast

or K,

time

A> hee

Fig. 27.—Diagram illustrating consecutive reactions in which a substance M is formed by
the reaction A —> M and decomposed by the reaction M—>

as it is decomposed and has the constant value 2700, and
that M has the constant value 90. On transferring from
sea water to NaCl 0.52 M the production of A ceases, but
A continues to break down toform M and B. The velocity
constants*! in NaCl are taken as K,—0.018 and K,—0.540.

We may now calculate the resistance, putting Net
Resistance=M-+10, ‘because the base line of the death

“ Osterhout (1916, D).

MECHANISM OF PROCESS OF DEATH — 59

curve, as shown in Fig. 28, is not zero but ten. M is
therefore equal to the resistance of the living tissue after
the resistance of the dead tissue and the resistance of
the apparatus has been subtracted. We may call this the
residual resistance, while the resistance of the living
tissue minus that of the apparatus is called the
net resistance.

100%

100 200 MINUTES

Fig. 28.—Death curve of Laminaria agardhit in NaCl 0.52 M. The curve shows the cal-
culated value of the resistance: the observed values are shown by the points (©, ©). All
readings were made at 15° C. or corrected to this temperature. Each point represents the
average of ten or more experiments. Probable error of the mean less than 10% of the mean.

In our calculations we may employ the methods used in
calculating the decomposition of radioactive substances.*”

If we start with A and M in equilibrium in sea water
according to the scheme A —> M —>B, and if the value
of A is called A, and that of M is called M,, the amount of
M which is formed in each unit of time is A,K,, and the
amount of M which decomposes in each unit of time is
M,K,. Since at equilibrium, 4,K,—M,K., the value of
M remains constant.

If the tissue is transferred to NaCl 0.52 M we have at

* Rutherford, E. (1913) p. 421.

60 INJURY, RECOVERY, AND DEATH

the instant of transfer the following values: A, = 2700, -
M,=90, K,=0.018, and K,—0.540. As no more of A is
produced A, diminishes and at the end of time 7 has the

value Ap= Aen Bt During exposure to NaCl a cer-

tain amount of M is formed from A. Some of this dis-
appears during exposure. The amount which remains
at the time 7 may be called M,. At the time T the
amount of M,, which disappears in unit time is M,, Ko.
At this time the amount of M produced in unit time is

— K,T
ATK, = Ae) OR

The change in M,, occurring in unit time, which we may
eall pt ,is equal to the difference between the amount
formed and the amount decomposed, or

The solution of this equation is of the form

Mr=A, ag OE aaa ee
By substitution it is found that a= Hr
| y substitution it is foun a 0 eae
Since M,= 0 when, T=), b— 2s Ky
_(Ki—K;)

Thus M,=Ao Gate ) (eiP_ ga)
("Ee Ba oh ta 8
ae )

We must also consider that at the beginning of the
exposure to NaCl 0.52 M there was present a certain

or M,=2700

MECHANISM OF PROCESS OF DEATH Gi

amount of M/ (this was called M,) which diminished dur-
ing exposure, and the amount remaining at the time 7

is M,e pen If this is added to the amount of M pro-

duced from A during exposure we get (substituting the
value M,=—90)

Total amount of M =2700¢¢ ) (e~ Kal __.— Kal) 4 99.—KaT
and since Net Resistance == V7 -++ 10 (because the base line
of the curve is taken as 10) we have **

Ky) | -KT_|-K,

T =i
K,—K, ( ash ) + 90e

T
Net Resistance = 2700 ( +10

If we calculate the resistance by means of this for-
mula we get the curve given in Fig. 28, which shows a
close agreement between the observed and calculated
values. It is therefore evident that, whether our picture
of the underlying mechanism is correct or not, it leads
to an equation which enables us to predict the death
curve with considerable accuracy. The predictive value
of the equation is quite independent of the assumptions
which led up to it, and while it creates a presumption
in favor of these assumptions, it of course does nothing
more. Itis hardly necessary to emphasize that equations
which enable us to predict the course of vital pro-
cesses are a prime necessity in biology, since they make
it possible to employ the methods by which the exact
sciences have been able to make rapid progress.

It is evident that we are able to follow the progress of

The values e aaa and e ‘ry may be obtained from Table IV
in the Smithsonian Mathematical Tables, Hyperbolic Functions, by G. F.
Becker and C. E. Van Orstrand, 1909. See also Mellor, J. W. (1909) pp.
16, 98, 118.

62 INJURY, RECOVERY, AND DEATH

death in this case just as a chemist follows the progress
of a reaction in vitro. This has also been found to be the
case in experiments with a great number of toxic sub-
stances and seems to be of very general applicability.

TABLE I.*

Net electrical resistance of Laminaria in NaCl 0.52 M. The resistance in sea
water (the normal environment) is taken as 100 per cent.

Resistance. Ks.

mene. Observed, Culoulated. From observed | From calculated

values. values.
min. per cent per cent

10 87.50 87.76 0.0065 0.0064
20 73.01 74.96 0.0077 0.0071
30 62.51 64.26 0.0078 0.0073
40 55.30 55.32 0.0075 0.0075
50 48.81 47.86 0.0073 0.0075
60 40.21 41.62 0.0079 0.0076
70 36.79 36.41 0.0075 0.0076
80 32.41 32.06 0.0076 0.0076
90 27.92 28.43 0.0079 0.0077
100 24.69 25.39 0.0079 0.0077
110 23.00 22.86 0.0076 0.0077
120 22.82 20.74 0.0071 0.0077
150 16.51 16.26 0.0076 0.0077
180 14.54 13.65 0.0072 0.0077
PVCURGE S| nn oe gate eicriite as een See 0.0075 0.0075

* All readings were made at 15°C., or corrected to this temperature.

If we were unaware that the death curve in NaCl 0.52
M represented two consecutive reactions, and supposed
it to represent a simple monomolecular reaction (M —>
B), we should calculate its velocity constant (which we
may call K,) by the usual formula:**

1
ee zoe (

If we make this calculation, employing for this pur-

a
a—-2Z

“Common logarithms are used for convenience, We put a = 100 — 10
anda — x — M — 10.

MECHANISM OF PROCESS OF DEATH 63

pose the calculated values given in the third column of
Table I, we obtain the values of the velocity constant
K, given in the fifth column of the table.

It is evident from an inspection of these values that
the velocity constant K, falls below the average value at
the start.

The amount by which it falls below the average value
will depend on the relation K,~ K,. When K, and K,
are nearly equal, the velocity constant falls a good deal
below the average value at the start, but as the difference
between them is increased the velocity constant K, will be
found to fall less and less below the average level at the
start.*> This is easily shown by assuming various values*®
of K, and K,.

From this it follows that we can tell something about
K,—K, from the experimental values of K,. It is
evident that in the present case the experimental values
of K point to the relation K, ~~ K,—30 (or K, + K,=
30). This relation was actually assumed by the writer, in
order to fit, not the NaCl curve, but antagonism curves*?
in various mixtures of NaCl-+ CaCl,. It is therefore a
striking confirmation of the general correctness of the
underlying assumption that we are also able by means of
this assumption to fit the NaCl curve so closely.

In general, where a chemical reaction is slower at
the start than is expected, we may suspect that we have

* It should be noted that we get the same result (as regards K, falling
below the average at the start) when K,~K,—=30 as when K,—EK,

= 30. With certain relations of K,~ K, the constant K, may be above
the average value at the start.
* When the values of K, and K, are changed, the concentrations of A
and M must also be changed in such a way that Cone. A Cone. M=
1+ K, if we wish the concentrations of A and M to remain constant
in the normal environment.
See Chapter IV.

64 INJURY, RECOVERY, AND DEATH

to do, not with a simple reaction, but with consecutive
reactions of the kind here described.*8

This explanation also applies to a considerable num-
ber of other cases of toxic action.

It is of interest to note that in all these cases death be-

25 50 75 HOURS

Fia. 29.—Curve showing the net electrical resistance of Laminaria agardhii in CaCl: 0.278 M.
Unbroken line, observed values; broken line, calculated values. All observations were made
at 18° C. or corrected to this figure. Average of ten or more observations. Probable error
of the mean less than 10% of the mean.

haves as a reaction which is continually going on, but at a
very slow rate until accelerated by the toxic agent. We
have assumed this acceleration to consist partly in the
increase of the velocity constant and partly in the stopping
of the reaction O —>A, causing a decrease in the sub-
stance (M) to which normal permeability (and perhaps
other normal properties) are due.

* Mellor, J. W. (1909), Chapter VI.

MECHANISM OF PROCESS OF DEATH 65

It may prove to be generally true that death behaves
as a monomolecular reaction, which is inhibited (or
accelerated) at the start. The assumption of consecutive
reactions affords an explanation not only of the inhibi-
tion (or acceleration) at the start, but also of the fact
that up to a certain point the reaction appears to be

TABLE II.
Net electrical resistance of Laminaria in CaCl, 0.278M. The resistance in sea
water (the normal environment) is taken as 100%.

Per cent. of net resistance in

Time in hours.

Obs. Cale.
1 154.0 152.9
2 152.1 148.9
3 143.2 136.5
4 128.8 123.8
5 117.2 112.2
6 101.8 101.8
10 67.76 69.59
25 23.90 21.79
50 GE 10.79
80 11.0 10.07

* The measurements were made at 15° Centigrade or corrected to this
figure. Each experimental figure is the average obtained from 6 or more
experiments. Probable error of the mean less than 10 % of the mean.
reversible. The latter fact will be fully discussed in a
subsequent chapter.

It is evident that if the theory of the writer is sound,
the equation which allows us to predict the death curve
in experiments with NaCl should apply equally well in the
case of experiments with CaCl,. This is the case, as is
evident from Fig. 29 and Table II (in this case we put
K,= 0.0018 and K, = 0.0295).

When the curve has a maximum, the height of the

66 INJURY, RECOVERY, AND DEATH

maximum may be approximately ascertained by means
of the formula.*®

2
Maximum resistance = 2830 (Zz ) Ky — Ky + 10
2

In the present case K,—K.,—0.0018—0.0295—1 16.3889.
Hence we may, for convenience, put K,—1 and K, =
16.3889. We then have Maximum resistance == 2380

16.3889
1 _\15.3889 oe
(im) + 10 = 153.98.

The actual maximum found by calculating the curve
is close to 153.94 (which occurs at 75 minutes). Such
a close approximation must not, however, be expected
in most cases.

Where the maximum of the curve is known and it is
desired to find the relation K,— K, (as a preliminary
step toward ascertaining the values of K, and K, by
trial) we may plot a series of values of K,+K, as
ordinates, and maxima (obtained by calculation)
as abscisse, and thus approximate graphically to the
desired figure.

When the height of the maximum is known, the time
at which the maximum occurs may be found as follows:
When the maximum is attained the value of M may
be called M max and the value of A may be called Ay.

——

* This may be regarded as an approximation formula. We consider
that the value of A before any of it has decomposed to form M and B,
is 3050 and if this is substituted for 2830 in the formula it will give
exact values, provided the constants are not changed as M increases from
0 to the maximum. But if M increases from 0 to 90 with one set of
constants and then from 90 to the maximum with another set, the formula
no longer holds and the approximation formula may be used. Cf. Mellor,
J. W. (1909) p. 115, In the formula as given by Mellor a misprint occurs.

MECHANISM OF PROCESS OF DEATH 67

Since at this time M is formed as rapidly as it is decom-
posed, we have
AK, = M max K,
A, =M max kK,
Ky

At the start (in sea water) the value of A was 2700, but
it has now diminished to a fraction represented by

M max K;

it requires to reach this value, for, as the reaction A —> M
is monomolecular, we may write
—K.T _M maz K

) 2700. We can easily find out how long

AT =27006e K,
é —KiT M max K;
~ oS I
Knowing the values of M max, K, and K, we can find

the value of 7 by looking up the value of ef? in
a table.*°

In the present instance we have M max = 153.94 —
10 = 148.94

Hence

(143.94) 0.0295 _ — (0.0018) T

(2700) 0.0018 ©
and 0.87372=e — (0.0018) 7
We find from the table that
e ~ 0-135 ___, — (0.0018) 75 __ 9 g7a79
Whence T = 75
It should be noted that multiplying K, and K, by the
“E.g., Table IV in the Smithsonian Mathematical Tables, Hyperbolic

Functions, by G. F. Becker and C. E. Van Orstrand, 1909. See also
Van Orstrand, C. E. (1921).

68 INJURY, RECOVERY, AND DEATH

same factor is equivalent to dividing all the abscisse by
the same factor and that in the case of a curve which
rises and falls, this does not change the height of the
maximum. If, therefore, both reactions have the same
temperature coefficient, raising the temperature is equiv-
alent to multiplying both K, and K, by the same factor
and the maximum will not be changed. But if the reac-
tions have different temperature coefficients this will not
be true.

Perhaps it may be desirable in this connection to add
a word regarding the measurement of life processes. The
development of quantitative methods in biology depends
largely on finding means of measuring the speed of life
processes. In most cases the absolute rate is of less
importance that the relative rate (e. g., the normal veloc-
ity compared with that observed under the influence of a
reagent). Examination of the literature shows that the
determination of relative rates is frequently made in a
faulty manner, which might easily be avoided by a slight
change of method.

As an illustration of this we may consider the processes
shown in Fig. 30. In the case of Curve A the process is
twice as rapid as in the case of Curve B. This is shown by
the fact that the abscisse of A are everywhere one-half
those of B. This means that the velocity constants of A
are twice those of B.41. In other words the velocity con-
stants are inversely proportional to the abscisse, or in-
versely proportional to the times required to bring
the reaction to the same stage*? (e.g. one-half com-
pleted). This is true for chemical processes in general,
not only for reactions of the first order (where a

~ “Gf, Osterhout (1918, B).
“Of. Osterhout (1918, B).

MECHANISM OF PROCESS OF DEATH — 69

single substance decomposes) but for reactions of
higher orders (where two or more substances combine)
as well as for consecutive reactions*® and autocatalysis.**

It follows that when a chemical process proceeds at
different rates under different conditions, we can com-
pare the velocity constants by simply taking the recip-
rocals of the times required to bring the reaction to the
same stage, so that if we wish to know merely the relative

100

50 S

fa)
0 40 80 MIN.

Fig. 30.—Curve A represents a process which proceeds at twice the velocity of B. The
abscisse of B are everywhere double those of A, but nosuch relation holds for the ordinates.
C is obtained by averaging the absciss# of A and B; D is obtained by averaging their ordinates.

rates (as is usually the case in biology) it is not necessary
to determine the actual velocity constants at all.
Whenever the initial conditions are the same with
respect to concentration we need only compare the times
required for equal amounts of work, since these bring
the reaction to the same stage. If, on the other hand, one
attempts to arrive at the relative rate by comparing the
amounts of work performed in equal times (as is fre-
quently done in biological research) he can easily fall
“The principle holds for consecutive reactions in case all the constants

are multiplied by the same factor, otherwise not. Cf. Osterhout (1917, Z).
“Of. Mellor (1909) p. 291.

70 INJURY, RECOVERY, AND DEATH

into serious error. This is evident from Fig. 30 which
shows that while the abscissa of A at any point is just
half that of B, no such relation obtains among the ordin-
ates.15 For example at 40 minutes, the ordinate of B
is twice as great as that of A, while at 4 minutes, it is
less than 1.1 times that of A. Hence it is evident that
we should compare abscisse rather than ordinates. (1.e.,
times required to do equal amounts of work rather than
amounts of work performed in equal times).

The principle is sufficiently obvious where successive
determinations are made and curves are drawn. But
there is a common type of experimentation in which, for
various reasons, a single observation at one rate is com-
pared with a single observation at another rate. The
principle in question is then easily overlooked. In some
cases this leads to serious errors.*®

It is therefore evident that when we average time
curves, we should, whenever possible, average abscissx
rather than ordinates. Thus for example, in Fig. 30 the
average of Curves A and B would be Curve C, obtained by
averaging the abscisse of Curves A and B: this gives a
curve whose velocity constants are the arithmetical mean
of those of A and B. On the other hand, by averaging
ordinates we obtain Curve D, which does not follow the
formula characteristic of the other two curves.

It may be desirable to point out that these methods
may be advantageously applied to the measurement
of toxicity.*

*“We cannot avoid the difficulty by comparing the rates of the two
processes at a given time; for the rates so obtained will bear no constant
ratio to each other. Only when they are compared at the same stage of
the reaction will they show a constant relation; this gives the relation
between the velocity constants.

“For a discussion of this see Osterhout (1918, B).

“ Of. Osterhout (1915, @).

MECHANISM OF PROCESS OF DEATH

71
One striking result of the investigations on toxicity
earried out by the writer is to emphasize the fact that the
apparent toxicity of two substances may depend very

largely upon the stage of the reaction at which the meas-
140%

100%

\ NORMAL

\
\
\

‘55% . HALF DEAD
‘
‘

‘

5 HOURS

Fic. 31.—Curves showing changes in the net electrical resistance of tissues in two toxic
solutions, A and B (the latter causes a rise followed by a fall in resistance).

Toxicity may be
measured by determining the time required to carry the reaction to a definite stage, as, for

example, to 55% which is half way between the normal condition and the death point.
urement is made

This is evident from an inspection of
the curves in Fig. 31.

These represent the electrical
resistance of Laminaria in sea water and in two toxic
solutions. i

If the tissue be placed in a solution of NaCl
of the same conductivity as sea water, the resistance falls

somewhat as shown in Curve A, until it reaches the death

72 INJURY, RECOVERY, AND DEATH

point. If, on the other hand, the tissue be placed in a
solution of some substance which causes a rise, followed
by a fall in resistance, we may get a curve somewhat like
that shown at B.

The most common method of measuring the toxicity of
a solution is to determine the time necessary to cause
death. But it is evident from an inspection of the curves
that it is impossible to determine the precise moment of
death, since the death curves approach the axis asymptot-
ically. This is doubtless true of death in all cases. It
is therefore obvious that the death point does not offer
a perfectly satisfactory criterion of toxicity.

We may avoid this difficulty by taking as a criterion
the time needed to reach any convenient point on the
curve, as, for example, 55% (half way between the normal
condition and the death point). This may be determined
with a good deal of precision by the measurement of elec-
trical resistance or by any method which permits us
to follow the reaction accurately from moment to moment.
But where this cannot be done, we may employ other
criteria. We may assume that as the reaction goes on,
certain phenomena appear at definite points on the curve,
such, for example, as changes in metabolism, cessation
of motion, or loss of irritability. The employment of
such criteria may give trustworthy results in many cases
if proper precautions be taken.

In the employment of any of these criteria, except
that of death, we may meet the difficulty that the relative
toxicity of two substances may vary greatly according to
the point in the curve at which the comparison is made.
Let us suppose that two toxic substances are so chosen
that they produce death at about the same time, giving
curves as shown in Fig. 31. They must be regarded

MECHANISM OF PROCESS OF DEATH ~— 73

as equally toxic if we adopt death as the criterion,
but as unequally toxic if we take any other criterion. For
example at 90%, A appears to be seven times as toxic as B.

It is clear that we cannot escape from this difficulty
by comparing the effects produced in equal times.

In view of these facts it is obviously undesirable to
compare results obtained by the use of unlike criteria,
as is often done.

Another method is to measure the degree of recovery
which is found where tissues are taken from toxic solu-
tions and replaced in sea water. This will be explained
more fully in Chapter III. It has many advantages which
entitle it to serious consideration. The writer has found
that death in many toxic substances, as measured by the
electrical method, follows approximately the course of a
monomolecular reaction. In such cases the constants
‘which express the reaction velocities of the two reactions
afford a measure of their relative toxicity. In cases
where such constants cannot be used, but where the com-
plete curve can be obtained, it would be possible to adopt,
as an arbitrary standard, the time necessary for the
reaction to proceed half way to the death point. But,
when the curves are related to each other as are A and B
in Fig. 31, it may be desirable to use some other criterion.
It is in any case desirable to give the whole curve, when-
ever possible, so that the reader may apply his own
criterion. The ease with which complete curves can be
obtained by determining electrical resistance may render
this method useful, especially since the writer has found
it possible to apply it to all sorts of plant tissues as well
as to some animal tissues.

The electrical method is not restricted to solutions
of the same conductivity. For example, we find that

74 INJURY, RECOVERY, AND DEATH

NaCl 0.52 M and CaCl, 0.278 M have the same conduct-
tivity as sea water. If we wish to compare the toxicity
of NaCl 0.278 M with that of CaCl, 0.278 M we may dilute
the sea water until it has the conductivity of NaCl 0.278
M. Tissue placed in this may be used as a control. At
the outset we make the resistance of the control equal
to that of the tissue in NaCl 0.278 M, or we divide the
resistance of the control by a figure which reduces it to
the same value (and divide all subsequent readings
of the control by the same figure). We then express all
readings of the tissue in NaCl 0.278 M as per cent. of the
reading of the control which is taken at the same time.
All readings of the tissue in CaCl, 0.278 M are likewise
expressed as percentage of the readings of a control in
sea water having the same conductivity as CaCl, 0.278 M.
Stronger solutions may be treated in the same way, using
sea water which has been concentrated by evaporation.

Attention may be called to a further difficulty in deter-
mining toxicity. If tissue of Laminaria be transferred
from sea water to pure solutions of toxic salts their
relative toxicity sometimes appears to be different from
that which is observed when the same substances are
added directly to the sea water. Similar considerations
may be found to apply to animals and plants which live
on land or in fresh water, in which cases Ringer’s solu-
tion or the water of soils and rivers may play the same
role as the sea water in experiments with marine forms.
These differences depend largely on the antagonistic
action of salts, which will be discussed in Chapter IV.

It may be added that in some cases variations in the
supply of oxygen may cause changes in relative toxicity ;
and in view of the fact that the temperature coefficient
is not the same in all cases of toxic action it seems

MECHANISM OF PROCESS OF DEATH = 75

desirable to carry out determinations as far as possible
at a standard temperature, preferably at 18°C.

In conclusion, attention may be drawn to the effects of
temperature* upon consecutive reactions such as are here
assumed to be responsible for the phenomena with which
we are dealing. The temperature coefficient of death in
NaCl 0.52 M, and CaCl, 0.278 M is not far from 2.

The temperature coefficients of life processes have
within the last few years attracted a good deal of atten-
tion. Interest has chiefly centred about the question
whether life processes have the temperature coefficients
of ordinary chemical reactions and whether investiga-
tions of this sort enable us to distinguish between
chemical and physical processes (on the ground that
in general, the latter possess lower temperature coeffi-
cients than the former).

In these discussions of life processes it is generally
assumed that we are dealing with simple chemical reac-
tions. A little consideration shows that this cannot always
(or even commonly) be the case. Most substances formed
in the organism are also broken down, and the amount
present must depend on the relative rates of formation
and of decomposition. Change of temperature may affect
consecutive reactions in an entirely different manner from
simple reactions (in which the substance formed is not
at once broken down). This may be made clear by a
concrete illustration.

Let us take for this purpose the death curve in NaCl
(Curve I, Table III) and consider the effect of raising the
temperature 10° C. If both reactions have the tempera-
ture coefficient 2, K, becomes 0.036 and K, becomes 1.080.

* Of. Osterhout (1917, H#). For the temperature coefficients of living
and dead tissues in sea water see p. 37.

76 INJURY, RECOVERY, AND DEATH

The values of M under these conditions are given in Table
IIT (Curve II). Inspection of the table, and of the curves
in Fig. 32, shows that at the higher temperature it re-

140

loo

204

20 60 Minutes

Fia. 32.—Curves showing the value of M when the velocity constants have the values
abe below.

Curve 1 Kz
I 0.018 0.540
II 0.036 1.080

IV 0.036 0.648
Curves II, III and IV are derived from Curve I by assuming that the temperature is raised
10° C.; if the two reactions have the temperature coefficient 2 we obtain CurvelI]; if the
coefficients are 1.2 and 2 respectively we obtain Curve III; if the coefficients are 2 aud 1.2
respectively we obtain Curve IV

quires just half as long to produce the same amount of
chemical action as at the lower. Hence the consecutive
reaction appears to behave in this instance like a
simple reaction.

MECHANISM OF PROCESS OF DEATH 77

The result will be quite different if the two reac-
tions have different temperature coefficients. Let us
suppose that the speed of the reaction 4 —> WM is deter-
mined by diffusion (as happens in some heterogeneous
reactions) and has in consequence a low temperature
coefficient which we will assume to be 1.2. Assuming that
the reaction M—>B has a temperature coefficient 2 we

TABLE III.
Value of M.
Time Curve I. Curve II. Curve III. Curve IV.
Ki = 0.018 Ki = 0.036 Ki = 0.0216 Ki = 0.036
K:= 0.540 K:= 1.080 K:=—= 1.080 K:= 0.648
min
10 87.76 74.96 64.39 120.70
20 74.96 55.32 53.82 87.31
30 64.26 41.62 45.31 63.94
40 55.32 32.06 38.45 47 .63
50 47 .86 25.35 32.92 36 .26
60 41.62 20.74 28.47 28.32
90 28 .43 13.65 19.62 16.22

find than on raising the temperature 10° C., K* becomes
0.0216 and K, becomes 1.080. The values under these
conditions are given in Table III (Curve III).

Let us now consider the effect when the temperature
coefficient of the first reaction is 2 and that of the second
is 1.2. On raising the temperature 10° C. K, becomes
0.036 and K, becomes 0.648. The values are given
in Table III (Curve IV). The form of the curve is quite
different from that of the others in that there is first a
rise followed by a fall. In experimental work a short
period of rise might be overlooked or regarded as due
to experimental error or some disturbing (‘‘inhibiting’’)
factor, such as is commonly assumed to account for delay
at the beginning of a reaction.

If the observer supposed that he had to do with a

78 INJURY, RECOVERY, AND DEATH

simple reaction of the type M—>B8 and proceeded to
calculate the velocity constant, he would obtain the values
given in Table IV.

A consideration of these values is very instructive.
It is evident that when the relation K ,-+ K , has a certain
value (as in Curves I and II where K ,.— K , = 30) the

TABLE IV.
Apparent velocity constants obtained on the supposition that the process 18 a
simple reaction.

Apparent velocity constant.

Time Curve I. Curve II. Curve III. Curve IV.
Ki= 0.018 Ki = 0.036 Ki = 0.0216 Ki=0 036
Ki1= 0.540 K:1= 1.080 K:= 1.080 Ke=0 48
min
10 0.0064 0.013 0.022
20 0.0071 0.014 0.016 0.0033
30 0.0073 0.015 0.014 0.0074
40 0.0075 0.015 0.013 0.0095
50 0.0075 0.015 0.012 0.011
60 0.0076 0.015 0.012 0.012
90 0.0077 0.015 0.011 0.013

reaction appears to proceed as a monomolecular reaction
which is somewhat ‘‘inhibited’’ at the start,*® while with
other values it may appear to be greatly inhibited at the
start (Curve IV, K ,-- K , = 18) or to go much faster in
the beginning than is expected (Curve III, K ,+~K ,=50).

These facts deserve consideration in interpreting the
temperature coefficients of consecutive reactions, to
which category many life processes undoubtedly belong.

Mellor (1909), p. 113.
” Of, Loeh (1912, D), p. 212.

CHAPTER III
INJURY AND RECOVERY

Aw investigation of the process of death leads us
naturally to a study of the power of the organism to
recover from exposure to unfavorable influences.

An interesting aspect of this subject is the connection
between injury and permeability. In the opinion of some
writers permeability is a relatively fixed property of the
cell which changes only as the result of injury, and is then
altered? irreversibly, while others assume that reversible
changes in permeability may form a normal part of the
activities of the cell.2 In view of the fact that such
changes may control metabolism it seemed desirable to
the writer to investigate them by determining conduc-
tivity, since (as will be shown in Chapter VI) an increase
in conductivity indicates an increase in permeability, and
since it is also possible to calculate the increase in proto-
plasmic conductivity (and hence of permeability) as dis-
tinguished from the increase in the conductivity of the
tissue as a whole.

The following will serve to illustrate the method of
experimentation. Tissue which had in sea water a net
resistance of 770 ohms was placed in a solution of NaCl
0.52 M. In the course of 5 minutes the resistance fell to
580 ohms, or 75.32% of the original resistance.t When

* Of. Héber (1914) Kap. 8, 9 und 13.

?Cf. Osterhout (1912, B).

> Cf. Osterhout (1915, B).

*Complete recovery after such a large increase of conductance is not
always obtainable unless the material is in good condition and is freshly
collected. Even in such material a lot will occasionally be found in which
recovery is poor.

80 INJURY, RECOVERY, AND DEATH

the tissue was replaced in sea water the resistance soon
rose to normal and so continued during the remainder
of the day.®

In this case the conductance of the tissue at the start
was 1—770—0.0013 reciprocal ohms and this changed to
1-+580—0.00172 reciprocal ohms, an increase of 32.3%. It
would be more convenient to say that the conductance at
the start was 1 - 100 and that this increased to 1 ~ 75.32.
The resistance of the protoplasm at the start® (as distin-
euished from that of the tissue as a whole) would
then be 140, decreasing to 96.03, a loss of 31.4%. The
conductance at the start would be 1~140— .007143
increasing to 1 ~ 96.03 = .010418; or, if we call the proto-
plasmic resistance at the start 100, it decreases from
100% to 68.60% and the conductance increases from
1 +100 —.01 to 1~ 68.60 = .014578, a gain of 45.78%."

In order to see whether this increase is accompanied
by permanent injury, an experiment was made in which
the same piece of tissue was exposed to the action of
NaCl several times during the same day. The net resist-
ance of the tissue in sea water was 810 ohms; after 5
minutes in NaCl the resistance fell to 84% of the original
resistance; the tissue was then placed in sea water and
a reading 10 minutes later showed that the resistance had
risen to 100%. In this case the fall of protoplasmic
resistance was (100 — 78.94) + 100— 21.06% and the
increase in permeability (conductance) was [ (1-78.94)
6 Shearer (1919. A) obtained similar results with bacteria.

°For the method of calculating this see p. 217.

"It might be objected that this increase is not necessarily the result of
increase in permeability, but may be due to the fact that the protoplasm
is more permeable to NaCl than to CaCl, and since the number of
Na-ions is increased the conductance also increases. But the increase in

Na-ions is much too small to account for the effect since it amounts to
about 1%.

INJURY AND RECOVERY 81

— (1+ 100) ] + (1+ 100) = 26.68%. During the next 95
minutes it showed no change. It was then placed in NaCl
for 5 minutes and the resistance fell to 82.8%. It was then
replaced in sea water; a reading taken 10 minutes later
showed that it had returned to normal, where it remained
for 90 minutes. It was then placed in NaCl for 5 minutes.
The resistance fell to 86.42% and returned to normal dur-
ing the ensuing 10 minutes in sea water. After 105

° 1 2 s HOURS

Fic. 33.—Graph showing the fall of net electrical resistance of Laminaria agardhii in NaC
0.52 M (unbroken line) and recovery in sea water (broken line). All readings were made at
2 . or corrected to this temperature. The graph represents a single experiment.

minutes in sea water (during which no change occurred)
it was again exposed to NaCl for 5 minutes. The resist-
ance fell to 82.8% and returned again to normal during
the following 10 minutes in sea water. On the following
day its resistance was only 30 ohms below the resistance
of the control, which at the beginning of the experiment
was 810 ohms. The results are presented in Fig. 33.

The successful outcome of this experiment led to an
attempt to carry on such an experiment for several days
in succession, giving the tissue one treatment daily with
NaCl. The material was selected with especial care. The
fronds were fairly thick, without reproductive organs.
The experiment was made at Woods Hole, Mass., in July,
at which time such fronds may be easily obtained. The
disks cut from these fronds were slightly curved, so that
when placed in the apparatus they separated spontan-

82 INJURY, RECOVERY, AND DEATH

eously, thus allowing the running sea water in which they
were kept to circulate freely between them. Care was
taken to keep them only about two-thirds submerged, so
that they had free access to air, but ran no risk of
drying up. The tissue in sea water had a net resistance
of 780 ohms at 20° C. As the temperature of the

WANA

0 ne 30

15 30
MIN

Fria. 34.—Graph showing loss of 'net electrical resistance of Lasantiree agardhiit in NaCl

0.52 M (unbroken lines) and recovery in sea water (broken lines) on 15 successive days. All

readings were madefat 20° C. or corrected} to this temperature. Each graph represents a
single experiment.

sea water varied but slightly from this during the experi-
ment, all readings were taken at 20°C. On being
placed in NaCl 0.52 M, the resistance fell in 5 minutes to
83.3% ; the tissue was then placed in sea water, and a
reading taken 10 minutes later showed that it had risen
again to the normal. The tissue was then placed in run-
ning sea water, with the precautions mentioned above.
At the end of 22 hours the resistance was 780 ohms. An
exposure of 5 minutes to NaCl resulted in a drop to 87.2%,
with complete recovery within 10 minutes. The same
treatment was given once each day for 15 days. On the
tenth day the resistance began to fall off, but as this

INJURY AND RECOVERY 83

falling off was also shown by the control, which remained
in sea water through the experiment, it was not due
to NaCl but to other causes. The results are shown
in Fig. 34.

Electrolytes may also cause a reversible decrease in
permeability. The simplest way of demonstrating this is
by means of the following very striking experiment. The
net resistance of a cylinder of living tissue in sea water
was found to be 500 ohms. It was tested an hour later
and found to be the same. Sufficient La(NO,), was then
added in solid form to make its concentration® in the sea
water 0.02 M. After 5 minutes the resistance rose to
130%. In order to ascertain whether this change in per-
-meability is reversible, the tissue was replaced in sea
water. In the course of an hour its resistance returned
again to the original value. The experiment was
repeated three times on the same lot of material with
practically the same result; it was then allowed to stand
over night in sea water. On the following day there was
no appearance of injury, and the resistance was the same
as that of the control, which had remained in sea water
throughout the experiment. The tissue was then placed
in the sea water plus lanthanum and left until its resist-
ance had increased 100 ohms; it was then put back into
sea water and left until the resistance fell to nearly
normal. This was repeated three times, and the tissue
was then allowed to stand over night in sea water. On the

* The concentration was reduced by the precipitation of a small amount
of La,(SO,),; this had practically no influence on the subsequent result,
since the outcome is the same if we use in place of sea water a mixture
of 1000 c.c. NaCl 0.52 M + 20 e.c. CaCl, 0.278 M, in which case no
precipitate is formed. It should be noted that the addition of lanthanum
chloride has the same effect as the addition of lanthanum nitrate.

*If the material is left in sea water plus La(NO,), the increased re-
sistance is maintained for a long time.

84 INJURY, RECOVERY, AND DEATH

third, fourth and fifth days, the same experiment was
repeated four times. On the fifth day the tissue appeared
to be in as gogd condition as the control, and had a
resistance which was slightly higher. There was no

reason, therefore, to suspect that the changes in permea-
DAY 1 2 3

100. \ ye
% \ / ‘\ \ \
\ \ \ \
\ \ \ X.
80
0 1 2 5 10
HOURS

Fic. 35.—Rise of net electrical resistance of Laminaria agardhui in 1000 cc. sea water plus

sufficient La(NOz): to make the concentration 0.002 M (unbroken line) and subsequent fall

on replacing in sea water (broken line). Lower horizontal broken line represents the control

in sea water. The same lot of tissue was exposed four times daily on five successive days to

the action of La(NOz):. All readings were made at 20° C. or corrected to this temperature.
Each curve represents a single experiment.

bility had been attended by any permanent injury. The
results are shown in detail in Fig. 35.

Similar experiments were performed in which CaCl,
was used in place of La (NO,),. In this case 3.3 gm. CaCl,
were added to each 1000 ec. of sea water. Owing to the
fact that the rise in resistance took place more slowly’®

SS nn ood ‘
.

Tf in place of solid CaCl, a strong solution ia added, the rise is more
rapid and reaches a higher figure.

INJURY AND RECOVERY 85

than when lanthanum was used, the experiment was per-
formed twice daily on each of the five successive days.
On the sixth day the material was in as good condition as
the control, and had the same resistance.

It is evident, therefore, that the conductivity may be
greatly decreased and then restored to the normal several
times on successive days, without any trace of injury.

Experiments on dead tissue (killed by heat or by
formalin or allowed to die a natural death) showed that
the results described above are due entirely to the
living cells.

A very marked decrease cf permeability may be pro-
duced by a considerable variety of other salts. The
addition of these salts in solid form simultaneously
increases the conductivity of the solution and decreases
the conductivity of the tissue. This affords the most
convincing proof that the change in the conductivity of
the tissue in these experiments cannot be due to any
cause other than a change in permeability; for the concen-
tration of the ions of the sea water remains unchanged,
and if they were able to penetrate as freely as they did
before the addition of the salt, the resistance would not
increase. It would, in fact, diminish on account of the
increased conductivity of the solution held in the cell
walls, as is clearly shown by experiments on dead tissue.

It may be remarked incidentally that these experi-
ments effectually dispose of the possible objection that the
current passes between the cells, but not through them.
Were this objection well founded, the decrease in con-
ductivity could be explained only as the result of a
decrease in the size of the spaces between the cells. This
decrease could not be brought about except by greatly
reducing the thickness of the cell walls. Both macroscopic

86 INJURY, RECOVERY, AND DEATH

and microscopic measurements show most conclusively
that this does not occur. The contrary effect would be
produced by the addition of salts in solid form, for they
would tend to produce plasmolysis and thereby increase
the space between the cells.

As these remarkable changes in permeability seemed

oe DAY 2

MIN.
Fie. 36.—Extreme alterations of net electrical resistance produced by placing Laminaria
agardhit alternately in CaCl 0.278 M (unbroken line) and in NaCl 0.52 M (broken line)
and then in sea water (broken line with dots). The experiment was repeated with the same

lot of tissue on the second day. All readings were taken at 18° C. or corrected to this tem-
perature. The control in sea water remained constant during the two days.

to produce no bad effects, it occurred to the writer to
see whether the protoplasm could endure still more violent
alterations without permanent injury. In order to test
this the following experiment was performed. A lot of
tissue was found to have in sea water a net resistance
of 750 ohms. It was placed in CaCl, 0.278 M, which had
the same conductivity as the sea water. At the end
of 10 minutes a reading was taken which showed that the
resistance had risen to 168%. ‘The material was

INJURY AND RECOVERY 87

then placed in NaCl 0.52 M, which had the same conduc-
tivity as the sea water; at the end of 10 minutes the resist-
ance was 85.4%. The experiment was continued by
placing the material for 10 minutes alternately in CaCl,
and NaCl, with the results shown in Fig. 36. After 80
minutes the material was placed in sea water, where it
soon regained its normal resistance: 24 hours later the
resistance was found to be unaltered, and the experiment
was repeated. After 80 minutes of alternate exposure
to CaCl, and NaCl, the material was placed in sea
water, where it soon regained its normal resistance,
which it maintained for 3 days, when the experiment
was discontinued.

Similar results}! were obtained with Ulva (sea let-
tuce), Rhodymema (dulse) and Zostera (eel -grass).
Recovery was also observed with frog skin.”

The fact that protoplasm is able to endure such vio-
lent alterations of conductivity throws a new light on the
normal life processes of the cell. In the course of met-
abolism a great variety of substances are produced which
affect the permeability of the protoplasm. Since it is
clear that the permeability may be greatly increased or
decreased without rendering a return to normal permea-
bility impossible, it is evident that considerable fluctua-
tions in permeability may form a normal part of the
life processes of the protoplasm. In this way the whole
course of metabolism may be controlled, since this depends
on the exchange of substances between the cell and
its environment.

It is a striking fact that normal specimens of
Laminaria are quite uniform in respect to electrical resist-

“Of. Osterhout (1919, A).
2 Of. Osterhout (1919, C).

88 INJURY, RECOVERY, AND DEATH

ance,'* but if plants have been subjected to unfavorable
conditions" their resistance is below the normal. This is
of considerable practical value, enabling the experimenter
to reject abnormal material, and is also theoretically
important, for it provides us with a measure of what
we may call the normal condition, or normal vitality, of
the organism.

Although the idea of normal condition (or normal
vitality) is one of the fundamental conceptions of
biology, it has never been precisely formulated: nor does
it seem possible to attempt this without the employment
of quantitative methods. The writer’s studies in this field
have led to a quantitative treatment of injury and recov-
ery, which may now be discussed.

In practice, we determine the condition of material
by measuring the resistance of ‘pieces of tissue or of
intact organisms. These investigations show that it is
often difficult to judge of the condition of an organism by
its appearance. Tissues were found to be capable of
losing much of their vitality without betraying it by
their appearance. (This was particularly the case with
the eel grass, Zostera, which retained its normal
green color and appearance for some days after
electrical measurements showed it to be dead). On the
other hand, material of doubtful appearance often
turned out to be much better than that which looked to be
in sound condition.

Material collected in the same locality and examined
as soon as taken from the ocean gave a very uniform
resistance. To make the comparison as accurate as possi-
ble disks of the same average thickness were used in the

mer” I.e., when the fronds are of about the same thickness, etc.
“Of. Osterhout, (1914, D).

INJURY AND RECOVERY 89

experiments. Under these circumstances the net resist-
ance at 18°C. did not vary much from 1070 ohms. For
example, in a series of determinations of 10 different lots
of tissue, the highest reading was 1090 ohms, and the
lowest 1055 ohms. These lots of tissue were allowed to
remain in the laboratory under different conditions.
Some were in running salt water, some in quiet salt water
in pans of various sizes, a part being placed in direct
sunlight (where the temperature rose to an injurious
point) while others were kept in a cool place, in partial
shade. At the end of 24 hours, there was no difference
in the appearance of these lots, but their net electrical
resistance varied from 200 ohms to 1090 ohms. All were
then placed side by side in the same dish. Those with
the lowest resistance were the first to die. The others
died in the order indicated by their electrical resistance.

Determinations of the resistance made it evident that
in no case did visible signs of death make their appear-
ance until twenty-four hours after death occurred, and
subsequent experiments showed that in some cases (espe-
cially at low temperatures and in the presence of certain
reagents) they may not appear until several days
after death.

It was found that material from one locality showed
a low resistance, and subsequent examination showed
that it was contaminated by fresh-water sewage. The
appearance of the plants was not such as to lead to their
rejection for experimental purposes. They did not sur-
vive as long in the laboratory as plants of normal resist-
ance taken from the other localities.

It may be taken for granted that vitality, whatever
else it may signify, means ability to resist unfavorable
influences. When organisms which are of the same kind,

90 INJURY, RECOVERY, AND DEATH

and similar in age, size and general characters, are placed
under the same unfavorable conditions, the one which lives
longest may be said to have the greatest vitality;'® the
one which lives next longest may be rated second in this
respect, and soon. Determination of the electrical resist-
ance of these individuals enables us to predict at the
outset which will live longest, which next longest, and so
on through the entire group.

It is therefore obvious that determinations of electri-
eal resistance afford a means of measuring vitality and
in the course of an extensive series of experiments it has
been found that this method may be relied upon to give
accurate results. |

The fact that determinations of electrical resistance
afford an accurate measure of vitality enables us to
attach the same sort of quantitative significance to nor-
mal vitality as we attach to normal size or to normal
weight. For this purpose we may construct a variation
curve and determine the mode in the usual way.

There is no reason to suppose that the vitality of
an individual organism is constant any more than its
weight is. There is probably some fluctuation which
usually passes unperceived unless a quantitative meth
of detecting it exists.

The writer finds that all substances (whether organic
or inorganic) and all agents (such as excessive light,
heat, electric shock, mechanical shock, partial drying,
lack of oxygen, ete.) which alter conductivity of the
protoplasm shorten the life of the organism. This is
equally true whether the alteration consists in an increase

*It might be expected that this individual would also excel in other
respects. A discussion of these is unnecessary from our present stand-
point: in so far as they can be quantitatively treated they form proper
material for a supplementary investigation.

INJURY AND RECOVERY 91

of conductivity, or in a decrease of conductivity (followed
by an increase), as is the case when certain reagents
(such as CaCl,) are applied. This is a very striking
fact and its significance in the present connection seems
to be perfectly clear. It shows in a convincing manner,
that electrical resistance is a delicate and accurate
indicator of normal vitality. |

Since it is evident that a fall of resistance indicates
injury, it seems reasonable to assume that the amount
of fall is a measure of the amount of injury. This may
be expressed as per cent. of the total possible loss (this
would correspond to the amount of loss of the substance,
M, as previously discussed). If tissue which has been in-
jured by exposure to a toxic solution be replaced in sea
water, it may recover a part or all of the resistance which
it had lost. If the resistance should fall to 70%, then
recover in sea water to 90%, and remain stationary, we
might call the temporary loss of resistance temporary
injury and the permanent loss permanent injury. In this
case the temporary injury'® would be 30--90=33.33% and
the permanent injury 10--90—11.11%. If we calculate the
protoplasmic resistance in this case we find that starting
at 100% the resistance decreases to 62.50% and recovers
to 86.4%. In the case of protoplasmic resistance the total
possible loss’? is 92.65, the temporary injury is there-
fore (100 — 62.5) + 92.65 — 40.5% and the permanent
injury is (100 — 86.4) + 92.65 = 14.68%. In this manner
we arrive at a quantitative basis for the study of injury
and recovery.

* We divide by 90 because if the resistance starts at 100 the total -

possible loss is 90. This is merely another way of saying that we

subtract 10 from 100 because the base line is taken as 10 (see p. 56).
“I.e., the base line is 7.35%. —

92 INJURY, RECOVERY, AND DEATH

It is evident from Fig. 37 that in the earlier stages
of the death process, recovery may be complete (7. e., the
normal resistance may be completely regained), but this
is not the case in the later stages. In other words we
see that as temporary injury increases, permanent injury
also increases.

Another interesting aspect of the subject?® is illus-
trated by the results obtained in mixtures of NaCl and

1007.4 0:0-0-9----0--O----- ------------- 27 ---- ++ +--+ O- -- 2+ == 2 = neon eee eee nee O--n---*

0 300 600 g00min.

Fia. 37.—Curves showing net electrical resistance of Laminaria agardhit in NaCl 0.52 M
(unbroken line), and recovery in sea water (dotted lines). The figure attached to each
recovery curve denotes the time of exposure (in minutes) to the solution of NaCl.

CaCl,. Curve C in Fig. 38 shows the behavior of tissue
placed in a solution containing 97.56 mols of NaCl to
2.44 of CaCl,; its electrical resistance falling in 37.5
hours to 72.87% of the original value in sea water.
In a solution containing 85 mols of NaCl to 15 mols of
CaCl, (Curve A) the resistance fell in the same time to
practically the same point (72.47%).

When these two lots of tissue were replaced in sea
water they behaved differently. The resistance of the

% Of. Osterhout (1920, A, B; 1921, A, B, C).

INJURY AND RECOVERY 93

first lot rose to 78.2% (Fig. 38, upper dotted line),
but the resistance of the second fell (much more rapidly
than if it had not been removed to sea water) and eventu-

140%

100

40
1000 2,000 3000 min.

Fic. 38.—Curves showing net electrical resistance of Laminaria agardhii in a solution con-

taining 97.56 mols of NaCl to 2.44 mols of CaCl (Curve C) and in a solution containing 85

mols of NaCl to 15 mols of CaCle (Curve A). The dotted lines show recovery in sea water.

Curves B and D show the levels to which the resistance rises when the tissue recovers in sea

water after exposure to these mixtures; their abscisse denote the times of exposure. Curve

B pertains to the first mixture (belonging with Curve C), while Curve D pertains to the
second mixture (belonging with Curve A).

ally became practically stationary at 38.1% (Fig. 38,
lower dotted line).

If we plot the curve of permanent injury (2. e. level
to which the resistance rises after replacing the tissue
in sea water) after various periods of exposure to the
first mixture, we get Curve B (and for the second mix-
ture, Curve D).

94 INJURY, RECOVERY, AND DEATH

If we use the term recovery for the rise of resistance
which occurs when tissue is transferred to sea water
from certain solutions (such as the first mixture) there
seems to be no good reason why it should not be applied
to the fall of resistance which occurs when tissue is trans-
ferred from certain other solutions (such as the second
mixture) to sea water.1® The amount of recovery after
any given period of exposure is equal to the vertical
distance between Curves B and C, in the ease of the first
mixture, and between Curves A and D in the case of the
second mixture.

It may be asked whether Curves B and D are better
criteria of toxicity than Curves A and C. The question
involves the definition of toxicity. Since this term is used
in a variety of ways, it is desirable that it should always
have a precise quantitative significance. In the present
case it is evident that we need not only A and C but also
B and D for a complete description of the facts. It seems
possible that this may be generally true in the study of
toxicity, although at present we may be unable to con-
struct similar curves in many cases because suitable
methods of measurement are lacking.

The fact that recovery is never complete except at
the beginning (as shown by Curves B and D) might also
be explained as due to the death of certain cells; for if
some of the cells are killed by exposure to a solution of
NaCl the complete recovery of the surviving cells cannot
restore the resistance to its normal value. This hypoth-

* Substances which cause increase of resistance commonly produce per-
manent injury; this is apparent when the tissues are replaced in sea water.

It would therefore seem that any alteration of resistance (increase or
decrease) may produce permanent injury if sufficiently prolonged. In
spite of this it seems preferable to restrict the term temporary injury to

the fall of resistance observed in toxic solutions without coining a new
term to express the injurious action accompanying rise of resistance,

INJURY AND RECOVERY 95

esis would in no way invalidate the conception developed
above, that an individual cell may lose part of its
resistance and subsequently regain it, either partially or
completely. But there are serious objections to this
hypothesis. The appearance of the cells under the micro-
scope indicates that they all die at about the same time.
Moreover, Inman (1921, B) has recently obtained striking
experimental evidence that recovery may be far from
complete when practically all the cells are alive. In his
experiments he employed a unicellular alga, Chlorella,
which does not stain readily with methylene blue as long
as it is alive, but stains intensely as soon as it dies. Cells
were treated with hypertonic salt solutions until the rate
of respiration was greatly diminished. When they were
replaced in the normal culture medium, the respiration
did not return to normal, but the rate appeared to be
permanently lowered. In order to determine whether
this was due to the death of a part of the cells they were
carefully stained with methylene blue. The percentage of
dead cells®® was practically the same as in the normal
culture before treatment with the salt solution. In other
words, the incompleteness of the recovery seems to be due
to the fact that the metabolism of each cell is permanently
lowered. Similar results were obtained when the cells
were treated with chloroform; in this case a great
depression of respiration was not followed by recovery,
but by a greatly lowered metabolism which was perman-
ent and which was not due to the death of a part of
the cells.

Some recent experiments of Inman (1921, A) indicate
that we obtain similar results whether we use electrical
resistance or respiration as the criterion of partial recov-
ery. He found that in NaCl 0.52 M the rate of production

®The cells were counted with a hemocytometer.

96 INJURY, RECOVERY, AND DEATH

of carbon dioxide by Laminaria steadily decreased. If
the tissue was replaced in sea water after exposure to
NaCl the recovery (as judged by the rate of production
of CO,) was either partial or complete according to the
degree of depression which the rate had undergone. The
results are shown in Fig. 39. It will be seen that they

20
10
° HOURS a

Fig. 39.—Curves showing rate of respiration of Laminaria agardhii (expressed as per cent.

of the normal). The normal rate represents a change from pH 7.78 to 7.36 in from 1% to 2

minutes, depending upon the amount of material used. The solid lines show rate of res-

piration during one hour of exposure to isotonic sodium chloride (0.52 M for Woods Hole sea

water). The dotted lines show stages of recovery after the tissue was put back in normal
sea water. Each curve represents a typical experiment.

offer a striking parallel to those obtained by measuring
the electrical resistance.

Similar results are observed where we employ hyper-
tonic or hypotonic solutions in place of NaCl. When
Laminaria is placed in dilute sea water, or in sea water
concentrated by evaporation, injury may occur, and recov-
ery may be partial or complete. This is true whether we
use electrical resistance or rate of production of CO, as
the criterion of injury and recovery. The results obtained
by Inman (1921, 4A) with hypertonic solutions are shown
in Fig. 40. |

The fact that in the case of Laminaria and Chlorella
recovery may be either partial or complete, according to
circumstances, raises the question whether this is also
true of other forms. It is certainly true of all the plants

INJURY AND RECOVERY if

investigated by the writer, such as the green alga, Ulva
(sea lettuce), the red alga, Rhodymenia (dulse), and the
flowering plant, Zostera, (eel grass). It seems to be also
true of frog skin as far as the experiments of the writer
have gone.”?_ In physiological literature it seems to be
generally assumed that when recovery occurs at all it
is practically complete, as though it obeyed an ‘‘all or
none’’ law.22. It is evident that partial recovery might

100
%

Fie. 40.—Curves showing rate of respiration of Laminaria agardhii (expressed as per cent.
of the normal). The normal rate represents a change from pH 7.78 to 7.36 in from 1%
minutes to 2 minutes, depending upon the amount of material used. The solid lines show
rate of respiration while tissue was exposed to hypertonic sea water (sp. gr. 1.130, A®° = — 9.37°
approximately). The dotted lines show stages of recovery after the tissue was put back in
normal sea water. Each curve represents a typical experiment. The figure attached to
each recovery curve denotes the time (in minutes) of exposure to the solution of hypertonic
sea water; thus the uppermost curve represents recovery after an exposure of 5 minutes.

* The recovery experiments on frog skin were few in number and dealt
chiefly with the effects of anesthetics.

* There are indications in the literature that partial recovery occurs.
Thus Leo Loeb and his collaborators Loeb, L. (1903) 1905; Corson-White,
E. P. and Loeb, L. (1910); Fleischer, M. S., Corson-White, E. P., and Loeb,

L. (1912); Ishii, O., and Loeb, L. (1914) observed that destruction of the
corpora lutea produces a permanently depressing effect on the ovary and

that the virulence of tumor tissue is permanently diminished by exposure
to heat or certain reagents. In both cases a condition is produced which
is intermediate between death and normal vigor. The diminution of the
virulence of bacteria by various means cannot be cited as an illustration
unless it is certain that it is not due to the selection of less viru-
lent individuals.

98 INJURY, RECOVERY, AND DEATH

easily be overlooked except in cases where recovery can be
measured with considerable accuracy, and it seems
possible that further investigation may show that incom-
plete recovery is a general phenomenon.

Let us now consider the cause of permanent injury.
If we assume that the death process proceeds according
to the scheme

| A—>M—>B
it is evident that in sea water A must be continually
renewed. Let us assume that this occurs by means of
the reactions O —>S—+> A and that O (the origin of all
the substances produced) is present in such large amount
that its concentration does not appreciably change dur-
ing the time of the experiment. If we start with O alone,
it will produce all the other substances according to
the scheme
O—>S—>A—>M—>B

and their amounts will increase until equilibrium is
reached, 7. e., until they are decomposed as rapidly as they
are formed. Their values will then remain constant.

We assume that when the tissue is placed in NaCl
0.52 M, the reactions O—> S —>A cease, while the reac-
tions A —>M___ B continue. In consequence the values
of A, M, and B steadily fall. If the tissue is now replaced
in sea water the reactions O —> S—>A recommence and
in consequence the values of A, M, and B will rise to their
original level (the level which is normal for sea water).
But if O is diminished by exposure to the solution of NaCl
it can no longer restore these values to their original level.
If, for example, it diminishes to one-half it can restore
them only to one-half the normal values. In this case the
permanent injury would amount to 50%. We therefore

INJURY AND RECOVERY 99

see that the permanent injury is an index of the
condition of O.

We may now calculate the curve of recovery?* after
exposure to a solution of NaCl 0.52 M. We assume that
when the tissue is transferred from sea water to the
solution of NaCl the reactions O —>S—»>A cease and
that the velocity constant K, of the reaction A —>M
increases from 0.0036 to 0.0180 while the velocity constant
K ,,of the reaction M—>B increases from 0.1080 to 0.540.
We may then calculate the resistance in the solution of
NaCl after any length of exposure by means of the
formula

Resistance = 2,700 \(e —KA TE, —KmTeE

fone val )-+90e7 *MTF +10 (1)

in which 7’, is the time of exposure in minutes, and e is
the basis of natural logarithms. 10 is added in the for-
mula because the base line is taken as 10 (not as 0) for the
reason that the resistance sinks to 10 (as shown in Fig. 28)
when the tissue dies.

We assume that when the tissue is replaced in sea
water the reactions 0—» S—» A recommence and that
the values of K , and K ,, become 0.0036 and 0.1080 respec-
tively, while the other velocity constants likewise acquire
the values which they normally have in sea water. Under
these conditions M will be formed faster than it is decom-
posed and the resistance will rise.

The fact that the rise does not reach as high a level
after a long exposure as after a short one indicates that
during the exposure O gradually diminishes; we assume
that this takes place by the reactions

N=>-O—>-P

We likewise assume that during exposure to the solu-
* See Chapter II.

100 INJURY, RECOVERY, AND DEATH

tion of NaCl the amount of S changes by means of
the reactions

R—>S—>T
and that on transferring to sea water S is rapidly con-
verted into A. In order to calculate the rate of recovery
we find by trial the most satisfactory values of the ve-
locity constants. The values thus found are given in
Table V.
TABLE V

Velocity Constants

Value at 15° C. in

Reaction Velocity constant NaCl CaCl
N—»O Kn 0.03 0.0045
O—»P Ko 0.0297 0.004455
R=» § K 0.04998 0.0145
See ay a K 0.02856 0.007
Aap KA 0.018 0.0018
M—»B Km 0.540 0.0295

As an example of the method of calculation we may
take the case of tissue exposed for 15 minutes to a solu-
tion of 0.52 M NaCl at 17°C. The net resistance in sea
water at the start was 960 ohms; in the course of 15 min-
utes in the solution of NaCl it fell to 775 ohms, which is
80.69% of the original resistance. The fall of resistance is
a little more rapid than in the ‘‘standard curve’’ previous-
ly obtained. If we assume that this is due to the difference
in temperature (these measurements were made at 17°C.
while those on which the standard curve is based were
obtained at 15°C.) we may introduce a correction by mul-
tiplving the abscissa by the factor®* 1.06, which makes it

* This agrees closely with the temperature coefficient as determined else-
where. - See page 37.

INJURY AND RECOVERY 101

15.9 minutes, and causes it to agree with the standard
curve. All the abscissx are multiplied by the same factor.?5
The effect of this is to make the process appear to proceed
at 15 instead of at 17°C. If the difference between the two
curves is due wholly to difference in temperature this
introduces no error, and if the difference is due in part
to other factors, the error, if any, is less than the usual
experimental error.

The advantages of this procedure are that we can
employ for our calculations the constants already obtained
for the standard curve and also compare the theoretical
curves which start from the same points. This procedure
has therefore been followed throughout and the corrected
results (2. e., the figures multiplied by a suitable factor)
are employed in the following description.

When the tissue was replaced in sea water the resist-
ance began to rise. At the end of 10 minutes it had risen
from 80.69 to 89.10%.2* Since, however, the abscissx
of the death curve have been multiplied by 1.06
the same thing must be done for the recovery curve and
in place of 10 minutes we must put 10.6 minutes.
Proceeding in this manner we obtain the recovery curve
which is labeled 15.9 in Fig. 41.

In order to calculate the course of the recovery we
must consider the reactions which determine the amount
of electrical resistance. When the tissue is placed in the

* This procedure may displace the points on the curve so that where
several curves are averaged it may be necessary to employ interpolation
in order to average points on the same ordinate. In many cases curves
were obtained by averaging the ordinates of death curves and recovery
curves before multiplying by the factor.

“In earlier experiments it was found that complete recovery was pos-
sible after the resistance had fallen to about 80%. This was not the

case in the present series; the difference may be due to differences in
material or in technique. Cf. Osterhout (1915, B).

102 INJURY, RECOVERY, AND DEATH

solution of NaCl the reactions which occur are: (1)
A—> M—>B; (2) R—»> S—+>T; and (3) N—>O->P.
Let us first consider the reactions 4d—»>M—>B. The
value of A in sea water is taken as 2,700 and that of M as
90. As previously explained the value of A will diminish

TU Se aoe es nae ioe J dss bi Seen 2
Orn sasenee O------------ O.----------------- OQ----------------------:

- 15.9

40.48
eececenna ag O=---------- O--- o-oo enone een seeeneeneee O--

ff ae
50
ee ae e
N20. aa es
: .
0 50 100 150min.

Fia. 41.—Curves showing the fall of net electrical resistance of Laminaria agardhii in 0.52 M
NaCl (descending curve) and recovery in sea water (ascending curves). The figure attached
to each recovery curve denotes the time of exposure (in minutes) to the solution of NaCl.
In the recovery curves the experimental results are shown by dotted lines, the calculated
results by the unbroken lines (the curves are extended beyond the last observed point shown
because of later observations which cannot be shown in the figure). The observed points
represent the average of eight or more Pree penis) probable error of the mean less than 10%
of the mean.

during exposure to NaCl according to the formula

dion. SA7* (2)

in which Tis the time of exposure to the solution.
Since K , = 0.018 (see Table V) the value of A after 15.9
minutes in NaCl 0.52 M is

2,700 e ney ae = 2,027.96

The value of M at the end of 15.9 minutes is the observed
resistance 80.69 less 10 (since the base line of the curve
is not 0 but 10).

INJURY AND RECOVERY 103

On replacing the tissue in sea water, therefore, we
start with M = 70.69 and A =2,027.96, but this value of
A is at once augmented by the conversion of S into A.
In order to find the amount of this augmentation we
must know the value of 8.

During exposure to NaCl the reaction R—»S—>T
occurs. The value of S may be easily calculated by
employing formula (1) and substituting the appropriate
constants. We thus obtain

KR
==1041.77 (aks) (-* Tre_—Ks ve) er ae TE (3)

The value of F at the start in sea water is taken as 1,041.77
and that of S as 2.7. In the solution of NaCl the values of
K , (the velocity constant of the reaction R—>S) and Kk,
(the velocity constant of the reaction S—> T) are taken
as 0.04998 and 0.02856 respectively (see Table V). Hence
the value?’ of S at the end of 15.9 minutes is 447.26.
When the tissue is replaced in sea water S is rapidly
converted into A so that the total value of the latter
becomes 447.26 + 2,027.96 — 2,475.22.

On replacing the tissue in sea water A = 2,475.22 and
M =70.69. The resistance due to A and M after any
given time 7 in sea water is obtained by modifying
formula (1) which becomes

K >
Resistance == 2,475.22 ee) f. api | Te_—KuTe )

+70.69 (Kure) 41 (4)

in which 7’, denotes the time which has elapsed after

* In general the greater the rise in recovery the greater the value of 8S,
while the greater the fall the less the value of S.

104 INJURY, RECOVERY, AND DEATH

replacement in sea water. The velocity constants K ,
and K ,, have the normal values in sea water, 0.0036 and
0.1080 respectively. Hence the resistance at the end of
10.6 minutes is 87.44.

We must likewise remember that on replacing the
tissue in sea water the reactions O —»S—»>A _ recom-
mence and produce a certain amount of A; this breaks
down to form M, which in turn decomposes. The resulting
amount of M may be easily calculated. It will be recalled
that in sea water all processes are so adjusted that the
amount of M remains constant; it is evident that if
the reactions O —> S—~» A were suddenly to stop, allow-
ing A—»M —+B to continue, the amount of M would
diminish. At the start the total resistance is 100. If O
should stop producing this would diminish and we may
call the loss of resistance L. Now if O were producing
normally it would just replace this loss, so as to keep
the resistance constant at 100: hence the amount pro-
duced from O in any given time will be equal to the loss
[, which would occur in that time if O were to
stop producing.

When tissue is exposed to a solution of NaCl, O di-
minishes according to the scheme N—> O—>P. Assum-
ing that at the start VN = 89.1 and O = 90 we find”s that the
value of O after any given time (7'z ) of exposure to a solu-

*This value of O is assumed merely for convenience in calculation,
without reference to other assumed values. Its real value must be much
greater than that of A, but it is not necessary to assign any definite real
value to it, since the only point of interest is to determine what per cent.
of O remains after any given time of exposure to sea water. It is assumed
that in sea water any change in the amount of O is so small as to be
negligible. This might be due to the fact that O is present in large
amount and decomposes slowly or to the fact that it is formed as rapidly
as it decomposes (by the reactions N —»O—~>P).

INJURY AND RECOVERY 105

tion of NaCl may be obtained by changing the constants
in formula (1) thus:

Kn
—~KwyTz —KoTr ~KoTr
aia ee a b oe ) — .

in which K,, (the velocity constant of the reaction N —>»
O) and K, (the velocity constant of the reaction O —> P)
have the values 0.03 and 0.0297 respectively (see Table V,
page 98).

We find by this formula that at the end of an exposure
of 15.9 minutes the value of O + 10 is 92.57; hence it can
produce only (92.57 — 10) -- (100 — 10) = 0.917 as much
of M in any given time as it could produce if it were
intact2® The amount it could produce, if intact, during
recovery in sea water is easily found by subtracting from
100 the resistance obtained by means of formula (1), when
K ,= 0.0036 and K ,= 0.1080 (these are the normal val-
ues in sea water). Bese these values we find that at the
end of 10.6 minutes the amount of resistance, as given by
formula (1), would be 98.55. Hence the loss during that
time would be 100 — 98.55 = 1.45, which is the amount O
could produce in 10.6 minutes if intact. This value may
be called Z and expressed as follows:

L=100—{ 2700 ona THATE RMS KaT.
4 kw okas\. iy +900 “MF +10

(6)
in which K , = 0.0036 and K ,,= 0.1080 (these are the nor-
mal values in sea water) and 7’, is the time which has
elapsed since the tissue was replaced in sea water.

* In other words, if S, 7 and A were completely removed, O could raise
the level of M to 100 — 10 = 90 in the course of time. But if, for
example, half of O is lost the remainder can raise the level of M to
only one-half its former value; i.e., to 45.

106 INJURY, RECOVERY, AND DEATH

But as O has diminished to 0.917 times its original
value it can produce in 10.6 minutes only (1.45) (0.917)
—= 1.33. By adding this to that obtained by formula (4)
we find the resistance after 10.6 minutes in sea water to
be 87.44 + 1.33 = 88.77.

The recovery formula may therefore be expressed
as follows:

ee Se Pe aie
Resistance = (A+S) (ie; ( Ka eae aH on?

+L (2) 10. (7)

Using this formula, we may find the resistance at any
given time after replacement in sea water. <A series of

TABLE VI.
Recovery in Sea Water after Exposure of 15.9 Minutes to 0.62mM NaCl.

Total time=time in sea raed Electrical resistance
Time in sea water
‘daaan cab Observed Calculated

min. min. per cent. per cent.
26.5 10.6 89.10 88.77
37.1 21.2 93.21 91.34
58.3 42.4 93.99 92.43
84.8 68.9 94.48 92.52

121.9 106.0 94.20 92.55

164.3 148.4 93.81 92.55

545.9 530.0 94.00 92.57

863.9 848.0 93.87 92.57

values so obtained is given in Table VI. It will be seen
that they are in good agreement with the experimental
values. The calculated and observed values are also
plotted in Fig. 41, in which the abscisse represent the time
in the solution of NaCl plus the time of recovery in sea
water (in the case just discussed this would amount to
15.9+-10.6—26.5 minutes).

INJURY AND RECOVERY 107

Proceeding in this manner with different times of
exposure we obtain the series of recovery curves shown in
Fig. 41. The number attached to each curve denotes the

1602
100
x... 636
0 |
0 200 400 600 800min

Fie. 42.—Curves showing the rise and fall of net electrical resistance in Laminaria agardhii

in 0.278 M CaCh (single curve which rises and falls) and recovery in sea water (descending

curves). The figure attached to each recovery curve denotes the time.of exposure (in min-

utes) to the solution of CaCl. In the recovery curves the experimental results are shown

by the dotted lines, the calculated results by the unbroken lines. The observed points repre-

sent the average of eight or more ng a Probable error of the mean less than 10%
of the mean.

time of exposure to the solution of NaCl. The observed
results are plotted as dotted lines, the calculated values
as unbroken lines.

It will be seen that the agreement is satisfactory
throughout. In general the greater the number of experi-

108 INJURY, RECOVERY, AND DEATH

ments which were averaged to obtain the result the nearer
it approached to the calculated curve.

Let us now consider the behavior of tissues transferred
from a solution of 0.278 M CaCl, (which has the conduc-
tivity of sea water) to sea water. In such a solution the
resistance rises and then falls. If tissue is allowed to
remain in the solution for a short time and is then replaced
in sea water the resistance falls rapidly, as shown in Fig.
42. This fall of resistance may be regarded as analogous
to the rise of resistance which occurs in the experiments
with NaCl and the term recovery may be used in both
eases. It is evident from the figure that as the exposure
to the solution of CaCl, lengthens the level which is
reached as the result of recovery gets lower. This is pre-
cisely what happens in the experiments with NaCl. It
would therefore appear as though the same mechanism of
recovery were involved. If this is so the same method of
calculation should enable us to predict recovery in both
eases. This is found to be true. Using the same formulas
which have already been employed in the experiments
with NaCl we are able to predict the course of the curves
obtained in experiments with CaCl,. This is rather strik-
ing in view of the fact that the two sets of curves differ
so fundamentally in appearance.

In calculating the eurves for CaCl, the constants given
in Table V (page 98) are employed. The results are
shown as unbroken lines in Fig 42 (the dotted lines show
the experimental results). It is evident that the agree-
ment is very satisfactory.

Some assistance in picturing the reactions which occur
during exposure is afforded by Fig. 43, which shows the
curve of O0+10 in NaCl (unbroken line) and in CaCl,
(dotted line). These curves are plotted from the caleu-

INJURY AND RECOVERY 109

lated values; the observed values are shown as points;
it will be observed that they lie fairly close to the
calculated curve. The figure also shows the calculated
values of S; in this case no observed values are given
because such values cannot be very precisely determined.
This is owing to the fact that the value of S affects only

“0 in Ca Cle

aed

-
me
“«
t+

‘Sin Ca Ch is,

O in Na Cl

ee
tS em
a eal we
meee ee

200 400 600 min.

Fic. 43.—Curves showing the values of 0+ 10 in NaCl (upper unbroken line) and in CaC

(upper dotted line); also the values of S in NaCl (lower unbroken line) and in CaCh (lower

dotted line). The ordinates give the values of O; these must be multiplied by 6.75 to obtain

the values of S. The observed points represent the average of eight or more experiments;
probable error of the mean less than 10% of the mean.

the speed of recovery (not the final level attained) and as
the speed is variable the only satisfactory procedure is to
assume such values of K ,and K, as cause the closest
approximation to the observed speed of recovery. When
these values have been found the value of S can readily
be calculated. The results of these calculations are plot-
ted in Fig. 43.

In this figure the ordinates give the values of O + 10:
these must be multiplied by 6.75 to obtain the values of S.
In all curves the value of S at the start is 2.7 (the value of

110 INJURY, RECOVERY, AND DEATH

S in sea water®°); this appears on the ordinate in the
figure as 2.7 + 6.75 0.4. The curves rise to a maximum
and then fall to zero. The curves for O + 10 start at 100
and fall to 10 (since the base line is taken as 10, just as in
the curve of M).

It is found that the rate of recovery is approximately
the same in all cases; this applies to the experiments with
CaCl, as well as with those in NaCl. In general it may be
said that it usually requires about 60 minutes for the
curve to complete nine-tenths of the total rise or fall which
occurs in recovery.

If the theory here developed is sound it should also
enable us to predict the behavior of tissue transferred
from one toxic solution to another. In order to put this
to a test a variety of experiments was made in which the
tissue was exposed to several solutions in succession.

I. Alternate Exposure to NaCl and Sea Water.

The procedure may be illustrated by a typical experi-
ment, the results of which are shown in Fig. 44.

The tissue was exposed for 20.8 minutes*! to 0.52 M
NaCl, during which time the resistance fell from 100 to
74.03%. After 20.8 minutes in the solution of NaCl
the value of 7, (in formulas (2), (3) and (5)) is 20.8
and the following results are obtained: A = 1856.80, S =
484.06, M — 64.03, O — 88.41. When the tissue has been

* The normal value of S in sea water is taken as 2.7 which is exceed-
ingly small as compared with the amount of O. The amount of S which is
produced from O in each unit of time is relatively large, but S is so rapidly

transferred into A that its amount in sea water never becomes greater

than: 2.7.
* This is corrected from 20 minutes (as previously explained) in order
to make it conform to the standard curve.

INJURY AND RECOVERY 111

replaced in sea water and left for 10.4 minutes*? the value
of 7, (in formulas (6) and (7)) is 10.4 and the value of L

is found to be 1.33.
| x Na Cl |
O Sea Water

100
0

Oo 400 MINUTES 800

Fig. 44.—Curves showing the net electrical resistance of Laminaria agardhii in NaCl 0.52 M

and in sea water. Unbroken line, calculated values; broken line, observed values. Average
, of ten or more experiments; probable error of the mean less than 10% of the mean.

Substituting these values in formula (1) we find that
when the tissue has been replaced in sea water the resis-
tance at the end of 10.4 minutes is 83.49. Proceeding in
this manner we calculate the resistance at various
intervals after replacement in sea water and obtain the
first (calculated) recovery curve shown in Fig. 44. It is
evident that.it is in fairly good agreement with the
observed values.

After 200 minutes in sea water (during which the
resistance rose to 87.10% and remained practically
constant) the tissue was replaced in the solution of NaCl.
In the course of 21.2 minutes** the resistance fell from

“This is corrected from 10 minutes (as previously explained )..
* The actual time was 20 minutes: the manner in which the corrected
figure is obtained is explained in a subsequent paragraph.

112 INJURY, RECOVERY, AND DEATH

87.10 to 64.18. It was then replaced in sea water. The
recovery curve may be calculated as before, the only
differences being as follows:

1. On replacing the tissue in sea water the destruction
of O (by the reactions N —> O —>P) ceases (or becomes
negligible) ; hence the value of O at the beginning of the
second exposure (if equilibrium has been reached) is
that of the observed resistance less 10, or 87.10 —10—
77.10. We find by means of formula (5) that when O at
the start equals 90 it loses 11.95 during an exposure of
21.2 minutes to the solution of NaCl, but as it only equals
77.10 at the start the loss will be 11.95 (77.10 90) =
10.23. Subtracting this from 77.10 gives 66.87, the value
of O at the end of the second exposure, and adding 10
(since the base line is 10) makes 76.87, the level to which
the resistance should rise after the second exposure.

2. At the start of the first recovery** S is rapidly con-
verted into A, but is partially restored during the
subsequent stay in sea water and at the beginning of the
second exposure equals 2.7 (O = 90) in which O has the
value given above (77.10).

3. During exposure to NaCl the value of FR diminishes
from R, to R, according to the formula

—KrTer — (0.0498) 42 .

Rh, = Roe = 1041.77 e (8)
in which R,=the value of R before the first exposure
(1041.77) and T,, equals the total exposure to NaCl (20.8
+ 21.2 = 42).

It is evident that unless # is restored during the
“Tf the value of O were 90, S would be completely restored to its orig-

inal value of 2.7, but since O has fallen to 77.10 it can only restore S to
2.7 (77.10 + 90).

INJURY AND RECOVERY 113

period in sea water the speed of recovery will fall off
somewhat with each successive exposure.

4. The value of M is the observed resistance (at the
end of the second exposure) less 10 or 64.18 — 10 = 54.18.

5. The value of A is obtained by multiplying by 30
the resistance observed at equilibrium (less 10). This is
based upon the following considerations:

Just before the beginning of the second exposure A
and M are assumed to be in equilibrium in sea water, in
which case as much of A must decompose in any minute
as of M (otherwise M would not remain constant). But the
amount of 4 which decomposes in 1 minute is AK, and
of M is MK,,; and since K,,is 30 times as great as K , it
follows that 430 M. At the beginning of the second
exposure M= 87.10—10= 77.10 and A= (77.10)
30 = 2313.

In order to ascertain how the resistance would change
during the second exposure if it conformed to the
standard curve previously employed, we may employ
the formula

: Ms Ka —KaTe -—KmMTe
Resistance = 2313 ( Ee; ( e as )

—KmTez
+77.1le +10 (9)

in which K , = 0.018, K ,,— 0.540 and 7',=— time the tis-
sue has remained in the solution of NaCl. Comparing the
values thus obtained with the observed resistance after
an exposure of 20 minutes we find that if the time is
multiplied by 1.06 (making it 21.2 minutes) the observed
resistance (64.18) agrees with the standard curve. This
figure is therefore adopted. The value of 7’, in formulas
8

114 INJURY, RECOVERY, AND DEATH

(2), (8) and (5) should now correspond to the total
exposure to NaCl, and is 20.8 + 21.2 = 42.

These data were employed in calculating the second
recovery curve and the results are shown in Fig. 44. The
third recovery curve was calculated in the same fashion.

Instead of waiting for the establishment of equilib-
rium we may replace the tissue in NaCl after it has
been for a short time in sea water. During the fourth
recovery, after the tissue had been 10.2 minutes in sea
water and the resistance had risen to 54.92%, it
was replaced in sea water: the subsequent fall in resist-
ance was calculated by means of formula (9). For the
value 77.1 in this formula we must substitute the observed
resistance less 10, or 55.89 — 10 = 45.89; and in place of
2313 we must substitute the present value of A. We
assume that at the beginning of the fourth exposure to
NaCl equilibrium had been reached in sea water: hence
as the resistance was 68.10 the value of A (which we call
A,) is, A, = 30 (68.10— 10). During the fourth exposure
to NaCl (lasting 20.4 minutes) the value of A, diminished
to A, according to the formula

— (0.018) 20.4

1€ isa
On replacing the tissue in sea water A, was augmented
by the conversion of S into A. The value of S is found
according to formula (3) in which 7 , is equal to the total
time of exposure (20.8 + 21.2 + 20.8 + 20.4 83.2). We
may call this S,. Hence the value of A immediately after
replacement in sea water is A, —=A,+S,. During the
subsequent 10.2 minutes in sea water A, diminished to
A, according to the formula
— (0.0036) 10.2 :

3¢

But at the same time it received an addition from the

INJURY AND RECOVERY . 115

decomposition of O; the amount of this may be found as
follows: The loss of A in sea water under normal condi-
tions®® in 10.2 minutes is

Loss = 2700— ( 2700e ys: (0.0036) 10.2 ) = 97.26

and this could be completely replaced by O if O were
intact. But since O has diminished*® from 90 to 50.86
it can supply only 97.26 (50.86 ~ 90) = 54.95. This must
be added to A giving 4,=—A,+ 54.95. The value of
A, must be substituted for 2313 in formula (9). This
enables us to calculate the fall of resistance after the last
recovery (of 10.2 minutes). Fig. 44 shows the values
so obtained and also the observed values.

II. Alternate Exposure to CaCl, and Sea Water.

When the tissue of Laminaria is transferred from
sea water to a solution of CaCl, (of the same conductivity
as sea water) the resistance rises and then falls as
shown in Fig. 45. When it is replaced in sea water the
resistance falls (much more rapidly than if left in the
solution of CaCl.) and eventually becomes stationary.
This fall of resistance may be spoken of as recovery,
since it may be regarded as analogous to the rise of
resistance which occurs when tissue is transferred from
NaCl to sea water.

* The principle upon which this formula is based is explained on page
103 in discussing the loss of M and its replacement by O. In the present

case the effect of S is negligible since the amount of S in sea water is
only 2.7.

* This is calculated as follows: at the beginning of the fourth exposure
_ O =68.10 — 10 = 58.10. If its value were 90 it would lose 11.23 during
an exposure of 20.4 minutes to NaCl. Since 0 = 58.10 the loss will be
11.23 (58.10 ~ 90) 7.24: subtracting this from 58.10 we have 50.86.

116 INJURY, RECOVERY, AND DEATH

Recovery after exposure to CaCl, may be calculated in
precisely the same manner as recovery after exposure
to NaCl. The only difference is that in formulas (2), (3),
(5), (8) and (9) we must employ for the velocity con-
stants (K,, K,, K,, K,, K, and K,,) the values given
for CaCl, in Table V, page 98. In formulas (6) and
(7) the values of the velocity constants are always the
same (AK ,— 0.0036 and K,,— 0.1080) since these are the
values which are normal for sea water.

o Ca Ch
oO Sea Water

Vile) IS Oe Beet Sie pe

5) 400 MINUTES 800

Fic. 45.—Curves showing the net electrical resistance of Laminaria agardhii in CaCl 0.278 M
and in sea water. Unbroken line, calculated values; broken line, observed values. Average
of ten or more experiments; probable error of the mean less than 10% of the mean.

Results of such calculations are shown in Fig. 45
together with the observed values.

III. NaCl, Sea Water, CaCl,, Sea Water, ete.

It seemed desirable to test the theory further by vary-
ing the experiments in the manner shown in Fig. 46. The
calculations are made as already explained. It will be

INJURY AND RECOVERY 117

noticed that in this and in some other experiments the
resistance rises rather more rapidly in CaCl, than the
ealeulations would lead us to expect. This is due to the
N x Na Cl

o Ca Cle
O Sea Water

100

° 400 MINUTES 800

Fic. 46.—Curves showing the net electrical resistance of Laminaria agardhii in NaCl 0.52 M,

in CaCl 0.278 M and in sea water. Unbroken line, calculated values; broken line, observed

values. Average of ten or more experiments; probable error of the mean less than 10%
of the mean.

fact that the ‘‘standard curve’’ for CaCl,, which was
based upon previous experiments made under different
conditions, seems to be a little too low for the
present material.

IV. CaCl.,, NaCl, Sea Water, etc.

A series of experiments was made to determine the
effect of CaCl, followed directly by NaCl. The results
are shown in Fig. 47. The rise in CaCl, during the first
91.8 minutes is calculated in the usual manner. In order
to calculate the subsequent drop in NaCl we must substi-
tute for 77.1 in formula (9) the value of M; 1ze., the
observed resistance (less 10) at the beginning of exposure
to NaCl. In place of 2313 we must substitute the value
of A, which is A, =2700e —(.0018)91.8

118 INJURY, RECOVERY, AND DEATH

During the exposure of 60.6 minutes to NaCl the value
of ‘A changes from A, to 4e—= Ayer

100

x NaCl
oO Cathe
o Sea Water

oO 400 MINUTES 800

Fic. 47.—Curves showing the net electrical resistance of Laminaria agardhii in NaCl 0.52 M,

in CaCl: 0.278 M and in sea water. Unbroken line, caleulated values; broken line, observed —

values. Average of ten or more saa probable error of the mean less than 10%
of the mean.

This value must be substituted for A in formula (7) in
calculating the recovery in sea water.

In finding the value of S (by means of formula (3))
we must remember that during the 91.8 minutes in CaCl,
the value of R (which at the start is R , = 1041.77) dimin-
ishes from R, to R, according to the formula

—91.8KR
R,= Roe

Kp, in CaCl, = 0.012532 (See Table V, page 98). Dur-

INJURY AND RECOVERY 119

ing the 60.6 minutes in NaCl R, diminishes to R, accord-
ing to the formula

—60.6KR
R.= Rie

Ke in NaCl = 0.04998.

We must also bear in mind that O diminishes during
the exposure. Since this process is 6 times as rapid in
NaCl as in CaCl, we may consider 91.8 minutes in CaCl,
to be equivalent to 91.8 + 6 15.3 minutes in NaCl and
the total exposure to be equivalent to 60.6 + 15.3 = 75.9
minutes in NaCl.*7 The value of O may then be found by
means of formula (5).

V. CaCl.,, NaCl, CaCl., NaCl, Sea Water, etc.

A series of experiments was performed in which
tissue was placed in CaCl, for 30 minutes, then in NaCl
for 10 minutes, then in CaCl, for 60 minutes. The tissue
was allowed to recover in sea water, after which it was
placed in CaCl, for 360 minutes, and then in NaCl
(Fig. 48).

In this case the observed time was not corrected (2.e.,
was not multiplied by a factor) as in the previous calcu-
lations. In consequence the calculated and observed
values do not correspond at the beginning of each expos-
ure, the only exception being after recovery in sea water,
in which case it was assumed*® that equilibrium had been

** This involves the assumption that O is not restored to any extent dur-
ing recovery in sea water. This assumption may not be correct, especially
at the start, but even in that case the present calculation would not be
appreciably altered.

*In this case the tissue did not remain long enough in sea water to
establish equilibrium, but it was so nearly established that only a very
small error is involved in regarding it as complete. In cases where it is
not completely established the final equilibrium may be approximated
by extrapolation.

120 INJURY, RECOVERY, AND DEATH

reached and that in consequence A,—30 M (the value
of M being that of the observed resistance less 10). This
value of A was taken for the subsequent calculations.

x NaCl
rota Oe! et
oO Sea Water

loo

re) 400 MINUTES 800

Fic. 48.—Curves showing the net electrical resistance of Laminaria agardhii in NaCl0.52 M,

in CaCl: 0.278 M and in seawater. Unbroken line, calculated values; broken line, observed

values. Average of ten or more caer probable error of the mean less than 10%
of the mean.

During the subsequent exposure to CaCl, A, diminished
to A, according to the formula

— (0.0018)360
A = Aoe

and this value was used in calculating the fall of resist-
ance during the final exposure to NaCl.

Experiments similar to those shown in Figs. 44, 45, 46,
47, and 48 have been made, in which mixtures of NaCl
plus CaCl, have been used in a variety of ways. In this
case we employ for the calculations the constants appro-

INJURY AND RECOVERY 121

priate for each mixture, as given on page 140. In general
the agreement is satisfactory.

With so large a number of constants it might seem
possible to fit any sort of curve, and hence the significance
of the actual accomplishment might be lessened. This,
however, is by no means the case.

It should be noted that we do not employ new constants
to fit these curves, but that in every case we use the con-
stants already determined as the result of other and
quite different experiments. In view of this the results
have a special significance.

If we accept the conclusions stated above we are
obliged to look upon recovery in a somewhat different
fashion from that which is customary. Recovery is usu-
ally regarded as due to the reversal of the reaction which
produces injury. The conception of the writer is funda-
mentally different; it assumes that the reactions involved
are irreversible (or practically so) and that injury and
recovery differ only in the relative speed at which certain
reactions take place.

It would seem that these experiments, and those pre-
viously described, afford a sufficient test of the theory.
It has been found that the agreement between the calcu-
lated and observed values is satisfactory whenever a
sufficiently large number of readings are averaged in
arriving at the observed values.

In the foregoing account many details are necessar-
ily omitted, owing to lack of space. These, however, are
not essential to the main purpose, which is to show how
the process of injury and recovery may be analyzed and
subjected to mathematical treatment. Starting with cer-
tain assumptions we have formulated equations by means
of which we can predict the behavior of the tissue. If the

122 INJURY, RECOVERY. AND DEATH

predictions are fairly accurate it is natural to infer that
the assumptions are in accordance with the facts. It is
evident from an examination of the figures that the equa-
tions enable us to predict with considerable accuracy the
behavior of tissues in solutions of NaCl and CaCl,, as well
as the recovery curves after any length of exposure to
either of these solutions. But we must not lose sight of
the fact that the predictive value of the equations does
not depend on the validity of these assumptions and would
in no way be impaired if they were to be given up. The
equations have a permanent value which is quite inde-
pendent of assumptions.

The mechanism which has been postulated in devel-
oping these equations consists essentially of a series
of catenary reactions. There can be no doubt that, as
Loeb (1912, D) has emphasized, catenary reactions play
a large part in life phenomena, and it would seem that
the role assigned to them in the present discussion
involves no unreasonable assumption.

A substance which acts as a member of such a caten-
ary system may, as Hopkins (1913) has remarked, be of
great importance in the organism even if present in very
small amount.

It may be desirable to call attention to certain features
of this mechanism which are of general interest from
a theoretical viewpoint. It is evident that by means of a
simple catenary system we can account for practically all
the processes which occur in the organism. If such a
system is present in the egg we can easily picture all of
the subsequent development as due to this system, without
the introduction of any new reactions. All that we need
to postulate is that during development the relative rates
of the reactions change. The processes involved in irrita-

INJURY AND RECOVERY 123

bility, as well as those concerned in injury and death, may
be accounted for in this same way. We thus arrive at a
very simple conception of the underlying mechanism of
life processes, which may be useful in formulating a
theory of living matter.

If life is dependent upon a series of reactions which
normally proceed at rates bearing a definite relation to
each other, it is clear that a disturbance of these rate-rela-
tions may have profound effects upon the organism. It
is evident that such a disturbance might be produced by
changes in temperature (in case the temperature coeffi-
cients of the reactions differ) or by chemical agents. The
same result might be brought about by physical means,
especially where structural changes occur which alter the
permeability of the plasma membrane or of internal
structures (such as the nucleus and plastids) in such a
way as to bring together substances which do not nor-
mally interact.*®

This investigation of fundamental life processes shows
that they appear to obey the laws of chemical dynamics.
It illustrates a method of attack which may throw some
light upon the underlying mechanism of these processes
and which may assist materially in the analysis and con-
trol of life-phenomena. |

“Or which normally react to a lesser degree.

CHAPTER IV.
ANTAGONISM.

When one toxic substance acts as an antidote to
another, we speak of this as antagonism. If the antagon-
istic substances are mixed in such proportions that tox-
icity disappears we have a physiologically balanced
solution as defined by Loeb.

In seeking an accurate measure of antagonism the
writer made experiments on growth. It was found that
both NaCl and CaCl, are toxic to plants, as shown by the
fact that in solutions of these substances there is less
growth than in distilled water.2. In a series of experi-
ments on wheat, it was found that the growth of roots
in NaCl 0.12 M was practically the same as in CaCl, 0.164
M. These solutions were therefore regarded as
equally toxic. |

On mixing equally toxic salt solutions, we may
encounter one of the following conditions :3

1. The toxicity is unaltered, that is, the toxic action
of the two salts is additive. Each salt produces its own
toxic effect precisely as though the other were not present.
This is expressed by the horizontal dotted line LJM in
Fig. 49.

It is evident that we cannot get increased growth by
mixing two such solutions unless the salts have an antag-
onistic action. If we mix equal volumes of A 0.1 M and

*Of. Loeb, J. (1906, B). For the literature of antagonism see Loeb
(1909), Robertson (1910), Héber (1914).
* This statement does not apply to very dilute solutions.
°Of. Osterhout (1914, B, O; 1915, F).
124

ANTAGONISM 125

B 0.1 M the dilution of A from 0.1 M to 0.05 M is
exactly compensated by the introduction of molecules of
B. Or, to put it in another way, the toxic effect depends
on the number of molecules present (if both kinds of mole-
ecules are equally toxic and
there is no antagonism) and
it makes no_ difference
whether the solutions are
pure or mixed.

If the toxic effect depends
on ions, rather than on mole-
cules, then, since the number
of ions may be somewhat
increased by mixing solu-
tions, the toxicity may be
correspondingly increased;
but the amount of this
increase would ordinarily be

negligible.
2. The toxicity is dimin- A100 75500 %
ee thatern, the effect ig BO 7 2

antitoxic. We then get a Fic. 49.—Curves showing the growth of

roots in mixtures of equally toxic solutions

curve rising somewhere of two salts A and B: the ordinates represent

growth; the abscissae represent the composi-

above the dotted line, such tion of the mixtures, thus A 50, B 50 means

a ag gy in bpm = dissolved molecules

as the unbroken line LKM. #° 7 (Ei iesewls ie ieee
Fae 2 ° would occur if there were no antagoni

3. The toxicity 1s 1N- (additive effect); LKM is the ee ee i

curve; LHM, curve expressing increased

creased. We then get a curve toxicity (opposite of antagonism); the quan-

which somewhere falls below vont Bis KJ soe” **
the dotted line, such as the line interrupted by cir-
cles LHM.*

The considerations here set forth apply in all cases
where two equally toxic solutions are mixed, whether their
ee ae

* See page 177.

126 INJURY, RECOVERY, AND DEATH

concentration is the same or not. Thus, if a solution of
A 0.05 M is just as toxic as a solution of B 0.1 M, mixtures
of the two will give a horizontal straight line (as in Fig.
49) provided their effects are additive.

iimphasis should be laid upon the fact that the method
of mixing two equally toxic solutions eliminates disturb-
ances due to variations of osmotic pressure. If a mole-
cule of A is twice as toxic as a molecule of B, a solution
of A 0.05 M will be just as toxic as a solution of B 0.1 M,
provided there are no other factors to be considered. But
if the osmotic pressure of the 0.05 M solution of A is less
than that of the 0.1 M solution of B, there will in many
cases be better growth in the 0.05 M solution of A. In
order to make the solution of A appear equally toxic with
the solution of Bb, the concentration of A must be some-
what increased, say to 0.055 M. We thus compensate for
the variation in osmotic pressure, and this compensation
is not destroyed when the 0.055 M solution of A is mixed
with the 0.1 M solution of B. If the effects of the salts
are additive, we must therefore get a horizontal straight
line, as shown in Fig. 49.

It is evident that this straight line furnishes a quanti-
tative criterion of antagonism. All that is necessary is
to determine what concentrations of A and B are equally
toxic, mix these solutions in various proportions, and
determine the amount of growth. The antagonism in
‘any mixture may then be expressed in a very simple
manner. In the. curve LKM (Fig. 49) the antagonism
in a mixture in which the molecules are 50% A and
\0% B may be expressed as KJ+JE. JE is the
growth which would have been obtained if the effect of
the salts had been additive (that is, if there had been no
antagonism, but each salt had produced its effect inde-

ANTAGONISM 127

pendent of the other.) KJ is the increased growth due to
antagonism; it is best expressed as percentage of JH or as
KJ —JE X 100.

In the same way increased toxicity (when the mixture
is more toxic than either of the pure solutions) may be
expressed as JH —JE. This sometimes occurs, but it is
much less common than antagonism.°®

~—PABLE VII
MIXTURES OF EQUALLY TOXIC SOLUTIONS
Wheat (growth during 30 days) (NaCl 0.12 M+CaCl 0.164 M)

Aggregate length of

Culture solution roots per plant | Additive effect Antagonism
keg AES Se Pa 55 BOOT) (teens treet mie abe
os Nacl...i| 1 | nO
0 Nach) 189 | ag ar
mB NaCl) 8 ha ag atl
6“ Nel) Che oe
6 MG) ‘ae ec eels
Pn ne ea Dar pale
al aint eis a os 3 55 Sage Ger nae Co fae

The percentages refer to molecular proportions; that is, 75 per cent. CaCl2+ 25 per cent.
ag ae peween in which 75 per cent. of the dissolved molecules are CaCl2 and 25 per
cent. are NaCl.

As an illustration of this method the results given in
Table VII may be cited. In this case the growth in the
various mixtures was in part determined directly and in
part was calculated from results obtained by growing

* See page 177.

128 INJURY, RECOVERY, AND DEATH

plants in mixtures having almost the same composition
as the solutions given in the table.

In another method® of measuring antagonism we may
look at the matter from the following standpoint. A cer-
tain amount of growth occurs in distilled water as shown
in Fig. 50; when salts are added to distilled water the

GROWTH

WATER

A .10 05 0
B O 05 -10

Fia. 50.—Curve showing antagonism between two salts, A and B. The additive effect is

GH. Antagonism at the ordinate J may be expressed as 100 X FH +GH; the opposite of

antagonism as 100 X EH ~+GH. ‘The dotted line represents growth in distilled water. The
absciss# represent the molecular concentrations of the salts.

growth is lessened. The lessening of growth due to the
action of the salts where no antagonism (or its opposite)
occurs is regarded as the additive effect. When the salts
are antagonistic growth is less hindered. The additive
effect is then GH; the antagonism is FH and may be
expressed as 100 * FH ~ GH. The opposite of antagon-
ism is 100 x HH ~ GH.

*Of. Osterhout (1918, A),

ANTAGONISM 129

We may now consider the effect of mixing two solu-
tions which are not equally toxic. Suppose solution A 0.1
M to be twice as toxic as solution B 0.1 M. The effect
of mixing these, if the effects were equally additive, would
be the same as mixing a solu-
tion of A 0.1 M with another
solution A just half as toxic,
or in other words, would be
the same as decreasing the
concentration of A. In this
ease the curve expressing
purely additive effects would
not be a straight line, but
would assume the form of a
eurved line, convex to the
horizontal axis, similar to
VTW in Fig. 51. This is evi-
dent’ from the curves given
by Magowan,showing growth y
in toxic solutions of various A100 75
concentrations. 2 eee

Beer Pe possible: 0) sie. 5 — Cure showing growth in mis:
Be Ue additive Curve termcmpcliion'd tie iiseares et Fie 90

experimentally, and then to the dotted line V7W expresses the growth

which would occur if there were no antago-

express antagonism quanti- nism (additive effect); VUW, antagonism

curve; VSW, curve expressing increased

tatively ; for example, at the ean Be ageoncee Teac ee
point P it would be expressed peice Vata
as UT —TP. But the labor would be much greater than
by the method of mixing equally toxic solutions. The
additive curve would be determined by growing plants,
not in mixtures of A with B, but in mixtures of A with
another solution of A having the same toxicity as B. Or
*Magowan (1908).
9

50 75 100 %

130 INJURY, RECOVERY, AND DEATH

we might use mixtures of B with another solution of B
having the same toxicity as A. The two methods might
not give exactly the same result. This is an additional
argument in favor of using equally toxic solutions.

An illustration of this method is found in the results
given in Table VIII. The growth in the various mixtures
(additive and antagonistic) was in part determined
directly and in part was calculated from results obtained
by growing plants in mixtures having almost the

TABLE VIII
MIXTURES OF UNEQUALLY TOXIC SOLUTIONS

Wheat (growth during 30 days) (NaCl 0.12 M+CaCl, 0.12M)

‘ A l h of
Culture solution Folin ae oat ; Additive effect Antagonism
In mm.

Oe Ae LO rey | see rea 85 85. |... 4e0s
75 uh cant. 61 8) SE 125—75
mo NG) a 5 | BE - 7
ro Sager O° Os SAIS AL 195 —66.5
Paci, eee aie a ane Ob
7: 3 Se GN Osi Sr a 310—60
Wa atl oak 310 60 60 = 4.17
i © 6h PU ee 380 —58
Backs f) | IRR ete vos 380 58 5g 9.55

TN Soames 7. ©, &| MS ae 438 —56
Ba MA te 428 ne 5G SCO 8.82

: Wn eae. ©"! @. TS areamane te 300—55

0 55 =

BO yr ert VEIN GT a Motels a 55 ae
MO Gre ara\t cian tals 7, oh 55 555s sw oth yr

The percentages refer to molecular proportions; that is, 75 per cent. CaClz +25 per cent.
NaCl means a solution in which 75 per cent. of the dissolved molecules are CaCl and
25 per cent. are NaCl.

same composition as the solutions given in the
table mentioned.

For the sake of completeness it may be mentioned
that other types of antagonism curves are found; for

ANTAGONISM 131

example, flat-topped curves and also curves with two
maxima, as shown in Fig. 52.

If instead of ‘mixing two equally toxic solutions we
keep the concentration of one salt constant while varying
that of the other, it becomes very difficult to determine the
additive curve, especially when variations in osmotic
pressure influence the result. It is therefore difficult to
obtain an accurate quantitative expression of antagonism
by this method, and in critical
cases it may be impossible to de-
cide whether antagonism exists
or not.

Emphasis should be laid on the
fact that the growth of parts
not in immediate contact with
the solution does not furnish a
trustworthy criterion of antagon- x B
ism. Thus the leaves of wheat fic. 52—Types of antagonism
(which are not in contact with the srowth; the ixenee, carton ‘the
solution) often grow well at the ee es ss
start in solutions of toxic substances because the latter
are held back by the roots.

The method of mixing equally toxic solutions has also
a great advantage when three solutions are employed. As
an illustration of this we may take mixtures of NaCl +
KCl + CaCl. In the case of wheat it was found that the
roots grew equally well in solutions of NaCl.12 M, KC1.13
M, and CaCl, 0.164 M. Mixtures of these solutions were
prepared and the growth of the roots in these mixtures
was measured after a period of 30 days. In order to show
the results graphically, the composition of the solutions
may be conveniently expressed by means of a triangular
diagram as drawn in Fig. 53,

132 INJURY, RECOVERY, AND DEATH

The diagram consists of an equilateral triangle, the
apices of which represent equally toxic pure solutions.
Thus the point A represents pure CaCl, (0.164 M), B
represents pure KCl (0.18 MW), and C represents pure
NaCl (0.12 M). All points on the sides of the triangle
represent mixtures of two solutions only, the composition
depending on the position of the point. Thus the point

ED EEE EIEN SATA LEO rate eee Neto nieve nen eee EE
0 25 50 75 100 NACL
KCL 100 75 50 25 - 0

CACL2

Fia. 53.—Diagram representing the composition of various mixtures of g's rt NaCl + CaCl:
this serves as the base of the solid model shown in Fig. 5

H represents a solution made by mixing the equally toxic
solutions NaCl 0.12 M and KCl 0.13 M in such proportions
that in the mixture 50% of the dissolved molecules are
NaCl and 50% are KCl. In the same way G represents
a solution in which the molecular proportions are NaCl
25% + KCl 75%; I represents NaCl 75% + KCl 25%;
E represents KCl 50% + CaCl, 50%; K represents NaCl
50% + CaCl, 50%.

All points in the interior of the triangle represent mix-
tures of the three equally toxie solutions NaCl 0.12 M,
KCl 0.13 M, and CaCl, 0.164 M. Along the line FJ are
represented mixtures in which the dissolved molecules are

ANTAGONISM 133

25% CaCl; the line EK represents mixtures in which
the dissolved molecules are 50% CaCl.; the line DL mix-
tures in which the dissolved molecules are 75% CaClo.
In the same way F'G means 75% KCl; EH, 50% KCI; DI,
25% KCl; GL, 25% NaCl; HK, 50% NaCl; and JJ,
75% NaCl.

The point M is on the line FJ, meaning 25% CaCl,;
it is also on the line EH, meaning 50% KCl; and likewise
on the line GL, meaning 25% NaCl. It therefore repre-
sents a mixture of the three equally toxic solutions,
NaCl 0.12 M, KCl 0.13 M, and CaCl, 0.164 M, in which the
dissolved molecules are 25% CaCl, + 50% KCl + 25%
NaCl. In the same way the point O represents a mixture
in which the dissolved molecules are 50% CaCl, 25% KCl
+ 25% NaCl.

It is obvious that the composition of any solution can
be represented by selecting a suitable point on the dia-
gram. At any such point an ordinate may be erected ex-
pressing the growth of the plant in that solution. When
this has been done for a sufficient number of points, a
solid model may be constructed which gives a complete
description of the growth of the plant in all the solutions.
Such a model is shown, in Fig. 54. The ordinates represent
the aggregate length of roots per plant of wheat at the end
of 30 days. The ordinates in the pure solutions are equal
(55 mm.), showing that the solutions are equally
toxic. The ordinates were in part determined directly by
experiment and in part calculated from data obtained by
growing plants in solutions of approximately the same
composition as those represented.

From such a model the antagonism in any solution
may be determined at once by measuring with calipers
the height of the ordinate at the required point, subtract-

134 INJURY, RECOVERY, AND DEATH

ing 55, which is the amount of growth in the pure solutions,
and in this case (since all the pure solutions are equally
toxic) the amount of growth which would occur if the toxic
actions of the salts were
additive (that is, if each
salt exerted its own toxic
effect independently of the
other salts); the result
should then be divided
by 90.

In this case the addi-
tive effect is represented
by a plane surface parallel
to the plane which forms
the base of the model. The
height of this plane is
indicated by the shading
in the figure.

Other methods (as
mixing unequally toxic
solutions or keeping the
concentration of one salt
constant while varying
Fic. 54.—Solid model showing the forms of that of the ‘others ) wil

the antagonism curves in all possible mixtures

of NaCl0.12 M,{KC10.13 M,and CaCl. 0.164M. give for the additive
effect a curved surface very difficult to determine and
not easily represented or measured on the model.

With solutions of more than three components the
results cannot be expressed in a solid model; but a
graphical expression may easily be obtained in the follow-
ing way. Let us suppose that equally toxie solutions
of A, b, C and D are to be mixed. A mixture of the first

ANTAGONISM 135

three may be made and ealled solution 1 (different mix-
tures may be called solution 2, ete.). To solution 1 various
amounts of D may be added and the results plotted as
shown in Fig. 55, in which the additive effect is expressed
by the dotted line and the growth in the mixtures by the
unbroken line. Antagonism at any point may be easily
expressed. For example, the antagonism at the point
M is (MO — MN) ~ MN.

By the method of mixing unequally toxic, pure solu-

Sol. 1 100 7% 50 25 0%
DO 25 50 75 100 %

Fig. 55.—Method of expressing antagonism 7 mixtures containing more than three com-
ponents: three of the components (A, B and C) are combined into solution I and various
amounts of the fourth component (D) are added; the ordinates represent growth; the abscis-

s@ represent the composition of the mixtures; thus at the point M the mixture contains 62.5
0.0. of solution 1 to each 37.5 c.c. of solution D; the antagonism at MisON+MN.

tions or by the method of keeping the concentration of one
salt constant while varying that of the others, the dotted
line would become a curved one.

If we mix such solutions as NaCl 0.12 M and CaCl,
0.164 M the antagonism curve resembles the one in Fig. 49.
If, however, we reduce the concentration by one-half
there will be less toxicity and in consequence the antag-
onism will appear less pronounced. In order to illus-
trate this, curves have been prepared which are diagram-
matic composites of the curves obtained by the use
of several pairs of salts; these composite curves are shown

136 INJURY, RECOVERY, AND DEATH

in Fig.56. For the sake of simplicity they are represented
as having been obtained by the use of one pair of salts,
which are designated as A and B. The curve CDE, there-
fore, represents diagrammatically the growth of roots

A.001M +B 915

G H i |
A_100 ib 50 25 0 %
BO 25 50 75 100 %

Fia. 56.—Effect of dilution on the forms of antagonism curves: the ordinates represent the

growth of roots in solutions, the composition of which is represented by the abscisse; for

example, on the curve CDE the ordinate at G represents growth in a mixture of A 0.1 M ‘and

B 0.12 M in such proportions that 75% of the dissolved molecules are A and 25% are B; on

the curve which lies immediately above CDE the ordinate at G repre growth in a

mixture of A 0.05 M and B 0.06 M in such proportions that 75% of the dissolved molecules
are A and 25% are B.

in mixtures of equally toxic solutions of two salts, A and
B. The abscisse represent molecular proportions; thus
the point G represents a mixture in which the dissolved
molecules are 75% A and 25% B; the point H a mixture
in which the dissolved molecules are 50% A and 50% B

ANTAGONISM 137

The ordinates represent the growth of roots in the
various mixtures.

The antagonism at any point is the total growth
minus the growth which would have taken place if no
antagonism existed. This antagonism is best expressed
as percentage of the growth which would have taken place
in the absence of antagonism. Hence the antagonism at
the point G is expressed as 100 (GD — FG) - FG.

The figure shows in a diagrammatic way the effect of
dilution on the form of an antagonism curve. The lowest
eurve CDE shows the effect on growth of various mix-
tures of two equally toxie solutions A 0.1 M+ B 0.12 M.
The next curve shows the form of the antagonism curve
when all of these mixtures were diluted by the addition
of an equal volume of water (4 0.05 M+ B 0.06 M). The
next curve was produced by growing plants in mixtures of
A 0.0025 M+ B 0.03 M. The topmost curve was obtained
with mixtures of A 0.001 M+ B 0.0012 M.

The pairs of pure solutions were in each case equally
toxic, as is shown by the fact that the two ordinates at
the ends of each curve are equal in height.

It will be observed that as the solutions become more
dilute, the antagonism curve becomes flatter, and it is
evident that at still greater dilutions it must tend to
become a horizontal straight line.

In order to give a complete description of the changes
in the antagonism curve as dilution increases, it is neces-
sary to construct a solid model. This might have as its
base a triangular diagram as previously described. The
apices of the triangle would in that case represent, A, B,
and H,0.

It is more suitable for our present purpose to employ
a square as the base and to represent the composition of

138 INJURY, RECOVERY, AND DEATH

the solutions according to the scheme shown in Fig. 57.
In this figure the abscisse have the same significance as
in Fig. 56, while the ordinates represent various dilutions
of the mixtures. Thus all points on the line CD represent
distilled water, while a point such as E, halfway between
ah} A and C, represents a mixture

c D containing equal quantities of
distilled water and of A 0.1 M.

The points on the line EF,

E F therefore, represent the same
mixtures as the corresponding

points on the lowest line, ex-

cept that the concentrations

A 100 50 0 % are in all cases just one-half
Bo C—O’ as great as those represented

Fic. 57.—Diagram representing the nh 7 my

composition of solutions (this serve on the base line. It is evident
t of the solid model shown in °

Fig. 58). the lowest line represents that the growth in any concen-

various mixtures of solutions of two P

salts, A and B; the line EF represents tration may be expressed by

the same mixtures diluted with equal

volume of water; any line drawn erecting at the proper point a

parallel to EF will express the same

mx res ted tem ftheline whe ~=ne perpendicular to the plane
the dilution onthe line CD all pointe Of the paper. In this way, we

ner ee ae may obtain a solid model which
gives a complete description of the changes in growth
produced by diluting the various mixtures. Such a model
is shown in Fig. 58.

In all of these cases the measurements are made after
erowth has ceased and in consequence they represent a
final condition of development. If, however, we use
electrical conductivity as a criterion of antagonism, we
obtain curves which change constantly. (See Fig. 78).
In this case the best method of procedure is to construct
the time curves of the death process and to compare the

ANTAGONISM 139

4
'
4
i
i}
{

| ea
| a
|

“Ny
My)

a eG
i ad
nt

Fia. 58.—A solid model which gives a complete description of the changes produced in the
form of the antagonism curve by altering the concentrations of the solutions.

velocity of the process in various mixtures. A series of
such time curves is shown in Fig. 59.

140 INJURY, RECOVERY, AND DEATH

In the preceding pages a theory is developed which
enables us to predict the behavior of Laminaria when
transferred from sea water to certain solutions, e.g., NaCl

* 100. NACL +0. CaCre
G 9859 » 1.41 »
Vv 97. 56 » ~ 244 »
O 9524 » 4.76 »
A 65 5: »
YY (6a 2" 33. ud
X 38 » 62. ”
©® oO » 400. ”

°
Q 2000 4000 MINUTES

Fia. 59.—Curves showing the net electrical resistance of Laminaria agardhit in 0.52 M

NaCl, in 0.278 M CaCh, and in mixtures of these (the figures attached to the curves show

the molecular per cent. of CaCl: in the solution). The curves show the calculated values

(from constants obtained by trial) which are given in Table X, the points show the observed

values (some are omitted in order to avoid undue crowding) ; each represents the average of
6ix or more experiments. Probable error of the mean less than 10% of the mean.

0.52 M and CaCl, 0.278 M. If the theory is sound it
should also enable us to predict the behavior of the
tissue in mixtures of these solutions.

In order to test this theory experiments were made
with a variety of mixtures. The solutions employed are

ANTAGONISM 141

given in Table IX. The electrical resistance of the tissue
in these solutions, is shown in Fig. 59. The curves are in
rood agreement with the formula

ala NB ech —KmyT
Resistance = 2,700 +. 90 e + 10
m perp

This is evident from Figs. 59 to 63, which show the curves
calculated by means of this formula and also the
observed values.

As previously explained, this formula is based upon
the assumption that the electrical resistance is propor-

TABLE IX.
Composition of Miztures.

Molecular proportions in the mixture

0.52 M NaCl 0.278 M CaCl;

NaCl CaCl:

ce. cc. per cent. per cent.

973 27 98.59 1.41
955 45 97 .56 2.44
914 86 95.24 4.76
751 249 85.00 15.00
496 504 65 .00 35.00
247 753 38.00 62.00

tional to a substance, M, whichis formed and decomposed
by the reactions

0—>S— A— M-——> B

We assume that when the tissue is transferred from sea
water to NaCl, or to CaCl., or to a mixture of these two
solutions, the reactions 0O—>S-—~>A cease, while the
reactions 4—> M —>B continue. By assuming various
values of K , (the velocity constant of the reaction 4 —>
M) and of K, (the velocity constant of the reaction M—>
_B), and employing these in the formula, we obtain curves
which closely approximate those which we find by experi-

INJURY, RECOVERY, AND DEATH

142

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ANTAGONISM 143

ment. The values of the velocity constants which are
thus obtained, are given in Table X.

It is evident from Table X, that as the per cent. of
CaCl, in the mixtures increases (beginning at 1.41%

CaCl,) the value of K,, first falls and then rises, its

25 ye 75 HOURS

Fic. 60.—Curve of net electrical resistance of Laminaria agardhii in 95.24 NaCl+4.76

CaCl: (unbroken line) the trial curve (broken line) calculated from the velocity constants Ka

= .000245 and Km=.00590. Each observed point represents the average of six or more

experiments: probable error of the mean less than 10% of the mean. All readings were
made at 15° C. or corrected to this temperature.

minimum value occurring in 97.56 NaCl + 2.44 CaCl,
(which is the mixture in which the tissue lives the long-
est.). It seems reasonable to assume that in each
mixture a substance is formed which reduces the value
of K,, Wemay assume that the decrease of Kis directly
proportional to the amount of this substance, which may
be assumed to occur in maximum amount in 97.56 NaCl +
2.44 CaCl,.

The simplest assumption which we can make is that
NaCl and CaCl, combine with some constituent of the

144 INJURY, RECOVERY, AND DEATH

protoplasm, as X,, to form a compound.* If we suppose
that the compound is Na,XCa, formed by the revers-
ible reaction
4 NaCl -+ XZ, + CaCl, = NaiXCa + 2 ZCl;

we can calculate the amount of Na,XCa which is formed
in each mixture of NaCl and CaCl,.

If antagonism really depends on the production of a
salt compound, it is evident that some mechanism must
exist which insures that an increase in the total concen-

ee 25

Fia. 61.—Curve of net electrical resistance of Laminaria agardhii in 85 NaCl+15 CaCsl

(unbroken line); the trial curve (broken line) calculated from the velocity constants Ka =

.000364 and Km =.0073. Each observed point represents the average of six or more experi-

ments: probable error of the mean less than 10% of the mean. All readings were made at
15° C. or corrected to this temperature.

50 75 HOURS

tration of salts can have but little effect as compared with
that produced by a change in their relative proportions.

*It is assumed that XZ,, Na,XCa, and ZCl, are in solution. Since the
per cent. of XZ, which is transformed to Na,XCa is negligible, the con-
centration of XZ, may be regarded as constant.

ANTAGONISM 145

It is easy to see how such a mechanism must exist if
the formation of the salt compound takes place at a
surface (at the external surface of the cell or at internal
surfaces). In a surface, substances usually exist in a dif-
_ ferent concentration from that which they have elsewhere

25 5° 75 hours

Fic. 62.—Curve of net electrical resistance of Laminaria agardhii in 65 NaCl+35 CaCle
(unbroken line), the trial curve (broken line) calculated from the velocity constants Ka =
-000481 and Km=.00859. Each observed point represents the average of six or more experi-

ments: probable error of the mean less than 10% of the mean. All readings were made at
15° C. or corrected to this temperature.

in the solution. If NaCl and CaCl, migrate into the sur-
face, so as to become more concentrated there than in the
rest of the solution, their concentration in the surface must
increase as their concentration in the solution increases
until a certain point (called the saturation point) is
reached. Beyond this point an increase in their concen-
tration in the solution produces no effect on their
concentration in the surface.

When this stage has been reached the formation of

146 INJURY, RECOVERY, AND DEATH

the salt compound, if it takes place in the surface, will
not be affected by an increase in the concentration of the
salts in the solution. It will, however, be affected by
changes in the relative proportions of the salts. The

25 50 75 HOURS

Fig. 63.—Curve of net electrical resistance of Laminaria agardhit in 38 NaCl+62 CaCl
(unbroken line) and the trial curve (broken line) calculated from the velocity constants
Ka = .000530 and Km=.0090. Each observed point represents the average of six or more
experiments: probable error of the mean less than 10% of the mean. All readings were
made at 15° C. or a Sai: to this temperature.

number of molecules of salt in a unit of ‘surface will
remain nearly constant, but if the proportion of NaCl in
the solution be increased some of the CaCl, in the surface
will be displaced by NaCl.

Below the saturation point the relative proportions
of the salts will be of less importance than their total
concentration: this is the case at low concentrations in

the region of the so-called ‘‘nutritive effects.”’

ANTAGONISM 147

It has been pointed out by the writer® that while va-
riations in concentration affect the form of the antagonism
curve, they do not in general affect the proportions which
are most favorable for life processes.

It is therefore evident that if we wish to preserve the
favorable character of a mixture when the concentration

TABLE XI.
Amount of Na:XCa.
% Molecular proportions
In the solution In the surface Anon 5 rg
NaCl CaCl NaCl CaCl:

per cent per cent. per cent per cent.

100.0 0 100.0 0 0 0
98.59 1.41 87.5 12.5 0.000902 0.000855
97.56 2.44 80.0 20.0 0.000936 0.000889
95.24 4.76 66 .67 33 .33 0.000870 0.000823
85.0 15.0 36.27 | 63.73 0.000488 0.000441
65.0 35.0 15.66 84.34 0.000177 0.000125
38.0 62.0 5.78 94.22 0.000047 0

0 100.0 0 100.00 0 0

of any antagonistic substance is increased we must at
the same time increase the concentration of the others
in the same proportion.

In discussing the results of his experiments on ani-
mals, Loeb’® states that the law of direct proportionality,
found in such cases is in reality Weber’s law.!! In
regard to the significance of this, Loeb says:

‘*Since this law underlies many phenomena of stimu-
lation, if appears possible that changes in the concentra-
tion of antagonistic ions or salts are the means by which

” Loeb, J. (1915, A).
“Of. Osterhout (1916, C).

148 INJURY, RECOVERY, AND DEATH

these stimulations are brought about, as suggested by my
ion-protein theory and by the investigations of Lasareff.’’

In view of the importance of these relations it seems
desirable to point out that the hypothesis of the writer
explains the mechanism which makes one proportion
better than others and preserves this preéminence in
spite of variations in concentration.

We assume that CaCl, accumulates in the surface to
a greater degree than NaCl. The increase in concentra-
tion of CaCl, in the surface is supposed to be ten times
as great as the corresponding increase of NaCl, so that
the proportions in the surface are those given in Table XI.
For example, when the proportions in the solution are
97.56 NaCl + 2.44 CaCl., the proportion of NaCl to CaCl,
in the surface is as 97.56 to 24.40, which is equivalent’ to
80 NaCl + 20 CaCl,.

We ealeulate the amount of Na,XCa by the
usual formula:

rt (CNasXCa) (Czc1)?
~~ (Cnaci)4 (Ccach) (Cx2Z:)

but since 2Cnaxca = Czc, we may write

(CNacXCa) (2 CNasXCa)?
(Cnaci)4 (Ccach:) (CXZa)

Putting K —4(1071") and Cxz,=—0.1 we get

ie (CNasXCa) 8
~~ (80)4 (20) (0.1)

whence Cnaxca = 0.000936.

“ Ag previously explained it is assumed that the reaction takes place
in a surface which is saturated with respect to NaCl and CaCh so
that while one of these may be displaced by the other (in case their
relative proportions in the solution are altered) the total concentration
does not change; for convenience this concentration is taken as 100 and
the sum of NaCl 4- CaCl, is therefore always equal to 100.

10—17

ANTAGONISM 149
Proceeding in the same manner with the other mix-

tures, we get the values given in Table XI. Starting with

A Decrease in KM
O ” » Kn

> 4 ” v Ko
” » Ks

0.000500

Al 4 62
244 —+% Ca Chg in solution,

Fic. 64.—Shows the increase of a hypothetical salt compound NasXCa (see Table XI) and the
corresponding decrease of the velocity constants Ky, Ko, Ks and Ky (these constants are

given in Table X). The figures on the abscisse give the molecular per cent. of CaCh in the
mixture. The mixture containing 62.0% CaCl is taken as the standard of comparison:
proceeding from this to the mixtures containing less CaCle we find that NasXCa increases
and the velocity constants decrease as shown by the ordinates. In order to facilitate com-
parison the values of K ,, have been multiplied by 0.989; of K 9 by 0.991; of K > by 0.383; and

of K y,by 0.251.

the lowest value (that in 62.0% CaCl.) we observe that
there is an increase as the per cent. of CaCl, decreases
until 2.44% is reached (the amount of this increase is

150 INJURY, RECOVERY, AND DEATH

shown in Column 6 of the table). Conversely we find
(Table X) that the velocity constants are higher in 62.0%
CaCl, than in any other mixture and that they decrease
as the per cent. of CaCl, decreases to 2.44%. Thus in
the case of K,,, the value in 62.0% CaCl, is 0.009, in 2.44%
CaCl, it is less by 0.00354, while in 15.0% it is less by
0.0017, and in 35.0% by 0.00041; if we multiply these num-
bers by the constant factor 0.251 they agree very closely
with the figures for the increase in Na,XCa. These values
are plotted in Fig. 64, which shows that the decrease in
K,, is directly proportional to the increase in the amount
of Na,XCa. Hence we assume that Na,XCa acts as a
negative catalyzer or inhibitor of the reaction M—>B.

An inspection of Table X shows that the value of K4
fluctuates with that of Km, except that as CaCl, increases
the value of K arises more rapidly than that of Ky. This
is also obvious from Fig. 59, which shows that the greater
the per cent. of CaCl, in the mixture, the greater tne
maximum attained. Since this maximum increases as the
value of Ka Ky increases, it is evident that the value
of Ka ~ Kw» must rise as the per cent. of CaCl, becomes
greater. The value of Ka K min the solution containing
1.41% of CaCl, is 0.03333 while in the solution containing
62.0% CaCl, it is 0.05889, an increase of 0.02556. If we
calculate this increase for the other mixtures and plot the
values so obtained against the per cent. of CaCl, in the sur-
face, we obtain a straight line as shown in Fig. 65. This
indicates that CaCl, catalyzes! the reaction A —> M; for
if this were not the case the value of Ka and K ywould rise

*In the absence of Na,XCa it would appear that NaCl catalyzes the
death process, since death is more rapid in NaCl 0.52 M than in 0.26 M.

ANTAGONISM 151

and fall in such a way that the value of K,4-- Ky would
remain constant.

It is evident from Figs. 64 and 65 that the values of Ku
and Kyare determined by the amount of Na,XCa and by
the per cent. of CaCl, in the mixture, and that when
these values are experimentally determined for any two

Ka
Increase in Kn O
Kr

x
"oft

° Ks 0 10
oo:
()
Oo )
oO
” 05
1
>
x °
"ss 1250 200 33.38 6375 =e ee ae

CACiy in surface

Fig. 65.—Graph showing the increase of K_, + K,,and the value of K, + Kas the molecular

per cent. of CaCl: increases. The figure shows that CaCl acts as a catalyzer of the reaction
A— > M (which has the velocity constant K_,) and also of the reaction R—>S (which has the

velocity constant K,). The figures on the ordinate at the right show the values of
Kp ~+K.; those on the ordinate at the left show the increase in the value of K , + Ky,

over the value found in the mixture containing 1.41% CaCh. The abscisse denote molecular
per cent. of CaCle in the surface (not in the solution).

mixtures they can be calculated for any other mixture.
When this is done we can calculate the course of the death
curve in that mixture.

Having thus accounted for the death curves, we may
turn our attention to the process of recovery. We find
that, when tissue is removed from a mixture of NaCl and
CaCl., and replaced in sea water, the resistance at once
rises or falls and after a time becomes stationary. This
rise or fall of resistance may be called recovery.

In order to account for the facts we suppose that when

152 INJURY, RECOVERY, AND DEATH

we replace the tissue in sea water the reactions 0 —>_
S —> A—> M —>B proceed at the rates which are nor-
mal for sea water. The manner in which the rate of
recovery is calculated has already been explained in
detail. It is assumed that during the exposure
to any of the mixtures the following reactions occur: (1)
N —0—>P; (2) R-——>S —T ; (8) A— Mae
assuming values of the velocity constants of these
reactions we can approximate the observed results. The
velocity constants thus found are given in Table X. An
inspection of the table shows that all these velocity
constants behave like K, and Ky in that as the per cent.
of CaCl, in the mixture increases (beginning with 1.41%
CaCl.) the value of the velocity constant first falls and
then rises, and that this value in every case reaches its
minumum in the mixture containing 97.56 NaCl + 2.44
CaCl,. It would therefore appear that the reactions
N —> O—>P and R—> S—T are inhibited by Na,XCa
in the same manner as the reactions dA —>M—>B. This
is borne out by an inspection of Fig. 64, in which the
decrease’ of the velocity constants is plotted, together
with the increase of Na,XCa.

* By the decrease in the velocity constant is meant the decrease which
we observe as we pass from the solution containing the highest per cent.
of calcium (38.0% NaCl + 62.0% CaCl,) to mixtures containing smaller

per cents of calcium. Thus the decrease of K ,,= 0.009 — K uthe decrease
of E y= 0.00134 — Ky; the decrease of Ko= 0.0013266 — Ko; and the
decrease of Ky = 0.00319 — K,. In the same manner we find that the
increase in the amount of Na,XCa—= amount of Na,XCa — 0.000047.
The decrease of the amount of K A and Kp is not shown in the

figure because it depends not only on Na,XCa, but also on the per cent.
of CaCl,.

The fact that even in the presence of the maximum amount of Na,XCa
these velocity constants are greater than in sea water is of course to
be attributed to the other substances present in sea water.

ANTAGONISM 153

We have seen that the value of K4-- Km increases as
the per cent. of CaCl, increases and we interpreted this
to mean that the reaction A —> M is catalyzed by CaCl,.
In the same manner we infer that the reaction R —> S is
catalyzed by CaCl., since we find that the value of Kr-+
Ks increases with increasing percentage of CaCl., as
shown in Fig. 65. It is not certain that the curve does
not reach a minimum in the mixture of 97.56 NaCl +
2.44 CaCl., but for practical purposes we may, for the
present, regard it as a straight line.?®

The relation between Ky and Kois taken as constant
in the proportion of 100 to 99.

It is evident that when the constants have been empir-
ically determined for two mixtures the constants for
any other mixture can be calculated at once, since all of
them depend in a definite manner on Na,XCa (Ku and Kr
also depend on the per cent.of CaCl,). The agreement
between the constants thus obtained by calculation
and those found by trial is fairly close, as is evident from
Figs. 64 and 65.1°

It has already been shown that the height to

* Since in pure NaCl or CaCl, the salt compound Na,XCa is not formed,
we should expect that in these solutions all the reactions would be more
rapid than in the mixtures. That this expectation is fully realized is
evident from Table X.

The velocity constants are somewhat higher in NaCl than in Ca(Cl,;
this is not explained by the assumptions already made, but it does not
seem desirable at present to make additional assumptions for this

purpose. We might expect the values of K, + K,, and Kp~ K, to
reach a maximum in CaCl,. This is actually the case. It might perhaps
be expected that these values would fall to a minimum in NaCl. This
is the case with K,+ Kj, but not for Kp+ Kg

*The constants obtained by calculation would fall exactly on the

graphs in these figures while those found by trial are indicated by
the points given.

154 INJURY, RECOVERY, AND DEATH

which the recovery curve rises depends on the value of
O: the value of O + 10 is shown for all the solutions!” in
Fig. 66, which shows the agreement between observation
and calculation in respect to the final level reached by
the recovery curve, but not in respect to speed of recovery,
which depends more on the value of S than on that of O.
The rate of recovery seems to be about the same in the
mixtures as in the pure salts. In general it is found that

100. NaCi + O. CaCLo
9859 » 141 »

Rm
Oo
Vv
O
A
iV;
x
)

0 2000 4000 MINUTES

Fic. 66.—Curves showing the value of O0+10 in 0.52 M NaCl, in 0.278 M CaCh, and in
mixtures of these (the figures attached to the curves show the molecular per cent. of CaCh in
the solution). The ordinates give the relative values of O +10, the value in sea water being
arbitrarily taken as 100%. These values are ;obtained by exposing tissue to toxic
solutions and then finding the level to which the resistance rises or falls after the tissue is
replaced in sea water: they are therefore a measure of permanent injury. The abscisse give
the length of exposure to the toxic solution. The curves show the calculated values (using
the velocity constants given in Table X). The points show the observed values; each repre-
sents the average of six or more ements Probable error of the mean less than 10% of
the mean.

the rise or fall is nine-tenths completed in about an hour.
Fig. 67 shows the calculated values of S; observed
values are not given because they cannot be very pre-
“The values of O + 10 for solutions containing 2.44 and 15.0%
CaCl, differ slightly from those given earlier for the reason that the
curves here presented include a larger series of experiments. 10 is added

to the value of O because the base line is taken as 10, just as in the
case of M,

ANTAGONISM 155

cisely determined. This is owing to the fact that S affects
only the speed of recovery (not the final level attained)

100 62

9° 1000 2000 MINUTES

Fic. 67.—Curves showing the (calculated) values of S in 0.52 M NaCl, 0.278 M CaCh, and

in mixtures of these (the figures attached to the curves show the molecular per cent. of CaCl,

in the solution). The curves show the values calculated from constants obtained by trial

which are given in Table X. The abscisse represent the time of exposure to the toxic solu-
tion. The value of S at the start is in all cases 2.7.

and, as the speed is variable, the most satisfactory pro-

cedure is to assume such values of Kr and Ks in
the equation’®

a Kr — KrT — KsT — KsT
4 Ks —Kr é —— + Soe

as cause the closest approximation to the observed speed
of recovery. The values of S thus obtained for each
solution are shown in the figure. In general, the speed
of recovery, as calculated from these values of S, is in
satisfactory agreement with the observations.

By means of the equations already given and of the
itmaion(s)jpzi. |. |.

156 INJURY, RECOVERY, AND DEATH

velocity constants in Table X, we are able to calculate
the recovery curves for any solution after any length
of exposure.

Lack of space prevents a tabulation of the observed

—--0------0--0---

bso?

° 1000 2000

MINUTES
Fic. 68.—Curves showing the net electrical resistance (descending curve) of Laminaria
agardhii in a mixture containing 97.56 mols of NaCl to 2.44 mols of CaCl: and recovery in sea
water (ascending curves). The figure attached to each recovery curve denotes the time of
exposure to the toxic solution. In the recovery curves the experimental results are shown by
the broken lines, the calculated results by the unbroken lines. The observed points represent
the average of six or more experiments. aha ige error of the mean less than 10% of
the mean.

and calculated values, but it is possible to exhibit graphi-
cally the data for three mixtures and for this purpose
one in which recovery consists in a rise of resistance (Fig.
68), one in which it shows a moderate fall (Fig. 69), and

3
wwe wee Qeree<-Gesseeee

° 1000 \ 2000. .

— = .

MINUTES.
Fia. 69.—Curves showing the net electrical resistance (curve which ascends and descends)
of Laminaria agardhii in a mixture containing 95.24 mols of NaCl to 4.76 mols of CaCl: and
recovery in sea water (descending curves). The figure attached to each recovery curve
denotes the time of exposure to the toxic solution. In the recovery curves the experimental
results are shown by broken lines, the calculated results by unbroken lines. The observed
points represent the average of six or more experiments. Probable error of the mean less

than 10%. of the mean.

one showing a very decided fall (Fig. 70) are presented.
In general the agreement between observation and caleu-
lation is satisfactory for all the solutions employed in
the investigation.

ANTAGONISM 157

It might be thought that the number of constants is
sufficient to make it possible to fit any sort of experimen-
tal curve and that the consequent agreement between
observed and calculated results is less significant than
would otherwise be the case. But, as a matter of fact,

100

es 1000 2000
MINUTES

Fic. 70.—Curves showing the net electrical resistance (curve which ascends and descends)

of Laminaria agardhii in a mixture containing 38 mols of NaCl to 62 mols of CaCh, and re-

covery in sea water (descending curves). The figure attached to each recovery curve denotes

the time of exposure to the toxic solution. In the recovery curves, the experimental results

are shown by the broken lines, the calculated results by the unbroken lines. The observed

points represent the average of six or more experiments. Probabie error of the mean less
than 10% of the mean.

the constants are so related to each other and to the salt
compound, Na,XCa that the whole set of curves fits into
a consistent scheme, so that when the constants are
determined for any two mixtures the theoretical curves
for all the other mixtures are thereby fixed. Under these
circumstances the close agreement in the six different
mixtures (ranging from 1.41 to 62.0% CaCl.) seems to

be significant.

158 INJURY, RECOVERY, AND DEATH

There seems to be no doubt that the behavior of the
tissue is such as to indicate an underlying mechanism
which is the same in all cases.12 We have assumed that
this mechanism consists in the production and decompo-
sition of a substance, M, the amount of which, in the mix-
tures, depends largely on a compound Na,XCa formed
by the combination of Na and Ca with a constituent X
of the protoplasm. It is not necessary to discuss these
assumptions more fully at present. But it may be
pointed out that two things seem to be fairly well estab-
lished; (1) a consistent mechanism underlies the entire —
behavior of the tissue, and (2) its operation can be pre-
dicted with a fair degree of accuracy by means of the
equations which have been developed. The predictive
value of these equations may be regarded as permanently
established, since it does not depend on our views regard-
ing the underlying assumptions.

The results of these experiments may be summarized
as follows:

1. The equations which serve to predict the injury of
tissue in 0.52 M NaCl and in 0.278 M CaCl, and its sub-
sequent recovery (when it is replaced in sea water) also
enable us to predict the behavior of tissue in mixtures of
these solutions, as well as its recovery in sea water after
exposure to mixtures.

2. The reactions which are assumed in order to
account for the behavior of the tissue proceed as if they

” This is shown, for example, by the fact that the rapidity of permanent
injury (as observed after replacement in sea water) corresponds through-
out with the rate of death, and that the rate of change of M corresponds
throughout with the rate of change of O, S and A. In other words if we
change the solution in such a way as to increase (or decrease) one of

the reactions on which the resistance depends we simultaneously increase
(or decrease) all the others in a definite and predictable manner.

ANTAGONISM 159

were inhibited by a salt compound, formed by the union
of NaCl and CaCl, with some constituent of the proto-
plasm (certain of these reactions are accelerated
by CaCl,).

3. A quantitative theory is developed in order to
explain: (a) the toxicity of NaCl and CaCl.,; (b) the
antagonism between these two substances; (c) the fact
that the optimum proportions do not change with altera-
tions of concentration, and (d) the fact that recovery (in
sea water) may be partial or complete, depending on the
length of exposure to the toxic solution.

It may be appropriate to call attention to some appli-
cations of this theory. Antagonism has been explained
by Loeb, and by the writer on the ground that antagon-
istic substances prevent each other from entering the
cell. As the writer has repeatedly pointed out®®, this
explanation encounters a difficulty in the fact that
antagonistic substances penetrate the cell in a balanced
solution (although the penetration is much slower than
in unbalanced solutions). The proof of this has been
obtained by the writer by means of the method of plas-
molysis*! as well as that of electrical resistance?’; it has
recently been confirmed by Brooks** by means of the
method of tissue tension as well as of diffusion through
a disk of living tissue and by direct determinations of the
penetrating substances made by the writer (see page 216).

It is obvious that antagonistic substances must
penetrate from a balanced solution since otherwise the
cell could not obtain the salts necessary to its existence.

7 Cf. Osterhout 1911. See page 214.

“Cf. Osterhout (1912. A. C; 1915, C).
* Of. Brooks (1916, A. C.; 1917, B). See pages 206 to 209.

160 INJURY, RECOVERY, AND DEATH

As a way out of this difficulty, the writer has sug-
gested?* that the slow penetration of salts may produce
effects quite different from those produced by
rapid penetration.

This difficulty completely disappears if we adopt the
standpoint outlined above in developing a dynamical
theory of antagonism. From this point of view, we
regard the slowness of the penetration of salts in balanced
solutions, not as the cause of the antagonistic action but
rather the result of it; or we may regard both the slow
penetration and the increased length of life (or growth,
etc.) by which we measure antagonism, as the results
of certain life processes which are directly acted on by
the antagonistic substances.

The essential feature of the explanation lies in the
behavior of these life processes rather than in the manner
or rate of penetration.

We assume, as explained above, that certain life pro-
cesses may consist of consecutive reactions of the type
Sg a A eee.
in which M is a substance which determines the rate of
penetration of salts and the electrical resistance of

the protoplasm.

If the antagonistic substances are NaCl and CaCl,
it appears that CaCl accelerates the reaction 4 —~M,
while both 4A—>M and M —B are inhibited by a salt
compound formed by the union of NaCl and CaCl, with
a constituent of the protoplasm.

From this standpoint the slow penetration of antag-
onistic substances should not have unfavorable results
provided these substances are properly balanced at the

* Of, Osterhout (1911, 1912, A, C; 1913, B; 1916, H; 1917, B).

ANTAGONISM 161

start and remain so (2. e., if their relative proportions are
not too much changed by unequal speed of diffusion,
precipitation, chemical union, ete.) after they enter the
cell. For they must affect the life processes mentioned
above in quite the same way in the interior of the cell
as at the surface, and these life processes will go on in the
normal way so long as the antagonistic substances within
the cell remain properly balanced.

The result will be the preservation of normal per-
meability as well as of all other properties essential
to life.

It has been shown?® that the normal permeability
may be regarded as a sensitive and accurate indicator
of health and vitality. All factors which disturb it bring
about temporary or permanent injury and eventually
produce death if the action be sufficiently prolonged. It
is therefore evident that the life processes which pre-
serve normal permeability are of peculiar importance
and that the manner in which they are influenced by
antagonistic substances is of especial interest.

We may now turn our attention to another aspect
of the subject. Explanations have been suggested by
Loeb and others to account for the antagonistic action
of various snbstances on living protoplasm, but none of
them have thus far developed to the point where they
enable us to predict what substances (including both elec-
trolytes and non-electrolytes) will antagonize each other
and what degree of antagonism may be expected.

This kind of prediction is apparently made possible
by a hypothesis formulated by the writer, as the result

* Whatever effects are found at the outer surface of the cell may doubt-
less be found also at many of the internal surfaces, such as the surfaces of

vacuoles, plastids, microsomes, etc. See Chapter VII.
11

162 INJURY, RECOVERY, AND DEATH

of his investigations on the conductivity of protoplasm. |
Substances which alter the conductivity of protoplasm
may be divided into (1) those which cause an increase,
but not a decrease, of conductivity and (2) those which
ean produce a decrease of conductivity (followed by
an increase).7°
The hypothesis states that substances belonging to
the first class will antagonize those belonging to the
second, and vice versa. In order to predict which sub-
stances will antagonize each other it is only necessary
to determine to which of these classes the substances
belong. The amount of antagonism may also be pre-
dicted; at least to a considerable extent, since the greater
effect of the substances on permeability, the greater
will be their antagonistic action. This relation may be
obscured by secondary causes, so that the predictions
which it allows will not be of equal value in all cases.?7
As we have seen above, NaCl belongs to the first class,
being able to increase conductivity but not to decrease
it, while CaCl, belongs to the second class, as it is able to
decrease conductivity. It was found that the antagon-
ism between NaCl and CaCl, in the case of Laminaria is
well marked. These facts led the writer to formulate the
hypothesis as stated above. The next step was to test the
hypothesis by the investigation of other salts. Magnes-
ium seemed of especial interest for this purpose, as in
most of the writer’s previous experiments (on other
plants) it had shown no antagonism to sodium, though it
might be expected on chemical grounds that magnesium
and calcium would behave alike. To the surprise of the
7 Of, Osterhout (1915, A).

*The decrease is followed by an increase if the exposure is suffi-
ciently prolonged.

ANTAGONISM 163

writer, it turned out that magnesium was able to decrease
conductivity, though its effect was much inferior to that
of calcium. The antagonistic relations for Laminaria
were then investigated, and it was found that MgCl, was
able to antagonize NaCl, though its antagonistic action
was much less than that of CaCl.,.

This unexpected and striking result strengthened the
writer’s confidence in the hypothesis and led to further
investigations. One of these which was of special interest
related to acids. For a number of reasons it was sup-
' posed that acid would not cause a decrease in permeabil-
ity. But investigation showed that such a decrease actu-
ally occurred in the presence of HCl and it was then a
simple matter to predict that antagonism would be found
between NaCl and HCl. This turned out to be the case,
the amount of antagonism corresponding to the amount
of decrease of conductivity.

These results are also of interest in view of the fact
that Loeb? has shown that salts are antagonized by acids
and has pointed out that this has a special significance
for the theory of permeability, since it indicates that the
permeability of the plasma membrane (for water and
substances soluble in water) depends on the presence
of protein rather than of lipoid substances. The investi-
gations of the writer show that similar (though less
striking) antagonism occurs in plants. This affords
evidence of the protein character of the plasma mem-
brane in plants and is in harmony with the fact that (as
the writer has shown) various ions pass through the
plasma membrane of plants,®?° which would not be
expected if it were composed of lipoid.

® Loeb, J. (1899; A, B; 1912, A, B; 1917): Loeb and Wasteneys
(1911, B; 1912).
*° Of. Osterhout (1912, A, B; 1913, B).

164 INJURY, RECOVERY, AND DEATH

In carrying out these investigations a solution of HCl
having the same conductivity as sea water (about 0.119
M HCl) was prepared. Various amounts of this were
added to a solution of NaCl 0.52 M (which had the same
conductivity as sea water). Several lots of tissue were
prepared with a view of making them as much alike as
possible. One lot of tissue was placed in each of the mix-
tures of NaCl + HCl; other lots were also placed in pure
NaCl and in pure HCl.

The results are shown in Fig. 71. It will be seen that
in pure NaCl and in pure HCl the resistance fell rapidly,
indicating injury; while in a mixture in which the dis-
solved molecules are 99.09% NaCl and 0.91% HCl, the
resistance fell less rapidly, indicating that this mixture
was less injurious than either of the pure solutions. In
other words, the salt and the acid have an antagonistic
action. This antagonism may be expressed quantita- |
tively (as previously explained*:) in the following man-
ner: The ends of the antagonism curve are connected by
a straight line®* and an ordinate is erected at the point
on the curve which is to be measured. For example, the
ends of the 30 minute curve in Fig. 71 are connected by
the dotted line. The antagonism at the point A (repre-
senting a mixture in which the dissolved molecules are
99.09% NaCl and 0.91% HCl) is expressed as AB ~ BC.

The rise in resistance at the end of 1 minute in pure
HCl agrees with the results previously described.**

+See pages 122 to 129.

* This should in many cases be a curved line, provided the pure solu-
tions are not equally toxic. But in the present case the curvature would be
small, and at the maximum point of the curve very small indeed. This
line expresses the additive effect; i.e., the effect which would be produced
if there were no antagonism, and each component of the solution acted

independently. (See page 72).
* See page 48.

ANTAGONISM 165
130 PER CENT

100

NaC1100 75 50 25. GO:
HC: O 25 50 75 100

Fie. 71.—Antagonism curve of Laminaria agardhit in NaCl 0.52 M, in HC1 0.119 M, and in
mixtures of these. The ordinates represent net electrical resistance (expressed as per cent. of
the normal net resistance); the abscisse represent the molecular proportions in the mix-
tures. Thus NaCl 50, HCl 50 means a mixture of NaCl 0.52 M and HCl 0.119 M in such
proportions that 50% of the dissolved molecules is NaCl and 50% is HCl. Each curve repre-
sents a single experiment. All readings were taken at 18° C. or corrected to this temperature.

A reading taken at the end of 18 hours showed that the
tissue was dead in all the solutions. The plants can be
kept alive much longer than this in mixtures of NaCl +
CaCl,; it is also noteworthy that the degree of antagon-

166 INJURY, RECOVERY, AND DEATH

ism, as shown by the electrical resistance, is greater in
NaCl-+ CaCl, than in NaCl + HCIl.*4

The hypothesis was further tested by investigations
on other salts, the most interesting of which are those
which (in contrast to those just mentioned) are more
effective than CaCl, in decreasing permeability, such as
La (NO,;)3, Ce (NO;)3, ete. Here also it was found that
the degree of antagonistic action could be foretold by
observing the amount of decrease of permeability pro-
duced by the pure salts. The results of these investiga-
tions afford strong support to the hypothesis.

The soundness of this point of view is indicated not
only by the fact that we are able to predict both qualita-
tively (and to a considerable extent quantitatively) the
effect of combinations of salts*® but also by the very

*4# See page 140.

*It should be noted that mixing solutions of two salts which belong
to different classes does not produce an effect which is merely intermediate
between the two. For example, tissue may be killed by an exposure of
24 hours to NaCl or to CaCl,, but not in a mixture of these in the proper
proportions.

The writer has found cases in which two substances which can decrease
permeability are able to antagonize each other. So far as the writer’s
experiments with Laminaria have gone there is no great amount of
antagonism in such cases and what there is may perhaps be correlated
with the fact that all substances which decrease permeability do not act
alike, some producing a much greater decrease than others. Moreover
these substances will, if the exposure be sufficiently prolonged, alter their
action and increase permeability. The rapidity of this change varies
with different substances, and this may be related to the fact that some
of these substances antagonize each other to some degree.

Experiments on some plants (in which the criterion of antagonism is
not electrical resistance, but growth) show a fairly strong antagonism
between magnesium and calcium. It is possible that for these plants mag-
nesium belongs in the first class.

It will be noted that the hypothesis, as here set forth, says nothing
about the mutual relations of substances belonging to the same class, but
merely states that substances of one class will antagonize those of the
other. In this form the hypothesis is completely justified by all the
experiments, including those on organic substances.

ANTAGONISM 167

significant fact that we are able to extend this conception
to organic compounds and to show that non-electrolytes
which decrease permeability can also antagonize such
substances as NaCl. These facts indicate that the hypoth-
esis may perhaps be applied in a general manner so as
to include both electrolytes and non-electrolytes.

As an example of antagonism between salts and
organic substances we may cite some experiments with
bile salts. The writer found, very early in the course of
his experiments, that Na-taurocholate increases the elec-
trical resistance of Laminaria. This was somewhat
striking in view of the fact that agents which increase
permeability have long been known, but the discovery of
substances which have the opposite effect, is compara-
tively recent. The number of such substances known at
present (especially organic substances) is very small and
it is therefore of interest to find that bile salts possess
this property.

The experiments were made by determining the elec-
trical conductivity of Laminaria in solutions to which
Na-taurocholate was added.*®

In the first experiments the bile salt was dissolved in
sea water. The amounts added to 1,000 cc. of sea water va-
ried from 0.8 to 1.5 gm. If the Na-taurocholate were pure,
1 gm. in 1,000 ecc., would make the concentration about
0.002 M, but as its purity is doubtful the concentration can-
not be accurately determined.

After dissolving the Na-taurocholate the sea water
was restored to the normal conductivity and made
approximately neutral to litmus.

At all the concentrations employed there was an
immediate increase in resistance®? followed by a fall.

* Of. Osterhout (1919, B, C.).
** See page 55.

168 INJURY, RECOVERY, AND DEATH

Under the conditions of the experiment (temperature
19°+2°C.) the rise lasted about an hour. The effect is
comparable with that of anesthetics*®* (ether, chloroform,
and alcohol) as described by the writer. An increase
in resistance was also observed with Ulva rigida and with
Rhodymenia palmata.

In the experiments on antagonism ane tissue was
placed in a solution of NaCl 0.52 M to which various
amounts of Na-taurocholate were added (all the solu-
tions having the same conductivity as the sea water
and being approximately neutral to litmus). The tem-
perature was 18.5+2.5°C.

The results are shown in Fig. 72. There is a gradual
fall of resistance in all the solutions which continues
until the death point (10%) is reached. In the solution
containing 1,000 «ec. of NaCl 0.52 M+ 0.52 gm. of Na-
taurocholate the fall of resistance is much slower, indi-
cating that this is the most favorable mixture.

It should be emphasized that the effect is not an
intermediate but an antagonistic one. By this is meant
that the resistance is not merely the algebraic mean
between a rise in resistance produced by the bile salt and
a fall produced by NaCl. A consideration of the lowest
curve shows that at 180 minutes the tissue is dead in
NaCl 0.52 M as well as in 1,000 c.c. of NaCl 0.52 M+ 10
om. of Na-taurocholate, but in the mixture containing
only 0.5 gm. of taurocholate it is not yet half dead, its
resistance being much higher than in the other mixtures.*®

The result serves as a striking confirmation of the
idea that antagonistic relations can be predicted, to a
considerable extent at least, by ascertaining the effect

% See Chapter V.
*” At the end of 180 minutes the resistance of the control in sea water

was 100%.

ANTAGONISM 169

upon permeability of each substance taken by itself,
inasmuch as substances which decrease permeability
antagonize those which increase it.

Similar investigations were made upon alkaloids.
Antagonism between salts and alkaloids has been reported

100%

Fic. 72.—Curves showing antagonism between NaCl and Na-taurocholate. The

ordinates represent the net electrical resistance of Laminaria agardhii (expressed as per cent.

of the control in sea water which is taken as 100%). The abscisse represent the amount

of Na-taurocholate added to 1000 c.c. of NaCl 0.52 M. Average of two experiments;
probable error of the mean less than 5% of the mean.

by several authors, the most extensive investigation
being that of Robertson.*

The alkaloids studied were nicotine, caffeine and
cevadine. They were added in varying amounts to NaCl
0.52 M*1, and their effect upon the electrical conductivity
of Laminaria was determined.

The results obtained with nicotine are shown in Fig.

“For the literature see Robertson, T. B. (1906, 1910, pp. 238, 311).
Also Sziics (1912).
“ All the solutions had the same conductivity as sea water.

170 INJURY, RECOVERY, AND DEATH

73. The lower curve shows that after 1814 hours the
resistance of the tissue has dropped to 10% (the death

901%

)

(<)

2 Firs:
18% Hrs.
o)

10% 02 04 ; 06M

Fig. 73.—Curves showing antagonism between NaCl and nicotine. Ordinates re resent

net electrical resistance of Laminaria agardhii (expressed as per cent. of the normal); absciss

represent concentrations of nicotine added to 0.52 M NaCl. The resistance of the control

at 18% hours was 94%. Average of two experiments; probable error of the mean less than
3% of the mean.

point) in 0.52 M NaCl, as well as in 0.52 M NaCl to which
sufficient nicotine has been added to make the concentra-
tion 0.01 M. In NaCl 0.52 M plus nicotine 0.002 M the
resistance has dropped to 49.5% (1.e., the tissue is about '

ANTAGONISM 171

half dead). It is evident that nicotine antagonizes the
action of NaCl by inhibiting the fall of resistance which
occurs in pure NaCl. The upper curve (2 hours) shows
even more pronounced antagonism.

The results with caffeine (Fig. 74) are similar except
that the curve does not fall as rapidly with increasing

807%

O O02 04 06M

Fia. 74.—Curves showing antagonism between NaCl and caffeine. Ordinates represent net

electrical resistance of Laminaria agardhii (expressed as per cent. of the normal); abscissz

represent concentrations of caffeine added to 0.52 M NaCl. The resistance of the control

at 18% hours was 96%. Average of two experiments; probable error of the mean less than
: 5% of the mean.

concentrations of alkaloid. With cevadine (Fig. 75) the
curve falls much more rapidly, the maximum being in the
neighborhood of 0.005 M cevadine sulfate. Here death

172 INJURY, RECOVERY, AND DEATH

is more rapid, the tissue being killed in 18 hours or less,
even in the most favorable solution.
The experiments with cevadine were carried out dur-

807 % SQ)

€0

ss in
%)
Gs
@)
50 NX
6)

i 005 om

Fia. 75.—Curves showing antagonism between NaCl and cevadine sulfate. Ordinates

represent net electrical resistance of Laminaria agardhii (expressed as per cent. of the normal) ;

absciss# represent concentrations of cevadine sulfate added to 0.52 M NaCl. The resistance

of the control at 150 minutes was 100%. Average of two experiments; probable error of the
mean less than 5% of the mean.

ing the day at 15 +2°, and the time curves in the various
solutions follow more or less closely a monomolecular
course. In the case of nicotine and caffeine (where the
experiment ran during the day and the following night)
this is not the case, except in the earlier part of the reac-

ANTAGONISM 173

tion. This is perhaps explained by the fall of tempera-
ture which occurred during the night and retarded the
speed of the process. It should be noted that all the experi-
ments in any set were begun at the same time, so that all
shared equally in the variations of temperature; in
consequence the form of the antagonism curve is not
greatly affected by such variations.

In order to determine whether these alkaloids pro-
duce a decrease in permeability they were added to sea
water. The experiment was not successful in the case of
nicotine, owing to the formation of a visible precipitate,
which was apparently due to the presence of calcium and
magnesium in the sea water. In the case of caffeine (0.01
to 0.04 M) and of cevadine sulfate*? (0.0006 to 0.0025 M)
a distinct decrease in permeability was found (as shown
by the rise in resistance); this was followed by an
increase. In this respect they resemble CaCl, which also
produces a decrease in permeability when added to
sea water. |

~The idea that substances which have opposite effects
on permeability can antagonize each other seems to apply
to alkaloids as well as to salts.

The question arises whether the decrease of conduc-
tivity and the antagonistic action produced by organic
substances are of the same nature as those produced by
salts. Are they, in terms of the theory outlined above,
due to an increase in the thickness of the layer of M at the
surface of the cell? The writer is not prepared to answer
this question at present, but there is no reason to suppose
that their effects may not differ from those produced by

“This is regarded as two molecules of cevadine united to one molecule

of H,SO,. It was purchased from Merck under the name of veratrine
sulfate (C;.H,,.NO,)..H,SO,. Cf. Osterhout (1919.D).

174 INJURY, RECOVERY, AND DEATH

salts. We may assume that they may act upon the
hypothetical substance M, and increase its resistance by
changing properties other than the thickness of the layer.

In order to ascertain whether these results have gen-

Per cent
100

ae) ee
Nact 100 85 65 38 O
CaCl O 15 35 62 100

Fig. 76.—Curves showing antagonism (after an exposure of 24 hours) between NaCl 0.52
M and CaCh 0.278 M in Laminaria agardhii (upper curve) and Rhodymenia palmata (lower
curve). The ordinates denote net electrical resistance. The abscisse denote molecular
proportions of the solutions (all the solutions having the conductivity of sea water). Thus
NaCl 85, CaCh 15 signifies a mixture of 75 ¢.c. NaCl 0.52 M+ 25c.c. CaCk 0.278 M in which
the molecular proportions of Na to Ca are as 85to15. Temperature 17.5°+5°C. During
the 24 hours the resistance of Laminaria in sea water remained practically unaltered while
that of Rhodymenia fell to 84.5%. Average of six experiments. Probable error of the
mean less than 5.2% of the mean.

eral validity, experiments were made upon other plants
and upon animals. In general the outcome (as far as the
experiments have gone) is similar to what has been
described for Laminaria. Thus antagonism between
NaCl and CaCl, was observed in the cases of Ulva (sea
lettuce), Rhodymenia (dulse) and Zostera (eel grass).**

As was to be expected, the most favorable proportions
were not always exactly the same for the different plants.

“QOsterhout (1919, A).

ANTAGONISM 175

Thus it was found that in the case of Rhodymenia it
required more Ca to antagonize Na than it did in the case
of Laminaria. It was also observed that in the case of
Rhodymenia (Fig. 76) the antagonism was not so great
as in Laminaria and this appears to be correlated with
the fact that less decrease of permeability is produced
by Ca in Rhodymenia. In other words, the effect
of such a substance as Ca upon permeability not
only indicates what substances it will antagonize but
also, to some degree at least, the amount of antagonism.

It may be added that Rhodymenia affords an interest-
ing confirmation of the value of the electrical method
in measuring antagonism, since the plants begin to change
color soon after injury occurs. It was found that the
relative rates of death as indicated by color changes in
NaCl, CaCl,, and the various mixtures, correspond with
the results obtained by determining conductivity.

Antagonism between NaCl and CaCl, was also
observed in the case of frog skin.*4

Shearer (1919) in an experiment on bacteria finds
that NaCl and KCl decrease, and that CaCl, increases
the resistance, but that a mixture of these (Ringer’s
solution) in the proper proportions preserves the
normal resistance.

Thus far, we have confined ourselves to the consid- .
eration of antagonism among kations. Numerous cases
of this are known, but the search for similar relations
among anions has achieved little result. Some cases have
been described by Loeb*® and Miss Moore (1901, 1902).
Lipman and his associates** have reported antagonistic

. action of anions as the result of studies on bacteria in

“Osterhout (1919, C).

“Loeb, J. (1905, 1912, A, B) and literature there cited.

“Lipman, C. B. (1912-13, 1914). Lipman, C. B., and Burgess, P.
S. (1914, 1914-1915).

176 INJURY, RECOVERY, AND DEATH

which salts were added to the soil, but it is very difficult
to separate the effects of the added salts from those

100
\°

80
o
5)
3 70 ;
Q
® A
ba
ee OU
o ba
g =
130 x
S
A, 3B
40 : xC
x
D
30
aE

20
ig th tines 25 45 65 85 105
Fira. 77.—Curves showing the resistance of Laminaria agardhii in 1.1 M sodium acetate, in
0.36 M sodium sulfate, and in mixtures of both: A in equal parts (by volume) of acetate and
sulfate; B in acetate 75, sulfate 25; C in acetate; D in acetate 25, sulfate 75; E in sulfate.
Ordinates represent net electrical resistance (expressed as per cent. of the original resistance in
sea water which is taken as 100%). Each point represents the average of ten experiments:
probable error of the mean less than 5% of the mean.

of salts already present in the soil. Miyake (1913) found
some antagonism among anions in studying the growth of
rice. Fenn (1918) has called attention to the fact that this

kind of antagonism is commonly met with in experiments
on gelatin.

ANTAGONISM 177

Using electrical conductivity as a criterion, Raber
(1920) has found well marked antagonism between

100
Min.
es
: ane
= 80 a
~
Q
WY
gy
oj
e——
Oo
i \evase-necam OF
©
oO
q,
av)
AY
45
‘ ‘i 65
Awe 6
S405
20
Acetate 100 75 50 25 0

Sulfate 0 29 50 79 100

Fie. 78.—Antagonism curves showing the net electrical resistance of Laminaria agardhii
in 1.1 M sodium acetate, in 0.36 M sodium sulfate, and in mixtures of both. Ordinates repre-
sent resistance (expressed as per cent. of the original resistance in sea water which is taken
as 100%); abscisse represent volumetric proportions of the two salts. The dotted line
connecting the ends of each curve shows the approximate additive effect; the vertical dis-
tance of the curve above this dotted line may be regarded as a measure of antagonism.

Na-acetate and Na.SO, in experiments on Laminaria, as
shown in Fig. 77.
On placing tissue in the pure acetate we observe that
at the end of 134 hours, the resistance has fallen to about
12

178 INJURY, RECOVERY, AND DEATH

40% of the original and in the pure sulfate it has fallen
to about 25% of the original, while in the mixture com-
posed of equal volumes of the solution of each salt, the
resistance has fallen only to about 60%. If no antagon-

NaCl 100% 75% 50%, 25%

%
NaCit 00% 25% 50% 75% 100%

Fia. 79.—Increased toxicity shown by curves of the electrical resistance of Laminaria
agardhii in NaCl 0.52 M, Na-citrate 0.58 M (approximate) and in mixtures of these (the
proportions, representing c.c. of the component solutions, are indicated on the abscissee.
Curve A, observed values, after an exposure of 15 minutes to the solution, Curve B values.
expected on the supposition that neither salt affects the action of the other (additive effect).
The increase of toxicity is measured by the vertical distance between the curves.

readings were made at 23° C. or corrected to this temperature. Each observed point pre-
sents the average of 10 experiments: probable error of the mean less than 10% of the mean.

ism were present, the resistance in the mixture should
drop to about 35% (additive effect).

Fig. 78 shows the antagonism curves after various
intervals, using resistance for ordinates and salt propor-
tions as abscisse. Here the antagonism is clearly evident.

Similar experiments with NaCl and Na-citrate, by

ANTAGONISM 179

Raber (1917) gave quite the opposite result. In this case
a distinet increase of toxicity occurs on mixing the com-
ponents. This is evident from Fig. 79. Similar results
were obtained when Na-citrate was combined with Nal,
NaSCN, NaNO, or Na,SQ,.

Cases which show increased toxicity (as judged by
other criteria) have been reported by Lipman‘? and
by Loeb.*®

It may be of interest to call attention to certain
phenomena in non-living matter which bear at least a
superficial resemblance to some of the facts dis-
cussed above.

In the course of experiments on Laminaria, the writer
frequently observed that fronds kept in NaCl become
softer,#® but that in CaCl,, and in LaCl,, they become
harder. The changes in viscosity are so great as to
suggest that they are fully capable of explaining the
fall of electrical resistance which occurs when tissue
is placed in NaCl and the rise of resistance which occurs
in CaCl, and LaCl, (which is always followed by a fall
of resistance).

In the hope of throwing some light upon this process,
sections of tissue were observed in CaCl. under the
microscope. It was then seen that after a time the proto-
plasm assumed a coagulated appearance: it seemed
obvious that the process which increased the viscosity
might produce a coagulation of the protoplasm or some
other change in its structure whereby it became
more permeable.

This conception led the writer to expect decreased
resistance in tissues placed in NaCl (because of decreased

“Lipman, C. B. (1909, 1912).

*Loeb, J. (1911, A; 1912, B; 1916, D).

“© The cell walls not only soften, but eventually go partly into solution.
Of. Hansteen-Cranner, B. (1910, 1914); Lillie, R. S. (1921).

180 INJURY, RECOVERY, AND DEATH

viscosity) while in CaCl, we should expect to find
increased resistance (due to increased viscosity) followed
by a fall of resistance (due to coagulation or other struc-
tural change in the protoplasm).

It soon became apparent that there were several
serious objections to this conception. The most impor-
tant of these may be briefly stated as follows :°°

1. If to a solution of NaCl we add CaCl, until the
increase of viscosity produced by one salt is just balanced
by the decrease produced by the other, the resistance
should remain stationary. This is not the case: there
is always a fall, or a rise followed by a fall, of resistance.

2. If more CaCl, be added there should be a rise of
resistance: this should after a while become stationary,
provided there is not enough CaCl, to produce the coagu-
lation or other structural change which decreases
the resistance. This does not occur: the tissue never
maintains its increased resistance, but shows a fall of
resistance which begins soon after the maximum
is reached. |

3. If still more CaCl, be added, so as to produce the
coagulation or other structural change which decreases
resistance, we should expect to find in all cases the same
viscosity (and consequently the same maximum of resist-
ance) just before the fall begins. Still further increase
of CaCl, would only hasten this process without changing
the maximum. This does not correspond with the facts.
The maximum steadily rises as the proportion of CaCl,
increases, so that the greatest maximum is found in
pure CaCl.,.

4. If the fall of resistance in CaCl, is due to coagula-
tion, or to some other structural change, it might be
expected to be irreversible almost from the start; but this

“Of. Osterhout (1916, B).

ANTAGONISM 181

is not the case. Only when it has proceeded a good way
toward the death point, does it become irreversible. On
the other hand, the fall in NaCl (due to liquefaction)
might be expected to be reversible at every stage. But it
ceases to be wholly reversible after it has proceeded
one sixth of the way (or less) to the death point.

5. Since the changes in viscosity occur in dead as
well as in living tissue we should expect to find in both
cases similar changes in resistance. It is found that in
tissue which has been killed in such a manner as not to
alter the properties of the cell wall, the decrease in vis-
eosity in NaCl produces no appreciable effect on resis-
tance. Hven when the process goes so far that the tissue
is reduced to a very soft jelly, there is little or no change
in resistance.*? The hardening in CaCl, produces some
rise in resistance, but it is much too small to account
for the great changes which occur in living tissue.

It might be supposed that the reason that no change
in resistance occurs in dead tissue is because the hard-
ening and softening do not proceed as far as in living
plants, but this is not the case. Moreover, it is found that
the decrease of viscosity in NaCl is accompanied by
absorption of water, while the increase of viscosity in
CaCl, is accompanied by loss of water, and these pro-
cesses take place in the same way in living and dead tissue.

It would seem that these and other important objec-
tions must be removed before we can accept the idea that
changes in permeability are determined by changes
in viscosity.®”

These objections apply to the theory advanced by

In a liquid a change of viscosity alters the resistance, but this is
not necessarily the case in a gel. Where a gelatin gel is converted to
a sol, the change in resistance is very slight.

* It would appear that the term viscosity is loosely applied to a variety
of phenomena which may be produced in different ways.

182 INJURY, RECOVERY, AND DEATH

Spaeth (1916) to account for variations in permeability
under the influence of salts.

The great variations in the electrical resistance pro-
duced in living protoplasm by the action of salts seem
to the writer to depend on the fact that living protoplasm
is in a state of dynamic equilibrium so that the —
material of which it is composed is. constantly chang-
ing. This constant change is due to a succession of
chemical processes which may be easily influenced so
as to produce great changes in electrical resistance ©
which appear to become irreversible if carried beyond
a certain point.**

In dead protoplasm, as in gelatin, such processes do
not occur, or at least they go on much more slowly. As
a result we cannot expect such great variations in electri-
eal conductivity. If we wish to imitate these it would
seem advisable to work with systems, which, like living
protoplasm, are in dynamic equilibrium.

Clowes (1918), states that he has prepared emulsions
of oil in soap, which change their electrical resistance
under the influence of NaCl and CaCl., in a manner
similar to that observed in Laminaria. It remains to be
seen whether this parallel extends to the effect of
other substances.

The writer obtained similar results some years ago,"
with the shells of the Horse Chestnut (Aesculus) which
had been killed by boiling or by soaking for 24 hours in 5%
formaldehyde. He was not able to convince himself, how-
ever, that the factors involved here are the same as in
living protoplasm.

8 This apparent reversibility finds a ready explanation on the theory
of successive reactions. See page 121.

* A brief account of these was given at the Boston meeting of the
American Physiological Society, in 1915.

ANTAGONISM 183

The writer has experimented with a great variety
of materials in order to determine whether it is possible
to imitate by means of non-living materials, the change
in permeability found in living cells. In some cases
membranes have been found which show an increase of
conductivity when transferred from sea water to NaCl
and a decrease when transferred from sea water to CaCl,
or LaCl,.°> But in no case was the alteration great enough,
nor produced by a sufficient variety of substances, to
justify the author in concluding that the effects were
really the same as those found in living material. The
relatively small changes found in dead material,
in so far as they are due to the cell walls (or intercel-
lular substance), must in the living conditions be
superimposed on the changes due to the activities of
the protoplasm.

Until we succeed in finding a membrane (or other
static system) which imitates qualitatively and quanti-
tatively the permeability of the living protoplasm, the
author is inclined to regard a dynamic equilibrium
as essential.

% The solutions of NaCl, CaCl, and LaCl, had the same conductivity
as sea water. When transferring from sea water to another solution a
temporary rise or fall may occur which is due to diffusion. See page 28.

CHAPTER V.
ANESTHESIA.

In order to ascertain the effect of anesthetics on con-
ductivity, experiments were performed with ether,
chloroform, chloral hydrate and alcohol.2, Subsequently
alkaloids were employed.®

The method may be illustrated by the following
experiment with ether. Tissue was transferred from sea
water to a mixture consisting of 990 c.c. sea water + 10 ¢.c.
ether + 5 ¢.c. sea water which had been concentrated by
evaporation until its conductivity was about double that
of ordinary sea water. This mixture contained approx-
imately 1% by volume of ether (= .099 M) and had the
conductivity of sea water. In 10 minutes the resistance
had risen to 113.4%,1 but, in 10 minutes more it had fallen
to 109.4%. It continued to fall until it had reached 98.8%,
after which it fell very slowly (at about the same rate as
the control). The fact that it fell below the starting
point is not necessarily to be attributed to any injury,
but rather to the fact that the exaporation of the ether
increases the conductivity of the sea water, which is
contained in the apparatus, and in the cell walls between
the protoplasmic masses. The results of the experi-
ment are shown in Fig. 80.

In order to see how the evaporation of the ether

*Since ether, chloroform, and alcohol deteriorate on standing, espe-
cially when in contact with metal or with cork stoppers, special care
must be taken to obtain pure reagents. Those used were Kahlbaum’s
or Squibb’s. Cf. Baskerville (1913).

Cf. Osterhout (1913, A; 1916, A).

Of. Osterhout (1919, D).

* All readings were made at 18° C. or corrected to this temperature.

184

ANESTHESIA 185

from the solution influenced the result, another experi-
ment was performed in which the solution was renewed
every 5 minutes during the first 60 minutes, and there-
after every 15 minutes. In this way the concentration of
ether was kept more nearly constant. It was then found
that the resistance rose as before, but did not fall during

A Sea Water
B Ether 1% (= .099M)

solution not renewed.
C » 1% solution renewed.

110

“i ‘

Oo _

100 o-A
B

ag 150 MINUTES 500

Fia. 80.—Curve A shows the net electrical resistance of Laminaria agardhii in sea water; B

in sea water containing 1% ether by volume (.099 M) from which the ether was allowed to

evaporate in an open dish; C in the same mixture in which the concentration of ether was
maintained by frequent renewal.

the first 80 minutes, and after this fell very slowly, so
that after 300 minutes it was still 80 ohms above that of
the control. At this point, the experiment was discon-
tinued. The results are shown in Fig. 80.

In order to see whether the effect of the anesthetic
could be quickly reversed, some tissue was kept in sea
water containing 0.099 M ether for 50 minutes (the
solution being renewed every 5 minutes). During this
time the resistance rose to 113.7%. It was then placed
in sea water. At the end of 10 minutes the resistance
had fallen to 100%. It was then left in sea water contain-
ing 0.099 M ether (the solution being renewed every 15
minutes). The resistance promptly rose to 113.7%, and

186 INJURY, RECOVERY, AND DEATH

remained there for an hour; 240 minutes later, when the
experiment was discontinued, the resistance was 111.4%.
The results are shown in Fig. 81.

The effect of higher concentrations of ether was next
investigated. Tissue was placed in a mixture of 970 c.c.
sea water + 30 cc. ether +15 c.c. of concentrated sea

94-96 il ae
110 B
I
% ; i
\ J
\ ! x Ether 1%
100 Aiur lee © Ea hatte 296%

90 Bee oe oC

O 200 MINUTES 400.
Fia. 81.—Curve A shows the net electrical resistance of Laminaria agardhii in sea water,
Curve B, unbroken line, in sea water containing 1% ether (.099 M), the solution being fre-

quently renewed, broken line in sea water; Curve C, unbroken line in sea water containing
2.96% ether (0.293 M), broken line, in sea water.

water, which was added to make the conductivity of the
mixture equal to that of sea water. The concentration of
the ether was therefore 2.96% by volume (= 0.293 M). In
the course of 10 minutes the resistance rose to 112% ; dur-
ing the next 10 minutes it fell to 105.3% ; it continued to
fall rapidly during the next 40 minutes, reaching 89.5% at
the end of this period. The tissue was then placed in sea
water; in the next 10 minutes, the resistance fell to 87%..
This fall in resistance was doubtless due to the continued
action of the ether, which required time to diffuse out of
the tissue. During the next 10 minutes, there was a rise

ANESTHESIA 187

of 2.5%, which was probably due, either wholly or in
part, to the fact that the resistance of the sea water was
greater than that of the mixture from which the ether
had partly evaporated. During the next 400 minutes
no rise occurred. The results are shown.in Fig. 81.

This outcome is very significant, for it shows that
the increase of permeability produced by ether is not
reversible, while, as we have seen, the decrease of
permeability is easily reversed. Since the essential
characteristic of an anesthetic is the reversibility of its
action, we must conclude that anesthesia is associated
with the reversible decrease of permeability and not with
the irreversible increase of permeability.

In view of the importance of this result the experi-
ment was repeated many times, the fall of resistance
(before placing in sea water) varying from 6 to 25%, but
always with practically the same result. On placing in
sea water there were sometimes irregular fluctuations
(amounting to 5% or less) but no recovery.

This result is the more striking inasmuch as material
of which the resistance has fallen as much as 5 to 10% in
NaCl recovers completely when placed in sea water,
and may even undergo this treatment daily for several
days in succession without injury.®

The fall of resistance below the normal may be taken
as a measure of the toxicity. The toxicity increases with
the concentration, and it should be noted that it is greatly
decreased if the material is allowed to stand in an open
dish, owing to the evaporation of the ether. If the
‘material be placed in a closed jar, oxvgen must be sup-
plied. The other alternative, frequent renewal of the
solution, is usually preferable.

° See page 82.

188 INJURY, RECOVERY, AND DEATH

A series of investigations on chloroform gave similar
results, the chief difference being that chloroform is
much more toxic, and that the concentration necessary
for long continued decrease of permeability is much
lower, being about 0.05% by volume (or 0.064 M). This
is evident from Fig. 82, which shows the results of an
experiment with a mixture containing 999.5 ce. sea water
+ 0.5 ¢c.c. chloroform + 0.25 ¢.c. concentrated sea water
(this mixture had the same conductivity as sea water).
In this experiment the solution was renewed every 5 min-
utes during the first 80 minutes, and every 15 minutes
thereafter.

If we increase the concentration of chloroform to
0.1% by volume (— 0.0128 M), the result is quite similar
to that obtained with 0.293 M ether. This is shown in
Fig. 82, which gives the results of an experiment contain-
ing 999 c.c. sea water + 1 c.c. chloroform + 0.5 ¢.c. concen-
trated sea water (this mixture had the conductivity of
sea water).® The solution was renewed every 5 minutes
during the first 80 minutes, after which it was kept in
sea water. There is no indication of recovery after the
tissue is replaced in sea water.

Experiments with chloral hydrate gave results very
similar to those obtained with chloroform, the corres-
ponding effects being produced in both cases by
approximately the same percentage concentrations,’
that is, chloral hydrate 0.1% (—0.006 M) acts similarly
to chloroform 0.1% by volume (= 0.0128 M).

*Stiles and Jérgensen (1914) report a decrease of resistance as the
result of exposure to chloroform. See also Waller, A. D. (1919).

™No effort was made to find the exact percentages which are required
to produce given effects, as this was not the primary object of the investi-
gation. The actual concentration of chloral hydrate may have been
somewhat lower than those given, owing to the presence of water in the
chloral hydrate.

ANESTHESIA 189

The experiments with alcohol lead to somewhat differ-
ent results. In the first place, aleohol is not so toxic as
ether, chloroform, or chloral hydrate, and _ higher
concentrations must be used to produce the same effects
on permeability. In sea water containing alcohol 0.051 M,
or 2.955% by volume, (the solution being renewed every

x ee _ %
a %

Oo 100 200 300
MINUTES

Fic. 82.—Curve B shows the net electrical resistance of Laminaria agardhii in sea water;
Curve A in sea water containing 0.05% chloroform, Curve C placed for 80 minutes
in sea water containing 0.1% of chloroform and then put back into sea water.

15 minutes) the results were much the same as in 0.099 M
ether (the solution being renewed every 5 minutes),
except that the rise in resistance took place more slowly,
sometimes occupying 30 minutes or more. It was found
that 0.2385 M, or 13.875% by volume, is decidedly toxic.

An interesting feature of the results with alcohol is
that the increase of permeability is reversible. If the
increase be carried too far it is not reversible (or at

190 INJURY, RECOVERY, AND DEATH

least recovery is incomplete); in the first experiments
this condition was unintentionally realized and led the
writer to suppose that alcohol behaves like ether. The
course of a typical experiment is shown in Fig. 83. The
tissue was first placed in a mixture containing 970 c.c.
sea water + 30 ¢.c. Squibb’s absolute alcohol + about 16

110 x Aleohol 0.269%
o ” 13.875 %
% uy O SeaWater.

100 @------ Q@------

Ta Ree 1
8) 160 200 300
MINUTES

Fic. 83.—Curves showing the net electrical resistance of Laminaria agardhii placed for 40
minutes in sea water containing 0.269% ethyl alcohol, then in 13.875% for 20 minutes and then
put back into sea water.

e.c. of concentrated sea water. The mixture had the
conductivity of sea water; the concentration of the
aleohol was 0.051 M (2.96% by volume). The net resist-
ance rose to 110% in the course of 40 minutes. The tissue
was then placed in sea water containing 0.2385 M alcohol
(13.875% by volume) ; and in the course of 20 minutes the
resistance fell to 87.6%. The tissue was then placed in
sea water and the resistance again rose to 100%.

The facts that recovery occurs in alcohol, and that
irregular fluctuations are often observed in experiments
on recovery from ether, suggest that the difference

ANESTHESIA 191

between the behavior of alcohol and the other anesthetics
investigated may be only one of degree. It is probable
that there is some recovery in ether, chloroform, and
chloral hydrate, but that it is so slight and so transitory
as to be difficult to detect.

It is evident that suitable concentrations of anesthetics
produce a marked decrease of permeability. This con-
dition may be maintained for a long time if the concentra-
tion is not too high; with higher concentrations the
period is shortened and may become so short as to be
observed with difficulty. This decrease of permeability
can be easily and quickly reversed by replacing the tissue
in sea water. It does not seem to produce any injury
if the concentration is not too high. The relative con-
centrations necessary to produce this result, correspond
closely with those required to produce anesthesia, being
least for chloral hydrate and greatest for alcohol.

On the other hand, the increase of permeability,
(except in the case of alcohol, within certain limits) pro-
duces permanent injury and is not reversible. It cannot
be regarded, therefore, as the characteristic effect of the
anesthetic. The characteristic effect must be regarded as
in some way connected with decrease of permeability.’

*The amount depends somewhat on the condition of the material.
Material in poor condition generally shows less rise in resistance than
good material.

*Winterstein (1916) says that these experiments are not convincing
because the anesthetic may act on the interior of the cell rather than on
the surface of the protoplasm. This objection can hardly apply, since the
interior of the cell is filled with cell sap: this is surrounded by a thin
layer of protoplasm (see page 197). If the anesthetic decreased the con-
ductivity of the cell sap to any marked degree, this effect would be observed
in the material immediately after death: this, however, is not the case;
if any rise in resistance occurs in dead tissue it is much less than in
living tissue. Loewe, (1913) states that anesthetics decrease the con-
ductivity of artificial lipoid membranes. See also Moore and Roaf (1905).

192 INJURY, RECOVERY, AND DEATH

It is easy to see how a decrease of permeability to
ions must hinder the production and the transmission
of stimuli in so far as these are dependent on the move-
ment of ions in the tissues, and there is abundant
evidence that stimulation is always accompanied by such
movements of ions in the protoplasm. It seems clear,
therefore, that a decrease of permeability may result in
the decrease of irritability, which is the characteristic
effect of an anesthetic.?°

These investigations are of interest in view of the
fact that a number of writers hold the view that anesthet-
ics increase permeability, while others believe that
anesthetics bring about a decrease of permeability.’! It
appeared desirable to clear up this uncertainty as a
necessary step toward a satisfactory. theory of anesthesia.

In order to see whether these facts are generally true,
the scope of the investigation was widened to include a
variety of material. Similar results were obtained in
experiments on frog skin,!? but the effect was much more
striking. The increase of resistance was greater and
occurred with lower concentrations.1* With respect to
recovery, the same difference was found between alcohol,
on the one hand, and ether, chloroform, and chloral
hydrate on the other.

“It might be expected on this basis that substances which decrease
permeability, such as Ca, La, etc. would act as anesthetics, To what
extent this is the case must be decided by future investigation.

“Of. Hiber (1914) pp. 466, 597; Lillie (1912, A, B; 1913, A, B;
1916, 1918); Lepeschkin (1911); Ruhland (1912, A); Katz (1918),
Weinstein (1916).

%¥For technique see page 33.

4% Of. Osterhout (1919, 0).

ANESTHESIA 193

Experiments were also made" to determine the effects:
of ether on a variety of plants. An increase of resistance
(followed by a decrease) was observed in Laminaria and
Ulva. In Rhodymenia ether (2.5, 3, 5 and 5.5% by
volume), and alcohol (1, 3.5, 7, 8% by volume) added to
sea water produced little or no rise. This is not surprising
in view of the fact that these substances always produce
less rise in Laminaria than does Ca and that even Ca
produces very little rise in Rhodymema. In respect
to recovery from the injury caused by these sub-
stances, Rhodymenia agrees with Laminaria in that
recovery is practically complete in alcohol (if the fall
in resistance has not gone too far), but is almost entirely
absent in ether and chloroform.

While the writer has found no records of similar
experiments made by other investigators, it may be
desirable to refer briefly to the work of Joel on the con-
ductivity of red blood corpuscles. When red blood cor-
puscles are repeatedly washed in an isotonic solution of
cane sugar and allowed to stand in this solution the
conductivity of the suspension gradually increases. This
is due in part to the exosmosis of electrolytes (which
increases the conductivity of the solution) and probably
in part to the fact that the permeability of the corpuscles
to ions increases. The experiments of Joel,!® show
that this increase in conductivity can be hindered
by the addition of ‘‘indifferent’’ narcotics (at certain
concentrations).

In order to determine whether alkaloids decrease the

* Of. Osterhout (1919, A).
*% Joel, A. (1915).

194 INJURY, RECOVERY, AND DEATH

conductivity of Laminaria, experiments were made by
adding small amounts of nicotine, caffeine and cevadine
sulfate to sea water (and then making the solution the
same conductivity as sea water). The experiment was
not successful in the case of nicotine, owing to the for-
mation of a visible precipitate, which was apparently due
to the presence of calcium and ‘magnesium in the sea
water. In the case of caffeine (0.01 to 0.04 M) and of
cevadine sulfate!® (0.0006 to 0.0025 7) a distinct decrease
in permeability was found !(as shown by the rise in
resistance) ; this was followed by an increase.

78 This is regarded as two molecules of cevadine united to one molecule
of H.SO,. It was purchased from Merck under the name of veratrine
sulfate (Ca,H,NO,) ..H,SO,.

CHAPTER VI.
CONDUCTIVITY AND PERMEABILITY

It is well known that death is accompanied by an
increase of permeability. Thus a slice of red beet kept
in water will live for a long time without giving off pig-
ment, but as soon as it is killed the color begins to escape
from the cells. In this case the coloring matter is dis-
solved in the large central vacuole which fills the interior
of the cell. In order to escape, it must pass out through
the layer of protoplasm which surrounds the vacuole.
As long as the protoplasm remains in its normal condi-
tion it is impermeable to the dissolved pigment, but as
soon as death occurs it become freely permeable and the
color escapes.

We meet the same condition if we study the penetra-
tion of substances from without. It is a matter of
common observation that cells may resist the penetration
of certain dyes as long as they ‘are alive, but absorb
them readily as soon as they are killed.

' The increase in permeability which accompanies
death is paralleled in a striking manner by a simultaneous
increase in electrical conductivity.1. This suggests that

1 An apparent exception to this statement is found in two articles by
Galeotti (1901, 1903) who states that death produces an increase in the
electrical resistance of muscle, kidney, etc., followed by a decrease. He
suggests that the increase is due to the fixation of electrolytes by the
tissues. In Galeotti’s experiments the tissues were not immersed in solu-
tions and in consequence the electrodes had to be applied directly to the
surfaces of the tissue. It is possible that his results were due in part to
faulty technique (bubbles of gas readily form in dying tissue, increasing
the resistance). The matter requires further investigation.

Kodis (1901) whose technique seems to be decidedly preferable to
that of Galeotti, (see page 21) found that dead frog muscle had less
electrical resistance than living. The writer has confirmed this, using the
method employed by Kodis.

195

196 INJURY, RECOVERY, AND DEATH

the two phenomena may be closely connected. If this is
the case it may be possible to use electrical conductivity
as a measure of permeability.

Let us consider this from the standpoint of the per-
meability of protoplasm to salts.

OOLOUW

Fig. 84.—Cross section of Monostroma latissima (X 450).

When an electrical current passes from a salt solu-
tion into a living cell, ions must enter the protoplasm.”
An increase in the permeability of the protoplasm to ions

Fia. 85.—Cross section of Ulva lactuca, var. latissima (sea lettuce). (X 450).

must decrease its electrical resistance, and vice versa.
The electrical resistance of the protoplasm may there-
fore be regarded as a measure of its permeability to ions.

?In this connection it should be noted that experiments have been
made with direct currents. Of. Stiles and Jérgensen (1914).

CONDUCTIVITY AND PERMEABILITY — 197

If we attempt to measure the electrical resistance of
the protoplasm we must first consider the structure of
the tissue.

Very useful for experiments on tissues are plants
which form membranes consisting of a single (Fig. 84)
or a double layer (Fig. 85) of cells. In measuring
the conductivity of these plants we obtain much the
same results as with the more complex tissues of
Rhodymenia (Fig. 86) and Lamimana (Fig. 87). We

OT LOIS

COS seed

Fig. 86.—Cross section of Rhodymenia palmata (dulse). (X 150).

may therefore conclude that the complexity of structure
is not a factor of importance in the interpretation of the
results. Asa matter of fact in the case of Laminaria the
resistance appears to be due chiefly to the rounded cells
lying at and below the surface, while the elongated cells
which occupy the center of frond have large spaces
between them through which the current can easily pass.
If we consider the structure of the individual cells,
we find that in Laminaria (as in the other plants employed
in the experiments of the writer) the protoplasm of

198 INJURY, RECOVERY, AND DEATH

each cell forms a thin layer, which surrounds a large
central vacuole filled with cell sap. When tissue (either
with or without previous treatment with liquid air) is
ground with powdered quartz (so as.to open the cells) and
a little sea water is added, and the juice is subsequently
expressed, it is found to have a little higher conductivity
than sea water. Since this expressed juice consists to a

POCOTDO DBO OOH SHSANABOOOVOE DODANNNFOOOUAE
TOPO ISS COOOL)
Done COL ORAZ TOS
e awasg: 262 CDS

Ss: SOOry
AQ as TICE <\.
PPR AON I Set

OOOO OOOO00CH

Fia. 87.—Cross section of Laminaria agardhii. (X 150).

considerable extent of cell sap we may conclude that the
‘conductivity of the sap does not differ very much from
that of sea water. The fact that the conductivity is
higher, may be due in part to evaporation during the
manipulation. When the juice is obtained by merely

CONDUCTIVITY AND PERMEABILITY = 199

heating the plants to 100° C in a tightly stoppered bottle
(without addition of sea water) the conductivity equals
that of sea water. |

This is confirmed by some observations on Valonia.
This marine alga forms a large, multinucleate cell consist-
ing of a cell wall, within which is a delicate layer of
protoplasm, forming a sac which encloses a very large
central vauole. By drying the exterior and pricking the
cell, the sap can be made to spurt out and may then be
collected for examination. It was found by Wodehouse
(1917) that the sap of uninjured cells gives little or no
test for SO,: the contents of each cell were accordingly
tested by this method by Dr. Crozier, who kindly collected
the sap for these experiments, and rejected those which
contained more than a minimum amount of SO,. Deter-
minations of the electrical conductivity of the sap, by the
writer, showed that it was not much higher (in no case
more than 20%) than that of the surrounding sea water.

Since the cell sap of Laminaria has about the same
electrical resistance as the solution which bathes the cell,
it is evident that if the electrical resistance of the cell
increases when itis transferred from sea water to another
solution of the same conductivity, the change must be due
to an increase in the resistance of the thin layer of proto-
plasm which bounds the cell. This has led the writer to
assume that the resistance is proportional to a substance,
M, at the surface of the cell; if M forms a layer at the sur-
face, it is obvious that an increase in the thickness of this
layer will increase the resistance, and vice versa. It is
therefore assumed that the resistance depends upon the
amount of M which is present in the surface.*

® See page 57.

200 INJURY, RECOVERY, AND DEATH

Since the protoplasmic masses (cells) are separated
from each other by thin layers of substance (cell wall) a
part of the current goes through the protoplasm and
another part passes between the protoplasmic masses,
in the substance of the cell wall. Consequently when we
employ the electrical method we must ascertain whether
we are investigating the permeability of the protoplasm
or merely that of the cell wall.

Obviously the best method of attacking this problem
is to kill the tissue by such means (e. g., partial drying,
heating to 35°C., weak alcohol, etc.) as cannot alter the
cell wall, and then investigate its behavior under the
influence of various reagents. We find that all of these
methods produce the same result. After death the tissue”
no longer shows the changes in resistance which are
observed when living tissue is subjected to the influence
of reagents. It is therefore evident that the changes are
due to the living protoplasm.

The cell wall appears in all cases to have practically
the same conductivity as the surrounding solution. If
we subject living tissue to solutions of the same conduc-
tivity, but of different chemical composition, the resist-
ance of the cell wall remains unaltered, while that of the
protoplasm undergoes great variations. If, for example, |
living tissue is placed in a solution of NaCl or CaCl, (of
the same conductivity as sea water) its behavior differs.
In NaCl the resistance falls; in CaCl,, it rapidly rises and
later falls toa minimum. We infer that the permeability

*As previously explained (Cf. Osterhout, 1918, C), a part of the
current must pass through the protoplasm; this is shown by the fact that
CaCl, (which has little effect on the resistance of the cell wall) raises the

resistance of the tissue and that the temperature coefficient of electrical
conductivity is not the same for dead as for living tissue.

CONDUCTIVITY AND PERMEABILITY = 201

of the protoplasm increases in NaCl; and that in CaCl,
there is a decrease followed by an increase.

« ‘This is in complete agreement with results obtained
when permeability is measured by such methods as
plasmolysis, specific gravity, tissue tension, exosmosis,
diffusion through living tissue and direct determination
of penetrating substances.

Fig. 88.—A vegetable cell showing plasmolysis. At left, normal; in the center, plasmolyzed;
at the right, nearly recovered.

In order to show the bearing of these measurements
on the problem, the methods employed will be
briefly described.

The measurement of osmotic pressure by means of
plasmolysis depends upon the fact that when the osmotic
pressure within a cell is greater than that of the surround-
ing solution, water is absorbed. In the case of plant cells,
there is usually a central vacuole, the contents of which
exert pressure against the protoplasmic sac which sur-
rounds the vacuole: in consequence the protoplasmic sac
is pressed against the cell wall. If, however, the osmotic
pressure of the external solution exceeds that of the
vacuole, water is withdrawn from the cell and the proto-
plasmic sac contracts. This contraction is called
plasmolysis (Fig. 88). At the moment when contraction
begins, the osmotic pressure of the solution within the
cell is regarded as slightly less than that of the external

202 INJURY, RECOVERY, AND DEATH

solution. We therefore obtain in this manner an approxi-
mate measure of the osmotic pressure of the cell.®
Strictly speaking, plasmolysis measures the osmotic
pressure within the cell without telling us anything
regarding the penetration of substances into the proto-
plasm. We may, nevertheless, learn something about
permeability by this method. When, for example, we
find that a substance (such as alcohol) fails to produce
as much plasmolysis as is expected, we infer that this
substance penetrates the cell so rapidly, as to partially
offset its own osmotic effect. Let us suppose that the
osmotic pressure within the cell is 10 atmospheres. On
placing the cell in a solution of alcohol, whose osmotic
pressure is 11 atmospheres, plasmolysis might be ex-
pected to occur, but if there is an immediate penetration
of aleohol, which raises the osmotic pressure within the
cell to 11 atmospheres, no plasmolysis will take place.
On placing the cell in a solution of alcohol strong
enough to cause plasmolysis we should expect the alcohol
to penetrate until the osmotic pressure of alcohol is the
same inside and outside of the cell. When this has
occurred, the osmotic pressure in the cell will be equal to

°There are a number of disturbing factors which interfere with such
measurements. Among these may be mentioned:

1. The plasmolyzing agent may have a toxic action and may
alter the permeability of the cell.

2. Plasmolysis may produce mechanical injury due to the
tearing of the outer layer of protoplasm.

3. Exosmosis of dissolved substances may occur during ex-
posure to the reagent.

4. The time necessary to produce plasmolysis is an important
factor which is frequently overlooked.

5. The shrinkage of the protoplasm away from the cell wall
is preceded by a diminution of volume of the entire cell. In some
cases, where the cellulose wall is considerably stretched by the

internal osmotic pressure, this may be of considerable importance. *»,

CONDUCTIVITY AND PERMEABILITY — 293

the original osmotic pressure, plus the increased pres-
sure due to the withdrawal of water (thus increasing the
concentration) plus the osmotic pressure due to the pene-
tration of alcohol. The total osmotic pressure will there-
fore be greater than that of the external solution and
water will accordingly be absorbed by the cell. The result
will be that the plasmolyzed cell will recover, and return
to its original expanded condition.® The time required for
recovery is usually regarded as approximately propor-
tional to the rate of penetration of the alcohol.

In this way we may obtain a rough measure
of permeability.

This method was employed by Overton (1895) in
the well-known studies on permeability which gave rise to
the lipoid theory. He came to the conclusion that there
is rapid penetration of alcohol, and of many other organic
substances, but that inorganic salts do not penetrate. He
attributed this to their insolubility in the lipoid layer, by
which he supposed the cell to be surrounded. The writer
repeated his experiments with salts, and came to the
opposite conclusion.?. It was found in experiments with
salts of NH,, Cs, Rb, Na, K, Li, Mg, Ca, Sr and Al, that
the protoplast which is plasmolyzed and left in the solu-
tion expands again to its normal size, showing that all
these salts penetrate the protoplasm.

The following experiment will serve to illustrate the
procedure. Filaments of Spirogyra were placed in 0.4
NaCl solution. Within two minutes the protoplasts of
the cells were so far plasmolyzed that they no longer
touched the end walls of the cells. Several of these were

*Since the effect of the alcohol outside the cell is exactly balanced
‘by that within the cell the final effect is the same as that of placing the
cell in pure water.

TOf. Osterhout (1911).

204 + INJURY, RECOVERY, AND DEATH

accurately sketched with the camera lucida and kept
under continuous observation. In the course of ten min-
utes, several of them had begun to expand and in thirty
minutes all had expanded so as to completely fill their
respective cells. To avoid the injurious action of the salt,
the filaments were then transferred to 0.18 M CaCl, solu-
tion, and this was gradually diluted until its osmotic
pressure was not greater than that of tap water. The
cells were then transferred to tap water. They were
examined the next day and found to be alive. On being
placed in 0.4 M NaCl, they were plasmolyzed and they
afterward expanded as before.

Certain facts may be worthy of mention which tend
to obscure these results and which may have caused
them to be overlooked.

In the experiment just described, the cells were trans-
ferred to a favorable solution as soon as expansion was
complete. If this precaution be neglected and the cells be
allowed to remain in the solution of NaCl, the injurious
action of the salt soon causes the protoplast to shrink.
In salts which are more toxic than NaCl, this contraction
may be more rapid and more pronounced. This shrink-
age, which may be called false plasmolysis,® may also be
produced by very weak (hypotonic) solutions and has
nothing to do with plasmolysis, but may simulate it in
very misleading fashion. If the cells are not continuously
observed, but only examined at intervals, the expansion
of the protoplast may easily be overlooked, and the sub-
sequent shrinkage may be easily mistaken for plasmolysis.
It is therefore desirable to keep the same individual
cell under observation during the entire course of
the experiment.

* Of. Osterhout (1908, 1913, C).

CONDUCTIVITY AND PERMEABILITY 905

It was found that recovery from plasmolysis is much
slower in CaCl, than in NaCl, indicating that the latter
penetrates more readily. This is in harmony with the
results of measuring electrical conductivity.

Other experiments indicate that the penetration of
NaCl is more rapid in a solution of pure NaCl than it is
in a mixture containing NaCl plus CaCl,, in such propor-
tions as to make a balanced solution. This is also in agree-
ment with the results of measurements on electrical
conductivity.

Another method of measuring permeability was used
by Loeb,°® who has shown that eggs of Fundulus will float
for a time in NaCl 3M, but not, as a rule longer than 3
hours. ‘‘Before sinking they lose water, as is indicated
by the collapse of the membrane and the shrinkage of the
yolk sac. Probably some NaCl enters the egg.’’ In CaCl,
1.25 M, they sink in about half an hour. If, however,
they are placed in a mixture of 50 ec. NaCl 3 M+ 2 ce.
CaCl, 1.25 M they float for three days or longer. Loeb
interprets this as showing that the membrane of the
Fundulus egg is practically impermeable to water and to
salts in a physiologically balanced solution. But when
transferred to a hypertonic non-balanced solution the
natural impermeability of the membrane is gradually lost,
so that water diffuses out of the egg and its specific
eravity is increased to such an extent that it sinks. This
action, and likewise the entrance of NaCl into the egg,
is prevented by the addition of small amounts of CaCl,.

A very interesting series of experiments was made by
S. C. Brooks, using the following methods: (1) diffusion
through living tissue, (2) exosmosis, (3) change of curva-
ture of strips of tissue.

®*Loeb (1912, C, D).

206 INJURY, RECOVERY, AND DEATH

In the first of these methods’ different solutions were
placed on opposite sides of a piece of tissue. The appa-
ratus used is shown in Fig. 89. The diffusion of salts
through the tissue was then measured. In the first
experiments, a solution of NaCl 0.52 M
was placed on one side, NaCl 0.26 M was
placed on the other: CaCl, 0.28 M and
0.14 M, as well as sea water and sea
water diluted with one volume of dis-
tilled water were employed in the same
way. As the stronger solution diffused
into the weaker the increase in the elec-
trical conductance of the latter was
measured. In Fig. 90, the rate am
change of the electrical conductance is
plotted against time. It will be observed
that NaCl diffuses through the tissue

Fic. 89.—Apparatus for

testing the rate of diff-
usion of salts through
living tissue. It consists
of two glass cells A and B
(the former is provided
with a cover, G) separat-
ed by a layer of living
tissue of Laminaria, E,
which is sealed to the
glass cells by a mixture
of vaseline and beeswax,
F. The lower cell is pro-
vided with a piece of
rubber tubing, C, and a
pinch cock, D.

more rapidly than sea water, while CaCl,
at first diffuses more slowly than sea
water and then more rapidly:

If dead tissue be substituted for
living, we find that the rate of diffusion
is very much more rapid in all cases,
and that all the solutions pass through
at about the same rate of speed.

These results are precisely what would be expected in
view of the results of the electrical experiments.
In the second of these methods," tissues of the dande-

lion (Taraxacum officinale) were placed for a short time
in a salt solution and the rate at which salts subsequently
diffused out of the cell was measured by placing the

” Of. Brooks, 8S. C, (1917, B).
1 Of, Brooks, 8. C. (1916, A).

CONDUCTIVITY AND PERMEABILITY — 207

tissue in distilled water and observing the increase in
conductance of the latter. The results are shown in Fig.
91, in which the ordinates denote the amount of exosmosis.
It will be seen that there is more exosmosis from tissue

a Change in Conductance ze
z
Sc

‘ 2 y] 4 6 8 10 12 Nours.
Fie. 90.—The ordinates denote the rate of diffusion of NaCl, CaCl and sea water through

living tissue of Laminaria. NaCl diffuses more rapidly than sea water, while CaCl at first
diffuses more slowly, then more rapidly than sea water.

which has been previously treated with NaCl 0.22 M than
from tissue treated with a balanced solution.!”
Tissue treated with CaCl, 0.17 M showed less exos-
mosis than that treated with the balanced solution.
These results are also in harmony with the experi-
ments on electrical conductivity.

“For exosmosis of pigments of Ithodymenia in relation to electrical
resistance see Osterhout (1919, C).

8 The concentrations of NaCl, CaCl, and of the balanced solution were
chosen so as to be approximately isotonic with the tissue.

208 INJURY, RECOVERY, AND DEATH

In the third method employed by Brooks," strips of
the peduncle of the dandelion were placed in hypertonic
solutions and the rate of penetration of the salt into the
protoplasm was calculated from the rate at which the

Conductance, Mhos X10?

1 2 3 4 Nour).
Fic. 91.—The ordinates denote the amount of exosmosis into distilled water from living
tissue of Taraxacum officinale which had been previously treated with various solutions and
then placed in distilled water. Treatment with NaCl produces more exosmosis than treat-
ment with a balanced solution, while treatment with CaCl: produces less exosmosis. The
experiments indicate that the measured exosmosis is largely due to salts present in the cells
before the application of reagents. If it were caused by the reagents (by diffusion out of the
cell walls and intercellular spaces) it would be greater in CaCl than in NaCl.

strips recovered their normal shape after being curved
by the action of hypertonic solution (the strips remaining
in the solution during recovery). This gives the same
kind of information as plasmolysis, but avoids the most
serious errors of that method.

In Fig. 92, the rate of recovery is plotted against time.
The more rapid rate of recovery in NaCl, KNO,, and
NH,Cl shows that in these salts penetration is more
rapid than in the balanced solution. On the other hand,

4 Of. Brooks, 8. C. (1916, B).

CONDUCTIVITY AND PERMEABILITY — 9209

the slower rate of recovery in MgCl,, CaCl,, Al,Cl, and
Ce (NO,), shows that in these salts penetration is slower
than in the balanced solution.

This is in complete agreement with the results of the

electrical experiments (even the order of penetration of

the salts of Mg, Ca, Al and Ce, is the same as that found
by the electrical method).

Fra. 92.—The ordinates denote rate of recovery from the effect of hypertonic solutions of

strips of the peduncle of the Taraxacum officinale. The rapid rate of recovery in NaCl,

KNOs: and NH.Cl shows rapid penetration of these salts. On the other hand in MgClh,
CaCh, AleCls and Ce (NOs)s penetration is slower than in the balanced solution.

Recently the writer has had an opportunity to test
some of these conclusions by direct determinations of
the penetration of substances into the cell sap. The
advantages of this are obvious, when we consider the
uncertainty of most indirect methods.

Some investigators have sought to avoid the diffi-
culties of indirect methods by making analyses of tissues,
but these must obviously include too much intercellular

material to be satisfactory. Analysis of the solutions
14

210 INJURY, RECOVERY, AND DEATH

in which the tissue lies is open to the objection that sub-
stances are absorbed on the surfaces of the cells and in
the spaces between them, and it is impossible to say what
actually penetrates the protoplasm. ’

Others have attempted to analyze the cell sap. The
most favorable cells for this purpose are those of plants,
since they contain, as a rule, vacuoles filled with sap. In
general, the method has been to crush the tissues and
express the sap, but it is obvious that this procedure
involves too many possibilities of error.’

The entrance of dyes has been extensively investi-
gated, but this method is beset by many pitfalls,?*® and the
results hitherto obtained are confusing.

** Among these may be mentioned contamination of the cell sap by
substances present in the cell walls or intercellular spaces and chemical
reaction between the cell sap and the crushed protoplasm and cell walls.
The degree of pressure used in expressing has a marked influence on the
concentration of the sap. Cf. Mameli (1908), Dixon and Atkins (1913)
Gortner and Harris (1914), Gortner, Lawrence and Harris (1916). The
investigation of blood and other body fluids is open to the objection that
we do not know to what extent substances penetrate between the cells
in reaching these fluids. In many cases penetration into these fluids
seems to present very special features.

“To a great extent the coloration of the cell by a dye shows the
extent to which the dye can combine with substances within the cell
rather than the rate at which the dye penetrates. Thus many cells contain
substances which combine with methylene blue so that it becomes far
more concentrated within the cell than in the external solution (Pfeffer
1900, 1:96). Unless the cell has this power it often fails to appear colored
even though it may contain the dye in the same concentration in which
it exists outside. In such cases it may sometimes be detected by plas-
molyzing the cell and thus concentrating the dye. A further complication
is that a cell may appear to have taken the dye into its interior when in
reality only the surface or the cell wall is stained. There are many
other difficulties, which need not be discussed here, such as toxic action
of the dye and changes in the dye (decolorization etc.) as it enters the
cell. A very serious objection to this method is that it does not give
quantitative results. A review of the literature will be found in
Héber (1914).

CONDUCTIVITY AND PERMEABILITY 211

The penetration of acids and alkalies has been studied
by employing organisms containing natural indicators,
or by introducing indicators into the cell." In some cases
the penetrating substance may cause a visible precipitate
with the cell: this is especially the case with alkaloids.'®
The absorption of Ca’® has been detected by observing
the formation of crystals of Ca-oxalate within the cell.
It is evident, however, that these methods have but
limited application and that in many cases they are open
to the objection that the penetrating substance injures
the cell.

The penetration of a substance may often be demon-
strated by observing its effect upon metabolism, but this
method is unsatisfactory from a quantitative standpoint.
Some investigators state that substances may produce
effects on metabolism by their action at the surface, with-
out actually penetrating the cell.

It is evident that the most satisfactory method would
be to place the cell in a solution containing the substance
whose penetration was to be investigated, and, after a
definite time of exposure, to obtain the cell sap without
contamination and test it for the presence of the sub-
stance. Experiments of this sort have apparently not
been carried out, though interesting results have been
obtained by Meyer,?° Hansen,?1 Wodehouse,?? and
Crozier,?* by comparing the cell sap of Valonia (which
can be obtained without contamination) with the sea

7 For the literature see Haas, A. R. C. (1916, B), Crozier (1916, A,
B; 1918).

* For the literature see Czapek (1911).

* Osterhout (1909).

Meyer (1891).

7 Hansen (1893).

= Wodehouse (1917).

% Crozier (1919).

212 INJURY, RECOVERY, AND DEATH

water. Janse?! found that filaments of Spirogyra which
had been kept for a time in a solution of KNO,, gave a
test for NO, after being rinsed and caused to burst in
a solution of diphenylamine. It is evident that in his
method, there is serious risk of contamination by the
substance in and upon the cell wall (or between the cell
wall and the protoplasm).

In order to avoid this difficulty, the writer has
employed the cells of a species of Nitella, some
of which reach a length of four or five inches and a diam-
eter of a thirty-second of an inch.

Within the cell wall the protoplasm forms a thin
layer in which are imbedded the chlorophyll bodies.
Inside this layer of protoplasm is the large central
vacuole filled with cell sap. It is possible to obtain the
cell sap without contamination in various ways. The
writer has made use of the following methods: The
cells are placed for the desired length of time in a solu-
tion containing the substance whose penetration is to be
tested. They are removed, washed in running tap water
(followed in many cases by distilled water) and dried by
means of filter paper. The cells are so large and turgid,
that this manipulation presents no difficulty. A cell is
then placed on a piece of glass or filter paper and pierced
with the point of a capillary tube (which has been drawn
out to a fine tip). The cell sap is drawn up into the tube.
(by capillary action) quite free from protoplasm or chlo-
roplasts.2> Another method, which is preferable in many
cases, is to suspend the cell by a pair of forceps attached
to the upper end, cut off the lower end and bring it in
contact with a glass slide, and then grasp the upper end

* Janse (1887).
* During the manipulation care should be taken to prevent the sap
from running out of the cell and coming in contact with its outer surface.

CONDUCTIVITY AND PERMEABILITY 213

gently with another pair of forceps, which is slowly moved
downwards. The cell sap then flows out on to the glass
slide in contact with the drop. By uniting the drops from
a number of cells it is possible to get a sufficient amount
for qualitative chemical tests, and in many cases approx-
imate quantitative results may be obtained.

Since in previous investigations the writer had
employed indirect methods of testing permeability, it
was of considerable interest to compare these results with
those obtained by direct tests of the cell sap. An investi-
gation was therefore made in which the permeability of
Nitella was tested by the direct method, as well as by
determinations of plasmolysis and of electrical conduc-
tivity. This may be illustrated by a series of experi-
ments?® with NaNO, and Ca(NQ,)>.

Experiments on plasmolysis were carried out by
placing the cells in a hypertonic solution and observing
the time required to recover from plasmolysis (without
removing the cells from the solution) on the assumption
that the more rapid the recovery, the more rapid is the
penetration of the salt.

In these experiments the smaller cells near the tip
of the plant were largely employed. They were observed
in Syracuse watch glasses or placed on glass slides and
covered with large cover glasses, the edges of which were
sealed with vaseline.

Plasmolysis may be harmful to many cells, even in a
balanced solution,27 while in an unbalanced solution there

* All the experiments were performed at about 19° C. All the
solutions were approximately neutral. Cf. Osterhout (1922).

* For this reason penetration may be more rapid than would otherwise
be the case. In order to reduce toxicity chemically pure salts should be
used and the water should be distilled from quartz (or from glass which

has been in use for some time), using cotton plugs in place of rubber
or cork stoppers and rejecting the first and last parts of the distillate.

214 INJURY, RECOVERY, AND DEATH

may be the additional injury due to the toxic action of
the salt. For this reason many cells which would recover
if very slightly plasmolyzed may not do so if plasmolyzed
more strongly, since recovery may require so long a time
that the process of injury gets the upper hand.

It was found that recovery was more rapid in NaNO,
than in a balanced solution of NaNO, + Ca(NO,), or in
Ca(NO,;). alone. Similar experiments with RbCl, CsCl,
and CaCl, gave the same result. This indicates that in a
solution of NaNO,, NaCl, RbCl, or CsCl penetration is
more rapid than in Ca(NO,),, CaCl,, or in a bal-
anced solution.

These results agree with those obtained in the study
of Smrogyra.?8

The experiments on conductivity were carried out by
means of the apparatus described on page 34. As it was
desirable to surround the cell by a solution of the same
conductivity as that of the cell sap determinations of the
latter were made by filling a small tube with sap and
inserting an electrode at each end (taking great care to
avoid the inclusion of air bubbles). It was found that
the sap had approximately the conductivity of sea water —
plus three parts of distilled water (this will be called
for convenience 0.25 sea water). The cells were accord-
ingly placed in this for some time before beginning the
determination of the conductivity of the living cell.
Under these circumstances it was assumed that altera-
tions in conductivity during the course of the experiment
must be due (in great part at least) to changes in the
protoplasm, rather than in the cell sap. |
In general, it was found that in 0.25 sea water, the
resistance remained unaltered for a long time, while in
NaNO, of the same conductivity it soon began to fall.

*See page 201.

CONDUCTIVITY AND PERMEABILITY = 215

This harmonizes with the results obtained with Laminaria.
Direct tests of the sap were made by determin-
ing NO,, since it was found that the cells normally
give tests for Na and Ca. The method employed was not
sensitive enough to detect NO, in the sap of the control
cells under any circumstances, so that if a test was
obtained after exposure to a solution containing NQ,, it
must have been due to penetration from without. The
sap was tested by placing it on a glass slide, adding a
drop of a solution of nitron in 10% acetic acid, and observ-
ing it under the microscope. If NO, is present, it may be
recognized by the formation of characteristic crystals.

Cells kept for 24 hours in 100 c.c. NaNO, 0.05 M + 10
e.c. Ca (NO,). 0.05 M gave no test, which shows conclu-
sively that the method is safe as far as contamination by
NO, on the surface is concerned. After 48 hours a test
was obtained. As the cells continued to live in this solu-
tion for 3 weeks (at which time the experiment was
discontinued) and as they appeared normal in every
way, it is evident that the penetration was not the result
of injury.

It is probable that in 24 hours some NO, penetrated
which was not revealed by the test. This, however, is
of no significance in the present investigation which does
not aim to determine the absolute amount of penetration,
but merely to compare the relative penetration in_bal-
anced and unbalanced solutions.

The results of such a comparison are very striking.
After 3 hours in NaNO, 0.05 M a good test was obtained.
The cells had lost some of their turgidity; if left in the
the solution of NaNO, or if transferred to tap water they
subsequently lost all their turgidity, indicating death.
It is therefore evident that this rapid penetration was
accompanied by injury. Similar results were obtained
by Mrs. Brooks (1922) with Li, Ba, and Sr.

216 INJURY, RECOVERY, AND DEATH

It may be remarked that the turgidity of the cells
affords good indication of their condition. It is easily
tested by lifting them partly out of the solution; if
turgid they appear stiff, otherwise they collapse or
appear flaccid. It is, however, necessary to distinguish
between loss of turgidity in isotonic or hypotonic solu-
tions, which indicates injury, and a similar appearance
in hypertonic solutions, which may indicate nothing of
the sort. In the latter case the cell promptly recovers
its turgidity when placed in tap water; in the former
it does not. |

Another criterion of injury is afforded by the appear-
ance of the chlorophyll bodies. In the normal cell they
are arranged in regular rows and are of a clear trans-
parent green color. Where injury occurs they lose their
regular arrangement and the color becomes more opaque.

In 0.05 M Ca (NO,),. the cells live for a week or more:
During the first few days, at least, penetration is not
more rapid (perhaps less so) than in a balanced solution.

Similar results were obtained with other salts.

The outcome of these direct tests is therefore an
unqualified confirmation of the results obtained by the
indirect methods. We find that penetration in injurious
solutions is relatively rapid as compared with penetration
in non-toxic solutions. This corresponds to the fact that
recovery from plasmolysis is more rapid in injurious
solutions as well as to the fact that conductivity increases
in such solutions.

It would therefore seem that we may regard deter-
minations of electrical conductivity, and, in some eases,
of recovery from plasmolysis as reliable means of detect-
ing alterations in permeability. It is, however, desirable
to go further, if possible, and analyze the factors involved
in electrical resistance.

CONDUCTIVITY AND PERMEABILITY 217

If we consider the behavior of the current from this
point of view, it is evident that in the simplest cases, where
the plant is a membrane only one cell thick (as in
Porphyra and Monostroma) and the current passes
through this membrane at right angles to its surface, we
need consider only a single cell and its adjacent cell wall,
as shown in Fig. 93, A. The part of the current which goes
through the protoplasm may be designated as Cp. while
that which traverses the cell may be called Cw.

Experiments show that the resistance of the living
tissue is much greater than that of tissue which has been
carefully killed with all possible precautions to prevent
any alteration of the cell wall2® We therefore feel con-
fident that the conductivity of the living protoplasm is
less than that of the cell wall.

In order to see how the current may distribute itself,
let us suppose the protoplasm to be replaced by a wire,°°
P, as in Fig. 93, B and the cell wall to be replaced by

* Osterhout (1918, C; 1921, D).

*° We might consider the protoplasm to be replaced by two wires,
one of which corresponds to the thin layers of protoplasm which are
traversed by the current in a direction at right angles to their planes,
the other corresponding to the similar layers of protoplasm in each cell
(around the edges of the cell shown in Fig. 93, A) in which the current
flows in the plane of the layer. It is evident, however, that these latter
may be neglected in our calculations since they occupy such exceedingly
small fractions of the cross-section.

If we neglect these we may say that in traversing a cell the current
passes through a thin layer of cell wall and then one of protoplasm (in
both cases at right angles to the plane of the layer), then through the
cell sap, and finally through a layer of cell wall and one of protoplasm
(at right angles to their planes). It is evident that in this case we may
neglect the effect of the cell wall and of the cell sap since their resistance
is very small in comparison with that of the protoplasm and is in
series with it. We may therefore consider the protoplasm to be replaced
by a single wire having a resistance equal to that of the two layers
of protoplasm which are traversed by the current in a direction at right
angles to their planes.

218 INJURY, RECOVERY, AND DEATH

a wire, W. The current flowing between the points X and
Y in the wire P may be called Cp; that in the other wire Cy.
The total current, C, flowing between X and Y will be the
sum of the partial currents, or,

C—Ce tw
We may consider the current (conductance) as equal to
the reciprocal of the resistance and write

ee a, |
R Rp' Rw ,

in which F is the total resistance between X and Y, Rp is
P 3

Fig. 93
the resistance of the wire P, and Rw, that of W. Apply-
ing this equation to Laminaria*! (and expressing the
resistance in the usual way as the per cent. of the normal)
we may calculate the values of Cw, Cp, Rw, and Re.
Under normal conditions in sea water, the resistance
is taken as 100 and therefore C — 1-100, but in certain

** So far we have considered only the simplest case, when the plant is
only one cell thick. But it is evident that these considerations also
apply when several membranes are placed together, forming a mass
comparable to the tissue of Laminaria. The only difference is in that
case the current would traverse a very thin layer of cell wall in passing
from one protoplasmic mass to the next, so that what we have spoken
of as the resistance of the protoplasm would be composed in part of
the resistance of these cell walls. When the protoplasm is dead the
total resistance is only 10.29 and the resistance of these cell walls must
be only a small fraction of this. Consequently their resistance in the
living tissue of Laminaria is undoubtedly less than 1 when that of the
protoplasm is 140. The resistance of these cell walls may therefore

be neglected. a

CONDUCTIVITY AND PERMEABILITY = 219

solutions (having the same conductivity as sea water)
the resistance may rise to 300 or more; and in this case
C would equal 1 — 3800 = .0038 (or less), and since some
of it must flow in the protoplasm the amount which trav-
erses the cell wall must be less than this. We are there-
fore safe in putting it as low as 1 + 350 = .002857.

All the experiments hitherto made indicate that the
eonductivity of the cell wall remains unaltered in spite
of changes in the chemical character of the solution, pro-
vided the conductivity of the solution remains the same.
We may therefore take .002857 as the fixed value of Cw.

Let us now consider what values Cp assumes as the
resistance changes. In sea water we have*? R — 100 and
ant
100
whence Cp= .007143 and Rp—=1 + Cp— 140. In the same
manner we find that when R — 90, Rep— 121.15, and when
H-=10, ip == 10.29.

The changes in resistance thus far discussed have
been treated as though they occurred in sea water; in this
ease the experiments indicate that the conductivity of the
eell sap remains practically constant and hence need not
be taken into account in our calculations. We may now
ask whether this is also the case when the changes in
resistance occur in other solutions. In order to investi-
gate this, experiments were made with solutions of NaCl
and CaCl, (of the same conductivity as sea water). The
tissue was placed in these solutions and removed after
various intervals of exposure. It was cut into small bits
and ground (so as to open the cells) and in some cases

C = .002857 + Cp

“The total conductance of the protoplasm is greater than that of
the cell walls, but the protoplasm occupies a much greater fraction of the
conducting cross-section than the cell walls, so that the actual conduc-
tivity of the protoplasm is much less than that of the cell wall.

220 INJURY, RECOVERY, AND DEATH

the tissue was killed by heat: the conductivity of
the expressed juice was compared with that of sea
water. As no significant difference was found we may
consider that the conductivity of the cell sap does not
change sufficiently in these solutions to alter
our calculations.

Let us now consider the changes in protoplasmic resis-
tance which occur in toxic solutions. When tissue is
placed in NaCl 0.52 M ‘the net resistance falls rapidly.
The death curve may be obtained by means of the
formula*?

K —KaT —KwmT aH
Resistance — 2700 - € —e + 90e KuT +10
Ku—Ka

in which T is the time of exposure, K4 and Kym are con-
stants, and e is the basis of natural logarithms. We find ©
by means of this formula that in a solution of NaCl 0.52 M
(for which K4= .018 and K y—.540) the net resistance
after 10 minutes is 87.76% of the normal; after 30 minutes
it is 64.26, and after 60 minutes it is 41.62. Knowing the
net resistance we can calculate the protoplasmic resist-
ance, as explained above. After 10 minutes the proto-
plasmic resistance is 117.12% (corresponding to the net
resistance of 87.76%). Since it is desirable to express all
resistances as per cent. of the resistance in sea water we
divide 117.12 by 140 (which is the protoplasmic resistance
in sea water) and obtain 83.66%. Proceeding in this way
we find that after 30 minutes the protoplasmic resistance
is 56.22% and after 60 minutes 33.74%. In order to fit the
formula to these values we must change the constants, put-
ing K sp—0.0234 (in place of Ka— 0.018) and K yp== 9.702
(in place of Ku—0.54). It is therefore evident that in

“Yor the explanation of this formula see" toe 61.

CONDUCTIVITY AND PERMEABILITY = 221

changing from net resistance to protoplasmic resistance
we merely shift the value of the constants. The question
arises whether this affects the general conclusions drawn
from the study of net resistance. In order to decide this
question the constants for CaCl, and for various mixtures
of NaCl and CaCl, were ascertained; these are given in
Table XIT.**

TABLE XII.
Velocity Constants at 15°C.

mee | pastes ae Ky Kap K yp
per cent. per cent.
0 0 0.018 0.540 0.0234 0.702
1.41 12.5 0.000222 0.00666 0.000293 0.00878

2.44 20.0 0.000187 0.00546 0.000237 0.00708
4.76 33.33 0.000245 0.00590 0.00032 0.007136

15.0 63.73 0.000364 0.0073 0.0005035 | 0.00855

35.0 84.34 0.000481 0.00859 0.000678 0.00955

62.0 94.22 0.00053 0.009 0.000761 0.00989
100.0 100.0 0.0018 0.0295 0.002685 0.0323

There are two points of principal importance in the
consideration of these constants: (1) It has been shown*?
that the value of Ka Km increases regularly as the per
cent. of CaCl, in the surface of the cell increases. That
this is also true in the case of protoplasmic resistance is
evident from Fig. 94. (2) It was also pointed out that as
the per cent. of CaCl, in the solution decreases from 62 to
1.41% the value of Ky first decreases (reaching a mini-
mum at 4.76%) and then increases. It was found that the

* These are approximate values, obtained graphically. The con-
stants of the curves of protoplasmic resistance are designated as K,>p
(corresponding to K,) and K,,,,(corresponding to Kj,). The curves of
protoplasmic resistance may show less inhibition at the start than those
of net resistance.

% See page 151.

222 INJURY, RECOVERY, AND DEATH

amount of decrease corresponds to the amount of a hypo-
thetical salt compound (Na,XCa). This is also true in
the case of protoplasmic resistance, as shown in Fig. 95.°°

It would therefore appear that we arrive at the same
conclusions whether we study net resistance or protoplas-
mic resistance. When the solution is changed the con-

x
"

Increase in Ka Ky
02 o-= li " K ap +K mp

@) OR Rem) a |
¢) 12.50 20 33.33 63.75 84.34 94.22 100
CaClz in surface
Fic. 94.—Ordinates represent the increase in value of K , + Ky,and K4,,)+K yp. In each
case the value given represents the increase over the corresponding value in the solution con-

taining 1.41% CaCl (the per cent. in the surface being 12.5). | Abscissee represent per cent.
of CaCl: in the surface. In order to facilitate comparison the values of K 4p + K yp have

been divided by 1.685.

stants change in a corresponding manner in both cases,
the only difference being in their absolute values, but it
is evident that in this case differences in absolute values
are of no importance.

It should be emphasized that this general conclusion
would remain valid in case it should be found that the
values given here for Cp and Cw are incorrect. There
seems to be no doubt that the value of Cw is constant
under the conditions of these experiments and as long as

* A rough calculation shows that this is also true of K ypand Kop
(corresponding to the K and Kp mentioned on page 98).

CONDUCTIVITY AND PERMEABILITY = 2238

0009

0005

14476 15
244 Per cent of CaCl, in solution

Fig. 95.—Ordinates represent the amount of NasXCa and also the decrease in the value of
K y,/\ and of Kypo-as compared with the corresponding value in the solution containing

62%CaCh. Abscisse represent per cent. of CaCl2 in the solution. In order to facilitate
comparison the values of K ,, have been multiplied by 0.251 and those of K 4,p by 0.321.

this is true the conclusions drawn from the study of net
resistance apply also to protoplasmic resistance.
The results of this investigation may be summarized

224 INJURY, RECOVERY, AND DEATH

as follows: An electrical current passing through a liv-
ing plant flows partly through the cell wall and partly
through the protoplasm. The relative amounts of these
two portions of the current can be ecaleulated. The
outcome of such calculations shows that the conclusions
drawn from the study of the resis f the tissue_as
a whole apply also to the resistance of the protoplasm,
and consequently to the permeability of the protoplasm
_to ions. ey

If these conclusions are sound it is evident that per-
meability may be measured with considerable accuracy.
Measurements under a variety of conditions indicate that
marked fluctuations of permeability are possible, and,
when their duration is brief, no permanent injury results.
It is obvious that the effect of such fluctuations on
metabolism may be of great importance.

Some writers®’ seem to think that under normal con-
ditions the cell is quite impermeable to salts. This is at va-
riance with the results obtained from measurements of
permeability by the method outlined above. If the net
resistance of the tissue under normal circumstances is
taken as 100, we find that in certain solutions (having the
conductivity of sea water) it may rise to 300. The proto-
plasmic resistance under normal conditions may be taken
as 140. When the net resistance of the tissue rises to only
250, the protoplasmic resistance increases to 874.89, a gain
of 524.92%.

It is therefore evident that the permeability of the cell
is by no means at a minimum under normal conditions.
This conclusion is borne out by the results of experiments
on plasmolysis** carried out by the writer as well as by

* Of, Osterhout (1915, C).
* Of. Osterhout (1913, B).

CONDUCTIVITY AND PERMEABILITY = 295

the investigations of Brooks cited above. It would seem
that it is well founded, since if the cell were impermeable,
it could not obtain the salts necessary for its existence.*®
It is, of course, true that the electrical resistance of the
cell is much higher when alive than when dead, as is shown
by the work of Roth (1897), Bugarsky and Tang] (1897),
Stewart (1897), and Woelfel (1908), on blood corpuscles,
that of McClendon (1910), and Gray (1913, 1916) on sea
urchin eggs, that of Shearer (1919) on bacteria, as well as
the results of the writer.

The seat of this higher resistance might be sought in
the interior of the cell, or at the surface. Plant cells offer
especially good material for this sort of investigation,
since in most cases the protoplasm forms a thin layer
surrounding a large central vacuole filled with cell sap. It
has been shown above, (pp. 198 and 199) that, in the cases
investigated, the sap has a conductivity which does not
differ greatly from that of the external solution. It
would therefore seem that the cause of the high resistance
is to be sought in or near the surface. Hober (1914, pp. 383,
442) has reached this conclusion as the result of experi-
ments on red blood corpuscles and muscles. He employed
two methods for measuring the conductivity of the
interior of the cell. The first depends on the fact
that a conducting body increases the capacity of a
condenser when inserted between the plates. The
second is based upon the fact that a conductor placed
in the centre of a coil of wire diminishes the strength of
an alternating current in the coil. Using these methods,
Hober finds that the conductivity is higher than when it
is measured in the usual way (in which the current passes
through the cell). He therefore concludes that the surface

* Of. Osterhout (1916, #).
15

226 INJURY, RECOVERY, AND DEATH

has a higher resistance than the interior. It should be
noted, however, that in these methods the experimental
errors are so great that the results must be accepted
with caution.

The view that the surface layer of the protoplasm is
less permeable than the interior has long been current.
Such a layer need not be a visible membrane:?° on the
contrary, it need only have the thickness of a single layer‘
of molecules. This surface layer is commonly spoken of as
the plasma membrane, but the writer prefers the term cell
surface (since a morphologically distinct membrane is not
necessary in order to ensure selective permeability). If
it is not necessarily a visible structure, we may ask what
evidence there is for its existence and whether it is any-
thing more than a convenient fiction.

It is easy to understand how the idea of the plasma
membrane was accepted by botanists. In many eases
the interior of the plant cell is filled with cell sap around
which the protoplasm forms a layer, so thin as to be almost
invisible under the microscope, except under the most
favorable conditions. In such a case the whole of the
protoplasm might be looked upon as constituting the
plasma membrane.

When the layer of protoplasm is thicker, it may he
shown that there are differences between the permeability
of its inner and outer surfaces. de Vries found that

“A layer of liquid may serve as in the experiment of Nernst (1904)
where a layer of water is interposed between pure ether and benzene dis-
solved in ether; such a layer is permeable to ether, but not to benzene.
In the same way a layer of air may be employed, e.g., the layer of air
over an aqueous solution of cane sugar is permeable to water molecules,
but not to sugar. If we place under a bell jar two beakers, one con-
taining pure water and the other sugar solution, the water will pass
over, in the form of vapor, into the sugar solution.

“Of. Langmuir, I. (1917).

CONDUCTIVITY AND PERMEABILITY — 227

certain dyes penetrated thet? outer surface much more
readily than the inner.*? The experiments of the writer
show a difference in the two surfaces and emphasize the
conception that the permeability of the protoplasm is not
alike in all its parts.*4

The real question is whether a special layer exists at
the outermost surface of the cell which admits some sub-
stances, but not others. Furthermore, is it possible that
substances which penetrate the outermost layer with
difficulty can spread freely throughout the cell when they
have passed the outer layer?

It is a well-known fact that substances, which, like
protoplasm, contain a considerable amount of protein
readily form films at their surfaces when brought into
contact with liquid.t? By means of the ultra-microscope
Gaidukov** observed a differentiated film at the surface
of the cell. Such films or membranes have been shown to
exist in some cells by micro-dissection and there are indi-
cations that they also exist at the surfaces of vacuoles,*7
and of nuclei.*® The surface of Ameba and of some other
protozoa is covered with a thin membrane capable of

“de Vries (1885).

“The objection might be made that the dye cannot penetrate the
inner surface until the protoplasm has become saturated with it) and

this might be confused with a difference in permeability.

“See page 229.

“Niageli (1855) J, pp. 9, 10; Hanstein (1870); Pfeffer (19
Robertson (1908); Héber (1914) p. 65; Harvey (1912, A, B);
(1904); Kiihne (1864); Ramsden (1905); Rosenthal (1901);
(1904); Shorter (1909); Rhode (1906). For a general summary, see
Zangger (1908). }

* Gaidukov (1910); Marinesco (1912); Price (1914). /

“ Seifriz (1918); Chambers (1915, 1917, 1920, 1921); Kite (¥912);
Pfeffer (1900) 1:107; Overton (1895, 1900, 1901, 1902).

* Chambers (1921).

228 INJURY, RECOVERY, AND DEATH

forming wrinkles. What part these films or membranes
play in permeability is not known.

When a cell is crushed, so that drops of protoplasm
are extruded, it is often observed that each drop behaves
as if surrounded with a plasma membrane, and a rupture
is In most cases instantly repaired (as long as the cell
remains in the normal condition). This might be ex-
plained as due to the formation of films upon contact
with liquid.

Kuster (1909; 1910 A, B.) found that when the proto-
plasm of a cell was separated into several pieces by
plasmolysis the parts would fuse if brought together at
once, but if left for a time would no longer do so, indicat-
ing that a change had taken place in the surface film.

Kite (1913) states that a dye which could not pene-
trate the cell was able to spread freely in its interior,
when introduced by a Barber pipette.*®

\ The nature of the cell surface has been the subject of
much dispute. Overton based his view that it is lipoid
in nature on the ground that lipoid-soluble substances
readily penetrate, while those which are not soluble in
lipoid do not enter the cell, and stated that this was
particularly the case with inorganic salts. It was subse-
quently found, however, that cells are permeable to salts,”
and to other substances insoluble in lipoid. He found?!
an apparent confirmation of his theory in the behavior of
dyes. It had been shown by Ehrlich*? that basic dyes are
taken up by nerves and by lipoid substances. Overton
extended this notion to living cells in general and assumed

“Of. Pfeffer (1900) 1:107.
” See page 203.

™ Overton (1900, 1902).

* Ehrlich (1893).

CONDUCTIVITY AND PERMEABILITY = 229

that the penetration of dyes is dependent on their solu-
bility in lipoid. Subsequent investigations have brought
to light so many exceptions to this rule that it can no
longer be regarded as conelusive evidence in favor of
Overton’s views.°®

Overton’s views gained wide support through their
application to the explanation of nareosis. Overton™* and
Meyer” independently arrived at the conclusion that the
more soluble a substance is in lipoid, the more effective it
is as a narcotic. They explained this by saying that the
more soluble the anesthetic 1s in lipoid, the more easily it
penetrates the lipoid membrane.

Although this hypothesis has found wide acceptance,
there are serious objections to it.

If it be true that anesthetics are generally effective in
proportion to their solubility in lipoid,** it does not by any
means follow that the plasma membrane is lipoid. As
we have already seen,” the effectiveness of a dye in color-
ing the cell does not depend on its rate of penetration, but
on its ability to accumulate within the cell by combining
with substances in the protoplasm. If this is also the
case with anesthetics, lipoids in the interior of the cell
may be the determining factor, and there is no necessity
for the assumption of a lipoid membrane.

It is not the desire of the writer to enter into further

SI ee ee nen,

8 Huber (1914) 426 ff. Also p. 645; Kiister (1911) Ruhland (1912,
A, B); Schulemann (1912) ; Goldman (1912); Garmus (1912) ; Robertson
(1908); Kite (1913) ; Ruhland (1909).

* Overton (1901).

8% Meyer (1899).

* There are some substances which act as anesthetics (¢.9., magnesium
salts) which are only slightly soluble in lipoid.

St See page 210.

230 INJURY, RECOVERY, AND DEATH

discussion of the nature of the cell surface.*® Knough has
been said to show that there is considerable evidence that
there is a layer at the surface which is different from the
underlying protoplasm and that some substances pene-
trate it more rapidly than others. It is doubtful, whether
there are many substances to which it ean be regarded as
wholly impermeable. It is, however, able to protect the
metabolism of the cell from various kinds of interference
from without, and to provide for the differentiation of
multicellular organisms by making it possible to keep
various processes separate. The principal advantage
of cell division may consist in providing the semiper-
meable membranes, which make differentiation possible.

A good illustration of this differentiation is seen in
those cases where diverse chemical operations go on in
adjoining cells without mutual interference. In many
plants deeply colored cells are surrounded by colorless
ones, and the soluble coloring matter does not show any
tendency to diffuse into the surrounding cells. We may
even observe that the color is confined to the vacuole of
the cell, and does not diffuse into the surrounding proto-
plasm. In the same way we observe in some plant cells
colored plastids (chromatophores) containing soluble
pigments which do not diffuse out into the cytoplasm. A
cell of this sort is shown in Fig. 96.

In the case of Griffithsia, each of these plastids is _
surrounded by a semipermeable membrane which retains

* Czapek (1914) has suggested that the plasma membrane is com-
posed of soaps. Nathanson (1914) regards it as mosaic of lipoid and
non-lipoid particles. This would not provide an entrance for lipoid-
soluble and lipoid-insoluble substances into the cell-sap unless each ele-
ment of the mosaic extended continuously, without a break, from the

outer surface to the vacuole. For a general summary see Bayliss (1915), _
Hoéber (1914), and McClendon (1917).

CONDUCTIVITY AND PERMEABILITY — 231

the pigment. This can easily be shown by killing the cell,
whereupon the semipermeable membranes are destroyed
and the pigment at once begins to diffuse out. In this
ease, we have to do with variety of semipermeable mem-
branes, such as the plasma membrane, the surfaces of the
plastids, the vacuolar surface,"® and the nuclear surface.
It is to be expected that these surfaces may differ some-
what in permeability. Each of them is in contact with

Fic. 96.—A cell of Griffithsia Bornetiana (in optical section). a, cell wall; 6, protoplasm
c, chromatophore containing chlorophyll and a red pigment (phycoerythrin) which is soluble

in water; e, vacuole filled with cell sap. (Diagrammatic).

a somewhat different environment, and this, as we have
already seen, might produce differences in permeability.
That such differences really exist is indicated by treating
the cells with NH,Cl (neutralized by adding NH,OH)
which is not strong enough to plasmolyze. The vacuolar
surface then contracts while the plasma membrane main-
tains its original position. At the same time the surfaces
of the plastid become permeable and the red pigment
comes out: it cannot, however, pass through the plasma
membrane or the vacuolar surface. We see that all three
sorts of surfaces act differently, and to these we may add

® de Vries (1885) states that certain dyes penetrate the outer sur-
face more easily that the surface of the vacuole. It has been objected
that the dye may combine with the protoplasm and hence cannot penetrate

the vacuole until the protoplasm is saturated. This might cause an
appearance of a difference in permeability.

232 INJURY, RECOVERY, AND DEATH

a fourth, the nuclear surface, which does not agree in
behavior with any of the others.®® It is quite possible
that there are other surfaces within the cell which likewise
differ in their behavior.

If we suppose that these surfaces not only differ among
themselves, but that their permeability fluctuates under
normal circumstances, we shall probably get a fairly cor-
rect picture of the complex relations which obtain in the
cell. This conception is not as simple as that of the
‘reaction chamber’’ hypothesis of Hofmeister,*! but it
agrees more nearly with our present knowledge.*?

The conception that the cell contains a variety of mem-
branes which are capable of alterations in permeability,
is capable of explaining some important phenomena.
Among these may be mentioned certain effects of injury.
It is well known that mechanical injury is followed by
increased respiration:® this may be explained by the
increased permeability of membranes which have pre-
viously kept the oxidizable material from being attacked.
Increased respiration due to chemical agents** might be
explained in the same way.

An illustration of a different reaction is the bitter

Sema set

™ Hofmeister (1891).

“It may be added that while changes in the permeability of internal
membranes may affect the electrical conductivity of cells which are
filled with protoplasm (as in the case of most animal cells) they can
hardly play an important role in cells like those of Laminaria (and most
plant cells) in which the interior of the cell is occupied by a large central
vacuole. In the latter, however, the permeability of the vacuole mem-
brane must be of importance.

% Of. Czapek (1913-20); Tashiro (1917).

* For recent investigations see Brooks, M. M., (1918, 1919, 1920, 1921,
A, B); Gustafson (1918, 1919); Haas (1919, A, B); Irwin (1918, A, B);
Thomas (1918).

CONDUCTIVITY AND PERMEABILITY = 233

injury occurs, and the resulting reaction produces HCN.
Such illustrations might be multiplied indefinitely.

Another important question which may be considered
in this connection is that of mechanical stimulation. The
effects of certain kinds of stimuli can be referred directly
to chemical changes which they produce in the proto-
plasm, but there are other kinds which appear to operate
by physical means only. In the latter category are such
stimuli as contact, mechanical shock and gravitation.
While their action appears at first sight to be purely
mechanical, they are able to produce effects so much
like those of chemical stimuli that it appears prob-
able that in every case their action must involve
chemical changes.

The chief difficulty which confronts a theory of
mechanical stimulation appears to be this: How can purely
physical alterations in the protoplasm give rise to chem-
ical changes? It would seem that a satisfactory solution
of this problem might serve to bring all kinds of stimu-
lation under a common point of view, by showing that a
stimulus acts in every case by the production of
chemical reactions.

The writer has observed when one of the larger cells of
Griffithsia (Fig. 96) is placed under the microscope (with-
out acover glass) and touched near one end (with a needle
or a glass rod or a splinter of wood) a change occurs in
the chromatophores directly beneath the spot which is
touched. The surfaces of the chromatophores in this
region become permeable to the red pigment, which begins
to diffuse out into the surrounding protoplasm. This
change begins soon after. the cell is touched. As the red
pigment diffuses through the protoplasm it soon reaches
neighboring chromatophores and it may then be seen that
their surfaces also become permeable and their pigment

234 INJURY, RECOVERY, AND DEATH

begins to diffuse out. In this way a wave—which may be
compared to a wave of stimulation—progresses along the
cell until the opposite end is reached.

The rate of propagation of this wave corresponds to
that of the diffusion of the pigment. It would seem that
at the point where the cell is touched, pigment, and prob-
ably other substances, are set free, diffuse out and set up
secondary changes as they progress. These changes are
doubtless chemical in nature.

The important question then arises: How does the
contact initiate the outward diffusion of the pigment or
other substances?

It seems to the writer that this may be due to a
mechanical rupture of the surface layer of the chromato-
phore which is either not repaired at all or only very
slowly. Many cases are now known in which the surface
layers of protoplasmic structures behave in this way.®
If, therefore, such structures exist within the cell, it is
evident that any deformation of the protoplasm which is
sufficient to rupture their surface layers, will permit their
contents to diffuse out into the surrounding protoplasm.
A great variety of cellular structures (plastids, vacuoles,
‘‘microsomes,’’ inclusions, ete.), possess surface layers of
ereat delicacy, and it is easy to see how some of these
might be ruptured by even the slightest mechani-
cal disturbance.

It would seem, therefore, that deformation may rup-
ture the surface layers of certain structures and cause
their contents to diffuse out. If the diffusing substances
meet others, from which they were separated by the semi-
permeable surface layer before it was ruptured, it is

*In many cases rupture of the plasma membrane causes the proto-
plasm to disintegrate and mix with the surrounding medium. In other
cases the surface layer is at once reconstituted.

CONDUCTIVITY AND PERMEABILITY = 285

-easy to see that reactions may occur which may produce
the responses characteristic of mechanical stimulation.
The occurrence of such reactions seems probable, since
many cases are known where substances in close juxta-
position are prevented from reacting by the presence of
semipermeable layers: when these layers are destroyed
(by crushing the cells) the reaction at once takes place.
If these processes occur it is evident that purely
physical alterations in the protoplasm can give rise to
chemical changes. Responses to contact and mechanical
stimuli may thus be explained; and since gravitational
stimuli involve deformation of the protoplasm we may
extend this conception to geotropism.*®
In this conception of mechanical stimulation the
essential things are (1) substances which are more or
less completely prevented from reacting by semipermea-
ble surfaces, (2) a deformation of the protoplasm suffi-
cient to produce in some of these surfaces a rupture which
is not at once repaired, (3) a resulting reaction which
produces the characteristic response to the stimulus.
Some authors (particularly Lillie and Hober)® as-
sume that stimulation is always associated with a change
in permeability, whereby the cell surface (which is
“Small (1918) has made experiments on geotropism by inserting
electrodes (a short distance apart) near the tip of the root and measuring
the electrical conductivity before and during stimulation. He states
that when the root is placed at angle to the vertical the resistance of
both the upper and lower sides decreases, but the decrease is less in the
upper side. He interprets this to mean that the permeability of both
sides increases, but the increase is less in the upper side: in consequence
it is more turgid and thus causes the downward bending of the tip.
Owing to the source of error in this method it is difficult to judge of
the value of the results. It seems highly probable that upon changing
position of the root there would be a movement of liquids and gases in

the intercellular spaces which would change the conductivity.
“Lillie (1911, 1913, 1914, A, B); Hiber (1914).

236 INJURY, RECOVERY, AND DEATH

assumed to be permeable in the resting state to kations,
but impermeable to anions)** suddenly becomes permea-
ble to anions, and hence becomes electrically negative.
This hypothesis has been favorably received in some
quarters, but according to Loeb and Beutner,®® such as-
sumptions are unnecessary. The conclusions of Loeb and
Beutner are based on accurate quantitative experiments ~
and in addition they have succeeded in imitating artificially
some of the most important phenomena, both qualitatively —
and quantitatively.

It may be added that the facts mentioned above’? show
that the electric current passes readily through the proto-
plasm. This could not be the case if it were not permeable
to both anions and kations.

For further information, the reader is referred to the
literature quoted, as a detailed discussion lies outside
the scope of the present work.

The facts set forth in this chapter indicate that
changes in permeability may be followed by determining
electrical conductivity. These alterations are evidently
important, since they may affect all the fundamental life-
processes. It has been shown that a study of such changes
by means of electrical measurements makes it possible to
treat such conceptions as vitality, injury, recovery, and
death in a quantitative manner. It also enables us to
predict the behavior of tissues, especially in respect to
injury and recovery, and leads directly to a quantitative
theory of the mechanism of certain fundamental life
processes.

* The idea that the cell surface may be permeable to only one kind
of ions was suggested by Ostwald (1890).

* Loeb (1915, B); Loeb and Beutner (1911, 1912, 1913, A, B, 1914);
Beutner, (1912, A, B; 1913, A, B, O, D, H, 1920).

” See pages 46 and 200.

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237

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and @

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248 INJURY, RECOVERY, AND DEATH

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INDEX

Acid, antagonism of, 163
effect on conductivity, 50, 51
penetration of, 211

Acree, 31

Additive effect, 124 ff

Aisculus, 182

Aleock, 21

Alcohol, effect on conductivity, 189

Alkali, effect on conductivity, 42
penetration of, 211
Alkaloids, antagonism of, 169 ff
effect on conductivity,
169, ff 194
Alum, effect on conductivity, 49
Aluminum, penetration of, 203, 209
Ammonium, penetration of, 203, 208
Ameeba, 227
Anesthetics, effect on conductivity,
184 ff
‘in relation to
theory, 229
Anions, effect on conductivity, 40
Antagonism, 16, 124 ff
among anions, 175 ff
effect on penetration,
205
in relation to permea-
bility, 214
opposite of, 124 ff,
179 ff
Arsenate, effect on conductivity, 40
Autocatalysis, 69

Bacteria, antagonism, 175
effect of acid on the con-
ductivity of, 55
effect of calcium on the
conductivity of, 175
effect of cerium on the
conductivity of, 50
effect of lanthanum on
the conductivity of, 50
effect of potassium on the
conductivity of, 175

lipoid

Bacteria, effect of sodium on the
conductivity of, 175
measurement of conduc-
tivity of, 22
Barium, effect on conductivity, 49
penetration of, 215
Baskerville, 184
Bayliss, 230
Becker, 67
Bell, 31 |
Beutner, 236
Bile salts, see sodium taurocholate
Bitter almond, 232
Brooks, M. M., 215, 232
Brooks, S. C., 159, 205, 206, 208,
225
Bugarsky, 21, 225
Burgess, 176

Cadmium, effect on conductivity, 46
Cesium, penetration of, 203, 214
Caffeine, antagonism of, 171 ff
effect on conductivity,
171 ff, 194
Calcium chloride, penetration of, 206,
209
Calcium, effect on conductivity, 48,
49, 64, 65, 115 ff
effect on growth, 127
effect on viscosity, 179
penetration of, 203, 211,
214, 215, 216
recovery after exposure to,
83, 86, 107 “
Catenary reactions, 57 ff, 75, 98, 122
Cell sap, chemical tests of, 210, 215
conductivity of, 199, 200
Cerium, effect on conductivity, 49
Cerium chloride, penetration of, 209
Cevadine, antagonism of, 171 ff
effect on conductivity,
171 ff, 194
Chambers, 227
Chapman, 31

254 INDEX

Chlorella, 95, 96 Conductivity, as affected by iron, 47

Imeasurement of conduc-
tivity of, 21
Chloride, effect on conductivity, 40
188
Chloroform, effect on conductivity,
188

Clowes, 182
Cobalt, effect on conductivity, 46
Conductivity, as a measure of per-
meability, 17
as affected by
aleohol, 189
as affected by alkali,
42

as affected by alka-
loids, 169 ff, 194

as affected by alum,
49

as affected by anes-
thetics, 184 ff

as affected by anions,
40

as affected by arsen-
ate, 40

as affected by
barium, 49

as affected by cad-
mium, 46

as affected by caf-
feine, 171 ff, 194

as affected by cal-
cium, 48, 49, 64, 65

as affected by
cerium, 49

as affected by ceva-
dine, 171 ff, 194

as affected by
chloral hydrate, 188

as affected by chlo-
rate, 40

as affected by chlo-
roform, 188

as affected by
cobalt, 46

as affected by ether,
184 ff

as affected by
formate, 40

as affected by gela-
tion, 181

as affected by
hydroxylion, 42

as affected by
kations, 45

as affected by lan-
thanum, 47, 49

as affected by mag-
nesium, 49

as affected by man-
ganese, 46, 49

as affected by
molybdate, 40

as affected by
nickel, 46, 49°

as affected by nico-
tine, 169, 194

as affected by
sodium chloride, 59,
62, 92, 102 ff, 178

as affected by
sodium citrate, 178

as affected by
sodium iodide, 178

as affected by
sodium nitrate, 178

as affected by
sodium suphate, 178

as affected by
sodium sulpho-
cyanide, 178

as affected by stron-
tium, 49

as affected by sul-
phate, 40

as affected by
thorium, 47

as affected by tin, 47

as affected by
yttrium, 47

as affected by zinc, 47

in relation to permea-
bility, 17, 195 ff

measurement of, 21-39

of bacteria, 22, 50, 55,
175

of Chlorella, 21

of Huglena, 21

of frog skin, 49, 50,
55, 87, 97, 175, 182

of muscle, 21

of red blood corpus-
cles, 21

INDEX

Conductivity of Nittella, 35, 36
of Rhodymenia, 49, 50,
56, 87, 97, 174, 175,
193, 197
of sea urchin eggs, 23,
50, 225
of Ulwa, 35, 49, 55,
87, 97, 174, 193, 196
of Zostera, 35, 49, 87,
88, 97, 174
temperature coeffi-
cient of, 39
Corpora lutea, 97
Corson-White, 97
Crozier, 199, 211
Czapek, 232

Death, a normal part of life pro-
cesses, 16
monomolecular curve of, 15
Diffusion method of measuring per-
meability, 159, 206 ff
Distilled water, preparation of, 38,
213

Dulse, see Rhodymenia
Dyes, penetration of, 210
Dynamic equilibrium, 183

Eel grass, see Zostera

Ehrlich, 228

Error, probable, 38

Erythrocytes, effect of anesthetics on

conductivity of, 193

Ether, effect on conductivity, 184 ff

Euglena, measurement of conductiv-
ity, 21

Exosmosis as measure of permeabil-

ity, 206 ff, 207

Fenn, 176

Fleischer, M. S., 97

Formate, effect on conductivity, 40

Frog skin, antagonism, 175

effect of acid on the con-
ductivity of, 55

effect of anesthetics on
the conductivity of, 192

3 I

255

Frog skin, effect of calcium on the
conductivity of, 49
effect of lanthanum on
the conductivity of, 49
effect of magnesium on
the conductivity of, 49
effect of potassium on the
conductivity of, 50
effect of sodium on the
conductivity of, 50
partial recovery of, 97
recovery after exposure to
sodium and calcium, 87
Fundulus, 205

Gaidukov, 227

Galeotti, 21, 195

Garmus, 229

Gelation, effect on conductivity, 181

Geotropism, 235

Glucoside, 232

Goldman, 229

Gray, 21, 50, 225

Griffithsia, 230, 233

Growth, effect of calcium chloride
on, 127

Gustafson, 232

Haas, 43, 232

Hall, 31

Hansen, 211

Hansteen-Cranner, 179

Hanstein, 227

Harvey, 227

Henri et Calugareanu, 21

Hibbard, 31

Hober, 21, 79, 192, 225, 227, 229, 230

Hofmeister, 232

Hopkins, 122

Horse chestnut, 182

Hydrogen ion, effect on conductiv-
ity, 50

Hydroxy] ion, effect on conductivity,

44
Hypertonic solutions, 95, 96

256

Injury, 18, 79 ff

permanent, 92 ff, 98
Inman, 95, 96
Iron, effect on conductivity, 47
Irwin, 232.

Janse, 212
Joel, 193
Jorgensen, 21, 31, 188, 196

Kations, effect on conductivity, 45
Katz, 192

Kite, 227, 228, 229

Kodis, 21, 22, 195

Kiihne, 227

Kiister, 228, 229

Laminaria, conductivity of cell sap,
199
measurement of conduc-
tivity, 23-39
structure of, 197
Langmuir, 226
Lanthanum, antagonism of, 166
effect on conductivity,
47, 49
effect on viscosity, 179
recovery after exposure
to, 83
Lasareff, 148
Lepeschkin, 192
Life processes, effect of tempera-
ture on, 75
measurement of, 68
Lillie, R. S., 179, 192, 235
Lipman, 176, 179
Lipoid theory, 228 ff
Lithium, penetration of, 203, 215
Loeb, J., antagonism, 124
antagonism between acids
and salts, 163
antagonism between
anions, 175
catenary reactions, 122
electrical phenomena and
permeability, 236

INDEX

Loeb, J., explanation of antagonism,
159, 161
measurement of permeabil-
ity in Fundulus, 205
opposite of antagonism, 179
Weber’s law, 147
Loeb, L., 97

Loewe, 191
M
Magnesium, effect on conductivity,
49

penetration of, 203, 209
Mamelli, 21
Manganese, effect on conductivity,
46, 49
Marinesco, 227
McClendon, 21, 225, 230
Measurement of antagonism, 124 ff
of life processes, 68
of toxicity, 71 ff, 94
Mechanical stimulation, 233
Mellor, 64; 66, 69
Metcalf, 227
Meyer, 211, 229
Miyake, 176
Molybdate, effect on conductivity, 40
Monostroma, 196, 217
Monomolecular reactions, 57 ff, 62
Moore, 175, 191
Muscle, measurement of conductiv-
ity of, 21

Niageli, 227
Nathanson, 230
Nernst, 226
Newberry, 31
Nickel, effect on conductivity, 46, 49
Nicotine, antagonism of, 169
effect on conductivity, 169,
194
Nitella, 213
measurement of conductiv-
ity of, 35
Nutritive effects, 146
Nitron, 215

Osmotic pressure, 202
Ostwald, 236

Ovary, partial recovery of, 97
Overton, 227, 228, 229

‘INDEX

Penetration from balanced solutions,
205, 214
Permeability, as measured by con-
ductivity, 17, 195
as measured by dif-
fusion, 206
as measured by exos-
mosis, 206, 207
as measured by tis-
sue tension, 208
as a measure of vital-
ity, 161
in relation to stimu-
lation, 233 ff, 235
of unlike membranes
in the cell, 231 if
Pfeffer, 227, 228
Plasma membrane, 227 ff
Plasmolysis, 159, 201, 213
Pierce, 29
Polacci, 21
Porphyra, 217 ;
Potassium cyanide, effect on per-
meability, 55
penetration of,
203
nitrate, pene-
tration of,
; 208, 212
Price, 227
Probable error, 38
Protoplasm, conductance of, 219 ff

Raber, 40, 41
Ramsden, 227
Reaction, catenary, 57 ff, 98, 122
monomolecular, 15
Reaction chamber hypothesis, 232
Recovery, 19, 79 ff
after exposure to alcohol
190
after exposure to calcium
chloride, 107, 115 ff
after exposure to chloro-
form, 188
after exposure to ether,
186
after exposure to hyper-
tonic solutions, 95, 96

257

Recovery, after exposure to mix-
tures of sodium and
calcium, 151 ff

after exposure to sodium
chloride, 92, 102 ff,
110 ff
measured by rate of res-
piration, 95
partial, 92 ff
Red blood corpuscles, measurement
of conduc-
tivity, 21
effect of an-
esthetics on
conductiv-
ity of, 193
Reed, 31
Respiration, as affected by hyper-
tonic solutions, 95, 96
as affected by hypo-
tonic solutions, 95, 96
as affected by sodium
chloride, 95, 96
Rhode, 227
Rhodymenia, 49, 50
antagonism, 174
color changes of, 175
effect of aluminum on
the conductivity of,
49
effect of anesthetics
on the conductivity
of, 193
effect of barium on the
conductivity of, 49
effect of calcium on the
conductivity of, 49
effect of cerium on the
conductivity of, 49
effect of lanthanum
on the conductivity
of, 49
effect of magnesium
on the conductivity
of, 49
effect of manganese
on the conductivity
of, 49
effect of nickel on the
conductivity of, 49
effect of potassium on
the conductivity of,
50

258

Rhodymenia, effect of sodium on the
conductivity of, 50
effect of sodium
taurocholate on the
conductivity of, 56
effect of strontium on
the conductivity of,
49
partial recovery of, 97
recovery after expos-
ure to sodium and
calcium, 87
structure of, 197
Rivers-Moore, 31
Roaf, 191
Robertson, 169, 227, 229
Rosenthal, 227
Réth, 23, 225
Rubidium, penetration of, 203, 214
Ruhland, 192, 229

Schlesinger, 31
Schulemann, 229
Schiitt, 227
Sea-lettuce, see Ulva
Sea urchin eggs, effect of cerium on
the conductivity
of, 50
effect of lanthanum
on the conductiv-
ity of, 50
measurement of the
conductivity of,

Seifriz, 227
Shearer, 23, 50, 175, 225
Shorter, 227
Small, 21, 235
Sodium chlorate, effect on conductiv-
ity, 59
chloride, effect on conductiv-
ity, 62, 92, 102 ff
110 ff, 178
effect on growth,
127
effect on respira-
tion, 95, 96
effect on viscos-
ity, 179

INDEX

Sodium chloride, penetration of,
203, 206, 208,
214, 215

recovery after ex-
posure to, 86,
92 ff, 99 ff
citrate, effect on conduc-
tivity, 178
iodide, effect on conduc-
tivity, 178
nitrate, effect on conduc-
tivity, 178
sulphate, effect on conduc-
tivity, 178
sulphocyanide, effect on
conductivity, 178
taurocholate, antagonism
of, 167
taurocholate, effect on con-
ductivity, 55, 167
Spaeth, 181
Spirogyra, 203, 212
Stewart, 21, 225
Stimulation, changes in permeability
associated with, 233 ff,
235
Stiles, 21, 31, 188, 196
Stone and Chapman, 21
Strontium, effect on conductivity, 49
penetration of, 203, 215
Sulphate, effect on conductivity, 40
Sziics, 169

Tangl, 21, 225
Tashiro, 232
Temperature coefficient of life pro-
cesses, 75 ff
coefficient of electrical
conductivity, 39
Thomas, 232
Thorium, effect on conductivity, 47
Thornton, 22
Tin, effect on conductivity, 47
Tissue tension as measure of per-
meability, 159, 208
Toxicity, 131
measurement of, 71 ff, 94
Tumor tissue, partial recovery of, 97

INDEX

U
Ulva, antagonism, 174

effect of anesthetics on con-
ductivity of, 193

effect of calcium chloride on
the conductivity of, 49

effect of magnesium on the
conductivity of, 49

effect of sodium taurocholate
on the conductivity of, 55

recovery after exposure to so-
dium and calcium, 87

partial recovery of, 97

structure of, 196

V
Valonia, 211
conductivity of cell sap of,
199
Van Orstrand, 67
Vitality as measured by permea-
bility, 18, 161
quantitative study of, 88 ff
Vries, H. de, 226, 227, 231

W
Waller, 188
Washburn, 31
Wasteneys, 163

259

Weber’s law, 147
Weibel and Thuras, 30
Weinstein, 192
Winterstein, 191
Wodehouse, 211
Woelfel, 19, 225
Woods Hole, 44

4
Yttrium, effect on conductivity, 47
Z

Zangger, 227
Zinc, effect on conductivity, 47
Zostera, antagonism, 174
appearance after
88 ff
effect of calcium on _ the
conductivity of, 49
effect of magnesium on the
conductivity of, 49
measurement of conductiv-
ity of, 35
partial recovery of, 97
recovery after exposure to
sodium and calcium, 87

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