Marine Biological Laboratory Library

Woods Hole, /Massachusetts

Gift of the F. R. Lillie estate-1977

U'me^^/^

LIFE AND DEATH AT LOW
TEMPERATURES

by

B. J. LUYET

Professor of B'wlofjij

and

P. M. GEHEXIO

Instructor iti Biology

SAINT LOUIS UN1\'EESITY

No. 1 of a series of monographs on general physiology
edited ])v B. J. Luyet

Published by

BIODYNAMICA, Nouma.xdy. .Missonn

1940

The authors dedicate this hoo\ to all
those straining in the pursuit of Truth

•••(•-^©nQ^in^^)*

The contents of this monoi^raph with the exception of
the General Bibliography, are reprinted from Biody-
naniica Xo. 33, 1938, No. 48, 1939 and Xo. 60, 1940.

The honesty of the readers being beyond all suspicion, no author's
right is reserved in any country.

iNTi\()i)r(:Ti()N

Tlio |)r()l)leiiis to be disciissod in lliis inoiiot^raj)!! arc
])i'iiiiarily those of llic i)ros(M'vat ion of lil'i' and of the
iiicclianisni of dcalli al low Irnipcrat iii-c. ( )ii account of
llu'ir innncdiatc connection with the ([uestions ot* the
structure of li\-in,i;- matter and of the nature of life, these
pi-obh'ins are of funch-iniental phih^sophical importance,
ll is ])i-ecisely this funihimeiital aspect which will he our
main conceiMi t lii-ouii,hout this work. None of the practi-
cal applications of low ti'm])erature reseai-ch will be con-
sidered exce))! inasnmch as they directly involve some
phase of our topic. Though the lields of Cold 11 (ird'nicss
in Phnifs and of Jlcfrif/erafuni / iidtistri/ are <|uite closely
related to our subject we shall not include them; each
one of these fields constitutes a science in itself wdiicli will
be better reviewed by a specialist.

Our knowledge on death by cold consists of observa-
tions concerniiii;': 1. Which organisms are killed by or
survive \arious low temperatures; 2. How the organisms
affected are physically modified by the action of cold;
.'>. What are the causes and the mechanism of cold injury
and cold death. The study of these three (piestions will
constitute, respect ix'ely, Parts 1, II and III of this l)ook.

Bibli()grai)hical references which only incidentally con-
ceiii the subject treated will l)e incorporated in the text,
in italics; references dealing primarily with the topic
undei- discussion will be gi\-en at the end of each ])art
in which they were used; a fciihitlvely complete bib-
liogra])hical list including also references not utilized in
our work, will be appended at the end of the monograph.

Sdii/f Loii/s, S<'/)tcml)('r I'l, I'-Cit),

TllK AUTUOKS.

TABLE OF CONTEXTS

TNTT^ODTTTIOX "4

TAIUJO OF (;()NTENTS 5

PART I

THE LOWER LIMIT OF VITAL TEMPERATURES

DEFINITIONS AND PKFLIMINARY REMARKS 0

I. INFRACELLULARS 13

1. Vitamins and Hormones 1:1

2. Enzymes H

0. ICiizyiiKtids 16

4. Mrnses 17

n. MONOCELLULARS 19

Skciiox I. Protophyta 19

1. liacteria and Bacterioids 19

2. Yeasts 25

3. Monocellnlar Alf>ae 27

Section II. Puotozoa 29

L Rhizopods 29

2. Ciliates 30

3. Flagellates 31

III. OEILM CELLS, SPOKES AND SEEDS 32

1. Spermatozoa 32

2. Eggs 31

3. Spores 37

1. Seeds 40

IV. ISOLATED CELLS AND TISSIES.. 43

Skctiox I. I*LAXT ^Iateuial 43

1. Hair Cells 43

2. Epidermal Tissne 44

.'>. Tnberons Tissne 47

4. Tissne from Leaves, Stems, Roots, etc 49

Skctiox II. Aximal Material 54

1. Blood 54

2. Embryonic Tissne 55

3. Ciliated lOpithelinm 50

4. Mnscnlar Tissne 57

5. Nerve Tissue 59

V. METAPHYTA 01

L Fungi (51

2. Algae 00

5

0

'^. TJclit'iis 08

4. Mosses 08

:.. iii-iici- I'hiiiis 00

\' I . M \V\\\'/A >A 71

I. ( "nclciiierates 71

-. llclmiiitlics 71

:t. Kotilcrs 72

1. Annelids 7:>

:>. M.. Husks 74

• i. An lii-o|i(i(ls 7.")

7. Ani|»liil»i;i and Keptiles 82

5. Fishes 80

!>. I lonioidi lieinis 1)1

PART II

the physical states of protoplasm at
low temperatures

pri:limi\.\ry chapter
/■'I \n\\ii:\r.\L rh'ixcii'LiJs or in:.\T coxni criox

1. The ••riolMeni of ihe Wall." in llie Steady Stale KM
•2. The Pic.hleni of Ihe Wall, in the N'anable Stale.. ..107
:;. The rrohlein of the Coolinji' IJody 108

CH APT Ely' f
EllEEZIMl. THE EPOZEX STATE 1 \ /> \/ELTI\(l

\. IXITIATIOX OF CUVSTALLIZATION 112

A. The F()U.\i.\'iio\ oi' ( 'm stai.lim-: XrcM'.i 112

r>. TiiK Fi!i:i;zi.\(; INdXTS 1 14

1. (MiHure Media liri

2. i'lnnt .Inices IK;

'.\. I'.lood and IJody FInids; linlividnal MilVei-ences ;

Relation Itetween the l].\ternal and Internal
iMedinni 110

4. Ixu^s ; SpeciHc Differences; Individual Differ-
ences; (V(]e in the Freezing Points of Develop
iiiji I'W ':. lis

ri. IM-olojilasni 121

0. Tissnes; Ijvin<>' and Dead Tissues; Intluence of

Coolino; Velocity on Freeyanj;' Points; The Don
l)le Freezinii' Point of Ia\in<i Tissues 121

II. KKodlMOSS Ol-^ ("KVSTALLIZATIOX 128

A. Tin; (Iitow rii ok ('i;vstals 128

l». TiiK JMi.vsii Si;i'.\i:.\ rio.x V.\2

1. Solnllons and Snspensi(»ns K^.2

1*. ('olldids; ( 'niii|»;ict Sui-rncc Frcczinji' ; liitcnnit-

ti'iit l^'i-cczinii ; I >iss('iiiiii;ited Froezing K)3

:'.. ( \);iinul;it(Ml Miilcrinl _ 138

4. IN. rims Material 13S

."). Tissues. Surface Fi-eeziiig 13!)

0. Tissues, luiercellular l^reeziiig- 141

7. Cells. Surface^ Freeziug 144

8. Cellular Coustitueuts 145

C. The Frekzixg Curves 147

1. Freezing Curve of Water 148

2. Freezing Curves of Solutions .- 152

3. Freezing Curves of Colloids 155

4. Freezing Curves of Tissues; Living and Dead Tis-

sues; Eutectic Poiul ; (juaulitv of lee Formed. .150

III. CO.MI'LKTIOX OF (CRYSTALLIZATION 102

1. Susjieusions and Colloids.. 102

2. Tissues 104

IV. THE FKOZEN STATE 100

Properties of Ice and of Frozen Systems ...100

1. ^[eclianical Properties of Ice 100

2. Specific Gravity 108

3. Thermal Constants 108

4. Electrical Properties 170

5. Optical Properties 171

V. MELTING AND MELTED :MATERIAL.. 171

A. The Process of Melting 171

B. Alterations Observed ix Thawed Materiai — 172

1. Suspensions and Colloids 172

2. Tissues 179

CHAPTER IT
Sil'JJh'COOLlXd AVD THE ^L I'ERL'OOLED .STATE

I. THE SrBCOOLIN(J (UKVE 185

II. FA( 'T( )KS AFFECTING CRYSTALLIZATION IN A
STTKCOOLED LIQI^ID IJ^S

1. Temperature 188

2. Inoculation 190

3. The Time Factor 192

4. Mechanical Disturbance 193

5. ( 'aiiillarit y 190

0. Impurities 198

7. Other Factors 200

8

rli \i''ri:i; III
■riii: 1 ri L'l'jti s sr \'ri:. \ rrinric \ri<)\. ni:\ iriiiri-

(' \l'l()\ I \ /> I /77.'0 \l I.LTI \ <;
I. I'll VSICAI, SVSTi:.\IS 203

1. TIk" \illc..iis Sl;i|(' 203

L'. \iiriti("ilinii 207

• '.. iN'vilrilicnIicm 211

4. N'iiioiiicli iiiti' 216

II. MNIXC .MATTi:i{ 217

1. (M'licr.il I'roccdiiic 217

■2. McIIkmIs 217

:». lOxpciiiin'iils ;iii(l IJcsult.s 219

PART TIT

THE MECHANISM Ol INJURY AND DEATH BY
LOW lEMI^ERATURE

CHAPTER T

.\(Ti(>\ o/' ro/./> wirnoiT K'l-: rohwiATiox

I. ACTION OtM:M)IJ) IX THE Xi:i(!III!Oi:iIOOl) OF

THE FKEEZIX({ POIXT OF PROTOPLASM L»:50

A. Action ok Cold Ahovk tuk Fki:i;zix(; I^oixt "J'JO

1. Slow Tiijui-ious Action 231

-. R;i|»i(l liijuiious Aclioii 234

15. AcTiox OF ('oLi> IX TiiK Srp.cooi.Ki) Statk. 23(J

11. ACTION OF EXTREME COLD 241

CHAPTER J I
ACTIOX or COLD ACC0MI*A\JE1) liV ICE FONMATJOX

T. THEORY ATTRII51 TIXO I)I:ATH TO A MERE

\ViTm)I{A\\'AL OF ENEROV 2r)7

II. THEORY ATTRIRI'TINO DEATH TO THE AT-
TAINMENT OF A MINIMAL TEMPER ATl'RE.. .25!)

111. THEORY ATTRTP.rTING DEATH TO MECHANI-

C.\L IX.U'RY 201

I Y. THEOl{Y OF DEATH liY TOO RAIM D THA\YIXCt....2(;7

Y. TIIi:oRY OF DEATH P»Y DEHYDRATION 272

YL TIIi:oRV OF DEATH P.Y YARIOIS PHYSIOLOG-
IC.KL. I'HVSKWL OR CHi:.MIC.\L .VLTIMLV

TIOXS JS5

OENERAL lUIiLIOGRAPHY 296

SriMECT INDEX 329

ATTIIOR 1XDI:X 336

PART I

THE LOWER LIMIT OF VITAL
TEMPERATURES

INTRODUCTION
DEFINITIONS AND PRELIMINARY REMARKS

111 a study of the mechanism of cellular or of proto-
plasmic death by low temperatures, one needs to know,
first of all, at what low temperatures life is destroyed.
The following pages present a review of the literature on
this subject.

According to the effects that they produce on living
matter, the temperatures are classified into rital, lethal,
survival and auahiotic. If the same effects are obtained
over a range of temperatures, there must be distinguished
a maximum and a minimum in each range; thus there
would be a maximal and a minimal lethal, a maximal and
a minimal survival temperature, and so on. The multiple
aspects originating from so many distinctions on one
hand, and the overlapping of the ranges on the other, have
resulted in some confusion. But more important mis-
understanding has arisen from the widely different senses
in which the various authors use the terms just mentioned,
and from the impossibility of defining clearly a death
temperature, in the present state of our knowledge of the
cause of death by cold. This will be illustrated by the
following analysis of the notion of lethal temperatures.

The death point has sometimes been considered as a
definite temperature at which an organism passes from
the living to the dead state in somewhat the same manner
as, at the freezing point, a substance passes from the
liquid to the solid state. So understood, the death tem-
perature would be specific for each organism in the same
sense as the physical constants are specific for each sub-

9

10

stance. A clear and atlc(|ualc clcliiiilion could llieii be
established.

Much evidence, however, has been accumulated for an
entiri'Iy different notion of death temperatures. In some
l)hints, for example — and the same mii-ht be true of single
cells — death seems to result from the une(inal slowing
down by cold of the various metabolic activities. If, for
instance (in the case of a higher plant), less moisture is
absorbed by the roots than is liberated by the leaves, des-
iccation and death will follow. An exposui-e to a tem-
perature as high as 5° might become lethal if enough time
is allowed for the material to dry; a temperature a few
degrees lower will dehydrate the plant to death in a
shorter time. It is evident, then, that the time factor
must be introduced into the definition of the death tem-
peratures. On the other hand, the degree of moisture of
the atmosphere, the amount of light present, the water-
content of the soil, etc., will also affect the duration of
death and the temperature at which it occurs. The lethal
temperatures, therefore, represent simply conditions un-
favorable for life, in the same sense as the scarcity of light
or of oxygen or of water. In cases of this kind, the defi-
nition of death temperatures is rather involved; instead
of a lethal point one should use the notion of a lethal re-
gion ; the time necessary for death should be mentioned,
as should also the other conditions which hasten death or
delay it. The temperature at which the plant dies, by
desiccation, in an arbitrarily chosen very long time, can
be called the maximal lethal temperature. The lethal
zone extends from that maximum downward, but it has no
minimum since there is no temperature, however low,
which will kill the plant instantaneously by putting its
metabolic processes out of balance.

If such a plant is cooled rapidly to the freezing point,
the injury produced by the congelation of its juices may
suffice to cause it to die. But death results here from an
enti]-ely different cause, and the plant has, besides the
death zone mentioned in the preceding paragraph, a death

11

point which coincides with the freezing innul. A precise
definition can be given of that death point, as well as in
the case in which death was supposed to result from a cool-
ing to a specific minimum. The solidification of a given
proportion of moisture takes place at a definite tempera-
ture. The time factor has little importance ; it is simply
related to the total mass of material to be killed in the
same way as the time necessary to melt a certain quantity
of metal depends on the amount present and bears no rela-
tion to the melting point.

But an organism which is killed when left to congeal at
the freezing point might not be killed if brought rapidly
to some hundred of degrees below zero, where congelation
cannot take place on account of the high viscosity of the
material (cf. Luyet, 1937). Thus, temperatures below
the lethal point might be non-lethal.

One and the same organism may, therefore, possess a
zone of lethal temperatures above zero, a sharp death
point slightly below zero, and a zone of non-lethal tem-
peratures some hundred degrees below zero. And these
are only a few^ of the many possibilities that one can
imagine.

It is clear, then, that the notion of lethal temperature
depends on the idea that one has of the mechanism of
death and, therefore, is susceptible of multiple interpre-
tations. There being no possible general definition of the
death point, of the survival point, etc., we shall, in this
review, use these expressions in the sometimes unprecise
sense given to them by the various authors, without
attempting to define them more exactly.

The most important factors to be considered in a study
of the temperatures at which animals or plants, organs,
tissues, cells, or protoplasm, die, are the following: 1.
The species or the type of material studied; 2. The ex-
ternal temperature to which the material has been sub-
jected; 3. The time of action of this temperature; 4. The

l('iHl»('r;i1iii-c williiii ihc dyiiii;' nialcrial; 5. Tlic tiniL' of
action of this latter tcinpciat iiro ; (5. Tlie rate of cooling
and, if the material was thawed, tiie i-ate of warminji': 7.
The presence or absence of ice in the tissne, in the cells or
in the ])rotoplasm; S. The state and the conditions of the
malei-ial, before and during' coolin.n', snch as, its acclimati-
zation to low temi)t'ratni-es, its water content, its thermic
insnlation, etc.

Inmost of tile older works on death by low temperature,
only the tirst two of these factors, that is, the typo of
organism and the external temperature, are recorded.
This literature, therefore, does not bring much substantial
information on the point at issue in the present work,
and we shall mention only some of the most outstanding
results obtained by the older investigators. Those papers
of later date which contain enough of the data mentioned
in tlie previous paragraph to be significant will be sum-
marized and some of them will be discussed briefly.

Immersion in liquid gases has been the usual method of
subjecting living matter to extreme cold. The liquefac-
tion temperatures of most of the gases used are given in
the following list and will not be indicated for each par-
ticular case in the text.

Absolute Zero -273° C.

Temperature of liquefaction of Helium - 269° C.

Temperature of liquefaction of Hydrogen - 253° C.

Temperature of liquefaction of Nitrogen -196° C.

Temperature of liquefaction of Air - 192° 0.

Temperature of liquefaction of Oxygen - 183° C.

Temperature of sublimation of Carbon di-
oxide - 78.5° C.

Temperature of liquefaction of Sulphur di-
oxide - 73° C.

Some conditions of the experiments, which follow as
natural consequences from other fully described condi-

13

tions, will often be omitted. For instance, when the in-
fracellnlars and the monocellular organisms are exposed
to the temperature of liquid air, in water suspensions, or
in nutrient media, for an hour or so, it is certain that the
medium is hard frozen and hence this will not be stated
explicitly.

The ordinary life stage and the usual culture conditions
are to be assumed if nothing to the contrary is mentioned.
For example, bacteria and yeasts are supposed to be in
the vegetative form and in aqueous media (solid or liquid),
if it is not specified that they have sporulated or that they
have been dried.

The data to be reviewed w411 be arranged in sections
according to the type of organisms studied, the generally
accepted classification of plants and animals being fol-
lowed. Within a section, except if otherwise indicated,
the order is that of decreasing temperatures. There
results an unavoidable separation of related genera or
species. But, since the subject-matter is indicated in
italics, the reader will be able, without much effort, to re-
establish the natural correlations. Finally, then, each
separate paragraph usually contains an investigation on
a given subject, at a specified temperature and by a given
author or group of authors.

The temperature shall always be given in degrees centi-
grade except if otherwise indicated.

The terms "freeze" and "congeal" shall be used exclu-
sively when it is meant that the fluids of the material
under experimentation solidify.

I. INFRACELLULARS

By our inclusion of the infracellulars with living matter
we do not mean to imply anything as to their real nature.

1. Vitamins and Hormones. The only information that
we found in the literature, on the action of low tempera-
tures on vitamins and hormones, was the observation that

14

Ihcvse subslaiices tlo iiul lose llu-ii- aclixily in cold storage.
Thus, Wright (J. Soc. Cliem. lu<J., 12, 509, 1923) reported
that pork, kept for 9 years in the frozen coiidilion, still
eontainod some active rifcun'ni A; "Weill, Alonriiiiiand and
Michel {C. r. ^uc. BioL 70, 189, 191G) showed that ritamin
B, in beef, was not affected by a storage of 8 months at a
temperature of - 8° to - 10° ; etc. It is generally thought
that non-protein substances should not be affected, as pro-
teins are, by low temi)erature and l)y the congelation of
the aqueous medium in which they are dissolved or sus-
pended.

2. Enzymes. Spoilage of refrigerated food is, in some
instances, attributed to the action of proteolytic enzymes
elaborated by bacteria. This type of enzyme has been
found by many authors to be active at sub-zero tempera-
tures, in some cases, even at -9°. (Cf. Glage, ZtscJir. f.
Fleisch. u. Milchhyg. 11, 131, 1901, and Talyract, Ann.
llyg. Pub. Set:, 45, 166, 1901.)

Kovchoff (1907) observed the conversion of protein
nitrogen into non-protein nitrogen in various plant tissues,
after they had been frozen for 24 hours. The plant pro-
teases, he concluded, survive freezing.

According to Pennington and Hepburn (J. Am. Chem.
Soc., 34, 210, 1911), lipase was active in the abdominal fat
of chickens after the latter had been kept hard frozen for
more than 7 years at temperatures of - 9.4° to - 12.2°.

Hepburn (1912) demonstrated the presence of oxidase,
peroxidase, catalase and reductase in the same material
maintained at the temperature indicated for several
months or even several years.

Gertz (1926), studying the oxidizing enzymes of the
alga Delesseria sangmnea and of potato tubers, found that,
after a stay of 24 hours in the frozen state at -15°, the
tubers yielded unimpaired oxidase, as did also the algal
sap, previously extruded, and kept frozen for 98 hours at
the same temperature. But, after an exposure of 1 hour
to -80°, the reaction of these enzymes was weaker.

15

Tliunberg (1920), who observed that the animal oxidases
are also impaired l)y exposure to - 80°, suggests that they
might be modified in their structure in such a way that
they act only on some particular fraction of the substances
on which they acted before.

According to d'Arsonal (1892), yeast, subjected for
24 hours to - 42° or for 45 minutes to about - 95°, furnished
an active sucrase; but if the enzyme, in a glycerin solu-
tion, was maintained for 45 minutes at -95°, it lost its
power of splitting sugar.

Pictet and Yung (1884) subjected yeast successively
for 108 hours to - 70° and for 20 hours to - 130° ; they
found that it lost its power to raise bread.

Yeast juice, exposed by Macfadyen (1900) for 20 hours
to the temperature of liquid air, furnished an active
zymase.

Buclmer obtained zymase by grinding yeast in liquid
air or by grinding a mixture of yeast with solid carbon
dioxide ("Die Zymasegahrung. " Munich and Berlin,
1903. pp. 67 and 226).

Rennet, in liquid preparations, exposed for 30 minutes
in liquid air, coagulated milk as effectively as did the con-
trols (Chanoz and Doyon, 1900).

Pozerski (1900) investigated the action of an exposure
to the temperature of liquid air on the following enzymes
in solution (in test tubes) : salivary enzymes from human
saliva, sucrase from yeast, sucrase, amylase and inulase
from Aspergillus niger, tryspin, pepsin (a glycerin solu-
tion) and rennet. The activity of all these enzymes, after
45 minutes exposure, was found to be identical with that
of the controls.

The coagulating enzyme of the blood was not destroyed
when oxalated dog blood, contained in a glass tube, was
exposed for 13 minutes to the temperature of liquid air ;
coagulation took place normally upon addition of calcium
chloride after thawing (Clianoz and Doyon, 1900). Since
the blood was oxalated, the fibrin-ferment itself was not
formed and the enzyme acted upon was the prothrombin.

k;

Ac'cordiiiji," to H('i)l)iii'ii and IJazzoiii (li)!.")), oxidase
was still active aftci' an cxposui-c of 3Vii hours to tlie
tompcratiire of li(iiiid air, wliile an aqueous solution of
urease rotaincd 96.29% of its initial activity after having
been kei)t in liquid air for 100 hours.

Bickel (1905) subjected pepsin (in i>astric juice) to
liciuid air temperatures for 22 hours, after which treatment
the proteolytic activity of the pepsin was not impaired.
Nor did repeated freezing in liquid air and thawing (up
to 6 times witliin an hour) produce any effect.

Solutions of injps'ni contained in glass tubes and frozen
in liquid air, then thawed rapidly in tap water, and re-
frozen in the same manner 12 times, showed 70% of the
trypsin inactivated when the 5% stock solution was diluted
1-500, and 17% when it was diluted 1-10 (Rivers, 1927).

Galvialo (1937) subjected catalase from human blood
and diastase from saliva (either undiluted or diluted with
water) to litpiid air temperatures; the catalase was not
affected, but the activity of the diastase was reduced to
one half.

The literature on the action of low temperature on
enzymes, up to 1913, has been reviewed by Hepburn
(1915).

3, Enzy molds. According to Tetsuda (1912), sera
frozen in an ice chest, for times varying from a few hours
to several weeks, and either kept continuously in the
frozen state or frozen and thawed repeatedly, did not show
any alteration of complement, of agglutliiui, of Jtemoli/slu,
of precipitin, or of the properties of inducing and of at-
tenuating anaphylaxis. (Freezing was used as a method
of concentrating these various substances in the sera.)

The toxin of Clostridium hotidluum showed no decrease
in toxicity after having been stored at -79° for 2 months
or at -16° for 14 months (Tanner and Wallace, 1931).

CompJement, frozen 12 consecutive times, in undiluted
guinea pig serum, or in 1-10 dilutions, was not affected;
l)ut it was inactivated when frozen the same number of

17

times in serum diluted 1-100 with physiological salt solu-
tion (Rivers, 1927).

Com pi cm cut, as well as awhocepter, in rabbit serum
immersed in liquid air for times varying from 10 to 30
minutes, kept their unaltered activity, according to Liidke
(1905).

Tetsuda (1912) also reported that complement and
antihodies were not affected by maintaining the sera for
several days in liquid air.

A 24 hour-old broth culture of Eberth bacilli, maintained
frozen in liquid air for 20 minutes, did not lose its
agglutinaUUty. Sheep serum, frozen in the same manner,
was as efficient in agglutinating these bacilli after the
freezing as before (Chanoz, Courmont and Doyon, 1900).
Macfadyen (1902a) used the juices extracted from
the Eberth bacilli by trituration in liquid air, to produce
immunizing sera. The latter possessed the normal anti-
toxic and antihacterial properties.

Cobra venom, in a 1% solution, maintained in liquid
air for 9 days, presented an unaltered toxicity (Lumiere
and Nicolas, Province mcdicale, Sept. 21, 1901).

According to Pictet (1893), pfomains were affected by
exposure to temperatures of - 100° to - 200°.

4. Viruses. A strain of bacteriophage active on B. coli
and one active on staphylococci, frozen at - 78° (with solid
CO2) and thawed 20 times consecutively, did not lose any
of their activity (Sanderson, 1925).

D'Herelle (''The Bacteriophage and Its Behavior," p.
300, Baltimore, 1926), however, reported that while the
phage (one for staphylococci and one for dysentery bacilli)
was not affected in young filtrates, it was inactivated by 1
to 3 freezings in liquid air, when treated in filtrates more
than 17 days old.

Rivers (1927), using 19- to 90-day-old filtrates of a
phage lytic for B. coli, observed that, after 12 freezings in
liquid air, the phage was completely inactivated when
physiological salt solution was used as a diluent, and that
it was partially inactivated when the diluents were Locke's

18

solution, distilled water, or hrotli, the dcni-cc ol" iiiactiva-
tiou decreasing in the order given, lie then cxixMiincuted
with dilutions of 1-10 and 1-1000 of tlie slock filtrate and
found lliat an increased dilution with salt and Locke's
solution increased the percentage of phage inactivated
while, on the coiiti'ai'N', increased dilution with distilled
water or brotii did not.

According to Stockman and Minett (li)2G), the virus of
foot and mouth disease was not destroyed by repeated
freezing in ammonia brine.

Pictet and Yung (1884) reported that the cow-pox vac-
cine was inactivated after two consecutive exposures of
respectively 108 hours to - 70° and 20 hours to - 130°.

According to Barrat (1903), an exposure of the rabies
virus to the temperature of liquid air for 1 to 5 hours did
not inactivate it.

Salvin-Moore and Barrat (1908) found that a stay of
30 minutes in liquid air did not affect the graftable mouth
cancer.

Gaylord (1908) obtained identical results with the
same material maintained in liquid air for 80 minutes.

Rivers (1927) observed that the herpes virus in a brain
emulsion was not inactivated by 12 freezings in liquid air
])ut was inactivated when frozen 24 times in an emulsion
diluted 1-20 or more with Locke's solution.

Similar results were obtained by the same author with
vaccine virus, which resisted 12 freezings in a testicular
emulsion diluted 1-10, 1-100, and 1-1000, Imt the titer
of the virus decreased after 24 freezings at. dilutions of
1-10,000 and 1-100,000 and the virulence was completely
destroyed after 34 freezings at a dilution of 1-100,000.

Rivers investigated also rims III and found it to be
readily inactivated after 12 freezings at dilutions of 1-10
of his stock emulsion.

The results on the infracellulars can be summarized
as follows: 1. The enzymes, toxins, bacteriophage and
viruses investigated are not affected by a freezing of their

19

solutions or suspensions in liquid air nor by a stay of short
duration at about - 190°. 2. Some of them are inactivated
by prolonged or repeated freezing at that temperature,
especially in very dilute solutions.

II. MONOCELLULARS
SECTION I. PROTOPHYTA

1. Bacteria and Bacterioids. The numerous investiga-
tions on bacteria will be classified into two groups : A.
Those in which the organisms were found unaffected by
low temperature ; B. Those describing lethal or injurious
effects.

A. Many authors have reported that bacteria support
temperatures of 0° or of a few degrees below 0°, as used
in cold storage; we shall not attempt to name all these
investigators.

McLean (1918) has decribed 4 species of bacteria which
he isolated from ice, from snow and from frozen algae of
the Antarctic where the mean annual temperature is about
-20°.

Smart (1935) observed that some bacteria, found alive
in fruit held for 3 years at - 9.4°, then isolated and sub-
jected in pure culture to a temperature of - 8.9° for 1
year, produced growth at this temperature.

Brehme (1901) reported that the cholera vibriones, in
bouillon cultures, survived a continuous freezing for 57
days at - 1° to - 16° ; nor were they killed when subjected
40 times alternately to - 15° and to 15°. In similar condi-
tions typhus bacilli survived freezing at -2° to -16° for
4^ months and were not killed when frozen and thawed
40 times at respectively -15° and 15°.

Tanner and Wallace (1931) found that spores of
Clostridium hotulinum survived freezing at -16° for 14
months, and that commercially frozen fruits and vege-
tables were not sterile even after 2 years at - 16°.

Citovicz (1928) put an emulsion of streptococci of scar-
let fever into capillary tubes, where they were subjected
to temperatures of - 17° to - 18° for at least two weeks ; the

20

()i\i;aiiisms wore not killcil. It is imjjorlaiit to remark lliat
ill liiose experiments, the emulsion was not frozezn but
subcooled.

Prnc'lia and Brannan (192(1) isolated //. ti/pJiosiis from
ice cream kept for 2S months at -20°.

(Madiii (ISiKS) froze //. prsfis at -20° daily for 40 days.
Such repeated freezini;' and thawing did not kill the
organisms.

Kasansky (lcS<)9) found no h)ss of viability in B. pestis
after a stay of SVo months at - 31°.

According to Pictet and Ynng (1884), bacteria, in sealed
glass tubes, did not show any impairment of their activity
after having been subjected for 108 hours to at least - 70°
and for 20 more hours to - 130°.

In 1893, Pictet reported that he had exposed 30 to 35
kinds of bacteria to - 200° and that they had apparently
suffered no injurious effects.

Bouillon suspensions of the vegetative form of B.
anthracis and of the bacillus of chicken cholera w^ere im-
mersed in liquid air for 15 hours by Belli (1902). No
change of the morphological characters nor of the viru-
lence of the organisms could be observed. In another
experiment the author placed in a sterilized test tube a
small piece of spleen from a rabbit wdiich had just died of
anthrax, and then immersed the test tube in liquid air for
15 hours. The treated s|3ecimen was found to be just as
virulent as another untreated specimen from the same
spleen. In a last experiment, small strips of filter paper
w^ere impregnated with bouillon cultures of B. anthracis
and of the bacillus of chicken cholera, and these strips
were then suspended in licpiid air for 8 hours. No diminu-
tion of virulence could be observed.

Paul and Prall (1907) found that dried StaphyJococcus
pyogenes aureus can remain alive for weeks in a mixture
of dry ice and ether, and for 125 days in liquid air.

Gonococci, held for 10 days at -20° and for 24 hours
at -195°, in liquid nitrogen, were found to be viable by
Lumiere and Chevrotier (1914).

21

Macfadyen (1900) exposed vegetative and spore forms
of 10 kinds of l)acteria {hacilU, spirilla, staphylococci and
a pliotohacterium) to liquid air for 20 hours. The cultures
were either in broth or on solid media (gelatin, agar,
potato). No effect whatsoever was observed; all the
physiological properties : curdling of milk, fermentation
of sugar, indol production, pigmentation, pathogenicity
and photogenicity, appeared to be normal.

In another experiment, Macfadyen liquefied the air of
his laboratory and cultured the bacteria present in it ; he
found several species of hacilli, cocci and sarcinae. The
temperature during the liquefaction of the air reached
about -210°.

Macfadyen and Rowland (1900a and 1902) left B. coli,
B. typhosus and Staphylococcus pyogenes aureus in liquid
air for 6 months. The bacteria, suspended in small loops
of platinum wire or on cotton-wool swabs, were directly in
contact with liquid air. No impairment of vitality was
observed in any of the organisms.

Lipman (1937) exposed for 48 hours to the temperature
of liquid air bacteria in the actively growing state. The
organisms were cultivated on agar slants in test tubes.
The latter were immersed in liquid air after having been
sealed. The 9 species of bacteria experimented upon sur-
vived and 8 of them presented an abundant growth. Par-
ticular attention was given to the choice of the species in
order to exclude the possibility of spore formation.

Macfadyen and Rowland (1900b) observed no sign of
injury on various bacteria left for 10 hours, in sealed
tubes, in liquid hydrogen.

De Jong (1922) summarizing the results of Beek (1919)
and Ongkiehong (1922), says that B. coli, B. faecalis
alcaligenes, B. lactis aerogenes, and the bacteria of
typhoid, of paratyphoid A and B, and of enteritis, resisted
equally well exposures to temperatures of - 20°, - 190°
and -253°.

Kadisch (1931) found that B. coli (in 0.85% NaCl
solution) could resist - 252° (liquid hydrogen) for 3 hours,

and that Sldph i/hndccus pi/iK/ciics ulhiis and llii' liihcrcle
hdcilhis could wil lislaiid llic same temperature I'oi* 50
hours. In I'urllici' ('.\])('rimeiits, the same invest i^'ator
re])orted tlial these ori;anisms were not killed after two
consecutive exposures of respectively 4 hours at - 268.8°
and li/> hours at - 271.8°. Cooling was relatively slow, it
rtM|uiiin,i;' al>out 24 hours for the entire experiment.

Zirpolo (1!).')2) observed no injurious effect when
lumiiK'scent l)acte]'ia {Bacillus and Micrococcus picran-
tonii), in the fresh vegetative state, were exposed for sev-
eral hours to the temperature of liquid helium.

Lipman (1936a) immersed in liquid helium, at tempera-
tures varying from 4.2° K to 1.35° K, spores of common
bacilli (species not mentioned), smeared on fragments of
cover glasses, and desiccated for 2 weeks. The prepara-
tions were kept 2 hours at the lowest temperature and 42
hours below 4.2° K. The spores presented as abundant
a bacterial growth as would normal spores.

We shall not review here the literature accumulated
during the last 3 years on the method of drying bacteria
at low temperatures for preserving them. We shall men-
tion only that, in these experiments, the physiological
properties of the organisms were found unimpaired (Cf.
the fundamental work of Flosdorf and Mudd, 1935).

B. Park, Williams and Krumwiede ("Pathogenic
Microorganisms," 8th ed. p. 698, New York, 1924) kept 21
strains of water-inoculated B. typhosus at about -2° to
- 7°. Six cultures were sterile after 5 weeks and all were
sterile after 22 weeks.

Hilliard, Torossian and Stone (1915) and Hilliard and
Davis (1918) subjected water suspensions of B. coli to
freezing for 3 hours at 0° or at - 15° and determined the
percent of survivors by the usual method of bacterial
counts. At 0°, 26.4% to 67.77r, and at -15°, 99.4% to
99.7% of the organisms were killed. After intermittent
freezing for the same length of time (4 freezings), the
number killed was slightly greater.

"}?.

In another series of experiments, the B. coli were sus-
pended in glucose solutions of ditferent concentrations
and then subjected to varying degrees of cold, from - 0.5°
to - 6°. The concentrations of glucose were such that the
solutions did not solidify at the temperatures used, so that
the effect of cold alone, without crystallization, could be
determined. The results were compared with those ob-
tained with tap-water suspensions of bacteria subjected
to the same temperatures. At - 0.5°, an average of 93.67o
of the organisms were killed in the frozen tap water,
while in the unfrozen glucose solution 46.47^ were de-
stroyed. At -6°, an average of 99.2% perished in the
frozen medium, and 49.5% in the unfrozen solution.
When both the glucose and the tap water suspensions
were frozen solidly at - 10° for 3 hours, the percent killed
in the former varied from 77.5% to 99%, while in the
latter it ranged between 98.1% and 99.8%.

Tanner and Williamson (1928) exposed 4 species of
common bacteria to temperatures of -13° to -15° for 2
to 16 weeks, and found that this prolonged freezing de-
stroyed some species completely, while others were still
alive after 160 weeks at - 15°.

According to Onorato (1902), the Hemophilus influen-
zae is destroyed in blood broth after a stay of 21/2 hours
at - 15° or 11/0 hours at - 20°.

Smith and Swingle (1905) exposed B. typhosus, in
bouillon cultures, to - 17.8° for 2 hours and found that,
on an average, 99.5% were killed.

Keith (1913) observed that when B. coli, in solidly
frozen tap water, were kept at - 20°, only a fraction of
one percent remained alive after 5 days, and that storage
of a few weeks at that temperature resulted in a complete
destruction of the bacteria. Moreover, when B. coli were
in pure milk or in milk diluted to various degrees with
water, a larger proportion of bacteria survived the freez-
ing of the undiluted than of the diluted milk.

According to de Jong (1922), the bacteria of dysentery
were killed after a relatively short exposure to -20°.

L'4

For certniii slraiiis, -190° was more lethal than -20°,
wliile I'or some others it Mas less.

Kasaiisky (1899) found that B. pestis, cultured on agar
and kept at -31° in the frozen condition for 4 months,
hut thawed 8 times during the interval, showed a weaken-
ing of its virulence.

Sanderson (1925) gives the following figures as repre-
senting the proportion of B. coli which were killed after,
respectively, the first, the tenth, and the fifteenth freezing
at-78°:167o, 86%, 94%.

According to Klepzoff (1895), bacteria are killed when
exposed to the temperature of liquid air.

D'Arsonval (1898) reported that the chromogenic func^
tion of B. pyocyanus was slightly impaired by exposure
to liquid air temperature. The effect ^vas more accentu-
ated if the cultures had previously been dried.

Macfadyen and Rowland (1900a) mention a slight
weakening in the activity of some species of bacteria,
after the latter had been left for 7 days in liquid air.

According to White (1901), a culture of bacteria, ex-
posed for 2 hours to liquid air temperature, presented a
large number of killed organisms; only the more resistant
ones survived.

Smith and Swingle (1905) subjected bouillon cultures
of B. typhosus to liquid air temperatures for 2 hours, and
found that, on an average, 99.3% were killed but the few
organisms which survived produced normal colonies.

Rivers (1927) froze a broth suspension of B. coli 12 suc-
cessive times in liquid air. Before freezing, there were
180,000,000 viable organisms per cc ; after the 12 freezings
and thawings, this number was reduced to 40,000. Greater
dilution of the suspension decreased the percentage of
survivors.

According to Park, Williams and Krumwiedc {loc. cit.,
p. 295) 30% of the staphylococci subjected to freezing in
liquid air for 30 minutes, remained viable.

A brief review of the investigations on pathogenic
bacteria has been pul)lished by Hampil in 1932.

_:;)

2. Yeasts. The investigations on yeasts, like those on
bacteria, will be arranged in two sections: A. Those re-
porting negative results; B. Those reporting injury or
death.

A. Several authors have observed that some yeast
species are physiologically active at sub-zero tempera-
tures. Thus, Berry (193-1:) found a species which repro-
duced actively in beer wort at -2.2°,

Salimovskaja-Rodina (1936) described 19 species of
yeast (!) isolated from colored snow {Arch. Biol. Nauk,
43,229).

Smart (1935) found that several species of yeast, cul-
tured on agar slants and left for 1 year at - 8.9°, produced
abundant growth when transferred to room temperature.

Molisch (1897) described a deformation and a shrink-
ing of about lOVf, but no loss of growth or of fer-
mentation power when yeast cells, growing actively in a
liquid medium, were frozen at -5° to -9° and observed
under the microscope during the congelation of their
culture medium.

Kadisch (1931) exposed several sorts of pathogenic
yeasts, suspended in 0.85% sodium chloride solutions, to
temperatures of - 20° to - 30° for 2 months, subjecting
them to 11 successive freezings and thawings during this
time. No harmful effects were observed.

Zopf (1890) made thin smears of vegetative cells and
of spores of Saccharomyces Hansenii on mica sheets and
subjected them for 4 hours 20 minutes to - 83° ; death did
not ensue. Furthermore, no harm was done when this
frozen material was transferred directly into water at
room temperature ("Die Pilze," p. 275, Breslau).

Melsens (1870), after having observed that yeast resists
-91°, investigated the combined action of pressure and
cold. He put a yeast suspension in a steel bomb calcu-
lated to resist a bursting pressure of 8,000 atmospheres
and froze the suspension at a temperature low enough to
burst the bomb; the cells remained active after this
treatment.

Doenu'Hs {Allg. Biau. ii. llopj. Ztg., V, 2225, 1897) ex-
posed beer yeast to the lemi)eratiire of liquid air for 6
mimiles; tlie yeast retained its vitality.

Kiii-clicr (lOni) found that Sacrliaronn/res cerevisiae.
cultured on agar slants, survived a 1 to 8 day action of a
temperature of -70°, as well as a 13 hour exposure to
-183° to -192° in liquid air.

Macfadyen and Rowland (1902) kept yeasts which had
been washed, pressed and wrapped in paper, immersed in
liquid air for 6 months; neither the vitality nor the
physiological properties of the organisms were impaired.

According to Kadisch (1931), pathogenic yeasts sus-
pended in 0.85'/ sodium chloride were not killed after 24
lionrs at -180°, nor after 3 hours at -252°, nor after 2
hours at -268.8°, though the latter treatment caused a
consideral)le delay in development. In a further test he
cooled the yeasts by degrees from room temperature to
-268.8°, where they stayed for 2 hours; thereupon he
brought them down to the temperature of -272°, which
was maintained for 14 hours. He then raised the tem-
perature to - 268.8° and kept it at this point for another
2 hours ; finally, lie l)rought the organisms by degrees back
to room temperature. There was no loss of vitality, but
merely a slowed development, and the author concludes
that cold alone cannot produce the death of the micro-
organisms.

B. Tanner and Williamson (1928) exposed ampullae
containing 2 cc. of a suspension of yeast in physiological
saline, to - 15° for varying lengths of time up to about 3
years; most of the yeasts were markedly injured after
that time.

Schumacher (1874) observed that in fresh yeast, ex-
posed for 15 minutes to -113°, many of the cells were
killed. The younger ones with smaller vacuoles and those
still devoid of vacuoles remained alive and were capable
of budding.

Pictet and Yung (1884) exposed Saccharomyces cere-

27

visae for 108 hours to - 70° and for a further 20 hours to
- 130°, after which the organisms were no longer capable
of raising bread, although no alteration, microscopically
visible, could be detected.

Doemens {Allg. Brau. u. Hopf. Ztg., 2, 2225, 1897)
reported that, when he exposed a suspension of yeast to
liquid air and thawed it too rapidly in water, the power
of development was impaired.

3. Monocellular Algae. It may be mentioned here that
several species of monocellular algae are found in the
encysted form in snow and give it a green or red colora-
tion. These organisms become active in the melting
snow. It is reported that some of them cannot resist
temperatures higher than 4°. We did not find any data
on the lower limit of temperature that they can support.
For the literature on this subject, we refer the reader to
a series of papers by Kol, E., and Chodat, F., in Bull. Soc.
Bot., Geneve, 25, 1934.

According to W. West and G. West, Closterium (a des-
niid) was found motile in water which had been frozen for
14 days. Microsterias (another desmid) was also found
alive under the same conditions. Since this last genus
was not observed in the cultures in the spore form, the
authors remark that it resisted freezing in the vegetative
state {Ann. of Bot., 12, 1898, p. 33).

Wisloucli (1910) exposed SticJwcoccus bacillaris, in
sterilized water, to various degrees of cold. A very small
number of cells survived a 2 hour exposure to - 75°, while
about 50% resisted a 6-7 hour exposure to -21°.

According to Pictet (1893), various species of diatoms
in water cultures, frozen at about - 200°, were uninjured
and, after the thawing of the medium, they were seen to
extend pseudopodia-like protrusions.

Edlich (1936) subjected Pleurococcus vulgaris, Apafo-
coccus minor and SticJwcoccus hacillaris on their natural
bark substratum to temperatures ranging from -20° to
-80° for varying lengths of time, after a previous ex-

28

})osiii-(' to (lilTeri'iil (Icgi'ct's ol" alinosphcric nioislurL' (rela-
ti\(' liuniidily 25% to 100%). Pleurococcus was found to
witlistaiul -80° for at least 24 to 26 hours, irrespective of
the humidity to which it had previously been subjected.
SficJiococcus and Apafococcus proved themselves less re-
sistant ; they were killed at - 80° in 1 wo hours and one hour
respectively. However, with the relative humidity re-
duced to 50% they were equally as resistant as Pleuro-
coccus to a temperature of -80°.

AVarl)iiri; (l!*!!)) maintained in li(|ui(l air for one hour
suspensions of ChlorcUa in Knopp's solution. The cells
sustained no injury from the treatment. The chlorophyll
granules were unaltered while they became enlarged and
showed structural changes in Euglena treated in a similar
manner {Bioch. Ztschr., 100, 234).

Kiircher (1931) exposed Stichococcus hacillayis, cul-
tured on agar slants in test tubes, to - 70° for 1 to 8 days,
or to - 183° to - 192° for 13 hours. The algae were not
killed.

Becquerel (1932d and 1936) found that Profococcus and
Pleurococcus grew normally after having been subjected
in the dry state to the lowest available temperatures. The
cells, obtained from cultures in synthetic liquid media (or
in a previous experiment from a piece of bark) were dried
on barium oxide for 3 months at + 35° ; they were then
sealed into glass tubes evacuated to 10"' mm. of mercury,
and subjected to -190° for 480 hours and to -269° to
- 271° for 71 hours. Some specimens had been kept dried
in the vacuum for 25 years before being cooled. In one
experiment a temperature of 1.84° K. was maintained for
one hour. The material so treated yielded living Pleuro-
coccus vulgaris, Chlorella vulgaris, Slichococcus hacillaris,
Hantzschia amphioxys, Pinnularia viridis, Chlorococcum
humicolum, and Palmella miniala. The same was also
true for cultures not subjected to high vacua, but exposed
directly to the action of the liquefied gases.

29

SECTION II. PROTOZOA

1. Bh'izopods. According to Kiihiic (1804), Amoeba
and Actinuphrys are killed when their culture medium
freezes, but they suffer no injury when they are kept at
0° for several hours, as long as the medium is not frozen.

Molisch (1897) observed under the microscope Amoeba
freezing in drops of water. He found that the organisms
die as soon as the ice forms in their proximity or within
them, that is, at a temperature slightly below zero.

Chambers and Hale (1932), who proceeded in a similar
manner, state that the internal freezing of Amoeba, which
they induced by inserting an ice-tipped pipette into the
interior of the organism, occurs at - 0.6°. Internal freez-
ing always kills these animals, while, if ice is not present
in them, temperatures as low as -5° produce no damage.
These authors describe how the ice spreads through the
protoplasm in the form of fine feathery crystals radiating
in all directions from the point touched by the pipette.

According to Deschiens (1934), dysenteric Amoebae, in
the vegetative form, were destroyed in a short time when
their medium froze, at a temperature of - 5°. They could
be kept alive, though immotile, for 56 hours, at 0.0°, in the
unfrozen medium.

Fol (1884) obtained growing Amoeba from dried earth
which he put to freeze in Pictet's apparatus at a tempera-
ture of about - 100°.

Becquerel (1936) revived several species of rhizopods
from dried soil cooled to the lowest available tempera-
tures. He kept in a vacuum, with barium oxide, at 35°,
for 3 months, samples of soils containing various micro-
organisms. When the soils were thoroughly dried he
sealed one portion of them in glass tubes evacuated to 10~^
mm. of mercury and placed another portion in glass tubes
closed with cotton plugs. Thereupon he subjected all the
tubes to liquid helium (-269° to -271°) for 7^ hours and
subsequently to liquid nitrogen (-190°) for 480 hours.
From both groups of soils he obtained, after this treat-
ment, living Amoeba proteus, Amoeba Umax, Amoeba
dacfylifera and Aci'mophrys sol.

DO

2. CiHatr,^. CJrci'k'V (li»()l) ohscrvcd llial wiu-ii cul-
tures of Stent or coc rill CHS were cooled slowly, ciliary
movement continued for 1 to 3 liours at 0°, after which
the cilia and i>ullet were absorl)ed, the ectosarc was thrown
off, and the endosarc transformed into a spherical cyst-
like ('(,'11. When the tempeiature was again raised, these
resting cells underwent a reverse process, and resumed
their normal activity. If the temperature was lowered
rapidly and the culture medium was solidly frozen, the
organisms were killed.

According to Efimotf (1924), Parawaeciuw can with-
stand freezing (Ausfrieren) at -1° for 30 minutes, but
is killed when frozen at the same temperature for 50 to
60 minutes. Various infusoria {Paramaecium caudatum,
Colpidium colpoda, Spirnsfomuni aml)ifjuum) die in less
than 30 minutes when exposed to temperatures below -4°.
Rapid and short subcooling (not l)elow -9°) produces no
injury, but, if it is prolonged, the paramaecia become
spherical and swell up, increasing their volume 4-5 fold,
while the other infusoria shrivel up and assume irregular
contours.

To study the effect of low temperatures on Para-
maecium in a subcooled medium. Wolf son (1935) enclosed
these organisms singly or in numbers from 2 to 116 in
capillary tubes and observed them microscopically during
cooling in a specially constructed, metal, glass-bottomed
well, cooled by stout metal leads connected with a brass
plate on which pieces of dry ice were placed as desired.
Warming was effected by means of a heating coil wound
about these same conducting leads. He found that both
bodily and ciliary movements could be observed down to
- 14.2°. Then, all bodily movements ceased, but the cilia
continued to beat for some time yet. While organisms
subjected momentarily to -16° were found to recover
completely, after a longer cooling at that temperature
morphological degeneration set in, manifesting itself in a
rounding up of the oi'ganism, an apparent increase in cell
volume and a marked visibility of the nucleus, followed by

31

complete disintegration of the organism. In no ease did
a paramaecium ever survive the freezing of its medium.

Fol (1884) isolated living ciliates from dried soil frozen
at about -100°.

Taylor and Strickland (1936) found that immersion in
liquid air for 13 i hours does not affect the viability of
air-dried cysts of Colpoda cucullus. In another experi-
ment these authors obtained some excystment (percentage
unknown) from cysts first subjected to a vacuum of about
10 ' mm. of mercury for 2 or 3 days, thereupon exposed
to liquid air temperatures for 12| days, and then warmed
to room temperature within 2 minutes.

Becquerel (1936) ioi\i\d\iy\i\g Paramaecium hursaria in
dried soil kept for 7? hours at a temperature of about
2°K.

3. Flagellate,^. Mainx {Arch. f. Protisten'k., 60, 387,
1928) did not find any evidence in favor of the belief that
Euglena can withstand freezing. He observed that par-
tially or completely frozen cultures of Euglena gracilis,
E. viridis and E. deses gave rise to very few living forms,
and to these only if cysts were present previously.

According to Jahn (1933), when Euglena cultures were
kept in the frozen state, at -4°, for 1 hour, most of the
organisms, but not all, were killed. A temperature of
-0.2° maintained for the same time, without freezing of
the medium, showed no harmful effect.

On the other hand, Giinther (Arch. /. Protistenlx., 60,
556, 1928) reported that he observed a specimen of
Euglena terricola which liad been frozen while in the
process of mitosis and which completed its division, on
the microscope, in a normal manner when the ice melted,
after having been congealed for 8 days at -12°.

Klebs {Ztschr. f. iviss. Zool., 55, 265, 1893) observed
that Euglena, in the free s^\^mming state, was not killed
by repeated freezing.

Trypanosoma gambiense, according to Gaylord (1908)
can resist being immersed in liquid air for 20 minutes, but
is killed after 40 minutes.

'r III ixniosnnifi Iciris'i was round alls'c aflcr iimnci'siou
of the ciillurc in liquid aii" lor 7") minnU's, 1ml it was killed
after 24 hours (aceordiiii;' to Dollein's "Leln-bncli dcr
Pi-otozoeiikiuide," 3rd ed., ]). 230, Jena, 11)11).

De Joiii;' (1922) reporled llie results obtained in liis
lal)ora1()ry l)y Zaiidberi>eii (11)22) on Trypanosoiua as
follows : Tri/jjaiiosoma Icwisi was killed at -20° in 8
minutes, at -30° in one hour and at -190° in 2 hours;
Trypanosoiua equiperdum lost its motility and pathoge-
nieity by a stay of 3] hours at -20°; at -39°, -65° and
-145° it lost its motility but not its pathogenicity; at
-191° it was still motile and pathogenic after 21 days.
Trypanosoma venezuelense and bnicei behaved in the
same manner at T. equiperdum.

Becqiierel (1936) found living Eugleua viridls in dried
soil subjected to liquid helium in a high vacuum.

A general survey of the monocellulars reveals that :
1. Most of the bacteria, bacterioids, yeasts, algae of the
type ChJorcUa or Stichococcus and flagellates of the type
Trypanosoma, in the vegetative state and in their aqueous
culture medium, are not destroyed by the lowest available
temperatures acting for some hours ; 2, When the bacteria
and bacterioids are affected by the temperatures of the
liquid gases, the influence of the factors: species, time of
exposure, concentration of the medium, and repeated con-
gelation has been emphasized but is not clear; 3. The
rhizopods, ciliates, and flagellates of the type Eugleua, in
the vegetative state, and in an aqueous medium, do not
resist more than a few degrees below zero and are usually
found dead after they had been congealed; 4. All the
monocellulars resist temperatures near the absolute zero
in the encysted and dry state.

III. GERM CELLS, SPORES AND SEEDS

1. Spermatozoa. Our knowledge of the resistance of
spermatozoa to cold is gleaned mostly from scattered data
obtained as secondary results in the study of other prob-

33

lems. Consequently some important details concerning
the method of exposure to low temperatures are often
missing.

H. Weber (1936) could maintain hidl spermatozoa
motile in vitro at 0° for 96 hours (Dissert., Leipzig, 1936;
quoted from Ber. /. ges. Physiol., 103, 294, 1938).

According to Spallanzani (1787), frog spermatozoa,
frozen hard for half an hour, are able, after thawing, to
initiate (^gg development, but if they are kept in the frozen
state for several hours, this power is lost.

The spermatozoa of Periplaneta orientalis (a cocJx-
roach), and those of man are capable of surviving freezing
for 10 to 11 hours, according to G. N. Pawlow (1927).

Schenk (1870) reported that the spermatozoa of the
frog and of the turtle, frozen at temperatures of -4° to
- 7°, resumed motility after being warmed to 38° or 40°,
but all attempts to fertilize eggs with these spermatozoa
failed. Resumption of motility was also observed on the
spermatozoa of rabbits and dogs after exposure to -6°.

Prevost (C. r. Ac. Sc, 11, 907, 1840) froze excised frog
testes at - 8° to - 10°, and then thawed them slowly in cold
water. The water was found full of motile spermatozoa.

Quatrefages (1853) made several observations on the
action of cold on fish sperm. His results can be sum-
marized as follows : 1. Carp's spermatozoa mixed with ice
water on a microscope slide stopped their motion immedi-
ately. Barbel's sperm, under the same conditions, main-
tained its motility for 50 seconds. 2. Pike's spermatozoa
could be observed to be motile after having been directly
in contact with ice for 50 hours. In a series of experi-
ments in which pieces of ice were put in a dish full of
sperm or in which the sperm was stored with ice in a con-
tainer devised to allow the water from the melting ice to
flow out, the organisms remained motile for 64 hours.
3. Pikes, dead for 24 hours and maintained in ice, fur-
nished motile spermatozoa, while dead fish kept at 13° to
15° showed less viable sperm. 4. Pike's spermatozoa left
to freeze overnight either on a plate or wrapped in moist

34

paptT, or in a dish full of water, were acliN'c llic next day,
after tliawiiii;'. 5. To kill Piko's sperm l)\' cold, it took
a 5-li()iii- exposure of the iee-sperm mixture to a lemi)era-
tiire of - 10° to - 12°. In the course of tlu'se expei-imenls
it was ()])ser\'ed also that: a. coni])let('ly uiatui'e s])ei'm
was less resistant than immature sperm; b. spermatozoa
ag'grei»'ated in clumps resisted longer than those dispersed
in water; c diluting the material was apparently more
injurious than cooling it.

Mantegazza {Reudir. r. Inst. Lomh., 3, 183, 1866), as
quoted from Davenport (1897), reported that human
spermatozoa were not killed by exposures to -17°.

2. Eggs. We shall classify the literature on eggs ac-
cording to species.

Gemmules of 2 kinds of sponges are said by Waltner
{Arch. f. Naturgesch., 59, 257, 1893) to have resisted
respectively 17 days and 59 days in ice.

Essex and Magath {Am. J. Eyg., 14, 700, 1931) placed
ova of the tapeworm, IJiphgltohothrium latum, in water
and subjected them to a temperature of - 10° for 48 hours.
This treatment killed them all.

Zawadowski (1926) reports that fertilized eggs of
Ascaris can withstand - 5° for 1 month, and show normal
development after subsequent warming. After 14 days
at - 20°, 50 per cent of the eggs degenerated ; after 2
months of exposure to - 24°, there was still a possibility
of development on warming, yet the embryos finally died.
An exposure of 1 j years to - 10° produced a similar
degree of injury. (From Belehradek, 1935.)

A wealth of data on the death temperatures of insect
eggs is found scattered in the observations of the entomol-
ogists of the last two centuries. Bakhmetieff (1901),
Bachmetjew (1901) and Uvarov (1931) reviewed the sub-
ject. "We refer the reader to these authors' works for
more complete information and we mention here only a
few of the more striking results. Some of them indicate
a high cold resistance, others a high cold sensitivity.

Spallanzani (1787) observed that the eggs of Bomhyx
morl can support an air temperature of -50° for 4 hours.

35

According to Pictet (1893), the eggs of Bomhyx can be
stored at - 40° ; this temperature is not only non-lethal to
the eggs but it is injurious to the germs which might
infect the eggs.

Donhoff (1872) exposed for 5 hours Bomhyx eggs con-
tained in small glass vials to the temperature of -15° to
-21° of an ice-salt mixture. The eggs were then placed
into little bottles tied shut with linen and the bottles put
into a box which Donhoff carried on his breast by day and
took to bed with him by night. After a few weeks all the
eggs hatched.

On the other hand, according to Pictet (1893), ant eggs
are extremely sensitive to cold. They are all killed be-
tween 0° and -5°. The sensitivity increases with the
stage of development and some more advanced eggs died
when exposed to 5° for a few hours.

Bach and Pemberton (J. Agr. Res., 5, 657, 1916) found
that no eggs of the Mediterranean fruit fy survived cold
storage at 4.5° to 7.2° for 7 weeks, or at 0.5° to 4.5° for 3
weeks or at 0.0° to 0.5° for two weeks. (From Uvarov,
1931.)

According to Hase {Ztschr. /. Parasitenli., 2, 368, 1930),
an exposure of bedbug eggs for 3 to 20 days to a tempera-
ture of 2° was harmful enough to prevent the hatching of
a number of eggs, in which only an initial development
was found to take place.

According to Eahm (1920 and 1923), the eggs of the
tardigrad, Echiniscus, in moist moss, survived two suc-
cessive freezings of 35 minutes each at - 81° and those of
another tardigrad, Maerobiotus, were not killed by a sud-
den immersion, in the moist condition, in liquid air or in
liquid hydrogen.

Schenk (1870) exposed fertilized eggs of Rana tempo-
raria to - 3° for one hour during w^hich time the jelly layer
had frozen hard; he found that, after thawing, the eggs
developed. Fertilized eggs exposed for one hour to - 7°
showed no further development after thawing. ^Mature
unfertilized eggs of Bufo cinereus, taken from the body of

•AG

lilt' inotlicf, and posscssiiio- a jelly layci-, were exposed to
-4° I'or 1 hoiii- and ihrii arl ilicially fei-tilized and placed
in favorable conditions for development. In the first
hours after tliawint;-, no externally perceptible chang-es in-
dicative of fertilization conld be observed, hnt 14 honrs
after fertilization, the cleavage process commenced and
thereafter ])roceeded at the normal rate.

P^ischer-Sigwart {Vlerteljahrscli. d. Naturf, Ges. Zurich,
62, 1897) records finding- masses of frog eggs which had
been frozen solid for 2 days, during which time the tem-
perature sank to -8°. When the eggs were gi'adually
thawed ont, they underwent a normal process of develop-
ment, though this was somewhat slower than usual.

Pictet (1893) says that he cooled frog eggs to -60° for
several hours and that they developed into tadpoles.

Several investigations are found in the older literature
on the death point of bird eggs, but many authors over-
looked the fact that it takes a relatively long time for the
interior of the larger eggs to reach the equilibrium of
temperature with the surrounding atmosphere. There
thus result some apparent contradictions which partially
vanish when one considers the temperature of the hath or
of the atmosphere in the various experiments, the time of
exposure, the size of the eggs and the other factors which
determine heat conduction. A short review of some of
the literature will be found in Moran (1925). We shall
describe here a few of the more typical investigations on
chicken eggs.

Lipschiitz and Illanes (1929) reported that of 33 lien's
eggs, exposed for 3 to 7 hours to an air temperature of

- 4° to - 6°, 20 developed embryos or yielded chicks, these
latter retaining their normal appearance throughout an
observation period of several months. In another series
of experiments, the temperature within the eggs was de-
termined by inserting a thermometer into a hole in the
egg. Temperatures of - 2.5° to - 4.5° were thus recorded.

Rabaud (1899) put to freeze in an ice-salt mixture at

- 18°, 30 sets of 18 eggs each. He left them in the mixture

37

for half an hour, after which time most of the shells were
cracked. Some of these frozen eggs were then incubated
immediately at 38°, others were kept in a cool chamber
and incubated after one day, and others, finally, were kept
for 3 days before being incubated. All of them, together
with 6 control eggs in each set, were opened after a 3-day
incubation. About one third of the eggs in each of the
different series of experiments presented embryos, al-
though badly deformed, while the other two thirds con-
tained blastoderms extended over the yolk, but showed no
embryo formation. The author concludes that freezing
did not prevent cell proliferation liut tended to inhibit cell
differentiation.

As a thoroughly worked out investigation on the death
temperatures of the chicken egg and on the time required
for death at various temperatures we shall describe Mo-
ran 's work (1925) . He placed 100 eggs in a constant tem-
perature room at - 2.9° and 100 in another room at - 4.6°.
Every few hours some eggs were taken out and tested, the
power of incubation being taken as the index of vitality.
In a previous determination of the velocity of cooling,
made with a thermo-couple, it was found that 24 hours
elapsed before the center of the egg reached - 2.9° and 30
hours before it reached -4.6°. The general results were
that some eggs were still capable of developing after hav-
ing been for nearly 47 hours at -4.6° and 118 hours at
- 2.9°. But even above zero the power of incubation was
soon destroyed. The limit of the time that the eggs could
be stored at 0.7° was 10 days and at 10.4°, 34 days. In
these experiments cooling and Avarming were graded and
slow. Moran concludes that the germination capacity is
probably destroyed immediately at -6° to -7°, while
at the higher temperatures death requires increasingly
longer times. It seems evident that all the eggs studied
were subcooled ; the question of whether or not the chicken
egg withstands congelation, is left untouched in this work.
3. Spores. Strasburger {Jena. Ztschr. f. Naturwiss.,
12, 612, 1878) observed swarm spores of the algae, Haema-

MS

fococciis and (InhiiiKnids inoxiii.i;- alxml in i)aiiially frozen
(li'ojjs of watci', anions,- the ice ci-ystals.

Acrordiiii*' lo llic same aiilhor, fully fio/cii ("viillig
oingofrorciie") sirann .v/^c^/tx of Uachtdlococcus, Uloth-
r'lx, Ih)f]iri/(liinn and CJiilonioiuis were fonnd dead after
tliawin.ii', in an experiment in which tlic 1eni])('rature of the
surroundings liad not drojjped l)elow - 1°.

Kjellmanu (quoted from Bof. Ztg., .7.'), 771, 1875) re-
ported that some marine algae form and discharge swarm
spores when the sea temperature lies between -1.5° and
-1.8°.

According to Wettstein {Sitztiiigsh. <L Wiener Al<a<J., .91,
33, 1885), the eonidia of BJiodomyces Kochii (a fungus
parasitic on man) are, for a large part, incapable of ger-
mination after having been exposed for 2 hours to - 7°.

Smart (1935) found that some species of the fungi, De-
matium, Monilia, Oidinm and PenicUlium failed to grow
after one year at - 8.9°. Other species of PeniciUium,
however, did grow after the same treatment.

According to Noack (1912), spores of fungi belonging to
the genera, Mucor, Tltcnnoascus, Thcrutoidunn , TJtermo-
myces, and Actinomyces, suspended in ha}' decoction in
test tubes, allowed to remain for several hours at room
temperature, and then exposed to -20° for 13 hours, did
not lose their germinating power.

Teodoresco (1906) subjected the zoospores of DunaJieUa
(a Volvocinea) to temperatures down to -32°, in concen-
trated salt solutions (in which they are known to thrive).
Some of these organisms survived freezing at -28° to
-29° for one hour. However, since many of them were
killed l)y this treatment, Teodoresco thinks that only those
zoospores survived that were able to tind interstices be-
tween the crystals of the hard-frozen mass.

Irmscher (1912) exposed water suspensions of moss
spores belonging to 4 genera to - 20° to - 21°, for 16 hours.
All of them germinated normally. In a second experiment
the spores of the same mosses were exposed, under the
same conditions, for 18 hours to - 32° to - 40°, after which
no germination occurred.

39

Chodat (1896) reported that spores of the mould,
Mucor, spread on agar or suspended in licinid Raulin's
medium and put for 2 hours in a freezing chamber at

- 70° to - 110°, germinated, but with a considerable delay
over the controls. The filaments originating from the
treated spores were 1 cm. long when those from the con-
trols were 2 to 3 cm.

Alexopoulos and Drummond (1934) exposed to the ac-
tion of liquid air for 1 hour the spores of the fungi, Melan-
conium, Comothijrium, Euroiium, and Cytospora, either
suspended in sterile water or inoculated on corn meal agar
slants (in both instances they were contained in test tubes)
or dried on a glass rod. In all cases, germination and
normal growth followed.

According to Kadisch (1931), the spores of some 10
species of pathogenic filamentous fungi {Epidermophytes)
could resist - 20° to - 30° for at least 34 days. In liquid
air, some species were killed in one hour, others in 24
hours, still others in 48 hours, while some were not at all
affected. Further experiments of Kadisch showed that
two species (called the Acli. Gyps, and Kaufmaun Wolf's
fungus) survived after respectively 3 and 50 hours at
-252° and were not killed either by a stay of 2 hours
at - 268° followed by 4 hours at - 268.8° and 1^ hours at

- 272°.

Becquerel made a series of observations on the germi-
nation of various spores and of pollen after exposure to
the temperature of liquid gases. His results are sum-
marized in the following paragraphs.

Spores of 4 kinds of phycomycefes {Mucor, Rliizopus,
Sterigfnatocystis and Aspergillus), thoroughly dried and
sealed in tubes where the vacuum was made to 10* cm. of
mercury, were immersed in liquid hydrogen for 77 hours.
They were then left 2 years in the vacuum. Germination
was normal (Becquerel, 1910).

Spores of 6 genera of mosses {Dicranella, AtricJium,
Hypnum, Leucohryuw, Funaria and Brachythecium), not
specially dried, were immersed for 10 days in liquid nitro-

40

yen, (lirrctly in coiitacl with llu' lluid. X()lliiii<4' abnonnal
was observed in liicir g'ermination. In anotlier experi-
ment, spores of 2 of the above genera, previously desic-
cated and maintained in a vacuum of 10 " mm. of mercury,
were immersed in li(|uid lielium at 4° K. for i) liours and
at 1.84° K. for 1 hour. No injurious effects resulted
(Becquerel, li);J2a).

The spores of the feni, Polystichuni filix mas, previously
desiccated, sealed in the highest obtainable vacuum and
maintained for () hours in liquid helium between 5° and 3°
K. and for f) hours at 3° K., germinated like the controls
(Becquerel, 1930).

Pollen gvsans of AiifirrJiiinou and Nicofiaiia, thoroughly
dried, in sealed tubes, in a vacuum of 10 ' mm. of mercury,
were put in liquid helium for 7 hours, the lowest tempera-
ture reached being 1.3° K. ; this did not weaken the ger-
minating power of the grains (Becquerel, 1929).

4. Seeds. Seeds have been a subject of predilection for
investigators of the effects of low temperatures.

Edwards and Colin (1834) subjected seeds of barley,
oafs and wheat for 15 minutes to - 40° (by the evaporation
of sulphur dioxide in the vacuum) and noticed no impair-
ment in their germinating power.

Wartman (1860 and 1884) exposed 9 species of seeds,
from 20 minutes to 2 hours, to temperatures from - 78°
to -110° (ether and car])on dioxide) and observed no in-
jurious effect in them. The seeds were enclosed in sealed
tubes.

De Candolle and Pictet (1879) subjected various seeds
to temperatures between - 40° and - 50° for about 4 hours,
and in another experiment, to -80° for more than one
hour; in no case did they observe any action of the cold
on germination. In a later experiment (1884), the same
authors subjected seeds of 8 species, dried in the air and
enclosed in sealed tubes, to a temperature lower than
-100° (by evaporation of carbon dioxide) and they ex-
tended the time to 4 days. This prolonged action of low
temperature had no effect on the seeds.

41

De Caiulolle (1895) investigated the action of repeated
freezing on seeds not previously subjected to a special
desiccation. The seeds, wrapped in tin foil and enclosed
in a sealed metal box, were exposed 118 times to a tem-
perature lower than - 100°, the duration of each exposure
varying from 8 to 20 hours. No harm was done to ivheat,
oats, and fennel by such treatment, but most of the lobelia
seeds did not germinate.

Pictet (1893) mentions exposure of seeds to -200°
without injury.

According to Brown and Escombe (1897), seeds of 12
kinds from 8 families, dried in the air, that is, still con-
taining 10 to 12% of water, were enclosed in thin glass
tubes and put in liquid air for 110 hours. Neither their
power of germination nor their growth was impaired in
any way.

In 1899 Moissan presented to the French Academy a
note received from Dewar where the latter stated that he
had succeeded in solidifying hydrogen and that seeds
cooled to the temperature of liquid hydrogen did not lose
their germinating power. (C r. Ac. So., 129, 434, 1899).

Thiselton-Dyer (1899) subjected to the temperature of
liquid hydrogen various seeds chosen for their diversified
nature (farinaceous or oily), for their various sizes and
shapes (ellipsoidal, flattened, etc.). The seeds, simply
air dried, enclosed in sealed tubes and cooled tirst in liquid
air, were left half an hour in liquid hydrogen (more than 1
hour it is said elsewhere in the paper). No change was
noticed and germination was normal. In a second series of
experiments, the seeds were immersed for 6 hours in
liquid hydrogen with which they were directly in contact.
No attempt was made this time to graduate the cooling.
All the seeds germinated in the normal manner.

Adams (1905) put in liquid air, for about 24 hours, 6
sorts of seeds, some of which were dry, while the others
had been buried in the soil for 3 days so as to absorb water.
All the dry seeds germinated while none of the moist ones
did, except timothy which is known to imbibe water with

42

diflii'iilly. 'I'lic author stales that seeds should eoutaiii
more tliau 12// water to l)e kiUed l)y tlio temperature of
liquid air.

Becquerel (IDO.j) showed tliat the hig'her the de^'ree of
desiccation llie more resistant are the seeds to tlie tem-
peratures of the li(iuid gases. In a lirst series of experi-
ments he soaked decorticated seeds in water for 12 liours
and immersed them in liquid air ; the seeds did not survive
the treatment. On seeds dried in the vacuum at 40° for a
montli and immersed for 132 hours in liquid air he did not
observe any injury. But, out of 16 kinds of seeds, simply
dried in the air, 6 only survived the immersion in liijuid
air and these were the species which contained less water
(6 to 12%). In later experiments (1909), in which Bec-
querel intended to bring- the seeds into a state of complete
inactivity, he desiccated them in the presence of barium
oxide for 2 weeks at 35°, then put them in sealed tubes
evacuated to 10 * mm. of mercury, and immersed the tubes
in liquid air where they stayed 3 weeks and in liquid
hydrogen where they stayed 77 hours and, finally, he
maintained them sealed for 1 3^ear. When he opened the
tubes, the seeds germinated in the usual manner. In a
last experiment (1925), he used liquid helium. Seeds
desiccated as described above, put in a vacuum of 10 " mm.
of mercury and kept immersed in liquid helium at 3.8° K.
for 10] hours germinated like the untreated controls.

Lipman and Lewis (1934) investigated the action of a
prolonged exposure of seeds to liquid air temperatures.
Nineteen sorts of seeds, previously dried over calcium
chloride and enclosed in glass tubes, were left in liquid air
for 30 days, in a first experiment, and for 60 days, in an-
other. Neither the germinating pow^er of the seeds, nor
their gro^vth, nor any of the characters of the plants grown
from them, differed from the controls.

Lipman (1936a) immersed in liquid helium 9 kinds of
seeds previously desiccated for 2 weeks over sulphuric
acid in a partial vacuum and contained in glass tubes 1
cm. w4de. The temperature was 4.2° K. for about 40 hours,

1.35° K. for 2 lioiirs and some intermediate temperature
for another 2 hours. He obtained 1.35° K. by application
of vacuum to liquid helium. The treated seeds grew like
the controls. The author gives photographs of the plants
obtained with the exposed seeds and he announces that he
intends to study the possible delayed effect of cold on the
next generations.

To summarize : 1. The spermatozoa, even those of the
homoiotherms, were found to resist sub-freezing tempera-
tures; they might well belong to the type of organisms
which, like the bacteria and some smaller flagellates, resist
the lowest temperatures, but we did not find enough evi-
dence in the literature to substantiate this view; 2. The
eggs of higher animals (birds, amphibia) were, in general,
found to be killed after having been exposed to a few
degrees below zero; the eggs of lower animals (insects,
parasite worms) often resisted -30 to -40°; 3. The few
motile forms of spores investigated were rather sensitive
(as were also the larger flagellates) ; 4. The spores of
fungi, ferns and mosses, and the seeds resisted, after
drying, and some of them even in the moist condition, the
lowest available temperatures.

IV. ISOLATED CELLS AND TISSUES
SECTION I. PLANT MATERIAL

1. Hair Cells. Klemm (Jahrh. f. iviss. Bot., 28, 641,
1895) exposed to low temperatures Triania and Momordica
hair cells in a test tube immersed in a freezing mixture.
The temperature was read on a thermometer placed in
the tube with the material. After 15 minutes exposure
to - 13°, the cells were dead, the protoplasmic streaming
had permanently ceased. After a shorter exposure or
at higher temperatures (-6°), the streaming was often
resumed; exposures to temperatures above -2° did not
stop the streaming.

Kiihne (1864) found that if isolated staminal hairs of
Tradescantia, placed in a drop of water on a slide, were

44

frozen on an icc-sall niixtmc, the jjiotoplasni was com-
pletely destroyed and, aftei- t hawinii', it l)roke nj) into
crumby coagulated clum])s. If the hairs were })nt, with-
out the addition of water, into a thin platinum crucible
and the latter lowered into the freezing- mixture, the proto-
plasm could be ke})t alixc for 5 minulcs at -14°; after
thawing, the ])rotoi)hismic streaming was resumed. At 0°
Tra(le.sca)iiia hairs could be kept alive for at least one hour.

Molisch (1897) who observed, mider the microscope in
a cold chamber, the freezing of Tradescantia hair mounted
in water, described the cessation of cytoplasmic movement
when the temperature reached -2° and the sudden con-
gelation of the cell content at al)out -6.5° (temperature
of the air within the freezing chest in which the material
had been for several hours). After thawing, the nucleus
was disorganized and the plasma coagulated. When the
hair cells were exposed directly to the air temperature of
the chamber, without water as a mounting medium, they
withstood 6 hours at - 5° to - 9°. When they were mounted
in oil, they did not freeze even at - 9° ; congelation and
death occurred only at -15°. In air and in oil the cells
could bo subcooled to a much lower temperature than
in water, and they stayed alive as long as they were in
the subcooled state. The epidermal hairs of Episcia,
Ageratum mexicanum and Pelargoniuw had a higher
resistance than Tradescantia hairs.

2. Epidermal Tissue. Molisch (1897) reported that in
sections of the epidermis of Tradescantia, mounted in
water between slide and cover-slip, and observed under
the microscope while exposed in an air chamber to a tem-
perature of - 5°, many cells were seen to freeze in sudden
flashes. On thawing, after 6 hours exposure, they were
dead, as judged by the disorganized, shrunken, and some-
times stainal)l(' nuclei, by the coagulated cytoplasm
and by the fact that the cell membranes absorbed the
anthocyanin present. Some cells did not freeze during
that time and at that temi)erature, which would indicate
that - 5° is wdthin the range of the lethal temperatures for
the epidermal cells.

45

Luyet and Gibbs (1937) repeated the experiments of
IMolisch, using the ei)idermis of the onion. They followed
this author's procedure in all details except that their
material was vitally stained with neutral red before being-
cooled, and that their low temperature chamber was set
to about - 10°. With the higher cooling velocity furnished
by this lower temperature, all the cells were observed to
freeze in 5 to 6 minutes. Sudden flashes during freezing
indicated that the individual cells were subcooled and that
they congealed at once. After thawing, they were all dead,
as shown by the release of the stain.

Zacharowa (1926), in a study of the effect of hydrogen
ion concentration on cold resistance, put to freeze epi-
dermal sections of red cahhage in test tubes immersed in
freezing mixtures and containing a little quantity of either
distilled water or a weak solution of sodium hydroxide
(0.01 to 0.001 N) or sulphuric acid (0.0001 to 0.00001 N).
The material, seeded with an ice particle, was left to freeze
and cool for one hour. It was then thawed and its vitality
was tested by plasmolysis. Half of the cells were found
alive after exposure to - 3.9° and one-fourth after expo-
sure to -5.75°, in distilled water. The sections in the
sodium hydroxide solution were slightly more resistant,
and the sections in the sulphuric acid solution less resis-
tant than those in distilled water.

Chambers and Hale (1932) followed under the micro-
scope the gradual freezing of single epidermal cells in
precisely known temperature conditions. Epidermal
strips of the red onion were placed, together with the tip
of a thermo-needle, in a hanging drop of liquid paraffin
and the temperature of the whole system was slowly low-
ered, in a cooling room, while inoculation with an ice-
tipped pipette prevented subcooling and subsequent too
rapid freezing. When the temperature reached -7° to
- 10°, ice was formed between the cellulose wall and the
protoplast but the latter was usually still capable of
plasmolysis. At - 10°, the cytoplasm and nucleus disinte-
grated, but the tonoplast still resisted. These phenomena

seem to indicalc that llii' lowest limit of iioii-lctlial tcm-
j)eratnro, uiidci' these coiidilioiis, is in the iiei,ii,lil)orlioo(l
ot'-10\

BecMiuerel (1!).')7) immersed in a li(|iii(l iiitroi>'on ])atli
oiiitnt epidermises mounted on thin glass slides juid lie left
tiiem at the temperature of that bath for 10 minutes, 24
hours, or 3 weeks. The cell contents, observed with the
microscope while they were in the liipiid gas, were con-
gealed and, after thawing, coagulation could be observed.
Death had always set in.

Lnyet and Thoennes, in an attemi)t to test a theory
according to which, in a very rapid cooling, the ])rotoplasm
would vitrify, that is, solidify without crystallizing, suc-
ceeded in maintaining alive onion epidermises after im-
mersion in li(|uid air. The material, vitally stained with
neutral red, was partially dehydrated by a rapid plas-
molysis in a sodium chloride solution ; it was then im-
mersed at once in liquid air and brought back into the
plasmolysing agent. After this treatment, the majority
of the cells held the vital stain and could be plasmolysed
to a further degree or deplasmolysed. (Paper in press;
Abstract presented at the Indianapolis meeting of the
Am. Soc. of Plant Physiol., Dec. 1937.)

Stomatal cells, which are known to be resistant to a
number of injurious agents, such as heat, drought, decay,
etc., have been found by several investigators to be also
exceptionally resistant to cold. Molisch (1897) put leaves
of different kinds {Primula, Nicotiana, CampannJa, Ilya-
cinthus, Episcia, Cyclamen) in test tubes in which he had
a thermometer and he immersed the tubes in cold mix-
tures. After two hours, when the temperature between
the leaves was - 7.5°, a plasmolysis test with 10% NaOl
revealed that the stomatal cells were alive while all or
almost all of the neighboring cells were killed. In Piper,
treated in a similar manner, the stomatal cells were also
killed at that temperature. In Maranfa, they were killed
by an exposure of one hour to - 6°. In Dahlia, all the cells
except those of the stomata were killed after exposure

47

to -3°, and in Pelargoniwn, after exposure to -6°. In
Nicotiaua, one hour at - 12° did not kill the stomatal cells ;
they died only when brought to -15° to -17°. In leaves
which die by exposure to temperatures of + 2° to + 3°, the
stomatal cells were also observed to be alive after the
other epidermal cells had been killed.

3. Tuberous Tissue. The potato tubers, on account of
the large masses of homogeneous tissue that they present,
were often used in investigations on the freezing point of
living material and on the effects of low temperatures.
The death point has been investigated, in particular, by
the following authors.

Goppert (1830) reported that when the temperature in
the interior of a potato (measured by inserting a ther-
mometer in it) had dropped by only one degree below
zero and all the sap had been converted into ice, the potato
was dead after thawing and turned brown.

According to Miiller-Thurgau (1886), potatoes exposed
to an air temperature of -6° for a time long enough to
bring the temperature in their center to - 1.5°, w^ere dead
throughout after thawing. A thermometer was inserted
in the middle of the potato. Death was diagnosed by the
blackening and the soft consistency of the tissue.

Apelt (1909) who Avas asked by his master, Mez, to test
a theory on the existence of a death temperature, specific
for each organism and lying below the freezing point,
found that each race of potato has its own death point,
although the latter can be raised or lowered by the condi-
tions of the previous storage. He determined the tem-
perature within the material with a thermocouple and
diagnosed the vitality of the cells by the plasmolysis
method, with potassium nitrate solutions containing a
few drops of methylene blue. The low temperatures were
produced by ice-salt mixtures. The death points regis-
tered extended from -1.71° to -3.63°. They were lower
than the freezing points ])y quantities varying from 0.10°
to 1.17°. The "hundredth of a degree," alw^ays scrupu-
lously given, and the idea of a death point determinable to

4S

the lliiid (l('('iin.-il ))l;ic(' li;i\c aimiscd some lalfr iiiN'csti-

Maximow (li)14) reiieatcd tlic exporiments of Apclt,
also using- plasmolysis as a criterion of vilalily. He
obtained the follow! iii;- i-csulls: 1. Potato tissue frozen to
-2.11° (internal tenii)('ra1ui-e) had all its cells alive after
thawing-; 2. The num])er of dead cells was quite consider-
able after freezing to - 2.26° ; 3. The cells were practically
all dead at -2.66°. The death temperatures would then
lie in the neighborhood of - 2°.

In other experiments, ^Maximow (11)14) showed that the
velocity of freezing was a factor to be considered in the
problem of death temperatures. A piece of potato, frozen
to -1.82° in 13 minutes, had almost all its cells alive;
another, frozen to the same temperature in 3 hours 52
minutes, had most of its cells dead. Measuring then the
amount of ice formed before death occurred he came to
the conclusion that a slower freezing, in the second experi-
ment, had caused more water to freeze in the tissue, and
that death depends, in the last analysis, on the quantity
of ice formed. These results fit with the notion of the
death point, according to which death is conditioned by
a number of variable factors and does not take place
Avithin sharply defined temperature limits.

Luyet and Condon subjected to slow freezing in an air
chamber potato tissue cut into cylindrical shells having a
wall thickness of 2 mm. and slipped around the bulb of a
thermometer; they stopped the cooling process in suc-
cessive experiments, after congelation had proceeded for
various lengths of time and they counted the number of
living cells, using the plasmolysis test combined with a
vital staining method. They found that as long as the
freezing cui-ve stays at an appi'oximately constant hori-
zoiilal level, in genei-al above - 2°, the cells are pi-aciically
all alive even after a freezing- of 12 to 15 minutes. Death
begins when the temperature drops below - 2°, and, if cool-
ing is continued at the same slow rate, the death of all the
cells requires about 10 moi-c minutes; Ihe temperature,

49

when all the cells are killed, is then in the neii>-lil)orhood
of -5°. The lethal temperatures extend, therefore, from
-2° to -5°. (Unpnhlished data; paper presented before
the Indianapolis meeting- of the Bot. Soc. of America,
Dec. 1937.)

4. Tissue from Leaves, Stems, Roots, etc. In this sec-
tion, we shall deal with plant organs rather than with plant
tissues. In some cases the investigators, in their studies
of organs, have endeavored to tind which tissues were
involved, but most of the time we have no such information.

The agricultural and horticultural literature is rich in
observations made in the open on death temperatures of
leaves, buds, flowers or stems of various species of plants.
Tables of observational and also of experimental data
have been published. The authors of most of these tables,
however, intended primarily to furnish the agriculturists
with instructions easy to follow, and they do not give such
factors as the time during which a plant organ can be
exposed to a given degree of cold or the internal tempera-
ture of the organ. Instead of quoting these survey tables,
we think it, therefore, more useful for our purpose to
describe some of the more significant experiments.

The leaves of some plants are killed by the action of
temperatures above zero. Molisch (1897) exposed the
isolated leaves of Episcia to temperatures of 2.5° to 4.4°.
After 24 hours, several leaves became bro^^i and spotted
and after 4 days, they were all brown. The plasmolysis
test revealed that the cells were dead. On another experi-
ment, the leaves were put in ice water between 0° and 1°.
In three hours, they exhibited brown spots and in 24 hours
they were entirely brown.

Miiller-Thurgau (1880), using as a death criterion the
change in color that the petals of some orchids undergo
during protoplasmic disintegration, put to freeze in a
cooling chamber petals of Phajus graudifolius wrapped
around the bulb of a thermometer. The temperature sank
first to a subcooling point, then rose suddenly to the freez-
ing point, stayed there for a short time and finally dropped

again slowly. Tlic aiiliior observed thai llie \vlii1(' petals
beeame l)lue wlicii llie Icinix'i'alui'c, in the last i)]iase of the
curve, began to fall below the freezing- point. The latter
was found to be - 0.8°.

The same author reported that an onion hulh, exposed
to an external temperature of -4° for 7 hours and hard
frozen, was found alive. A thermometer previously in-
serted in the middle of the onion showed that the tissue
had first been subcooled to -3.1'" and that it then fi-oze
for 4 hours between -0.7° and -0.95°. According to the
author's idea that death takes place when most of the
water content is frozen, the death temperature would lie
immediately below the freezing temperature registered.

Walter and Weismann (19oG), in studies on the differ-
ence in the position of the freezing i)oint in living and
in dead tissue, observed that in the roots of Daucus carota
frozen 3 times in succession (lowest temperature reached:
-1.28°) more than half of the cells were alive. The tem-
perature was read on a thermometer inserted in the mate-
rial, and the loss of vitality was ascertained by the stain-
ability of the tissue with eosin. In another series of 6
successive freezings of the same object, the lowest tem-
perature reached having been - 1.50°, a number of cells
were still alive. But repeated freezing, even at tempera-
tures becoming gradually higher in the successive experi-
ments, resulted in an increased number of dead cells.

According to Apelt (1909), the death point of potato
shoots lies between -2.16° and -2.74°. (For the details
of the experimentation see, above, the description of
Apelt 's work on potato.)

Maximow (1914) found that in the root of the red hect,
where death was diagnosed by the release of the pigment,
the freezing point lay at about -2°, the death point at
about - 3°, while ice continued to form, after death, at
lower temperatures. (The experimental procedure has
been described under the heading "Tuberous Tissue.")

According to Harvey (1919), leaves, petioles or stems
with waxy epidermal coverings resist lower temperatures

51

than the loss protected j^lant org-ans; they can be more
easily maintained in the subcooled state, the ice-inocula-
tion being prevented by the epidermal coverings. Sub-
cooling to 5 degrees below the freezing point was found
not uncommon in these less exposed plants.

The cold resistance of growing rye seedlings was investi-
gated by Zacharowa (1926). She exposed them, when
the rootlets were 1.5 to 2 cm. long, to various degrees of
cold in a freezing chamber, and occasionally determined
the internal temperature with a thermocouple. As a cri-
terion of death, the change in coloration and the facility
of the subsequent drying were used. The results were
that: 1. An air temperature of -2.9° for several hours
did not injure the roots ; 2. A similar exposure to - 2.9°
to - 3.9° resulted in the death of the cortex and the root
hairs ; 3. An exposure to - 5.75 for 1 hour killed the entire
root except 1 to 1.5 mm. at the tip; 4. The meristem of the
tip died after an exposure of 1 hour to - 7.8°, after which
time the interior of the root had reached that tempera-
ture ; 5. Without ice formation, exposures to - 11.1° were
harmless. Similar experiments on seedlings of wheat,
pea, corn and buckwheat showed a decreasing resistance
in the order given, the meristematic tip of the buckwheat
rootlets being killed at - 2.9°.

We have mentioned above Mez' theory (1905) accord-
ing to which death occurs at a minimal temperature spe-
cific for each plant or plant organ. Mez contended also
that congelation protects the tissue against death by re-
leasing heat and delaying the further drop of tempera-
ture. His disciple, Voigtlander (1909), claims to have
confirmed the theory in a variety of plant tissues. The
specific minimal temperatures that he obtained are in
general low, some of them being about 10 degrees below
the freezing point. The extreme cooling rapidity that he
used in his experiments (often 10 to 15 degrees per minute
at 0°) makes his results the most doubtful, since it has
subsequently been shown that the time the material takes
to freeze or the time that it stays at a given sub-freezing
temperature is to be considered in the damage caused.

.)_'

Tlic al)iiii<l;iiil litci'jit ui'c on wiiilcr liai-diiicss, wliicli wc
do not Ircnt in dclnil, is somclinu's snrainarizcd in llio
statement thai, in JHicif plant i issues, death occurs Avlien
the temperatnic diops a few degrees below zero, while in
conifers and several otlnr crergreeiis the death tempera-
tures are lower by some 10, 20 or more degrees. Some
investigations, however, like the following one by Winkler
seem to indicate tliat llic ])r()hh'm is not so simple, there
being important seasonal variations in the same species.

Winkler (1913) experimented on the cold resistance of
hiids, leaves, cambium, cortex and wood of branches, 5 to 6
years ohl, of nnnicrons species of trees. The branches
were cut into ])ieces 20 to 25 cm. long, split longitudinally,
and placed upright in a brass cylinder, with their lower
ends immersed in tap water in a glass dish set into the
bottom of the cylinder. The brass container was lowered
into an ice-salt bath and the temperatures recorded by a
thermometer suspended in the air between the twigs.
Subcooling was avoided by jjlacing a few ice crystals in
the water in the dish. After having been exposed for 12
hours, the material was thawed and its vitality investi-
gated by plasmolysis. The results were as follows: 1. In
experiments made in the Spring, the buds of 28 species of
deciduous trees, exposed to - 3°, were found uninjured,
only those of Quercus pedunculata having been killed at
that temperature; at -5°, all the buds were killed. Simi-
larly, the leaves or needles of 13 out of 14 species of ever-
greens were killed at approximately -4°. The cambium,
cortex, and wood of all the trees experimented upon sur-
vived exposure to -10°, but were all killed after 12 hours
at - 18°. 2. In the Summer, the cold resistance of decidu-
ous trees drops considerably, the death point of the cam-
bium, cortex and wood lying at about - 9°, while the resis-
tance of the same tissues in the evergreens undergoes
little change. 3. In the Fall, the resistance is on the up-
grade again, and it was found that in November, all the
buds of both deciduous and evergreen trees withstood
-17°, while those of 17 out of 29 deciduous trees, and 5

53

out of 14 evergreens were not killed at - 19°. 4. The
maximum resistance is reached in Jamiary, at which time
the buds of all the evergreens (14 species) and of 27 out
of 29 species of deciduous trees were not killed at - 20° ;
most of them survived - 22° as well. In none of the spe-
cies were the cambium, cortex and wood injured at this
time by -20° or -22°. 5. Branches, whose death point
had been determined as about -21°, were frozen repeat-
edly at - 13°, and, after each freezing, they were kept for
24 hours under a bell jar at 18°. Most of the trees were
killed by a 6-times repeated freezing at - 13° ; the leaves
and needles of the evergreens were, in general, found more
resistant.

While, as we have said above, the cells of juicy leaves
are generally thought to be killed when they are subjected
to temperatures of a few degrees below zero, Iljin (1933)
showed that sections of red cabbage leaves could be main-
tained alive after freezing at much lower temperatures if
the withdrawal and the reabsorption of water, concomi-
tant respectively Avith freezing and thawing, were gradual.
According to him, death results usually from a tearing of
the protoplast in a too rapid plasmolysis during freezing
or mostly in a too rapid invasion of the cell by water when
the ice melts. Freezing slowly and thawing in hypertonic
solutions would prevent such injury and maintain the
cells alive. So, he cooled the material by steps, letting it,
for example, one day at about -5°, one day at -8°, one
day at -11°, etc.; then he warmed it to about -5° and
immersed it in cooled salt solutions which were approxi-
mately isotonic with the frozen cell content ; he then let
the tissue thaw slowly in that solution. Proceeding in
this manner, he obtained living cells (capable of plasmoly-
sis) after an exposure of 21 hours to - 80° and after longer
exposure to the higher temperatures of - 15.8°, - 11.3°,
etc. If the observations of Iljin, which favor the old
theory that death is due primarily to a too rapid thawing
and not to congelation or to an injury at some given low
temperature, are confirmed, the classical notion of death

:)4

Icniltcraliirt' iinisl he ciitii'cly modified. AVliicli is llie
lowest Icniperature reaelied, liow long the material has
been at that temi)eralnie, and even how mnch ice has been
formed, become factors of secondary importance if death
results from a mechanical injury during thawing.

Becquerel (19o2b) investigated the action of the tem-
perature of liquid gases on the tuberous roots of some
RauuncuJaceae. In preliminary experiments he found
that the roots can be dried in the air for 6 months or in a
vacuum for 2 montlis without losing their ability to pro-
duce new si)routs when remoistened. The roots, dried in
the air and in a vacuum, withstood an immersion of 18
days in liquid nitrogen ; the roots, dried and subsequently
soaked so as to re-imbibe respectively 45, 98 and 160 per
cent of their previous water-content, died.

Lipman (1936b) immersed in liquid air for 50 hours
sealed tubes containing protonemata of 8 genera of
mosses, previously dried in a vacuum over sulphuric acid.
No injury resulted from the treatment, growth was nor-
mal and a microscopic examination revealed healthy cells,
and chloroplasts in a good condition. The author pub-
lished photographs of the plants grown from the exposed
protonemata.

According to Becquerel (1932c), sprouting seeds of
ifheat, rye, lucern and Helianthus, which can be dried in
a vacuum and I'evived, do not suffer from exposure in the
dry state to any low temperature. The material, consist-
ing of seeds with radicles no more than 10 mm. in length,
w^as first air-dried; then it was desiccated in a vacuum of
10 ' mm. of mercury in presence of barium oxide ; finally,
it was put for 18 days in li<iiiid nitrogen. Most of the
rootlets could be revived after this treatment. Some
specimens were immersed in liquid helium for 9 hours and
stayed one hour at 1.84° K. in a vacuum of 10 ' mm. of
mercury, without suffering any injury.

SECTION ir. ANIMAL MATERIAL

1. Blood. To study the action of low temperatures on
white blood corpuscles, Schenk (1870) placed the blood of

55

tritons, frogs or turtles in a shallow glass and set this on
a freezing mixture; the temperature \vas recorded by a
thermometer immersed in the blood. The white cells
cooled to 0°, -3°, -5°, or -7° for a short time, retained
their vitality and resumed amoeboid movement when the
temperature was again raised. If the blood was kept
for several hours at low temperature, movement was only
seldom resumed ; thus, a specimen kept for 8 hours at - 2°
to - 3°, did not resume its activity. The white blood cells
of the warm-blooded animals, especially of the rabbit,
retained their vital properties after exposure to - 3°, only
if the time of exposure was not more than 10 to 15 min-
utes. In Schenk's experiments the blood corpuscles were
apparently never congealed.

Pouchet (1866) reported that the red blood cells were
considerably altered by congelation. Frog's blood was
allowed to drop into a dish cooled to -15°. After thaw-
ing, the iniclei were found free in the plasma. In other
experiments, he froze entire animals such as frogs, toads,
an eel, a young cat, etc., and observed that the blood cor-
puscles were crenated and reduced in size. From these
observations he developed the theory that the damage
done in the body of an animal by freezing was in direct
ratio to the number of blood cells injured.

2. Embryonic Tissue. Simonin (1931) subjected frag-
ments of embryonic tissues of mouse, rat or ox, to tem-
peratures from 0° to - 15°, in boxes immersed in a freez-
ing mixture. He could obtain some growth in culturing
these tissues after they had been at 0° for 20 days or at
- 5° for 5 days, provided no ice had been formed in them;
- 15° was always lethal in a short time. Nerve cells and
liver tissue were most sensitive, while heart, lung, and
intestine tissue showed a greater cold resistance.

Gaylord (1908) observed that growing epithelium from
young mice embryos was killed after immersion in liquid
air.

The embryos of the birds were always found more re-
sistant to low temperatures than the adults. But almost

56

iiolliiiii;' is known on llic time at wliicli llic resistance
drops. Moraii (liJ'J.")) remarks in a general maiiiior that
l)artiall\" incnhalcd ej;g'.s were less resislaiil lliau i'resli
eggs to low-tenipcialni-e storage. According to Edwards
(J. PJii/.sioI., li, M.")!, 11)02), cliick euiJjryos responded like
cold-l)lo()de(l animals to temperature cliauges diii'ing tlie
lirsl 20 days of ineiil)atioii. This statement would indi-
cate a very late change in resistance.

On the more fundamental problem of the mechanism
of the decrease in cold resistance during incubation, as
related to the mechanism of the passage from the poikilo-
therm to the honioiotherm state, still less is known.

3. Ciliated EpifJu'lium. According to Schiff ("Lehrb.
d. Physiol, d. IMenschen," 1, VI, 1858), a ciliated frog epi-
thelinm, immersed for several hours in iodine serum at a
few degrees above zero and bronght into a warm room for
observation, presented a complete absence of movement;
but, with rising temperature, a slight motion began which
was soon followed by an intense ciliary activity. This
experiment could l)e carried out repeatedly on the same
preparation.

Koth [Arch. f. path. Anaf. u. Physiol., 87, 188, 1866)
made more accurate determinations of the temperature
of cessation of ciliary motion in Anodonta. The experi-
mental object, with a thermometer and enough water to
cover the bulb of the thermometer, was placed into a re-
agent glass, and this was set into an ice-salt mixture.
Recovery w^as found possible after congelation at - 3° to
- 4°, when this temperature was not maintained for more
than a few minutes. At - 6°, death always set in. Tem-
peratures above zero, w^hen they acted long enough, pro-
duced a transient cessation of motion; this happened, for
example, at 0.4° after 6 minutes. By freezing and tliaw-
ing, the endosmotic relations of the ciliated cells were
extraordinarily changed; the movement, temporarily re-
sumed, was soon definitely lost, and the cells sw^elled up;
very often the cuticle, with the cilia attached to it, sepa-
rated from whole cell rows and sometimes it rolled up

0/

into a tiny cylinder which, witli the protruding cilia,
looked like a t'ox-tail.

According to Pictet (1893) a ciliated epithelium from
the frog's mouth, frozen hard by exposure to a tempera-
ture of - 90°, resumed its beat after thawing, but expo-
sures to temperatures lower than - 90° killed the tissue.

4. Muscular Tissue. In describing the investigations
on the muscles and on the heart we shall follow the chrono-
logical order.

Kiihne (186-4) reported that frog muscles, frozen at
- 7° to - 10° for 3 hours, were irritable after thawing, and
that the irritability was observed to last for 6 hours at 15°.

Jensen and Fischer (1909) demonstrated the funda-
mental fact, often confirmed by later investigators, that
some water can be congealed in the muscle without injur-
ing the latter, but that the solidification of more water
induces death. The gastrocnemius of Rana esculenta
was cooled to its lowest subcooling point (about -9°),
then it was left to freeze till past the end of the horizontal
portion of the freezing curve, or to various points in the
subsequent drop of the curve. The cooling process was
then interrupted and the reactivity of the muscle tested.
The temperature was indicated by a thermocouple. Cool-
ing to about - 1° and solidification of the greater part of
the free water produced no serious injury. With further
cooling, the reactivity and conductivity of the muscle de-
creased rapidly and disappeared at about - 3°. Since, in
this temperature interval, the more firmly bound water
began to freeze out, the death of the muscle, it is sug-
gested, might be correlated with this process.

Brunow (1912) determined the death temperature of
the isolated frog's muscle in the following manner. He
drew the gastrocnemius of Rana fusca firmly into a nar-
row glass tube, centered this in a second wider tube,
letting an air space between them, and immersed the
whole into an ice-salt mixture, the temperature of which
was kept constant to within 1°. The temperature of the
muscle was determined bv means of a thermo-needle in-

58

sertcd into tlic middle of it. Isolated imisclcs lliiis 1 fcated
could be frozen and sid)jec'led lo a temperature of -2.9°
Avitliout losing- tlieir in'i1al)ility completely. The death
point would then lie at about - 3°.

In another sei'ies of experiments by the same author,
determinations were made on the frofi muscles in situ, for
which purpose the frog's leg- was pulled through a small
glass tube, as before, the frog being meanwhile fastened
to prevent movement. The muscle, not cut off from the
circulation by any ligature, was still irritable after hav-
ing been frozen to -4.06°. Its death point is given as
lying at about -4.1° to -4.2°.

Moran (1929) exposed to freezing temperatures excised
frog's muscles (sartorius and gastrocnemius) in constant
temperature rooms and, after freezing and thawing, he
tested the vitality of these muscles by observing their
response to an electric stimulus, their osmotic properties
and also their electric resistance. Cooling was very slow,
a long time being allowed for the establishment of the
temperature equilibrium. In a previous determination,
the freezing point of the material was found to be -0.42°.
Moran 's results can be summarized in the following
points : 1. The muscles brought to temperatures below
- 2° were killed ; 2. The muscles frozen hard but exposed
to temperatures above -2° for less than a day could be
revived after thawing; 3. The muscles left for a long time
in the frozen condition above the critical temperature of
-2° were killed; the lethal time of exposure was about
7 days at - 0.9°, 4 days at - 1.5° and 20 hours at - 2°. In
his attempts at rapid freezing, by immersion in liquid air
for example, Moran could never ol)tain revival. He thinks
that at the ci'itical point an irreversible change takes
place in an inappreciable time.

Chambers and Hale (1932) reinvestigated the problem
of the death temperature of isolated muscles, using some
of the methods that C^hambers had developed for micro-
mani|)ulation. Portions of muscle fil)ers, 3-5 mm. long,
from the sartorius of the /Vo// were ])laced in a hanging

59

drop of liquid paraffin. The coverslip with the hanging
drop was mounted on a chamber on the stage of the micro-
scope, and the temperature ascertained with a thermo-
couple inserted into the drop near the tissue. It was
found that muscle fibers could be subcooled at -6.5° for
2 hours or at - 15° for 3 hours without losing their power
of contraction. Surface freezing, induced by touching
the fiber with an ice-tipped pipette, occurred at - 1.2°, and
after a stay of about 15 hours at - 1.2° to - 1.3°, a slight
contraction could still be produced. Internal freezing
occurred at - 1.6°, but fibers in which such freezing was
complete were always found to be dead on thawing.

Heubel (1889) studied the effect of freezing on the
frog's heart in situ, as follows. He directed a stream of
ether from an atomizer upon the exposed, ligated and
therefore relatively blood-free heart, thus freezing it hard
through and through. The operation lasted 20 to 30
seconds. When the ligature was removed and the blood
forced into the heart, it soon started to beat and, shortly
after, it functioned normally again. Hearts subjected to
the cooling action of the ether for more than 1 minute
could never be revived, nor could hearts w^hich were
exposed for 10 or for 17 minutes (1 experiment in each
case) to an air-temperature of -18°.

According to Cameron and Brownlee (1913), excised
frog's hearts, placed in test tubes immersed in freezing-
solutions, recovered after having been frozen at tempera-
tures of -2.5° to -3° for two hours, but not after treat-
ment at lower temperatures. Recovery consisted in a
reestablishment of the beat after it had stopped. These
authors made about 25 determinations of death tempera-
tures on excised hearts.

Britton (1924) mentions a sJiate exposed for 16 hours
to -20° and solidly frozen, the heart of which resumed
its beat after thawing, though the fish did not recover.
The author states that the heart of the skate can be solidly
frozen for several hours and recover.

5. Nerve Tissue. Biihler (1905) observed that, when
the temperature of an isolated frog's nerve (Ischial of

CO

luiiKt Iciii jxn'di'Ki ;iii(l Itdi/d riridis) was lowered slowly,
the stimulus eoudueliou dropped im})ereeptil)ly down to a
certain ])oiii1 (a ))lieiiomeuoii not due to cold), then it sank
suddciilx lo a \ci>- small \alue, and finally it dropped
slowly auaiii 1<> coinplcle disappearance. The "critical
1emi)erature" a1 which the sudden drop occurred was
found to be between - 2 and - 10. With a thermocouple
inserted in the nerve it Avas possible to observe a remark-
able temperature rise coincident with the decrease in con-
duct ion. Kvidently the tissue was then freezing. Upon
warming, a slight conduction reappeared sometimes,
either below or above the freezing ])()int; the original con-
duction, however, was never reached again; and some
nerves did not recover at all. When there w^as a return
of conduction, it took place between - 11° and 7°. With a
too sudden cooling, convulsive twitches, due to stimula-
tion by cold, disturbed the experiments.

In 1932, Bahrmann repeated Biihler's experiments on
the same material, but he determined the electrical con-
ductivity of the nerve simultaneously with the stimulus
conduction at decreasing temperatures. He found that
there was a gradual increase in resistance and decrease
in stimulus conduction as the temperature was slowly
lowered down to about -6.4° to -7°, at which tempera-
ture there occurred abruptly a large rise in resistance and
simultaneously an abrupt drop in stimulus conduction,
followed by the absolute cessation of the latter. Thaw-
ing, as judged by the change in resistance, occurred be-
tween - 1.5° and 0°. Upon warming, stimulus conduction
w^as never resumed below - 1.8° and often it was not re-
sumed at all. As a whole, Bahrmann 's experiments
would show that death or serious injury results from
freezini>- after a subcooling of about 6°.

Summary: 1. Most of the tissues studied in this sec-
tion: plant epidermises, juicy tissue from leaves, stems
or roots, embi-yonic animal tissues, ciliated epithelium,
muscular tissue and apparently also nerve tissue, are

61

killed by frost at a few degrees below zero. Usnally some
ice can be formed in them bnt the solidification of a larger
proportion of their water becomes lethal. 2. Experi-
ments on plasmolysed material seem to indicate that some
tissnes can acquire an exceptionally high resistance by
partial dehydration. 3. Considerable seasonal changes
in cold hardiness are reported which might be due pri-
marily to a natural dehydration and rehydration. 4. The
tissues which can be dried withstand any low temperature.

V. METAPHYTA

1. Fiingi. We have already treated the action of cold
on the spores of fungi ; we shall deal here Avith the vege-
tative forms. But, in most of the investigations to be
summarized in this section, the authors did not attempt
to exclude entirely the presence of spores from their cul-
tures and in many instances the resistance attributed to
the mycelium should probably be ascribed to remaining
spores. When, in a research, it was evident that the ma-
terial experimented upon contained spores and that the
resistance observed was due to the latter, we excluded
such a research from this section and treated it in the
section ''Spores" even if the author did not present it
under that heading.

Growth of fungi at and below zero has been reported
by several investigators. But, inasmuch as this phase of
the problem is not directly related to death temperatures,
we shall give here only a few typical instances (the next
6 paragraphs), most of which are taken from a review of
the subject by Berry and Magoon (1934), to which we
refer the reader for further information.

Schmidt-Nielsen (1902) reported the growth of several
species of Actinomyces at 0°.

According to Horowitz-Wlassowa and Grinberg (1933),
some fungi of the genera Mucor, PeniciUium and Clado-
sporium can grow and multiply at -3°.

Brooks and Hansford {Food Inv. Bd., Spec. Ept. No.
17, 1923) found that Cladosporium not only grew, but also
developed fresh spores on meat at about - 7.8°.

02

According- to Smart (193;")), wlio exposed some species
of Poiiciiruini* cultured on agar slants, to -8.9°, a slight
growth could be observed at that temperature.

Bidault {C. r. Soc. BioL, 85, 1017, 1921) observed growth
in Peuicilliutn, CJadosporiinH and B(tti\i/tis l)etween 0° and
-6°, and in CJtocfostijliim and llurmodoidron at -10°.

Haines (1930) found that Sporotrichum carnis grew at
-5° to -7°, the lower limit of growth of this fungus on
supercooled Czapek's agar being near -10°.

Noack (1912) studied the cold resistance of thermo-
philic fungi belonging to the genera Mucor, Thennoascus,
Anixia, Thermoidiinn, Thermomyces and Actinomyces.
Spores suspended in hanging drops were germinated in
a thermostat, and when the germinating filament had
reached a length equal to \ to 10 times the diameter of
the spores, the cultures were exposed for 4 clays to 5° to
6°. After this treatment, granulations could be seen in
the filaments, and no further growth took place when the
cultures were put back into the thermostat. Vegetative
colonies of these fungi, either in fluid or on solid nutrient
media were also killed after an exposure of 2 to 6 days to
the temperature mentioned. In general the cold resis-
tance was found to be, to a large extent, independent of
the previous culture conditions, and it could not be raised
by an increased concentration of the medium.

Kiihne (1864) observed that when the myxomycete
Aethalium sept'icum, in the active, moving state, was
cooled by immersion of its container in ice water, it be-
came motionless; its contours presented many amoeba-
like protrusions, which, during the gradual rewarming,
constricted off as shiny spheres. The rest of the myxo-
mycete (apparently the uninjured central portion) re-
sumed its normal activity. However, when Aethalium
and Didymium were exposed to the lower temperature of
a freezing ice-salt mixture, they were killed.

Lindner (1915) subjected the submerged mycelia and
air hyphae of Aspergillus niger and Penicillium glaucum,
cultured on 3 per cent gelatin, to temperatures of - 10° to

63

- 13° for varying leiigtlis of time. After three hours of
freezing, 95 per cent of the cells were killed in the sub-
merged mycelia of a 24-hour-old culture, and after 12
hours all the cells were killed. Death was determined by
the inability to grow, which was observed to correspond
with the inability to plasmolyse. In a 48-hour-old cul-
ture, 90 per cent of the cells were destroyed after 4^ hours
and all were dead after 24 hours. So, the older cultures
had a higher resistance. When the submerged mycelia of
a 48-hour-old culture were kept subcooled at - 13° for 8
hours, only a few cells were killed ; after 24 hours of sub-
cooling many of the cells were dead, but a few older cells
still survived. The air liyphae showed no visible signs of
injury after 4^ hours at - 11°, while, after 7^ hours at
- 13°, disorganization occurred in the basal cells of the
hyphae, and after 24 hours all the air hyphae were dead.
According to Lindner, the duration of the exposure is an
important factor in causing death.

Molisch (1897) observed that Phycomyces nitens grow-
ing on bread and exposed over night to a temperature
which reached - 9°, presented the next day filaments which
were turgescent and growing. Hyphae of the same spe-
cies, mounted without water between slide and coverslip
and observed under the microscope during an exposure of
8 hours to - 10° to - 12°, showed no stiffening of the cellu-
lar fluids. When the temperature was lowered to - 17°,
ice crystals separated from the protoplasm, disaggregat-
ing the latter.

Rumbold {Naturw. Ztschr. f. Forst. u. Landw., 6, 110,
1908) found that the mycelia of Coniophora and of 3Ie-
ruleiis, in gelatine cultures, did not survive being hard
frozen at - 6° for 12 hours, while the gemmae of Coni-
ophora were not killed when kept over night in the open
at about - 20°, even though their aqueous medium was
hard frozen.

Bartetzko (1910) subjected germinating spores, with
mycelia 70 to 200 micra long, of Aspergillus, Penicillium,
Botrytis and Phycomyces to low temperatures in liquid

(;4

imlriciit media, in lesl liilx's. Xoiic of llic moulds were
injured by a ^-liour sul)e()olini;' at -14°. AspcrfjiUus sur-
vived subeoo]iiii>' for 4 days at - G° to -11°. Increased
concentration of tlie medium resulted in increased cold
resistance of liie suspended fuiis;i. In the frozen condi-
tion, ill 1 i)er cent <>hicose solution, AspergiUus was killed
after 2 hours at -12°, while in 50 per cent glucose, no
injury was apparent after 2 hours at -26°. The other
three fungi gave similar results, though their resistance
was not (piite so high. In genei-al, the resistance of the
hyphae increased with age.

Bartram (1916) exposed a number of fungi, cultivated
on agar slants of varying composition, to the cold of winter
for 4 months during which time a minimum temperature
of - 29° was reached. All the Sderotinia, Ceplialothecium,
Glomerella, Ventnria, and Ascophyta survived, while
Alteniaria, Cijlindrosporium, Plowriglitia and Pliifto-
ph tJwra survived on some media but not on others. Sphae-
ropsis and Fusarhnn were killed.

According to Richter (1910), the mycelia of Aspergillus
niger, cultivated in liquid nutrient media, survived a 24-
hour exposure to -10° to -13°. In another series of
experiments, he exposed the material to - 12° for two
days, then allowed it to grow for 3 days at + 30°, there-
upon exposed it to about -80° for 12 hours, again kept it
at ^ 30° for 24 hours, and finally exposed it over night to
-11° and let it thaw rapidly. No injurious effect of the
treatment could be observed.

Chodat (1896) exposed for 4 hours to temperatures of
- 70° to - 110° Mucor mycelium growing on agar or on
liquid medium. After thawing, the filaments seemed to
be dead, but some growth originated from them later. A
part of the mycelium which was entirely formed within
the liquid medium, showed, after thawing, a tendency to
roll into a ball as dead mycelium does, but, after a few
days, new growth originated from it.

ileldmaier {Zfschr. /. Bof., 22, 170, 1929) subjected the
mycelia of Schizophylluui and CoUyhia, cultured on agar

65

slants in test tubes, to - 5° to - 1° for 6 weeks, or to - 78°
for 7 lionrs, or to - 185° for ^ hour. The plants were not
killed.

Karcher (1931) cultured several genera of fungi on
agar slants in narrow test tubes, and, when the colonies
had spread over about j of the surface of the agar, she
exposed them to varying degrees of cold. Coprinus was
killed when the temperature of the agar was lowered to
-40°, even when it was thereupon immediately thawed.
Lepiota, Boletus, and the submerged mycelia of Phy-
comyces were similarly killed by a momentary exposure
to -60°. The mycelia of the following survived a 1- to
8-day action of -70° as well as a 13-hour exposure to
liquid air: Collyhia, Schizophyllum, Hypholoma, Clito-
cyhe, Placodes, Armillaria, Xylaria, Aspergillus (spore-
bearing, and young spore-free mycelia) and Phyconiyces
(mycelia with sporangia).

Lipman (1937), considering that the fungi used by
Karcher might have formed spores (the same objection
could be raised against most of the above described in-
vestigations), undertook a series of experiments in which
he made sure by direct observation that the fungi experi-
mented upon were in the actively growing state, that no
spores were present at the moment of the treatment.
Twelve species of fungi belonging to the genera, Asper-
gillus, Penicillium, Rhizopus, Mucor, Ahsidia, Mortier-
ella, Rhizoctonia, Armillaria, Trichoderma, Pythium and
Fusarium, were cultivated for 24 or 48 hours on synthetic
agar-media or on potato-agar. The cultures, on slants in
sealed test tubes, were immersed in liquid air for 48 hours.
The previous cooling and the subsequent warming were
gradual. In 14 cultures out of 26, and in 8 species out of
12, Lipman observed some grow^th, either in the tubes in
which the fungi were treated or after a transfer to fresh
media. A Rhizopus, a Rliizocionia, an Aspergillus and
an Armillaria were killed. A higher percentage survived
when exposed to low temperatures at the age of 48 hours
than when exposed at the age of 24 hours and the author

suggests tliat some cliaiigc took plac*' in ihc protoplasm
by aging.

2. Algae. According to W. West and (J. S. AVest,
Spirogyra cataeniformis was in excellent vitality after
having been imbedded in ice for 2 weeks while in the
process of conjngation {Ann. of Bot., 12, 33, 1898).

Cohn (1871) studied the vitality of Nitella syncarpa
cooled on a freezing stage on the microscope. Branches
of the alga were placed under a few mm. of water in a
shallow glass which was exposed to -20°. A thermome-
ter indicated the temperature of the water in the glass.
Active protoplasmic streaming could ])e observed at 0°
and slow movement could still be seen at -2°. After
exposure to lower temperatures, the cells were frozen and
killed, although, in 2 cases, cells taken from the ice at - 3°
were alive. Repeating these experiments with Nitella
branches not surrounded with water, Cohn observed the
protoplasmic streaming until the air temperature near the
alga registered - 2°. Ice formed in the cells between - 3°
and - 4° and the protoplasts shrunk. After thawing, the
cells were dead.

Molisch (1897) froze tilaments of Spirogyra between
slide and coverslip at - 3° to - 6° and found, after thaw-
ing, disorganized chloroplasts and swollen nuclei charac-
teristic of dead cells. Death was observed also in Clado-
phora frozen at -8° in the same conditions, and in Der-
besia and Codium frozen at - 11°.

Klemm (1895), using the method that we described
above under the heading ''Hair Cells," observed that
CJiara sprouts and Spirogyra filaments were killed after
an exposure of 15 minutes to - 13°, while protoplasmic
streaming could be resumed after exposures to higher
temperatures (-6°) or for shorter times and was only
slowed after a cooling to -2°.

Kylin (1917) experimented on the effects of freezing
on sea algae. The material, immersed in sea water, Avas
cooled successively to the temperatures of -2.9°, -4.0°,
-5.7°, -7.8°, -10.7°, -16.8°, and -18° to -20° and the

67

time necessary to kill the algae at any of these tempera-
tures noted. As a sign of death, he used the changing
color exhibited by the brown and the red algae and the
loss of the plasmolysing power for the green algae. He
found that these plants were never killed by low tempera-
ture in the subcooled condition, the pigment not being re-
leased when ice was not formed. The death tempera-
tures and the corresponding lethal times of exposure were
as follows: TraiUiella, -2.8°, 3 hours; Delesseria and
Laurencia, -4°, 10 hours; Laminaria (1 year old) -5.7°,
6 hours; Cerammm, -5.7°, 10 hours; Laminaria (several
years old) -16.8°, 3 hours; Chondrus, -16.8°, 10 hours;
CladopJiora, - 18° to - 20°, 3 hours ; PylaieUa, - 10° to - 20°,
10 hours; other species such as Nemalion, PorpJiyra,
Fucus, Enteromorpha were alive after 10 hours at - 18°
to -20°. Kylin obtained thus a surprisingly large differ-
ence in the death temperatures of the various kinds of
algae.

According to Karcher (1931), Pediastrum and Hor-
midium cultivated on agar slants in glass tubes, and im-
mersed for 5 hours in a freezing mixture at - 70°, were not
killed.

Becquerel (1936) investigated the vitality of dried
algae subjected to the temperatures of liquid gases. He
took samples of soils containing various algae and gradu-
ally dried them in a vacuum with barium oxide, at 35°,
for 3 months. He then placed a portion of the soils in
glass tubes closed with cotton plugs and sealed another
portion in tubes evacuated to 10' mm. of mercury.
Thereupon he subjected all of them to liquid helium
(- 269° to - 271°) for 71 hours, and to liquid nitrogen for
480 hours. Both groups of soils yielded living Oscilla-
toria, Glaeotila, Hormidium, Siphonema and Pediastrum.

According to the same author (1932b), the filamentous
alga Tribonema elegans, thoroughly dried on a piece of
bark, kept in a high vacuum for 22 years and exposed for
several hours to temperatures from 4° to 1.84° K., grew
after that treatment.

(IS

3. Lichens. Tlic lidiciis arc known lo be plants wliieli,
in the natnral condilions, arc anion*;- the most resistant to
low toni])t'ratnr('s. Tiicy flonrish in the northern climates
and on the hii;h monnlains in the proximity of the
glaeiers.

Jnmolle (181)0 and 1S!)2) attributed the high cold re-
sistance of the lichens to their ability to lose water at the
onset of the cold season. In confirmation of his views, he
gives the fact that the ratio between the fresh wxMght and
the dry weight of Phi/scia and Parmelia, collected during
the winter, was found to be as low^ as 1.10 to 1.14; also that
lichens of the same kind, soaked in water, absorbed an
amount of moisture equivalent to 3^ times their weight.
But determinations by the same author of the quantity of
oxygen and of carbon utilized by lichens previously
soaked in w^ater and then frozen resulted in the conclusion
that respiration was still active after 8 hours at - 8°, and
photosynthesis after several hours at -30° and -40°.
The author attributes this activity to the presence of free
water, not yet frozen at these temperatures. If a high
water content does not increase the injurious effect of
freezing, as the second part of Jumelle's work seems to
show^, one can hardly see hoW' a natural dehydration could
explain the high cold resistance, as it is assumed in the
first part.

According- to Becquerel (1932d), lichens of the genera
Parmelia, Xanthoria and Cladonia, desiccated in the air
and exposed for 18 days in liquid nitrogen, became green
again when brought back to favorable conditions, their
gonidia resumed grow^th,

4. Mosses. Irmscher (1912) studied the cold resistance
of about 34 species of mosses belonging to 27 genera. The
gametophytes, freshly gathered, were put into tubes 1.5
cm. in diameter, and the tubes immersed for 18 hours in
an ice-salt freezing mixture at - 5°, - 10°, - 15°, - 20°, and
-30°. Two i)ai'allel series of experiments were carried
on, one in which the material was in air, the other in
which it was in water. The leaf cells were then tested for

69

their vitality by plasmolysis. It was found that : 1. A stay
of 18 hours at temperatures down to - 10° caused no es-
sential injury; 2. At -15°, a considerable number of cells
in most of the species, and almost all in some species,
were destroyed; 3. At -20°, all the leaf cells of most of
the species were killed; 4. At -30°, they were all killed.
The apical cells and the cells of the dormant buds were
more resistant than the rest of the plant. The setae and
the protonemata did not differ essentially from the other
organs in their cold resistance. Gametophytes, previously
dried for 3 days in air at 20°, then made fully turgescent
again by immersion in water, and thereafter frozen,
showed a higher frost resistance than specimens not pre-
viously so treated. Repeated freezing at -10° to -15°
was found to increase the number of dead cells so as to
become equivalent to a single freezing at -15° to -20°.
Between the plants frozen in water and those frozen in
air there was no signiticant difference.

Becquerel (1932d) obtained some traces of growth in
the moss Hypnum, desiccated in the air and immersed for
18 days in liquid nitrogen. He thinks that cells of the
stem must be particularly resistant.

5. Higher Plants. The isolated organs or tissues of
higher plants, (in contrast to those of higher animals),
are capable of independent life. Consequently, almost all
the studies on death temperatures in plants are concerned
with the death of organs or tissues (while in animals it is
the organismal death which is considered primarily).
Since we have treated the tissues and organs previously,
little remains to be added here.

As stated above, most of the plant tissues are killed at
a temperature slightly below^ the freezing point of their
sap. But, since given tissues may have various freezing
points, depending on their water content, their structure,
their age, etc., they die at different temperatures; and
since their death influences the death of the whole plant,
the lethal temperature of the latter will vary to a great
extent and hardly any precise statement concerning it can
be made.

Of pai-ticular interest ai'e the i)lauts reported to die at
temperatures above zero. Molisch (1897), among others,
investigated their behavior. We refer the reader to tliis
aiillior's cliarts for details eoneeriiiiig tlie comparative
cold sensitivity of these phiuts. Most of them are from
tro])ieal countries and adajitation is tliouglit to play a
role in inducing sensitivity. As to the mechanism of death
by chilling, the commonly accepted view is that of a dis-
turbed balance between the various metabolic functions,
mostly trans])iration and al)soi-])tion of water by tlie
roots, the temperature coet!icient of these functions being
different.

Another group of higher plants of special interest for
their cold resistance is that of some conifers. The trees
of the Siberian forests {Larix sibirica in particular) are
often mentioned in the literature as typical in that re-
spect. Xot only do they withstand exposures of several
months to -30° or -40°, but they thrive in climates in
which such low temperatures are not uncommon. This re-
sistance can evidently not be attributed to any insulating-
layer, there is no known substance which would possess
the low heat conductivity required to protect the cambium
of these plants against the external temperature for that
length of time. Such cold hardiness is generally ascribed
to the nature of the colloidal cell contents and to the ability
of the protoplasm to undergo seasonal clianges in its com-
position and concentration. But the fundamental mecha-
nism of this phenomenon is still little known.

Summary: 1. Most of the fungi and algae are killed
when exposed to temperatures of - 10° to - 15°. 2. It
seems definitely established, however, that some fungi, in
tlie actively growing stage, can withstand the tempera-
tures of the li(piid gases, although they sliow less resis-
tance than the monocellulars to these temperatures. 3.
The algae which, like the Hormidimn, are more closely
related to the monocellular forms, also resist very low
temperatures. 4. The large majority of the higher plants

71

are killed when frozen at a few degrees below zero ; some,
however, resist - 30° or - 40° ; some others are killed by
cold at temperatures above zero.

VI. METAZOA

In the sections of this chapter in which the investiga-
tions are more closely coordinated toward the solution of
some fundamental problems, we shall follow the chrono-
logical order so as to show in a clearer manner the his-
torical development of these problems.

1. Coelenterates. Payne (1930) investigated the action
of repeated freezings on the coelenterates, Mnemiopsis
leidyi and Pennaria tiarella. She states that 3 successive
freezings and tha wings "break the colloid structure" of
Mnemiopsis, its constituent parts "going into solution,"
and that the animal "disappears," the disappearance
being sudden and definite. For Pennaria, freezing 5 to 7
times, or maintaining the temperature at - 10°, produced
the same effect. As described, this process, we think, is
unique in the literature.

2. Helminthes. Oliver {Lancet, p. 357, 1910-1) re-
ported that larvae of the JwoJi-worm recovered after hav-
ing been frozen solid in water and thawed slowly.

Kjava {Finska Laek. HandL, 55, 101, 1913) showed that
plerocercoids of DipliyUohothrium latum were killed when
kept for 48 hours at - 9°.

According to Magath and Essex {J. Prev. Med., 5, 239,
1931), all the larvae of Diphyllohothrium latum in 10
heavily infected fish {Stizostidion vitreum) were killed
when the fish were exposed to -15° for 24 hours. Ten
larvae that had been kept in fish at - 10° for 88, 40, and
16 hours respectively were fed to each of 3 dogs ; no worms
developed.

Schmidt, Ponomarer and Savellier (1915) studied the
resistance of encysted Trichina to cold. They found that
a temperature of 0° for 11 days does not affect this para-
site, that it can also support -6° for 10 days, that -9° is
sometimes but not always fatal, and that -15° to -16° is
always lethal.

The Ilelminthos, Monlezia, Ostertagia, Nematodinis
and TricJiostrojipifhis, wore reported ])y Griffiths (19.'37)
to resist the coUl ol' a Canadian winter in wliich the tem-
perature reached -26°, and to l)e eapal)k', llie next spring,
of infecting- the grazing sheep.

Augustine (1932) subjected to freezing f rich i nous meat
that he fed to experimental animals on which he studied
the subsequent infection. The temperature within the
meat was recorded with tliermocouples. lie found con-
siderable infection after exposure of the meat to - 21°, less
after exposure to - 27.6° to - 30.9°, and none after the
action of a temperature of - 33.7°.

Rahm (1920, 1921, and 1923) observed that the newa-
todes, Pled us, Tylenchus and Dorijlaiunis, in the dry state,
could be cooled without injury to tlie lowest available
temperatures (boiling helium), and that in the moist state,
they resisted a slow cooling to the temperature of liquid
hydrogen. The author did not mention the revival of any
nematode after a rapid cooling to the latter temperature.
The dried worms, heated to 136° or to 151° and then cooled
abruptly to - 190° or to - 81°, died; they supported a heat-
ing to 50° followed by a sudden immersion in liquid air.

3. Rotifers. The last-mentioned author {loc. cit.) ex-
posed to - 80° for 17 hours specimens of moss with their
natural fauna which consisted, among other organisms,
of rotifers belonging to the genera Callidina and Adi-
neta. The material, air-dried for periods extending from
18 days to 14 months, was immersed directly in a bath of
ether and solid carbon dioxide. All the organisms sur-
vived and resumed motion when remoistened. In other
experiments, the temperature of liquid air for ^, 5!, 25,
26 or 125 hours, that of liquid hydrogen for 26 hours, or
that of liquid helium (- 269 to - 271.88°) for 7^ hours was
found harmless to the dried animals. A previous desic-
cation of 3 months and a stay of a week in a vacuum of
1 10 mm. of mercury did not change their cold resistance.

While the ])receding observations of Rahm are in good
fitting witli tliose made by other investigators on dried

material, llie results that he reports on moist rotifers are
probably not paralleled in the literature. When the dried
animals had resumed their motion after immersion in
water, they were frozen either slowly, by steps, to - 81°,
to - 190° and to - 253°, or rapidly by sudden immersion in
liquid air and in liquid hydrogen. After having stayed
2 days and 1 day respectively in the two last-mentioned
liquids, they could be revived, although the rapid refrig-
eration seemed to have lengthened the duration of the
recovery and to have been somewhat more harmful to
some individuals than w^as the gradual cooling. The
author remarks that possibly some organisms, counted as
survivors, may have been hatched after the exposure to
low temperature. This would, of course, modify the
problem entirely.

In another series of experiments, Rahm succeeded in
maintaining alive some of the above-mentioned rotifers
after they had been thoroughly dried in air for a month,
then immersed in liquid air for 5 hours and finally heated
in an oven to 140° to 151° for 15 minutes. Heating the
dry organisms first and cooling them afterwards gave the
same results.

Becquerel (1936) found the rotifers. Ad met a gracilis.
Rotifer vulgaris, and Calliftiiia a)igiisticoUis, alive in sam-
ples of soil previously dried in a vacuum for 3 months and
exposed to liquid helium for 7| hours and to liquid nitro-
gen for 480 hours, either in cotton-plugged glass tubes or
in highly evacuated sealed tubes.

4. Anuelids. According to Doenhoff (1872), leeches,
exposed for 1 hour to a temperature of -1.5°, survived
though they had been frozen so stiff that they could be
bent only with difficulty. These animals, when cut with
scissors, exhibited a cross section whitish with ice. Simi-
larly, leeches exposed for 3 hours to -1.5° revived, but
could no longer crawl and died after a few days. Leeches,
subjected for a few minutes to - 6.25°, died.

According to Schmidt and Stchepkina (1917), earth-
ivorms could be revived after an exposure of 8 hours to

74

0° without coii^c'latioii, or an exposure of 2 to 3 liours to
- 1.2° ill the eoiigealed state; they were killed after having
l)('('ii frozen at -2° for 2 hours. The authors, who are
interested in the study of the anabiotic state, consider the
temperature range from 0° to -2° as the favora])le one
for that pur})ose. They reported also the somewhat sur-
prising fact that dried worms, that is, worms in which 30
to 40 per cent of the normal water-content was removed,
were killed when the temperature was lowered below
- 1.2°, though drying alone did not affect their vitality.
(The normal water-eoiilent was 82.8 per cent of the body
weight.)

5. Mollusl's. Koedel (1886), who exposed to low tem-
peratures land or water snails, either in the air or in
water, by immersing into freezing mixtures glass tubes
containing the animals, reported that PJauorhis and Lim-
}iaeus were killed in times varying from 15 minutes to 5
hours at air temperatures from -8° to -4°, the younger
and smaller animals offering less resistance, and that
Heli.r pomaiia was killed in 10 hours at -10°. None of
the mollusks investigated ever survived a complete freez-
ing. In the unfrozen state, in ice-water at 0°, Limnaeus
could be kept alive for days. It would follow from these
data that congelation is the main lethal factor.

According to Yung (1888), (cf. also Pictet, 1893) the
snail, Helix pomatia, survived after having been for 20
hours at an air temperature of -130° in Pictet 's refrig-
erating ''well" {pu'its frigorifique). This surprising re-
sult is always described in the literature among the several
achievements on reviviscence reported by Pictet.

Fischer (1930) found that Helix pomatia, kept for 8
hours at an air temperature of -5°, survived, while at
temperatures of -6°, -7° and -10° they always died in
less than 5 hours, in spite of all precautions for slow thaw-
ing. According to him, the air temperatures favorable
for hibernation are between 0° and - 5°.

Kapterev (1936) asserts that he "often succeeded in
reviving mollusks (Planorhis) that had been frozen in the

to

ice." Judging' l)y the Ihiekness of the ice from which the
animals were taken and from tlie climate of the country
(Far Eastern Russia) he thinks that tliese organisms
must have been subjected to - 20^.

Weigmami (19;>6) attempted to determine the body
temperature of snails, and of Helix pomatia in particular,
during death by cold. For that purpose he used a thermo-
couple inserted through the shell. The surrounding air
temperature was lowered slowly to a minimum where it
stayed for several hours. He found that when the snails,
with or without operculum, were frozen and when their
body temperature dropped to -3° to -4°, they were al-
ways killed. Whether they were also killed at higher
temperatures (between - 2° and - 3°) when ice was formed
in them, w^e cannot ascertain from Weigmann's paper.
He states, p. 310, that none of the species examined with-
stood freezing ("ein Einfrieren iibersteht also keine der
untersuchten Arten") and, p. 306, that some animals sur-
vived freezing at -2° (''ein Einfrieren bis auf -2° iiber-
leben"). Besides, a freezing curve is given (fig. 1) for
an individual which is reported to have survived (table 1).
As to the difference between operculated and non-opercu-
lated animals, Weigmann observed that the former could
resist for 4 to 5 hours air temperatures down to - 6°, while
the latter were killed in 4^ hours at - 4°. The water snails
Limnaea and BytJiinia are given as having a higher lethal
temperature than the land snails, namely, - 1.5°.

6. Arthropods. The action of low temperature on
arthropods has been the object of a very large number
of observations and of investigations. Several reviews
have been published, of which we shall mention two : that
of Bachmetjew (1901 and 1907), and that of Uvarov
(1901). The reader will find in the former a detailed his-
tory and compilation of the older works, and in the latter
a short compendium of the more important modern
contributions.

Although the larvae and the adult arthropoda present
a widely different cold resistance, we shall treat them
together.

Hi

As far l)ack as 1742, Kraininii', in liis famous "Momoii-cs
pour sei'vir a I'liistoire iiaturellc dcs insect es," described
experiments whicli confirmed tlic i)opular opinion that
insect larvae can be frozen ]ia)-(l willioul Ix'ini;- killed. He
froze caterpillars at -8.8° and icNixcd llieni. It is of in-
terest to notice that the method wliicli was nsed by most
of the investigators of low temperatnre effects and which
consists in putting the organisms into a glass tube and
immersing the latter into a mixture of ice and salt, was
tlie one used by this pioneer two centuries ago. Kf'aumnr
was ])ro])al)ly also tlie tirst to remark that some juices, in
the caterpillars, do not freeze at the freezing point of most
of the body lluids, he thought that they must congeal at
a lower temperature wiiich would be the death point. This
idea has been developed and studied experimentally dur-
ing the last 30 years.

From the middle of the eighteenth to the end of the
nineteenth century, the revival of hard frozen insects and
mostly of larvae has been asserted by a legion of natural-
ists. From a review of the literature of that period one
gains the impression that it was the most generally ac-
cepted view. However, there were divergent statements.
To explain the differences reported, one generally had
recourse to specificity. In the review lists we find silk-
worms, chrysalids, caterpillars, grasshoppers, mosquitoes,
bedbugs, scolopendrae, etc., among the animals reported
to have survived hard freezing, while ants, aphides, bnt-
tertiies, house flies, and particularly bees are perhaps
more often quoted among the sensitive insects. Bees
have been reported by many to die at a temperature of a
few degrees above zero (cf. Bakhmetieff, 1901). But
what role is played in cold resistance by specificity or by
the stage of development, the age, the season, adaptation,
and by other similar factors, has by no means been eluci-
dated in older works. The improved methods and the
more systematic studies of the last 50 years or so have
l)rought some light on the action of a few of the above-
mentioned factors.

Doeiihoir (1S72) placed hees, spiders and tucat flies on
the frozen ground, covered them with a wire cage, and
exposed them thus for 5 hours to a temperature of - 1.5°,
as indicated by a thermometer laid on the ground. The
bees were killed by this treatment, but the spiders and flies
soon recovered. It took an 8-hour exposure to - 2° to - 3°
under similar conditions to kill the spiders, while the flies
survived even a 12-hour exposure to -3.75° to -6.25°.
The flies were then put into a small glass, 1^ inches long
and ^l inch wide and so immersed into an ice-salt mixture
at -3.5° to -6.5° for 4 hours. They survived this treat-
ment also. Finally, they were killed by a 3-liour exposure
to -6° to -10°.

Plateau (1872) studied the cold resistance of aquatic
arthropod a. They were placed in glass tubes containing
a few cc. of water and a thermometer, and the whole was
immersed in a cooling mixture. The animals were little
by little surrounded by ice, and finally caught in it.
Plateau measured the time during which they were caught
in the ice and recorded their survival after thawing. He
gives the following figures as the longest periods sup-
ported in ice : 10 to 30 minutes for 3 coleoptera, 2 to 10
minutes for 2 liemiptera, 20 to 30 minutes for the larva
of a neuropteron, 2 minutes for the larva of an ortJiop-
terou, 10 minutes for the crustacean, Asellus, 1^ minutes
for Daphuia and 1 minute for Cyclops. In experiments
conducted in sugar solutions which froze at -2°, Asellus
always died in less than a minute after it was caught in
the ice, but it did not die at - 2° when no ice was present.
For Plateau it is evident, that the aquatic insects die at the
temperatures at which ice forms in the fluid medium
around them.

Roedel (1886) made experimental determinations of the
death temperature of some arthropoda with an apparatus
consisting of 2 concentric tubes, of which the external one
contained a freezing mixture and the internal one the
experimental animals, each tube being provided ^^dth a
thermometer. For slow cooling, he used the principle of

78

llio lowcriiii'- of tcraporaturo prodnccd l)v tlio evaporation
of waliT at llie fi'oe surf ace of a ])or()ns tube immersed in
iee water, lie (>l)serve(l: 1. Tliat <nil.^, exposed to an air
t('mi)erature of 0", soon lose their ability to eling- to the
^vall of a glass container, and in 15 minutes they are rigid,
but tliey are killed only by a stay of 3 hours at -15°;
'2. Thai hccs l)tH'()nie stiff in ;i shoi't linic in the neighbor-
hood of zero, and that they die after 'A}, hours at -1.5°;
."). That several coleoptera, as also the adult Bomhyx,
are killed in less than one houi- at -4° to -6°; 4. That
the mosquito, Culex pipiens, is killed in one hour at -4° ;
5. That house flies are killed in 5 minutes at - 12°, in 20
minutes at -8° and in 40 minutes at -5°; 6. That the
spif/cr, Tegenaria, dies after an exposure of 45 minutes
to - 1)°, or of one hour to - 6° ; 7. That the water spider,
Argifroneta, suffers no injury at - 5° as long as it does not
congeal, but dies in 3 hours at -4° when it is frozen
throughout; 8. That the water mite, Hydrachna, dies
after half an hour's exposure to -4° when its body is
congealed; 9. That, when the larvae of several species of
coleoptera were congealed all through at temperatures
lower than -6°, they never survived. (Actual internal
congelation was ascertained by sectioning the animals) ;
10. That some dipteran larvae could sometimes be revived
wlien they were taken out of a frozen medium l)ut never
when they were themselves frozen; 11. That the cater-
pillars of Smerinflius did not survive a lowering of tem-
perature to -10°. To summarize: Roeclel, whose work
covers most of the groups of arthropoda, never obtained
the survival of a com])letely frozen animal, not even in the
larval stage; he found that they all die in a range of tem-
perature extending to al)out 10° below zero and during
times of the order of an hour.

According to Bachmetjew (1901), death occurs when
an insect which has been subcooled to a certain tempera-
ture below zero and has congealed, has its temperature
lowered a second time to the subcooling point. The low-
est subcooling point that he observed was -15.7° and this

79

would be, consequently, the lowest death point. One does
not see for what reason death would correspond to a sec-
ond cooling to the subcooling point ; and, besides, the con-
clusions of Bachmetjew have against them that his experi-
mental results disagree with the conclusions in 27 per cent
of the cases (cf. Payne, 1926).

Revival after hard freezing was reported by Duval and
Portier (1922) for the caterpillars of Cossus cossiis.
These authors froze the caterpillars in test tubes im-
mersed in cooling mixtures. The temperature was
measured with a thermometer placed in the tubes in
between the animals. They found that death resulted
when the temperature dropped below -21°, while, at
higher temperatures, -15 to -17° and -20°, the caterpil-
lars could be frozen so hard as to be breakable, and be
revived on warming, even if the thawing was rapid (in
water at 40° ) . The portions broken otf in the frozen state
showed activity after warming. Caterpillars congealed
at -15° to -17°, then immersed in liquid air or exposed
to the temperature of boiling chloroform (-63°) for 50
minutes, or cooled only to - 22°, could not be revived. The
authors think that the rigidity of the frozen caterpillars
above -21° is due to the congelation of the hodif fluids,
while the cellular fluids would still be unfrozen. The lat-
ter would congeal at about - 21°, thus inducing death. As
a matter of fact, a sudden rise in temperature, indicative
of a congelation, was sometimes observed in the neighbor-
hood of -20°. The resistance of the caterpillars was
found to be less in the Spring than in the Winter.

A work which perhaps explains some conflicting results
of the older authors is that of Payne (1926a, 1926b and
1927). This author determined with thermocouples the
freezing and the subcooling points of various insects and
found these points to be always low in Winter and to cor-
respond to a low water-content. The freezing point
varied in one case from - 2.4° in midsummer to - 8° in
midwinter, and the subcooling point from about -6° to
-14°. The changes in freezing temperatures, and there-

80

tore in death U'liipcraliiix's, coukl be iiidueed artilicially
l)y exposure of llie animals to cold; changes in cokl resis-
tance were caused also by ex)»osui'e to moisture. AVJiile
a(|ualic insects consistently showed the same survival
temijcratures, insects which could be experimentally de-
hydrated presented a considerable lowering of the death
point. The Japanese beetle, for example, which can lose
half of its body weight in the form of water, exhibiled a
lowering of about 28 degrees in its survival tempei'ature,
when so dried. Several species of oak borers are self-
dehydrating during the winter and show a natural peri-
odicity in cold hardiness. Artificial softening, the reverse
of artificial hardening, could be accomplished by exposure
to high humidity or high temperature. As to the freez-
ing points, the author distinguishes two of them, the tirst
one being the freezing point of the blood and the second
probably that of some tissues, such as nerve tissue, or of
some tissue components. The insects were able to recover
from exposure to the first freezing temperature but not
from exposure to the second. The latter, therefore,
might be considered the death temperature. The time
factor was found to be important.

Another significant work along the same line is that
of Sacharov (1930). He determined, by the dilatometer
method, the quantity of ice formed in the body of insect
larvae at various temperatures during hibernation and
during the active state, and correlated these data witli
cold resistance. He found that, for example, in the
active caterpillar of the broioi fail moth, 44.85 per cent
of the water-content was frozen at -7.8°, while in the
hibernating stage only 5.06 per cent was frozen at - 11.1°,
and 15.22 per cent at - 17.35°. The total water-content
in the 2 stages was, respectively, 82.94 per cent and 71.83
per cent of the body weight. On the other hand, the
active caterpillars died after exposure to -7.8° while the
hibei'uating ones withstood - 17.35°. Sacharov also thinks
that the cold resistance bears some important relation to
the fat-content, which was about twice larger in the liiber-

81

iialing caterpillars. Adult hces were found to die when
their temperature was lowered to -0.7°.

Robinson (1926) insisted on the importance of the time
factor in death by cold. For exposures of Sifophilus
oryzae to temperatures from 1.1° to -17.70°, he obtained
lethal times decreasing from 98 to 1^^ hours. As to the
cause of cold hardiness, Robinson seeks it in the amount
of bound water, and the hardening- process would consist
in increasing water-binding.

According to Kalabuchov (1934), bees, humhle-'bees,
wasps, and larvae of beetles can revive after having been
subcooled for 48 hours at temperatures from -2.9° to
- 17.1° (body temperatures). Death usually ensues a few
minutes after the beginning of ice formation subsequent
to subcooling. Partial freezing, without preliminary sub-
cooling, is not fatal, nor is partial freezing following a
slight subcooling of short duration. Complete freezing
is always lethal.

Results, similar to those reported by Duval and Portier
and by Payne, were obtained by Lozina-Lozinskij (1935)
on the caterpiUars of a Pyrausta. These animals, frozen
at -21°, and breakable like glass, were revived after a
stay of 8 days in the congealed state. Considerable
changes in the position of the freezing point and in the
cold resistance took place during the passage from one
metamorphosic stage to the other.

Kapterev (1936) says that he "often" revived cy clops
taken from layers of ice 3 to 12 cm. thick. But, what is
much more remarkable, is the experiment in which he
claims that he revived a crustacean, Chydorus sphericus,
isolated from a piece of soil taken in the permanently
frozen ground, at a depth of 3.5 meters, in the valley of
the Great Never River (a tributary of the Amur). He
thinks that the "age of the layers whence these samples
were taken should be reckoned not in hundreds but in
thousands of years."

Some investigators have reported injurious effects or
death as a result of long exposures of insects to tempera-
tures above zero. For references, see Uvarov (1931).

6'. : ' ^. C>r ]ar abiliTy

to^e^..- . - - v.m the dry -• . ; . ..„ . . ..>.-: ..... action of
the lowest teinp»era tares when dried, the tardi grades
deserve to be mentioned in a separate seotion.

Tbe t; . ' ^ and Mii-

m^c^ivm. t ..-_.: . _ _ . . „_d 1923 ) to

the drying and cooling treatments that he used for rotifers
(see aboreL In the dry state, all these tardigrades snr-
vived a cooling down to abont 1' K: when moist, they
resisted a slow cooling to the t«n|>eratQre of liquid hydro-
g&L. and >ome Ma^robiotms likewise supported a rapid
cooling to that temperature. Moreover, the dried tardi-
grades eonld withstand a sndden et»oling in liqnid air fol-
lowed by abrapt heating to 14fJ' to 151'.

Bee*^nerel <1996) isolated the tardigrade, Macrobioins
Hvjtl^jK'^i. froan dried soils s"^ - for 71 honrs to

t«nj»eratiires of — 269* to — 271 . ^ . : the details of the
experiment, see above, nnder the heading Botifers.)

7. Amphibia amd Btptile?. That frogs and toads can
be revived -o'- _ " - i-n hard is a popular

stalefmex.* f: :. l. Many observations

and experiments are reported in the older biological lit-
erature which simply c-oirfinn this popular idea. Frogs

ortc»: " f " - _•: " ■ * --^ -^ i-^ ^md. or maintained

in Wi; . they l:>eeome inert

and risid- are described as resuming activity when warmed
iipu 7 - " " these rexx»rts we tried the experi-

n^^' - .... j:. ^-, wrapped in several layers of
cl :h- were pnt for S minntes into cavities dug in
solid earbon dioxide. The ^T^imalg became hard and rigid
like dead bc«"' " r. T:«eiiig re^ ". they regained

movement ai. -. t. _:j. As we - by the follow-

ing review, two important ste}>s were taken toward the
mjderstanding of the apparently high cold resistanc-e of
|,, — — -_--,-- _-. ^ ... -^ - ---;2:ators realized that a
r- . e a complete freezing,

"i.-n they began to determine the internal temp>era-
ture of the animaLs during cooling. The time necessary

83

for the temperature eqnilibTnini to be establiidied. betweei
the body of an animaT of the size of a fro^ and its oivi-
romnent. was f onnd to be nraeh longer than previoiLsly
'' .':' The body tempera*"" — ~^IL be above tise

: , ■ point when the anJTna . - : r a long trme at

an air tem.perature of several degrees beio^ar zero. Be-
sides, during congelation^ the tem.peTatiire stays for a
snrprisingiy long time in the n ' . ' ' —' - - - -

Himter. abont a century ag - ' -^ in-

ternal teniperature of frog? diirfng freeing expenments
and gave — 0.6' as the lowest point to which they •e

subjected without fatal results.

Mnller (1S72> investigated the effects of freezing on
the frog as follow?. He placed brown grass frogs in a
bottle almost iilled with water ai- - - '

the cold. The frogs were kept :-_-.-_ __ __;_-- -i
the water with a stick, till solidification tcK:»fc place. There-
upon the bottle was kept for a further 6 hours at an air
tempera trire of —6" to — S.7'. The frogs rec:~-~ - " -
after rapid and after slow iiiawing. Ji. '•! -r

water in the neck of the bottle froize brfore that in the
lower portion, the bottle burst and the frog was kxHeiiL
even though it !: ' ^ '" " ~ "~ ^ — - '"' ^ in the rigid

state. The auTl ~ - -r. the animal

has been injured internally by the aicticm of the pi-essure
developed by the ice.

Koch dS-' ^-~ -- - - -- -■---t the ^;_t _ ^ :^s

Miiller. H- _ ^ i?<i»a HriJi? sii - >

400 ce. beakers filled with water and exposed xhem. thms
to air temperatures of — -t". —10' and —15". ^'~ e

water was frozen, a hole was drilled through the -. -. .^d
a thermometer inserted to measure the ice temperature.
The frogs were killed by a 6-hour stay in iee at —6^. In
:• "' series of expe:-" - -- "t

:_ .....::ion of water. ^-; ,.._ r .._.. .. - _- :. _r

temperature of —4". They at first became very excited
and active, but. after one hour, even the most resistant
ones fell p~ - ' ' -' hard: sueh frogs

could never

S4

Kiiaiitlio (181)1) reported that frogs, laid on ice or
inil)edded in snow, mud, oi- moisi moss, became I'igid after
12 houi's at -1° to -5°. Animals, treated in tliis manner
and kept aflcrwards for several days at temperatures
between -0.2 and ()..')", reco\'ered completely on iliaw-
ing-, even tlioui^h, in the frozen state, tlieir heart had
ceased to beat and circulation had, to all appearances,
stopped. In niiol her <>roup of experiments, frogs (Ranidae
mid Ilijlidac) and toads {Bomhinatorldae and Biifonidae)
were frozen so stiff that their extremities could no longer
be extended, but not so hard that they could be broken,
and were left in this condition for several hours at -0.5°.
When they were thawed, only 10 to 157^ of the frogs, and
about 507f of the toads survived. None of the animals
could withstand a further congelation nor a repeated
freezing. Frogs and toads, frozen completely in water,
were always killed. (What is meant by ''completely"
is not clear.)

According to Pictet (1893), frogs could be revived after
having been frozen hard and brought to a temperature
of -28°. To kill them, it required an exposure to -SO""
to - 35°. The lack of information concerning the body
temperature of the frogs and the time the animals stayed
at that temperature deprives Pictet 's so often-quoted
observations of most of their value.

Kodis (1898) who investigated, among other subjects,
the effects of a considerable subcooling on vitality, re-
ported that whole frogs, with a thermocouple inserted into
the thigh, sutfered no injury when subcooled to - 10°.
Isolated frog muscles could be subcooled to - 18°.

Maurel and Lagriffe (1900) called attention to the
rigidity gradually acquired l)y frogs subjected to tem-
peratures above zero. They observed that the animals,
cooled slowly, were not capable of resuming their posi-
tion when set on their back, when their buccal tempera-
ture was 8° ; that they showed a complete absence of re-
flexes at 4°; and that they became rigid when tlu^y were
caught in ice, the buccal temperature (not actually mea-

85

sLirc'd) being near zero. They could be revived after this
treatment, l)nt not if the temperature was 2 or 3 degrees
lower.

According- to Harris (1910), pithed frogs, whose vis-
ceral temperature was maintained at 0.0° for 1 hour, sur-
vived; one frog died after having sustained for 100 min-
utes a visceral temperature of -2.1°. As to the external
temperatures supported, Harris summarizes his results
by saying that a frog weighing 10 to 12 grams could
probably withstand 1 hour but not 2, in water at -10°.
He states also that frogs which die after freezing experi-
ments, contain some subcutaneous or perivisceral ice,
those which survive do not.

The most extensive investigation on the resistance of
frogs to low temperatures is probably that of Cameron
and Brownlee (1913). They inserted the bulb of a ther-
mometer and, in some experiments, a thermocouple into
the stomach of the animals, and exposed them to a con-
stant low temperature in the air. They found that frogs,
the stomach of which showed - 0.5° for 8 hours, or - 0.5°
to - 1.0° for 1 hour, recovered, while those cooled to - 1.5°
to - 1.8° for 2 hours or to - 2° to - 2.4° for U hours, died.
The authors conclude from these experiments that - 1.8°
is the lowest non-lethal internal temperature which can
be maintained for a short time. The authors then deter-
mined the freezing temperature of the body of the frog
as a whole and found it to be - 0.4-1:°. The freezing tem-
perature being - 0.44° and the lethal temperature - 1.8°,
it evidently follows that ice can be formed in the frog
without killing it. Concerning the external air tempera-
tures supported, the authors found that occasionally -25°
could be withstood for 1 hour ; which shows how slowly
heat is withdrawn from the animal tissues by the sur-
rounding air.

According to Kalabuchov (1934), the toarl, Bufn hufo,
could not be subcooled below - 0.9° to - 1.0°. When frozen
without subcooling, it survived an exposure to sub-zero
temperatures for 20 to 145 minutes during which time its

8()

l)oily temporal ni"o rcaclicd a minimum of -0.5° to -0.15°.
Throe out of four toads were killed after a ir)5-.']02 min-
utes' exposure, the minimum body tompeialure having
1)0011-0.(55° to -5.75°.

The same author found that, in IS out of 22 experiments,
llio tortoise, Tcstudo iiorspchli, could be subcooled to
-2.6° to -5.3° and revived. If, however, ice-formation
sot ill after subcooling, the organisms were killed in a
few minutes, even though their body temperature had not
dropped below - O..")" to -1.4°. When the tortoises were
frozen without })rovi()us subcooling, they survived rela-
tively long exposures, during which the minimal body
toniiK'raturo reached was -0.5° to -0.7°. No completely
frozen animal could ever be revived.

Jecklin (1935) who investigated the cold resistance of
Salamandra maculosa reported tliat, when the tempera-
ture was lowered slowly, the animals could, without injury,
be subcooled to -1.7° or -2.2°; they could likewise with-
stand for a short time a cooling to -5.2°, even when they
had solidified; a temperature of -3.5°, with complete
rigidity, was supported for 70 minutes. The freezing
point of the blood was found to vary between - 0.5° and
-1.7°.

8. Fishes. The numerous reports, some of them rather
startling, about the revival of hard frozen fishes, would
make one think that the popular term *'fish stories " might
well have originated there. Indeed \vlien the possibility
of reviving hard frozen fish is mentioned to fishermen, it
sometimes "reminds" them that they actually saw it
themselves. Turner (1886) saw fishes revive in quite
extraordinary circumstances. He reports that the Alas-
kan black fish, Dallia pect oralis, kept frozen in grass
baskets for weeks, not only is fully alive on thawing, but
that "the pieces thrown to ravenous dogs are eagerly
sw^allowed; the animal heat of the dog's stomach thaws
the fish out, wheroui)()ii the movements soon cause the
dog to vomit it up alive." (Quoted from Borodin, 1934.)

87

As we sliall see, the results of the investigators diverged
widely on this very point of revival after hard freezing,
nntil good determinations of the body temperature of
fishes experimented upon were made.

Pouchet (1866) exposed to varions low temperatures
gold fishes, small s tickle-hacks, and eels contained in
tubes full of water, that he immersed in freezing mixtures.
He found that the eels were killed in 1 hour at - 14°, the
gold fishes in 2 hours at -19°, but that stickle-hacks,
the body of which was only half congealed after 2 hours
at - 19°, survived. He emphasizes that he never observed
the revival of a completely frozen fish, and that when the
animals were entirely caught in the ice they were dead.
He could observe some revived fishes carrying pieces of
ice attached to their body but the latter had been only
partially caught in the ice.

According to Knauthe (1891), fishes of the genera
Cyprinns, Carpio, Carassius, Rhodeus and Misgurnus,
laid on ice and covered with snow or ice w^ater, can be
maintained alive if the surrounding temperature never
drops below -4°, and if the animals do not stay in the
rigid condition for more than one hour.

Pictet (1893) says he could revive gold fishes and
tenches which had been left for 24 hours in water at 0°
and were then cooled slowly to - 8° to - 15°, at which tem-
perature they were brittle. He obtained no revival after
a cooling to -20°. Pictet 's experiments, which confirmed
the popular opinion of the reviviscence of "hard frozen"
fishes, have been generally considered as demonstrative
by the biologists until more recent determinations have
brought into evidence the fact that the body temperature
in fishes is often above the bath temperature for a time
much longer than it was generally thought.

The action of cold alone, ^^'ithout congelation, on fishes
was investigated by Regnard (1895). Carps, accus-
tomed to water containing 2.5% magnesium sulphate,
were cooled slowly in this medium. Cooling was achieved
by immersing the container in a refrigeration bath. Reg-

88

iiard obscrvc'd tliat when tlio tcmperatui'c of the mai»'-
lU'siiini water canic to 0°, tlie Hshes bccanu' little by little
iiiHiiotilc; when it rcaehcd -2° and -3°, the complete
cessation of motion gave the animals a dead appearance.
However, they were not rigid bnt ** perfectly supple."
On rewarming, they resumed motion and soon behaved
normally.

Britton (1924) made a somewhat extensive study on
the resistance of the skate, sculpiii, sea raven, flounder,
eelpouf, cod, foiicod and pollack to sub-zero temperatures.
The fishes were immersed in refrigerated tanks containing
sea watei-. The effects of cold, as exemplified in the case
of the sea raven, were in general, as follows. After an
exposure of 30 minutes to - 1.9°, the rectal temperature
w^as around - 1° ; the body then became rigid (not by con-
gelation but by some cold rigor ; the freezing point of sea
water being -1.6°), the respiratory movements, the jaw
and tin reflexes and finally the heart-beat ceased. When
re warmed, most of these fishes were dead. The lethal
body temperature was, then, that produced by an immer-
sion of the fish from 20 to 60 minutes in a bath at -1.9°.
At higher sub-zero temperatures, the fishes were also
usually killed, although after a longer time. Slow and
gradual cooling gave the same final result as rapid cool-
ing by abrupt immersion. The elasmobranchs were more
resistant.

Kalabuchov (1934) reported that the carp could not be
subcooled to temperatures below - 5°. When frozen with-
out subcooling, the fishes survived minimum body tem-
peratures of -0.2° to -0.5°; they were killed when the
latter dropped to -2° to -4.9°. Completely frozen fishes
could never be revived.

Borodin (1934) studied the effects of low temperature
on some 10 different kinds of fishes including, among oth-
ers, the mummkliog, eel, gold fish, young carp, perch, and
mild-minnow. The low temperatures were produced by a
Westinghouse household refrigerator within the freezing
chambers of which the temperature could be kept constant

89

to within |°. The fishes were put either directly into the
aluminum ice trays lined with cellophane to prevent ad-
hesion durino- freezing, or into a paraffined card-board
box. The ])ody temi)erature of the experimental animal
was ascertained by means of a needle thermocouple in-
serted into the muscles of the back of the fish behind the
dorsal fin. The various species were found to differ in
frost resistance; huUhead, 6 to 7 inches long, recovered
after f hour at a chamber temperature of - 18°, while
killifish, 4 to 6 inches long, survived 1 hour at -14° to
- 15°, these being the hardiest of the species used. In
general, the larger fishes of a species supported freezing
more easily than the smaller ones. Moreover, a rapid
freezing at a somewhat lower temperature proved less
fatal than a prolonged freezing at a higher temperature ;
thus, fishes frozen for 5 to | hour at - 14° to - 15° recov-
ered in most cases, while those frozen for f to 1 hour at
- 10° to - 12° generally did not revive. None of the fishes
could withstand a temperature below - 18° for more than
25 to 30 minutes. Death occurred when the body tem-
perature of the fishes dropped below -0.6° to -1° which
required 25 to 45 or more minutes depending on the tem-
perature of the chamber and the size of the fish. The
author concludes that the death point probably lies near
the point at which the body fluids freeze. In a few rare
cases, fishes with muscles, blood vessels and intestine
frozen hard, but with the heart not yet frozen throughout,
recovered wdien thawed. It is thought that only when the
heart is hard frozen and kept so for some time, does the
fish die. The author distinguishes between fishes that are
' * soft frozen, ' ' that is, stiff and apparently dead, but with
the body fluids still liquid as is evidenced by the bleeding
when they are dissected, and fishes that are ''hard frozen,"
that is, "frozen through as hard as stone." The former
recover rapidly and evince no ill effects of the frost, while
the latter, in most instances, do not regain their vitality,
or, if they should perchance survive, exhibit marked
injuries.

DO

Weigmaim (15).")()) stiulied dcatli Icnipcratures in stickle-
backs cooled slowly in water. Ho inserted a thormoconplc
into the body of a few specimens and showed that wiien
the temperatnre of the water was lowered, that of the
animal followed closely (how closely depends, of course,
on the cooling velocity and the size of the animal). As to
the death tem})eratnre, he observed that all the fishes died
when their body became surrounded with ice but before
being actually caught in ice. The maximal resistance
registered was 20 minutes at bath temperatures of - 2.5°
to -3.2°, which would give for the lethal body tempera-
ture, -2° to -3°. The author remarks that a velocity of
cooling varying from 0.02° to 0.6° per minute had no influ-
ence on death or survival.

Schmidt, Platonov and Person (1936) subjected to cold
carps maintained in air, determining their internal tem-
perature with thermocouples. They found that these
fishes could be revived if their body temperature, mea-
sured at a depth of 1.5 to 2.5 cm, from the surface of the
skin, did not fall below -0.72° to -0.92°. In the outer
surface layers of the body, - 3.33° was recorded. In an-
other series of experiments, the same authors revived
hJeaks and sticMehacks subcooled to -3.06° (body tem-
perature). None of the fishes experimented upon ever
survived a congelation of its body.

Luyet (1938), in an attempt to test a theory according
to which a rapid freezing would cause the formation of
smaller crystals less injurious to the cells, studied the
effects of a sudden immersion of gold fishes in liquid air.
He could never obtain any sign of recovery if the immer-
sion had lasted more than 15 seconds (for fishes 40 mm.
in length, not counting tlie caudal fin). However, after
that time, the fishes were not frozen throughout, it requir-
ing about 35 seconds for them to become breakable. Im-
mersions of 1 to 15 seconds caused gradually increasing
injuries, corresponding to the gradually increasing thick-
ness of body wall congealed. The author, who reports
that a juggler had as a regular item in his program the

91

revival of hard frozen gold fishes, remarks that for a
successful demonstration, the animals to be broken before
the audience should be left for 35 seconds in liquid air and
those to be revived should be withdrawn in less than
10 seconds.

9. Ilomoioihenns. It is well known that the warm-
blooded animals cannot support any considerable lowering
of their body temperature. Of the abundant literature on
this subject, w^e shall select some reports establishing the
general fact of the high cold sensitivity of homoiotherms,
a few others describing experiments made in some unusual
conditions, and still others which are of interest because
of the exceptionally low temperature reached.

Walther (1862), working with rabbits, found that when
the animals, immobilized but non-narcotized, were cooled
to a body temperature of 18° or 20°, they were unable
to recover spontaneously. However, the complete recov-
ery of such animals could be effected either by warming
them in air at about 40°, or by artificial respiration.

Coleman and McKendrick (1885) placed a rabbit whose
rectal temperature was 37.3°, pulse 160 per minute, and
respiratory rate 45 per minute, into a cold chamber at
an air temperature of -69.4°, for 2 hours. The animal
was then removed for a minute or two; it seemed unaf-
fected, though the rectal temperature had dropped to
34.5°. Thereupon it was returned to the cold chamber,
now at a temperature of -73°, for another hour, after
which it was again taken out. The animal seemed to be
comatose, reflex action had ceased, there w^ere jerking
movements of the limbs, the rectal temperature had
reached 6.1°, the pulse rate was 40 per minute, and respi-
ration was hardly perceptible. Placed in a warm atmos-
phere, the animal recovered completely.

Colin (1891) reported that rabbits could stay alive in
winter after 5 or 6 days at an atmospheric temperature
of - 10° to - 15°, in cages hanging on trees.

Pictet (1893) subjected a dog weighing 8^ kilos to an
air temperature of - 90° to - 100° in one of his ref rigera-

!)2

lion "wells." The metal walls of the latter were covered
with wood or cloth so as to avoid contact between the
animal and llic cold metal. The inguinal temperature
increased by about \ decree duiini*' the first 13 minutes,
it came back to normal during the following 12 minutes,
and decreased by I degree during the next 75 minutes.
Then it dropped rapidly, while at the same time respira-
tion and heart beat Ix'came slower and slower. When the
temperature reached 22° the animal was withdrawn, inani-
mate, and it did not recover.

The same author reported that a cat which fell into a
liquid refrigerating mixture at - 30° to - 35° died ''almost
suddenly." The more rapid cooling of the body when
in contact with a licjuid than when in contact with air,
is considered the cause of the I'apid death.

According to Winternitz (1894), a rahhit, whose body
temperature drops to 34° to 31°, begins to shiver, it tends
to fall asleep at 31° to 29°, and finally, when the tempera-
ture reaches 22° to 19°, breathing ceases and the animal
dies.

Simpson (1902) placed a monlxey {Macacus rhaesiis),
fully etherized, into a double-walled chamber made of thin
sheet-iron, and cooled wdth lumps of ice inserted into the
space between the walls. The animal could be observed
through a sliding glass door. The rectal temperature,
pulse rate and respiration rate were taken every 1 or ^
hour. After 3\ hours, the body temperature had fallen
to 14°, the heart beat had ceased and the respiration rate
was reduced to 2 per minute. The ice-water was then
removed and quickly replaced by hot ^vater till the cham-
ber temperature stood at 41.6°. When, after 5 hours, the
rectal temperature had risen to 37.7°, the animal was
removed from the chamber and placed in a room at 25°,
where it recovered completely in a further 2i hours.
Another monkey whose rectal temperature had been
lowered to 12.5°, under the same conditions, succumbed.

According to Simpson and Herring (1905), cats, whose
normal temperature is about 38°, can be cooled till their

93

rectal temperature reaches 16° and still recover, provided
they are then warmed artificially.

Tait (1922) reported that, while the excised, perfused
heart of an ordinary mammal stops beating at about 17°,
that of a hiheruailng hedgehog or woodchuck will cease to
beat only when it freezes.

The same author, in collaboration with Britton (1923),
found that, in the woodchuck, respiration ceases without
signs of asphyxia at 12° to 3° ; but, when warmed, the
animal may recommence breathing, and spontaneously
resume its former body temperature; with artificial res-
piration, it always recovers.

Britton (1923) repeated the earlier work of Simpson
and Herring, using a cooling apparatus similar to that of
these investigators, and working almost exclusively with
deeply anaesthetized cats. He called attention to the fact
that the ''deep rectal" temperature (the thermometer
bulb placed 8 cm. deep in the rectum) may be 0.5° to 2°
higher than the "anal" temperature (thermometer bulb
4 cm. deep). Cats, whose deep rectal temperature had
been lowered to 19°, could still spontaneously recover
their previous body temperature, even though very slowly.
Below 18°, recovery could be effected only by artificial
warming. The lowest deep rectal temperature at which
a cat gave evidence of being alive was 16°, while a guinea-
pig could be cooled to 14°.

According to Kalabuchov (1934), the hat Nyctalus
noctida can withstand a 5-15 minutes' subcooling to -2.9
to -5.9°. Of 10 bats of the same species, frozen after
subcooling, 7 were killed, though the minimal body tem-
perature reached only -0.5 to -1.5°. If, however, freez-
ing set in without a previous subcooling, the animals could
survive a 12-60 minutes' exposure, and a minimal body
temperature of -0.8 to -1.9°, but were killed after a 67-
145 minutes' exposure and minimal body temperature of
-1.2 to -5.5°. Myotis dauheutonii is not so resistant; 4
out of 5 bats of this species were killed when freezing with-
out subcooling occurred during a 17-90 minutes' exposure.

!)4

llic iiiiiiinial Ixtdy IciiiixTnlurc I'caclicd li;i\-iiin' been -0.9
lo 1..") .

Tlic saiiic ;uil linr i'('j)(>r1('(l t lint llic >y/o/rsr. .l//^s■ m use n I us,
was kilk'd when fiozcii till a body lemijcratiire of -0.9°
was reg'istercd, oi' wlicii sulu'oolcd lo-.S.T" 1o-7.1". (One
would natnrall>' expect it.)

Murigin (1937), repeating some old experiments of
Horvat (ISSl), in wliieli the f/roinid-sqiurrcJ , Cifflhis
siislicd. could be revived after its body 1em])ei-ature was
lowered to -0.2°, exposed 19 Cifclliis ))i/f/Hia('us i'ov 2 to 4
hours in cooling chambers to temperatures of -11° to
-19° and found that 47% of the animals could be revived
after their body temjierature, measured by a thermo-
couple, had sunk to -0.5° and -1°. When the body tem-
])erature went below - 1°, the animals died. Repeating a
cooling experiment on the same individual seemed to
result in a greater ability to resist a new lowering of
temperature; one specimen survived -0.5° 3 times, an-
other sui'vived - 1° twice. The smaller animals were more
resistant than the larger ones. The author compares the
special state of torpor which results from the lowering
of the temperature, to the anabiotic state; he describes
it as an "imaginarv anabiosis."

Summary: 1, The invertebrates were found, in general,
to be killed when frozen at temperatures of a few degrees
or of some 10 degrees below zero. 2. The rotifers, the
nematodes and the tardigrades, however, resisted, in the
moist condition, extremely low temperatures. 3. The
invertebrates which su])port desiccation could be cooled
to the lowest available temperatures without injury.
4. Some insect larvae were alive, although hard fi'ozen, at
some 20 degrees or more below zero, but they died at lower
temperatures, probably under a more complete congela-
tion of their cellular fluids. 5. The cold-blooded verte-
brates died when their internal tempeiature dropped a
few degrees below zero. They could su])|)ort the forma-
tion of some ice in their body. 6. Sub-cooling is not in-

95

jurious to the poikilotlierms. 7. Tlic warm-blooded ani-
mals died at above-zero temperatures except if, as in the
case of hibernators, they had developed some adaptative
properties.

GENERAL COMPENDIUM

By their resistance to low temperatures, the plants and
animals considered in this review can be classified into 3
groups : 1. Those which can approach the absolute zero
without being killed; 2. Those which die near the freez-
ing temperatures ; 3. Those wdiicli die above the freezing
temperatures.

The first group comprises : a) The forms wiiich support
desiccation, such as, the seeds, the spores (bacterial and
others), the protozoan cysts, the rotifers, the tardigrades.
the nematodes, b) A number of microorganisms of the
types, bacteria and yeast, some flagellates, some of the
lower fungi, and the infra-cellulars. All these resist
extreme temperatures without previous desiccation.

The third group, namely that of organisms wdiich are
killed at above-zero temperatures, includes only the
homoiotherms and some of the higher plants.

The large majority of plant and animal phyla belong,
then, to the second group, that is, to the kind of organisms
w^hich are killed at near-zero temperatures. There are,
it seems, two classes to be distinguished in this group:
1. The organisms which die Avhen their temperature is
lowered slightly below the freezing point. Usually these
organisms can support the formation of some ice in them
but they are killed wiien a larger proportion of their
w^ater-content solidifies. This has been observed in sev-
eral plant tissues, in some animal tissues and in entire
organisms, such as, the mollusks, the ami3hibia, the fishes,
etc. 2. The organisms wiiicli resist some 10, 20 or 30 de-
grees below freezing. Often a relatively large quantity
of ice can be formed in them, they become hard and break-
able, and they die, apparently wdien a last portion of their
cellular fluids solidifies. This is probably the case of

!»(;

iiiset'l larvae al crrtaiu i)erio(ls ol" Die year and, 1o a
certain extent, of the eold-liardened woody plants.

Between the lifsl and the second class, Dial is, between
the organisms which ai'e killed near the freezing })oint
and those which resist some 30^ below zero, there seems
to be a colli Innons gradation.

Jn short, one can perhaps summarize this entire review
as follows: With the exception of a few org'anisms which
are atifected by above-zero temperatures, all the })lants,
animals, tissues or cells either die in the neighborhood of
their freezini;' ])()int or are not killed at all by any low
temperature.

BIBLIOGRAPHY

ADAMS, ,T., Sc. Frnc. h'oy. Soc. Dublin, 2, 1-G, litli.l.

ALEXOPOULOS, C. J., and DRUMMOND, J., Ttoh.s. llUnoi^i Acad. ScL, 26,

G3, 1934.
APELT, A., Cohns Bcilr. z. Biol. d. Ffl.. D, 215-262, 1909.
AUGUSTINE, D. L., J. Parasit., 19, 175, 1932 (Abstract).
BACHMETJEW, P., Experimentelle Entomologische Studieii voni pliys-

ikaliscli-cliemisc-lion StandiJiinlct aus. Vol. I. Temperaturvcrluiltnisse

bei Insekteii. Leipzig, 1901; Vol. II. Einfluss der ausseren Faktoren

auf Insekten. Sopliia, 1907.
BAHRMANxV, E., Zt)<chr. f. Biol, 92, 366-372, 1932.
BAKHMETIEFF, P., Arch. Sc. biol. St.-Petersbourg, S, 242-264, 1901.
BARRATT, J. O. W., Chi. Bakt., 35, 769-775, 1903.
BARTETZKO, H., Jahrh. f. wiss. Bot., 47, 57-98, 1910.
BARTRAM, H. E., J. A//r.Res., 5, 651-655, 1916.
BECQUEREL, P., C. r. Acad. Sci., 140, 1652-1654, 1905; 14S, 1052-1054,

1909; 150, 1437-1439, 1910; 181, 805-807, 1925; 1S8, 1308-1310, 1929;

190, 1134-1137, 1930; 194, 1378-1380, 1932a; 194, 1974-1976, 1932b;

194, 2158-2159, 1932c.

, JRapport 6« Cong. Int. du Froid, Buenos-Ayres, 1932d.

, C. r. Acad. Sci., 202, 978-981, 1936; 204, 1267-1269, 1937.

BEEK, F. J., Invlood van tempcraturen tot 20^ C. boneden het vriospunt op

toxischc en atoxische dysonteriebacterien. Doctor diss., Leyden, 1919.
BELEHRADEK, J., Temperature and Living Matter. Berlin, 1935.
BELLI, C. M., Cbl. Bakt., 31, 355-360, 1902.
BERRY, J. A., Science, 80, 341, 1934.

BERRY, J. A., and MAGOON, C. A., Fliylupath., 24, 780-796, 1934.
BICKEL, A., Deuisch. med. Wochenschr., 31, 1383, 1905.
P.ORODIN, X. A., Zool. Jahrh., Aht. f. allg. ZooL, 53, 313-342, 1934.
JJRKIIME, W., Arch. f. Eyg., 40, 320-346, 1901.
BRITTON, S. W., Quart. J. Exp. Fhysiol, 13, 55-68, 1932.
, Am. J. Physiol., 67, 411-421, 1924.

97

BROWN, II. T., and E8C0MBE, F., Trac. Fot/. Soc. London, 62, lGO-165,

1897.
BRUNOW, II., Zlschr. f. allg. Fhysiol., 13, 367-387, 1912.
BuHLER, Arch. f. Anaf. u. riiysiol., Physiol. AM., 239-251, 190.5.
CAMERON, A. T., and BROWNLEE, T. I., Proc. Boy. Soc. Canada, 7, Sect.

4, 107-124, 1913.
CHAMBERS, R., and HALE, H. P., Proc. Boy. Soc. London, 110, 336-352,

1932.
CHANOZ, COURMONT P., and DOYON, M., C. r. Soc. Biol, 52, 764-765,

1900.
CHANOZ, and DOYON, M., Ihid., 52, 453-454, 1900.
CHODAT, R., Bull. Berh. Boisier, 4, 890-897, 1896.
CITOVICZ, B., C. r. Soc. Biol., 99, 1699-1700, 1928.
COHN, F., Bot. Ztg., 29, 723, 1871.

COLEMAN, J. J., and McKENDRICK, J. G., Chem. News, 52, 61-64, 1885.
COLIN, G., C. r. Acad. Sci., 112, 397-399, 1891.

DAVENPORT, C. B., Experimental Morphology. New York, 1897-99.
d'ARSONVAL, C. r. Soc. Biol., 44, 808-809, 1892; 50, 877-878, 1898.
de CANDOLLE, C, Arcli. Sc. Phys. Nat. Geneve, Se per., 33, 497-512, 1895.
de CANDOLLE, C, and PICTET, R., Ihid., 2, 629-632, 1879; 11, 325-326,

1884.
de .JONG, D. A., Arch. Nccrland. Physiol., 7, 588-591, 1922.
DESCHIENS, R., C. r. Soc. Biol., 115, 793-795, 1934.
DOENHOFF, Arch. f. Anat. u. Physiol., 724-727, 1872.
DUVAL, M., et PORTIER, P., C. r. Soc. Biol, 86, 2-4, 1922.
EDLICH, F., Arch. Mihrohioh, 7, 62-109, 1936.
EDWARDS and COLIN, Ann. Sc. Nat., 2<- ser., 1, 257-270, 1834.
EFIMOFF, A. W., Arch. Protistenk., 49, 433-446, 1924.
FISCHER, H. W., and JENSEN, P., Bioch. Ztschr., 20, 143-165, 1909.
FISCHER, P. H., C. r. Soc. Biol., 105, 369-371, 1930.
FLOSDORF, E. W., and MUDD, S., J. Immun., 29, 389-425, 1935.
FOL, Arch. Sc. Phys. Nat. Geneve, 3e per., 11, 327, 1884.

GALVIALO, M. I., Bnssian Physiol. J., 22, 224^227, 1937. (English
summary. )

GAYLORD, H. R., J. Inf. Bis., 5, 443-448, 1908.

GERTZ, O., Bot. Notiser, 263-268, 1926.

GLADIN, G. P., CM. BaTct., 24, 588-591, 1898.

GOEPPERT, Ueber die Warmeentwickelung in den Pflanzen, deren Gefrie-
ren, und die Schutzmittel gegen dasselbe. Breslau, 1830.

GREELEY, A. W., Am. J. Physiol., 6, 122-128, 1901.

HAINES, R. B., J. Exp. Biol, 8, 379-388, 1930.

HAMPIL, B., Quart. Bev. Biol, 7, 172-196, 1932.

HARRIS, D. F., J. Physiol, 40, LIII-LV (Proc), 1910.

HARVEY, R. B., Bot. Gas., 67, 441-444, 1919.

HEPBURN, J. S., Z7. S. Dept. Agr., Bur. Chem. Circ, 103, 1-12, 1912.

— — , Bioch. Bull, 4, 136-150, 1915.

HEPBURN, J. S., and BAZZONI, C. B., J. Franllin In.st., ISO, 603-605,
1915.

HEUBEL, E., Pfliigers Arch., 45, 563-568, 1889.

DS

II I I.I.IAIMi, C. M., ■roiv'OSSIAX. ('., .111(1 S'I'OXi:, U. p., Science, 42, 770-

77 1, i;ii.'..

llll.I.IAUM), ('. M., ;iii(l DANIS, M. A., ./. liarl., .}, 42;i-i:n, 1918.
IIOK'OWITZ-WLASSOWA, I.. M., un.l ( ; l.'l .\ IlKliM ;, L. D., T/W. JSaLI., 89,

r)4-G2, 1933.
HUNTER, J., Works. I'.iliiicr 's edit ion. Loiidoii, 1837.
ILJIN, W. S., Protoplu.sma, J(J, 105-124, 1933.
IRMSCHER, E., Jahrb. /. wiss. Bot., 50, 387-450, 1912.
JAIIN, T. L., Arch. ProlLslcnl:, 79, 249-262, 1933,
JECKLIN, L., Her. i>ni.s.se ZooJ., 42, 731-738, 1935.
JUMELLE, H., C. r. Soc. Biol. (Mem.), 42, 115-119, 1890.

, Bev. gin. Bot., 4, 266-272 and 3U5-316, 1892.

KADI sen, E., Med. Klin., 1074-1078, and 1109-1112, 1931 II.

KALABUCHOV, N., C. r. Acad. Sci. U.B.S.S., 7, 424-426, 1934.

KAPTEREV, P., C. r. Acad. Sci. U.R.S.S., 3, 137-140, 1936.

KaRCHER, H., Flanta, 14, 515-516, 1931.

KASANSKY, M. W., Chi. Bakt., 25, 122-124, 1899.

KIOITII, S. C, Science, 37, 877-879, 1913.

KLEMM, P., Jahrh. f. wiss. Bot., 28, 641-644, 1895.

KLEPZOFF, C, CM. Bait., 17, 289-295, 1895.

KNAUTHE, K., Zool. Anz., 14, 104-106 and 109-115, 1891.

KOCHS, W., Biol. Zhl., 10, 673-686, 1890.

KODIS, T., Cbl. f. Physiol., 12, 592-595, 1898.

KOVCHOFF, J., Ber. d. deutsch. hot. Ges., 25, 473-479, 1907.

KtJHNE, W., UntersiK'lnnigcn iibcr das Protoplasina iind die Contractilitat.

Leipzig, 1864.
KYLIN, H., Ber. d. deutsch. hot. Ges., 35, 370-384, 1917.
LINDNER, J., Jahrh. f. wiss. Bot., 55, 1-52, 1915.
LIPMAN, C. B., Plant Physiol., 11, 201-205, 1936a.
, Bull. Torrey Boi. Club, 63, 515-518, 1936b; 64, 537-546,

1937.
LIPMAN, C. B., and LEWIS, G. N., Plant Physiol, 9, 392-394, 1934.
LIPHCIIuTZ, A., and ILLANES, A., C. r. Soc. Biol., 102, 555-556, 1929.
LOZINA-LOZINSKIJ, L. K., C. r. Acad. Sci. U.E.S.S., 2, 331-332, 1935.
LuDKE, Cbl. Bakt., 40, 577-583, 1905.

LUMIfiRE, A., et CHEVROTIER, J., C. r. Acad. Sci., 158, 139-140, 1914.
LUYKT, B. J., and GIBBS, M. C, Biodyn., No. 25, 1-18, 1937.
LUYET, B., C. r. Soc. Biol, 127, 788-790, 1938.
MACFADYEN, A., Proe. Boy. Sne. London, 66, 180-182, and Lancet, 1, 849,

1900.

, Proc. Boy. Soc. London, 71, 76-77, 1902a.

MACFADYEN, A., and EOWLAND, S., Ibid., 66, 339-340, 1900a; 66, 488-

489, 1900b.

, Ann. Bot., 16, 589-590, 1902.

MAUREL and LAGRIFFE, C. r. Soc. Biol, 52, 432-435, 1900.
MAXIMOV, N. A., Jahrb. f. ^viss. Bot., 53, 327-420, 1914.
MELSENS, M., C. r. Acad. Sci., 70, 629-632, 1870.
MEZ, C, Flora, 94, 89-123, 1905.

99

MOLISCH, H., UntcrsiK'liimgen iiber das Erfrioren der Pllanzon. Jena,

1897.
MORAN, T., Froc. Hoy. Soc. Lomlon, B, 08, 436-456, 1925; lOo, 177-195,

1929.
MuLLER, W., Arch. f. Anat. u. Physiol., 754-759, 1872.
MULLER-THURGAIT, H., Landw. Jahrb., 9, 133-189, 1880; 15, 453-610,

1886.
MURIGIN, I. I., Bull. Biol. Med. Exp. U.E.S.S., 4, 100-102, 1937.
McLEAN, A. L., Nature, 102, 35-39, 1918.
NOACK, K., JahrT). f. iviss. Bot., 51, 593-648, 1912.
ONGKIEHONG, Pathogeiie darmbacterien en lage temperaturen. Doctor

diss., Leyden, 1922.
OXORATO, R., Chi. BaM., 31, 704-712, 1902.

PAUL, T., und PRALL, F., Arh. Kaiserl. Gesundheitsamte, 26, 73-129, 1907.
PAWLOW, G. N., Euss. Physiol. J., 10, 291-300, 1927. (German summary.)
PAYNE, N. M., Ecology, 7, 99-106, 1926a; 11, 500-504, 1930.

, Quart. Rev. Biol., 1, 270-282, 1926b.

, Biol. Bull., 52, 449-457, 1927.

PICTET, R., Mevue Scient., 52, 577-583, 1893.

, Arch. Sc. Phys. Nat., Ser. 3, 30, 293-314, 1893.

PICTET, R., and YUNG, E., C. r. Acad. Sci., 98, 747-749, 1884.
PLATEAU, F., Bull, cle I'Acad. roy. Belgique, 2e ser., 34, 291-309, 1872.
POUCHET, F. A., J. Anat. et Physiol, 3, 1-36, 1866.
POZERSKI, C. r. Soc. Biol, 52, 714^716, 1900.

PRUCHA, M. J., and J. M. BRANNON, J. Bact., 11, 27-29, 1926.
QUATREFAGES, A., de., Ann. Sci. Nat., (3), 19, 341-369, 1853.
RABAUD, E., C. r. Acad. Sci., 128, 1183-1185, 1899.

RAHM, G., K. Akad. Wetensch. Amsterdam, Xatuurkundige Afdeeliug, 25.
235-248, 1920; 29, 499-512, 1921.

— , Ztschr. allg. Physiol., 20, 1-34, 1923.

REGNARD, P., C. r. Soc. Biol, 2, 652-653, 1895.

RICHTER, A., cm. Bakt., 28, 617-624, 1910.

RIVERS, T. M., J. Exp. Med., 45, 11-21, 1927.

ROBINSON, W., Minn. Agr. Exp. Stat., Tech. Bull 41, 1-43, 1926.

ROEDEL, H., uber das vitale Temperaturminimum wirbelloser Thiere.

Diss., Halle, 1881. Also: Ztschr. f. Naturw., 59, 183-214, 1886.
SACHAROV, N., Ecology, 11, 505-517, 1930.

SALVIN-MOORE, J. E., and BARRATT, J. O. W., Lancet, 1, 227, 1908.
SANDERSON, E. S., Science, 62, 377, 1925.
SCHENK, S. L., Sitzungsb. Wien. Akad., 60, 25-30, 1870.
SCHMIDT-NIELSEN, S., Cbl Bakt., 9, 145-147, 1902.
SCHMIDT, P. J., PONOMARER, A., et SAVELIER, F., C. r. Soc. Biol,

78, 306-307, 1915.
SCHMIDT, P. J., et STCHEPKINA, F. V., Ibid., 80, 366-368, 1917.
SCHMIDT, P. J., PLATANOV, G. P., and PERSON, S. A., C. r. Acad. ScL

U.B.S.S., 3, 305-308, 1936.
SCHUMACHER, E., Sitzungsb. Wien. Akad.. 70, 157-188, 1874.
SIMONIN, C, C. r. Soc. Biol, 107, 1029-1032, 1931.
SIMPSON, S., J. Physiol, 28, XXXVII-XL (Proc), 1902.

TOO

SI.MI'SC)^. S., ;iiHl IIEKIUNG, 1". '1'.. fh'uL, :!;.', 305-:?ll, l!t();j.
►SMART, H. F., Science, 82, 525, lit.!.").

SMITir. E. F.. mid RWTXOT.K. I>. T.., CM. liahl.. .17. .'^57-.S.')n, 1905.
SP.\Lr..\NZAM. Tj.. Oi.iiscillfs (Ic iili_vsh|iir ;iniiii;ilc ct vi'{,M'tal('. 1*. .T.

Duplaiii, I'avio & Paris, 1787.
STOCK^rAN, S., and MTXKTT, F. C, ./. Camp. J'alli. an, I Tlurap., 39

I 1), l-.'iO, 10:2(i.

TArr, .r.. Am. J. riij/sioi.. .-.:>. W7, ist^-j.

TAIT, .1.. and r.KMTTOX, S. W., (Jiiarl. J. E.ip. Fhi/.sioL, 22G-227, 1923

(Su].].!.).
TANNER, V. W., and WALLACE, G. I., Proc. Snc. E.rp. Binl. aiul Mr, I.,

x-'i*, 32-;{4, i!t;;i.

TANNER, F. W., ,nid WILLIAMSON, V,. W., Ihld, 25. 377-381, 1928.
TAYLOR, C. v., and STRICKLAND, A. G. R., rin/siol. ZonL. !>, 1.5-26,

1936.
TEODORESCO, E. C, Bev. (;>„. ,1, B„t.. 18, 415-418, 1906.
TETSUDA, ITO., Zl.scin: f. Iinmini.. /.7. 97-116, 1912.
TIIISELTON-DYER, W., Tnx: lUni. Soc. London, 65, 361-368, 1899.
THUNBERG, T., Skondinar. Arch. f. Phi/.^iol., 40, 71-80, 1920.
UVAROV, B. P., Trar\.^. Ent,nn,d. Soc. Loudon, 79, 1-247, 1931.
VOIGTLaXDKR, H., Cohn.s Bcilr. z. Biol d. Pfl., 9, 359-414, 1909.
WALTER, IL, nnd WEISMAXN, O., Jahrh. f. iviss Bot., 82, 27.3-310, 1936.
WALTHER, A., Virchoto's Arch., 25, 414-417, 1862.
"WARTMAN, Arch. Sc. Phys. Nat. Geneve, Nouv. scr., 7, 277-279, 1860.

{Cf. also a brief report by Wartman in Arch. Sc. Phys. Nat., for 1884.)
WETGMANN, R., Biol. Zbl., 58, 301-322, 1936.
WHITE, A. C, J. Am. Med. As.sn., 36, 426-428, 1901.
WINKLER, A., Jahrh. f. wi.'<.«. Bot., 52, 467-506, 1913.
WIXTERNITZ, R., Arch. f. e.rp. Path. v. Pharmal'., 33, 286-304, 1894.
WISLOUCH, S. M., Bull. Jardin Impcr. Botan. St.-Petrr.shourg, 10, 166-

178, 1910. (German resume, pp. 179-180.)
WOLFSON, C, Ecology, 16, 630-639, 1935.
YUNG, E., Mem. Acad. Boy. de Belg., 49, 1-119, 1888.
ZACHAROWA, T. M., Jahrh. f. wiss. Bot., 65, 61-87, 1926.
ZANDBERGEX", K., Trvpaiiosomen en lage tempcr.-iturcn. Doctor diss.,

Leyden, 1922.
ZIRPOLO, G., Proc. Sblh Int. Congr. Cryoscopy. Bnrnos Aire.^, p. 450,

1932.

PART II

THE PHYSICAL STATES OF PROTOPLASM
AT LOW TEMPERATURES

A substance may exist in four states, as a gas, as a
liquid, as a crystal and as a glass. The relation between
these states and temperature is illustrated in the diagram
(Fig. 1), in which the temperatures are plotted on a hori-
zontal axis, with the absolute zero at the origin. "A body
is a gas at high temperatures, in the zone A ; it is a liquid
at lower temperatures, in the zone B ; it becomes crystalline
at still lower temperatures, in the zone C ; and it hardens,
but does not crystallize, that is, it takes the vitreous state,
if it is brought (^\dthout being previously crystalline) to
the low temperatures represented by the zone D. ' ' (Luyet,
1937, p. 1).

D , C , B , A

A.Z. R Q P

Fig. 1. Diagram representing the relationship between temperature and
the four states of matter. A.Z. : Absolute zero.

Some physicists (cf. Tammann, 1925, p. 2) for whom the
arrangement of the molecules in the aggregate is the essen-
tial feature in the detinition of the states of matter, name
only two of them : the crystalline state which possesses as a
specific character an ordered arrangement of the atoms,
and the amorphous state (gas, liquid and vitreous) in which
the molecules are distributed at random in space. Most
students of nature name three states : gas, liquid and solid,
and make of the vitreous state a subdivision of one of the
three. Some, considering the fact that a glass possesses
the hardness and cohesion of the solid bodies, classify it as
a form of the solid state ; others, considering the def orm-
ability presented by some glasses during long periods of

101

102

lime, jii'd'cr lo call tliciii li(|ui(ls of lii<^ii x'iscosily; slill
olliers, c'onsideriiij;' the met hod of ol)taiiiin^' glasses and
llieir amorplioiis iialure, insist on callini;' llicm sni)or-cool('d
li(|nids. Detinini*- a state by its relation to temperatnre (as
illnslraled in Fi.n'. 1), !)>■ the consistency of the matei'ial
and l)y the arrani-ement of the constituent jjarticles, we
shall distinguish four states of matter: vitreous, crystal-
line, liquid and gas.

This classification of the states requires a revision
of the classification of the transitions from one state to
another. The following list gives the various ])ossil)ilities.
(We coined a few names, when such wei-e lacking; they are
indicated by (inotation marks.)

1.

From

1'.

From

o

From

2'.

From

3.

From

3'.

From

4.

From

4'.

From

5.

From

5'.

From

(5.

P"'rom

6'.

From

C^HANOEs OF State

the vitreous to the crystalline state:
the crystalline to the vitreous state:
the vitreous to the liquid state :
the liquid to the vitreous state:
the vitreous to the gas state:
the gas to the vitreous state:
the crystalline to the liquid state:
the liquid to the crystalline state:
the crystalline to the gas state:
the gas to the crystalline state:
the liquid to the gas state:
the gas to the liquid state:

Devitrification

(Tmpossilile)
' ' Vitromelting' '

Vitrification
' ' Vitrosubliniation ' '
' * Gasovitrification ' '

:Nrelting

Crystallization

Sublimation
' ' Gasocrystallization '

Vaporization

Liquefaction

The liquid state can be maintained also at temperatures
below the f 7'eezing point ; one then obtains, besides the
four states just described, a supernumerary one (called
state in a broader sense), the supercooled state.

Of these five states there are only three which protoplasm
can take at low temperatures, the crystalline, the vitreous
and the supercooled; the liquid state obtains at ordinary
temperatures and the gas state is unknown in protoplasmic
substances. As to the changes of state, the most important
for the biologist are: crystallization (that is, freezing),
melting, vitrification, devitrification and "vitromelting."
Consequently we shall divide this work into the following
chaptei's: I. Freezing, the Frozen State and ^Melting; TI.
Sni)ercooling and the Supercooled State ; IIT. Vitrification,

103

the Vitreous State, Devitrificatioii and " Vitromelting."
A preliminary cliapter will hv devoted to tlie study of the
principles of heat eondiiction.

Within each section, after having discussed the funda-
mental laws, Ave shall consider the various biological mate-
rials which undergo the changes of state, in the order of
the increasing complexity of these materials : pure water,
solutions, suspensions, emulsions, colloids, dead tissues,
living protoplasm, living cells and living tissues.

IM^KLIMINARY CHAPTCR

FUNDAMEiNTAL PRINCIPLES OF HEAT CONDUCTION

In llic study of the slatos of l)i()l()i;ical material at low
tc'ini)e'ratures the investigators, most of tlie time, "apply"
principles "established" l)y the physicists, but often they
overlook I he numerous assum])tions on which the physicists
built up these princii)les. There results a considerable
amount of confusion, ])articularly in the discussions con-
ceniinii,' cooliui;-, freezini;' and snbcoolini;' curves, freezing
and eutectic points, and even in more common problems
such as the inter})retation of the readings of a thermometer.
]\rost of our knowledge on these questions is based on the
fundamental principles of heat transmission. We shall
here discuss briefly these principles.

In the last analysis, heat is understood to be the move-
ment of the constituent particles of matter. When, in a
body, a disturbance at one place causes the particles to
move faster than in the surroundings we are accustomed
to say that this point is warmer. The faster motion will
be transmitted by collisions to the neighbouring particles
and this is what we call heat transmission.

1. The ''Problem of the Wall/' in the Steady State. One
of the best-known methods of analysis of heat transmission
is that used in the so-called "Problem of the Wall"
(Fourier). Let W (Fig. 2) be a wall of homogeneous mate-

r s

w

e s

Fig. 2

rial, of thickness d, limited on two sides by two parallel
surfaces S and s and unlimited in the other directions.
Let T and t be the temperatures, respectively, at S and s, T
being higher than t and both temperatures being main-
tained constant. Heat will flow through the wall from S to

l(t4

105

s, gradually modifying the temporal lire of each inter-
mediate plane within tlie wall until a state of equilibrium is
established. Then heat will continue to tlow but the tem-
perature at each intermediate plane will stay unchanged.
Before the establishment of this equilibrium the system is
in a variable state; after the equilibrium is established the
system is in a steady state. In the variable state, the
amount of heat which enters through a given area on S is
different from the amount which comes out through an
equal area on s ; in the steady state these amounts are the
same. From theoretical considerations it has been con-
cluded that, in the steady state, the quantity of heat Q which
traverses the wall should be proportional to the difference
of temperature T-t, to the area A through which heat flows,
to the time z during which it flows and inversely propor-
tional to the thickness d of the wall :

Q = K^^ (1)

Experimental studies have confirmed this relation.

The constant of proportionality K in the formula (1) is
the coefficient of heat conductivity. It is defined as the
number of calories which, during one second, traverse an
area of 1 cm" of a wall 1 cm. thick, when the temperatures
of the two sides of the wall difi'er by one degree.

After the steady state is reached, each intermediate plane
p is at a temperature 0 which is proportional to the distance
X from the plane p to the cooler surface (t being taken as
the temperature origin, that is, t = 0) :

9 = ax (2)

The constant of proportionality a is the ratio of the
temperature difference T-t to the thickness of the wall d.

The formula (2) which is a necessary consequence of the
principles admitted in formula (1) has also been verified
experimentally.

The main assumptions in these relations are: 1. That
the temperature on each side of the wall is constant; 2.

106

'I'lial the sl(';ul\' slalc is I'cacln'd ; .'I. 'I'lial the malcrial of
the wall is lioin()i;('iu'()iis ; 4. Tlial llic wall is iiol lliiiilcd In
any (lirrctiou oliu'r than liu' diri'dioii of How of heal. In
any application of tlit' i'ormulas these assumptions need lo
be considered.

It should be noticed that the specific heat of the mate-
rial does not i)lay any role in the flow' of heat, in the steady
state. This is of importance in several ])ro])lems, as wiien,
for example, the passage of heat tlii'on,i;h water is to be
compared with the passage of heat through ice, two snl)-
stances which possess a very different specific heat.

In any practical case, the wall is in contact w'itli two
media, one on the warmer side, the other on the cooler
side. TIeat Hows from the warmer medium to the cooler
one through the wall. But the constant temperature of
these media (even if they are perfectly stirred) is not the
temperature at the surfaces of the wall. In other w^ords,
when two bodies are in contact, their surfaces of contact
are not at the same temperature or, what is equivalent,
the interface between two substances in contact has a heat
conductivity (sometimes called * 'contact-conductivity")
which is different from that of either of the two substances.
For having overlooked this point in his experiments on the
heat conductivity coefficient in metals, Peclet, a pioneer
physicist of the last century, was led to the evidently
erroneous conclusion that all the metals have the same con-
ductivity. Applications of the notion of contact-conductiv-
ity in biological investigations will be mentioned later.

Usually it is not with a single wall in contact with two
media that one has to deal but with several walls in contact
with one anotlier. Furthermore, these walls are rarely
limited by plane surfaces, they have any shape. Of the
numerous problems which result from the coml)ination of
walls of various forms, the simpler ones, in particular that
of the "multiple cylindrical wall" and that of tlic "nni]ti])l('
spherical wall," ai'e studied in the treatises on mathe-
matical physics.

As an application of the problem of the multiple

101

cylindrical wall we shall mention the analysis of the freez-
ing curves obtained with arrangements of the type repre-
sented in Figure 3. A cylindrical shell of living tissue AC,

Fig. 3. Arrangement of apparatus illustrating the application of the
' ' Problem of the multiple cylindrical wall. ' '

slipped around the bulb B of a thermometer, is placed in
the medium D contained in a tube E, which is itself im-
mersed in a cooling bath H. During freezing, heat is then
transmitted from the mercury of the thermometer and
from the freezing object to the external bath, through the
many cylindrical walls which are the glass of the ther-
mometer, the tissue, the medium and the tube E. In such
cases important errors have resulted from a too candid
use of thermometers as temperature-meters, as their name
suggests them to be, instead of as heat conductors which, in
the last analysis, they are.

2. The Problem of the Wall, in the Variable State. The
essential laws governing the relation between the tempera-

108

lure at a i^ivi'ii plane in llic wall, in Icrms of timo and in
terms of the distance rioni thai plane to the Avarmer side,
are deduced from a famous ('(juation establislied by
Fourier. Of the various conelusious he arrived at we shall
mention here only the following one which is of some im-
portance in the ])racli('al ai)i)licati()ns to l)i()thermometry.
The time y necessary for the establishment of the steady
state is inversely proportional to the coethcient of conduc-
tivity c of the material and directly proportional to its
specific heat s, to its specific gravity D and to the s(|uare
of the thickness d of the wall

y°^-^ (3)

So the factor, specific heat, which had no intluence on the
amount of heat traversing the wall in the steady state, plays
a role in determining the time necessary for the establish-
ment of the steady state and, in general, in various prob-
lems of the variable state.

3. The Problem of the Cooling Body. A particular case
of the problem of the wall is that in Avhicli a wall at a tem-
perature 9 is brought into contact, on its two sides, with a
medium at a temperature t different from S. The wall will
then warm u]) or cool down at a certain rate ; if it is in con-
tact with the medium not only on two sides but all around,
the problem becomes that of a body immersed in a warming
or a cooling bath. This problem led to the establishment
of the '*Law^ of Cooling."

Newton, in 1701, on the basis of theoretical considera-
tions, came to the conclusion that, when a body is exposed to
a constant temperatui-e bath, it should lose, during a unit
of time, a quantity of heat Q z (z being the time) propor-
tional simultaneously to the difference between its tempera-
ture 8 and that of the cooling bath t, to the area A of the
body, and to a certain coefficient C which indicates the
velocity with whicli heat is removed fi-oni the system:

-^ocAC(S-t) (a)

109

(One recognizes the same relation as in tlic problem of the
wall.)

Cooling will result from the withdrawal of heat at a
rate determined by the fact that every miit of weight of the
body requires the removal of a number of calories repre-
sented by the specific heat in order to be cooled by one miit
of temperature (say, from S to 9i). Calling m the mass
of the body and s its specific heat, one has

Q=(9-90ms (b)

Bringing the value of Q from (b) into (a) one obtains

^^cc^'(9-t) (4)

z ms

that is, the cooling velocity is proportional to the area of
the body, to the coefficient C, to the difference between the
temperature of the body and that of the bath, and inversely
proportional to the mass and the specific heat of the body.
A consequence of the assumption that the cooling velocity
is proportional to the difference between the temperature
of the object and that of the bath is that, when the time is
increasing arithmetically by one unit, the temperature
decreases to a set fraction of its own value, that is geo-
metrically; in other words, the cooling curve is an ex-
ponential. Calling 9^ the temperature at the time z and 9o
the original temperature of the object, one has

9. = 9„e-^^ (5)

where e is the base of natural logarithms.

The formulas (4) and (5) express two aspects of New-
ton 's ' ' Law of Cooling. ' '

The assumptions on which the law is based are funda-
mentally the same as those previously given for the prob-
lem of the wall. An additional condition is that the body be
entirely surrounded by the bath.

If no heat escapes by radiation or convection and if all
contact-conductivity can be overlooked, C is the coefficient
of heat conductivity of the object.

110

Till' law of (.'ooliiin' was soon suhjcclcd to cxpcriinciital
tests, 'i'lir classical vcrilicalioii is llial ot" Kiclimaiui (the
law itself is often called l\icliniaiiii's law). Marline, in
1740, ol)served that for a ^ood aureenient between the
theoretical law and the e\|)('riineiital data the tenipei'al ni'e
of t he object shonld not dilfer from that of the ])atli by more
than al)ont 50°. Dalton showed that the cooling velocity
is in reality more rapid than that indicated by Newton's
law. Dnloiii; and Petit, in 1817, nndi'rtook to establish
more accurately the influence of each factor on the rate of
cooling. They proposed corrections to render the formula
applical)le ov^er a larger range of temperatures. Other
corrections were introduced later by other investigators.
But the fundamental law established by Newton, although
correct only in a first approximation, is still usually em-
ployed in most of the practical applications, ((^f. Chajj-
puis and Berget : "Legons de Physique Generate." Paris,
1911, Vol. I, p. 622.)

Perhaps, when erroneous conclusions are drawn from
the application of the law of cooling, it is not so much on
account of its lack of accuracy but because some of the
conditions for its application are not fulfilled; for example,
the object to be cooled may not be entirely surrounded by
the bath, as when use is made of a too bulky thermometer-
stem or of too conductive thermocouple-leads which estab-
lish thermal connections with the outside.

All the problems so far treated have been analysed as
particular cases of the original assumption of an ideal wall
limited by two surfaces at constant temperatures. In many
instances one or both of these temperatures vary. There
results a large number of possible problems to be analysed
by further application of the method of simplifying
assumptions.

SUMMARY

The essential points l)rought out in the preceding ])ages
can be summarized as follows: 1. ]\Iost of the investi-
gations 01] thermometry and calorimetrv to be described

Ill

or discnssed later in this work arc but applications of tlit'
fundamGntal principles of heat transmission. 2. These
princii)les are briefiy discussed under the form of: a)
The "Problem of the Wall," in the steady state; b) The
' ' Problem of the Wall, ' ' in the variable state ; c ) The prob-
lem of the cooling- body. 3. The fundamental laws are
sufficiently well established theoretically and experiment-
ally in some simplified conditions, but in the more compli-
cated conditions which obtain in nature and in the labora-
tory and in which they are supposed to apply, they are not
always proven to be applicable. 4. The acceptance of
"established" laws without previous analysis of the prin-
ciples on wiiich these laws are based led to misinterpreta-
tions and omissions. A notion almost ahvays forgotten is,
for example, that of "contact conductivity."

CHAPTER 1

FREEZING, THE FROZEN STATE AND MELTING

T. TXITIATIOX OF CRYSTALLIZATION
A. THE FORMATION OF CRYSTALLINE NUCLEI

Pliysics has acquired a wealtli of information during the
last 30 years on the sti-ncture of crystals, but very little has
been learned yet on the mechanism of initiation of crystal-
lization.

The condition s'nic qua von for the molecules of a liquid
to be hooked up in a crystalline pattern is the reduction of
their mean distance to a certain minimum, that is, the low-
ering of their temperature to the freezing point.

When the liquid to be solidified is at a convenient tem-
perature and when a crystal of the substance is present,
crystallization proceeds at once, but if a crystal is not there
to initiate the process, that is, to give a direction to the
molecules, the latter have first to orient themselves and
build up the original nuclei. Crystallization nuclei are
understood to originate from the collision of molecules
coming in contact in certain detinite conditions of orienta-
tion. After a first successful collision, a pair of molecules
would be constituted, but this would not yet be the crystal
unit wanted; other successive collisions under definite
conditions would be required to complete the original
crystallite.

Though our knowledge of the mechanism of formation
of the first crystallization nuclei is to a large extent theo-
retical, their coming into existence as individual centers
is a well observed fact ; the centers can be photographed
and counted. Figure 4 represents the formation of such
centers in a solution of gelatine spread between two glass
slides and immersed in a bath at -25°. Tammann (1925,
p. 228) has shown that during a gradual decrease of tem-
perature (below the freezing point), the number of crystal-
lization centers formed per unit of time increases to a
maximum and then decreases, following a curve of the

112

113

Fig. 4. Formation of crystallization nuclei in a film of gelatin solution
enclosed between two thin glass plates and immersed in a bath at - 25°. The
lower left-hand corner was the first j)art of each of the two preparations to be
immersed in the bath. (Original, Luyet and Gehenio.)

type represented in Figure 5. Unfortunately, little is
known of the course of such curves for water, aqueous
solutions and aqueous colloids.

Some physical chemists consider the congelation of
water not as the crystallization of a liquid but as the pre-
cipitation of a solute from a saturated solution. What we
ordinarily call water would be a solution of ice (a trihydrol

40

20

Fig. 5. Xumber of crystallization nuclei formed in piperine at various sub-
cooling temperatures. (Curve drawn from Tammann's data, 1925.) Abscis-
sae: temperatures (the melting point of piperine, 135°, being the origin) ; or-
dinates : number of nuclei.

114

<»1' t'orinula ( I l.O) ) in licinid wnlcr (.-i dilixdiol of fdriniila
(II-())j): ;i1 0 tlic solution would he s;il iiralcd and ice
would prccipilnlc 'Plic nice) lan ism of inil ialion ol' crxslal-
li/alion assumed ahoN'c is consistcnf willi this llicoi'v oi'
water Ix'ini;' a solution as well as with llic usually accepted
view tliat water is a definite coinijound.

A new lii>hl lias Ix'cii tlirown on tlie sn])ject !)>■ the inves-
tii>ati()ns of Barnes ("Tec Eng-iiieerini;', " Montreal, lf)28,
]). 7), who attempted to observe under the microscope the
formation of ice in water. He states that : "The particles
of ice as soon as they can be seen are devoid of crystal
form and a])peai' as a true colloid in small disc like particles.
These tioccnlale and t>i'ow, passing through a crystal col-
loidal form to a true ice crystal." {(\f. F\g. 6). More

Fig. f). Disc-like jiarticlcs of (•olloidnl ivv. (From Barnes, 1928.)

experimental investigations in that direction might prove
highly significant for the knowledge of the mechanism of
initiation of crystallization.

Bernal and Fowler (1933) have suggested that liquid
water might possess a quasi-crystalline structure. Five
molecules would hook up together to foi-m a tetrahedral
figure (centered tetrahedron) ; these tetrahedra would ag-
gregate, according to a definite pattern, in a mass of whaler.
It is evident that, if the structure of liquid water is such,
our notions of the ])assage from the liquid to the crystal-
line state, and in particular of initiation of crystallization,
should be radicallv modified.

B. THE FREEZING POINTS

Most of the freezing point determinations of biological
material have been undertaken for the information that

115

they furnish on the osmotic pressure of organic fluids and
on the binding of tlie water molecule by protoplasmic sub-
stances. We shall summarize here some of the more rep-
resentative works, but we refer the readers to reviews on
the two subjects mentioned, for more complete data.

1, Culture Media. The freezing point of fresh water
was found to be, in general, 0.03 or 0.04 of a degree lower
than that of distilled water.

Backman and Runnstrom (1912) report that the water
from which they collected frog's eggs froze at - 0.060°.

Bialaszewicz (1912) found a freezing point of -0.01° to

- 0.02° for the tap water that he used for studying the
development of amphibian eggs.

The freezing point of sea water, determined by various
authors, mostly at marine biological stations, was found
to vary from -1.09° at Kiel (Dakin, Biochem. Jour. S,
258, 1908) to -2.29° at Naples (Bottazzi, Arch. Ital. de
Biol., 2t>, 61, 1897). At the American biological stations
of Woods Hole (Mass.), Pacific Grove (Cal.) and Beaufort
(N. C), Garrey (1905) obtained respectively -1.81°,

- 1.92° and - 2.04°. In a later work of the last mentioned
author (1915), the reader will find tables of the freezing
points of sea water at various dilutions and of solutions
of NaCl and MgCL at concentrations osmotically compar-
able to sea water.

Jensen and Fischer (1910) give -0.42° as the freezing-
point of the ** physiological solution," that is, of a 0.7%
solution of sodium chloride.

Of the substances used in culture media, sucrose was
found to lower the freezing point more than was expected
by the law of the freezing point depression. A gram
molecular solution freezes at -2.775°, according to Garrey
(1915). Loeb ("Artificial Parthenogenesis and Fertiliza-
tion," Chicago, 1913, p. 130) pointed out the biological
significance of this fact, which is usually attributed to a
hydration of the molecules of sucrose. For more data and
discussion see ^lorse, Frazer, Hoffman and Kennon {Am.
Che)u.Jour.,S0,^9,1906).

IK)

2. P!a}if Juiii's. A laruo list of freezing- points of ex-
trncled ]tlaiit juices is yiven ])y Dixon and Atkins (1910),
ill a table i('i)r()(liu'e(i in Tahiifac Hiolof/icae (7, 4.')l-434,
1925). The rrcc/.iiiii' i)()iii1s vary IVoni - 0.:i57° to -2.455°.

Harris, (Jortner, liofman and Valentine (1921) mention
the exceptional ease of a plant, Atrlplcr nuttaUii, which
was collected near tiie (Jreat Salt Lake and which had a
freezing jjoint of - 14.4°. The same authors obtained
excei)tioiuilly high fi-eezing points in cactuses.

According to Harris, (Jortner and Lawrence (1917), the
freezing point is higher in juices exli-acted from leaves
near the ground than in leaves taken higher up. The
higher osmotic pressure at higher levels is thought to play
a role in the ascent of sap.

3. Blood and Bodji Fluids. Collip (1920a) measured
the freezing point of the blood of animals belonging to
various orders. He found values varying in mammals
from - 0.48° to - 0.70°, in birds from - 0.55° to - 0.69°, in
reptiles from -0.46° to -0.70°, in amphibia from -0.44°
to -0.76°, in fresh water fishes from - 0.45° to -0.69°.

The blood cells, in Collip 's experiments, froze, on an
average, at a temperature 0.043 degree higher than the
serum.

According to various authors (quoted by ^McClendon and
Medes, "Physical Chemistry in Biology and Medicine,"
Philadelphia, 1925, p. 300), the fluids of the mammalian
body have, in general, a freezing point very near that of
the whole blood. Such is the case for milk, gastric juice,
pancreatic juice, bile, intestinal juice, cerebrospinal and
spermatic fluid. Saliva, and sweat were often found to
have a higher freezing point, reaching about - 0.1°. Urine
had a much lower value, sometimes as low as -2.6°.

Individual Differences. Some authors have
thought that the freezing points of body fluids or of tissues
might be specific characteristics of some animal (or plant)
grouiJ. Atkins (1909a) attempted to study the individual
differences in the freezing point of the blood of birds
within the same species. He found — quoting only the

117

results that he considers reliable — 0.1 of a degree differ-
ence, between the extremes, in the freezin<»' points of the
blood of 10 ducks and about 0.3 of a degree, between the
extremes, in 7 turkeys.

Relation Between the External and
Internal ^1 e d i u m. The problem of the adjust-
ment of the fluids of the body to the external medium has
given rise to a series of observations on the comparative
freezing points of sea or fresh water and of the blood of
marine, fresh water or land animals. A first review of
the subject has been made by Hober ("Physikalische
Chemie der Zelle und der Gewebe," Leipzig, 1902, Chap-
ters 2 and 12).

Since the publication of this review, Garrey (1915)
reported that the freezing point of the blood of several
marine animals, including large-sized fishes such as the
shark, was very near that of sea water. The freezing
point of fresh water animals, for example, fishes of the
Mississippi River, varied from -0.48° to -0.52° in 6 spe-
cies, and was, therefore, considerably higher than the freez-
ing point of the blood of marine fishes and definitely lower
than that of fresh water itself.

The blood of the marine turtles, unlike that of other sea-
animals, had a freezing point of - 0.69°, that is, markedly
higher than that of the water in which they were living,
which was -2.04°. To study the possibility of an adjust-
ment of the internal to the external medium, in these ani-
mals, Garrey measured the freezing point of the blood of
two marine species left for 2 months in a tank containing
fresh water. He found the same value as for the animals
taken directly from sea water. However, some adjust-
ment in other animal groups, such as fishes, has been shown
by Garrey himself in some of his previous investigations
(1905) and also by other workers, in particular by Scott
(1913).

The last mentioned author published, in 1916, a review
in which the results of numerous investigators are tabu-
lated. There are listed 111 species from the most impor-
tant phyla of the animal kingdom.

ns

Collip (llL'Oa) made ohsci-xal ions leading' to llic same
coiU'lusioiis as lliosc of (Jarrcy coiicci'iiiiiL;- the di tTcrciico
l)ot\vt'c'ii fivsli water and marine animals.

J)uval {(\h\ Sor. Biol., .s/y. 22, 192.S) sliowed that the
t'reeziiii;' point of the sernm of sea fishes was dne, 1o a hirge
extent, to factors otlier tlian the mineral salt content. The
freezing- i)oint of the serum was - 1.89° (that of sea water
- 1.84°) and that of a water solution of the ashes from the
dried serum, diluted to occupy the same \()liini(' as the
serum itself, was -1.06°. Tf there is an adjustment of
the internal to the external medium, therefore, the modifi-
cations of the blood should involve more than a change in
the salt concentration.

4. Eggs. Abundant data on the freezing points of insect
eggs will be found in Bachmetjew (1901 and 1907).

Bialaszewicz (1912) and Backman and Runnstrom
(1912) made several determinations on amphibian eggs
(see below).

The chicken egg, according to Atkins (1909a), freezes
at - 0.454°, there being no difference between the freezing
point of the yolk and that of the white.

Howard {J. Gen. Physiol., 16, 107, 1932), like Atkins,
reported that the yolk and the white had nearly the same
freezing point.

Straub {Bee. Trav. Chim. Paijs-Bas, 48, 49, 1929), on the
contrary, found that the yolk froze more than 0.1 degree
lower than the white.

Hale (1935), in a series of very accurate determinations,
observed that the yolk never froze above -0.57° and the
white never above -0.42°.

Hale made several other concomitant observations which
deserve mention: a) The various layers of white had the
same freezing point; b) The intact yolk froze at a slightly
lower temperature than broken yolk; c) When the vitelline
membrane was punctui'ed, ice started at the punctured
point; d) A yolk surrounded l)y a thin layer of white did
not freeze when the latter froze.

According to the same investigator, the discrepancies

119

between the results of the authors mentioned above miglit
be due to the fact that some worked witli intact and some
with stirred yolk.

Specific Differences, Bialaszewicz (1912)
found - 0.444°, - 0.446° and - 0.455° for the freezing points
of the ovarian eggs of, respectively, Rana fuf^ca, Bana
escidenfa and Bomhinaior igneiis.

Atkins (1909a) gives -0.454°, -0.452° and -0.420° as
the freezing points of the eggs of the chicken, the dnck and
the goose.

I n cl i V i d n a 1 Differences. The last mentioned
author reported a difference of 0.05 degree between the
extremes of 12 lien's eggs and 0.09 degree between the
extremes of 7 dnck eggs.

Moran (1925) observed individual differences of the
same order for both the white of egg and the yolk.

Cycle in the Freezing Points of Develop-
ing E g g s. Atkins (1909a), investigating the difference
between the osmotic pressure of the blood and that of the
eggs, in birds, as related to the exchanges between the egg
and the mother's body, obtained a freezing point 0.13
degree lower for the blood than for the eggs. He attrib-
utes this fact to a higher concentration of inorganic salts
in the plasma of the blood, as a determination of the chlo-
rine content showed.

Atkins (1909b) furthermore observed that the freezing-
point of the eggs drops, during incubation, by about 0.15
degree. The mixed content of the egg (after the separa-
tion of the embryo, at the end of the incubation period)
froze at -0.611°, that is, at nearly the same point as the
blood. The author suggests that the animals in the phylo-
genetic series might exhibit the same differences in the
osmotic pressure of their internal fluids as do the embry-
onic forms in the ontogenetic series. According to this
view, the birds, for example, should have body fluids of
lower freezing point than the reptiles from which they
descend.

Bialaszewicz (1912) gives the following figures for the

120

freezing points of tlic yolk of tlic cliicken (',«;■<;• (luriui;' its
formation and development :

Ovarian egg, 4 to 1 cm. in diameter : - 0.632°
Ovarian egg .'> em. in diameter : - 0.613°

Eggs half way down the oviduct : - 0.585°
Freshly laid eggs: -0.564°

Embryo at the 8th day of inc. : - 0.496°

Embryo at the 18th day of inc. : - 0.601°

Blood of the adult : - 0.635°

So, when it is detached from the ovary, the egg has about
the same freezing point as the blood. During the growth
period, the freezing point increases to a maximum. Dur-
ing embryonic development, it gradually drops again to
its original value.

The amniotic fluid froze at - 0.582° and this point did not
change significantly up to the 18th day of incubation. The
freezing point of the allantoic fluid, on the other hand,
rose from - 0.513° to - 0.431° from the 8th to the 18th day.
The egg white froze at - 0.458° in fresh eggs and at - 0.444°
after 8 days of incubation.

The same au,thor reported a change in the freezing point
of the frog's egg from -0.444° for the ovarian egg, to
-0.294° on the 3rd day after fertilization and to -0.382°
12 days later. The blood of the adult frog froze at - 0.479°.
According to Backman and Runnstrom (1912), the cycle
of changes of the freezing point in the frog's egg and
embryo {Bana teniporaria) is as follows:

Ovarian egg : - 0.48°

Fertilized, unsegmented egg : - 0.045°
Stage of crescentic blastopore : - 0.215°
Stage of spherical blastopore : - 0.215°
Embryo, 5 days old : - 0.230°

Larvae, 20-25 days old : - 0.405°

Serum of the adult : - 0.465°

These data confirm the general results obtained by pre-
vious investigators and, furthermore, they point out a
remarkable rise of the freezing point at the time of fer-
tilization.

121

5. Protoplasm. Chambers and Hale (1932) induced
freezing in amoeba proteus by seeding the supercooled pro-
toplasm with an ice-tipped micromanipulator needle at a
temperature of - 0.8° and below.

Gehenio and Luyet (unpublished work) determined the
freezing point of the living plasmodium of the myxomycete
Physarum poli/cephalum. Some 5 cc of protoplasm col-
lected from agar cultures were used in each determination.
The values found in 7 specimens average -0.17°.

6. Tissues. The determination of the freezing points of
living tissues by any of the methods used involves an ele-
ment of uncertainty. Ice-seeding, to prevent subeooling,
or at least to prevent a too considerable degree of subeool-
ing, is an essential procedure for any accurate freezing-
point determination. But ice-seeding through cell w^alls
is not always efficient ; only some cells might freeze after
seeding or spontaneously, others not ; the number of cells
frozen might not suffice to bring the temperature of the
subcooled object up to the freezing point. On that basis
one should, perhaps, question most of the results obtained
for the freezing points of living tissues.

Collip (1920b) determined the freezing point of the pulp
obtained by grinding various tissues of the dog. He found
values varying from -0.74° to -0.87° for the heart, the
spleen and the lungs, -0.91° for the liver, and -0.76° for
the brain.

Jensen and Fischer (1910) obtained, for the frog's
muscle, in the living state, freezing points varying from
- 0.46° and - 0.53°.

Cameron and Brownlee (1913) give -0.44° as the freez-
ing point of an entire frog exposed to the freezing tempera-
ture with a thermometer in its stomach.

Living and Dead Tissues. ^liiller-Thurgau
(1886) noticed that living tissues have a lower freezing-
point than dead ones, that is, than tissues killed by a first
freezing. He found - 0.98° and - 0.55°, respectively, for
living and dead potato tuber, and likewise, - 0.8° and - 0.4°
for living and dead Phaseolus leaves. For the determina-

122

lions on j)ot;(to, ;i mci-cnrv 1 lici'nioinctcr was iiiscrlcd into
a cavity bored in the tuber; for those on leaves, the bulb of
the tlu'rmometer was wra|)i)ed in tliese organs. The cause
of the differences observed is attributed to the fact that
the eai)illai\\' spaces Ix'twcen the intact niembi-anes of tlie
cells in the living tissues hold water more tirmly and rcndci-
freezing more (,lif^icult than in dead material.

■Maximov (1914-) obsei'ved. similar differences in red
beet; he obtained -2.15° and -1.25° for living and dead
material, respectively, and -1.21° for the extracted beet
juice. According to this author, the resistance presented
by the living cellular membranes to the outward passage
of water during freezing is responsible for the lower freez-
ing point of the living material. In dead tissue, the sap
forms a uniform fluid mass which freezes like a solution,
there being no membrane resistance to overcome ; in living
tissue, the sap has to be extruded from the cells before it
freezes ; it is extruded in small quantities at a time and the
heat developed by the freezing of such small quantities is
not enough to raise the temperature of the whole tissue to
its real freezing point.

Walter and Weismann (1936) found, in pieces of potato
frozen several times in succession, a rise of the freezing
point at each of the first few congelations (for example,
-0.89°, -0.76°, -0.72°, -0.69°, in a series of 4 successive
experiments). Thereupon the freezing points oscillated
in an irregular manner. These authors determined by the
1)rowning of the tissue and the loss of turgor the increasing
num])er of dead cells in successive freezings and they claim
that it is the proportion of living cells which determines
the level of the freezing point. Dead matter freezes first
and the heat produced during its congelation, being ab-
sorbed by the bulk of surviving material, cannot I'aise the
temperature of the whole to the same level as when all the
cells are dead. Besides, in completely dead material, the
freezing point may be high because the sap is diluted by
the water which was bound in the living state and which is
set free at death.

123

Jaccard and Frey-Wyssling (1934) reported a sharp
rise in the freezing point of the tissue of the root of Daucus
carota after the first congelation, hnt this rise was followed
by an irregular drop in subsequent freezings. In pieces
killed by heat, the freezing point remained constant. They
attribute the rise in the freezing point at the second freez-
ing to the fact that the water extracted from the cells during
the first freezing dilutes the extruded sap around the ther-
mocouple, which they used in their determinations. Wal-
ter and Weismann object to this interpretation, claiming
that the water extruded should be reabsorbed if the tissue
is still alive as Jaccard and Frey-Wyssling assume.

According to Luyet and Gehenio (1937), the freezing
point of living potato tissue is, on an average, by as much
as 1.5 degree lower than that of dead material. In a ten-
tative explanation of this difference, they distinguish three
kinds of water in protoplasm: 1. water which acts simply
as a solvent in the cell sap, 2. water which behaves as a
protoplasmic constituent, 3. water which, in dead as well
as in living matter, is bound in such a way that it cannot
freeze; and they attribute the higher freezing point in
dead tissue to the fact that, at death, protoplasmic water
is transformed into solvent, readily-freezable water.

This assumption, however, does not seem to be tenable
in view of new findings of Gehenio and Luyet (unpub-
lished) that protoplasm which has no cellular structure,
like that of the myxomycetes {Physarum polycepJialum),
does not present the difference observed between living and
dead tissues. So, finally, the lower freezing point of living
tissues seems to be due not to a binding of water in a proto-
plasyyiic structure but to a hindering of the water activity
by a cellular structure, probably the cellular membranes.

Jensen and Fischer (1910) observed that the freezing
point was lower in dead than in living muscle. They
attribute this fact to some binding of water at death.

Influence of Cooling Velocity on Freez-
ing Point s. The use of a high cooling velocity has
been found by many investigators to result in a lowering of

124

tlie t'r(H'zini>- ])()iii1, particularly in living- lissuos. This sub-
ject will be discussed later in the section on Freezhig
Curves.

The Double Freezing I * o i n t of Living
Tissues, Maximov (1914) described a very ])articular
course in the freezing curve of the living tissue of the
petiole of TussUago farfara. After subcooling, the tem-
l)erature rose rai)idly, then it sank a little and either stayed
at that level or rose again slightly (r/. Curves 1 and 2.
Fig. 7). There were apparently two freezing points
marked by two maxima on the curve. This author ob-
served furthermore that only the living tissues presented
the double freezing point, and that soaking the material in
water favored the i)henomenon, while drying the canal
through which the thermocouple was inserted into the tis-
sue prevented it. On the basis of these observations he
attributed the first freezing point to the congelation of the
extruded cell sap which surrounded the thermocouple.

Zacharowa (1926), also using a thermocouple and fol-
lowing Maximov 's procedure, observed the same phenome-
non in roots of rye seedlings (Curve 3, Fig. 7). She
admits Maximov 's interpretation.

Walter and Weismann (1936) confirmed the observa-
tions and interpretations of the previous investigators on
potato tuber. They used a mercury thermometer (Curve
4, Fig. 7).

According to Mez (1905), one can observe, in the freezing
curves of plant tissues, two plateaus which correspond
respectively to the freezing and to the eutectic point of salt
solutions.

Voigtliinder (1909), following Mez' views, presented a
vast amount of data which, wiien plotted, showed two
periods of retardation in the drop of temperature (rf.
Curve 5, Fig. 7). He took for granted that these periods
corresponded to the freezing and the eutectic points (F and
E in the figure).

Luyet and Gehenio (1937) made a special study of the
factors involved in the doubling of the freezing point

125

-4

15

20 MIN.

O 5 10

Fig. 7. The double freezing point as observed by various authors. For
the sake of comparison, all the curves have been plotted to the same scale.
(From Luyet and Gehenio, 1937.)

(Curve 6, Fig. 7). They used potato tissue in the form of
hollow cylinders slipped about the bulb of a mercury ther-
mometer. In order to control the cooling velocity and the

V26

llicfinal ui'adiciil Iliroiiuli llic iiialci'ial, llicx' scjtai'ali'd llu'
latlcr from the ('ooliiii'' hath hy media ol" \-ari()iis lliickness
and of various licat coiKhiclivil y, sucii as mercury or
])araf1iii oil. Their liiidiiii^s can he summarized as foUows:
1. The ohserxatious of ])i-evious investigators, according to
which dead tissues do not present a douhle freezing point,
were verified in two series of experiments : one on living
tissues in which IV.] out of 68 specimens showed the double
freeziuii' poiiil, and one on dead tissues (killed by previous
freezing) in which none of the 44 specimens experimented
upon presented the double freezing point. 2. The obser-
vations to the effect that soaking the tissues induces double
freezing, while di'ying prevents it, were also confirmed;
the per cent of cases })i-esenting a double freezing increased
from 49 to 90 by a soaking of the tissue in water for 30
minutes or more and decreased to 0 by a drying in the air
for 3 hours or more. 3. The first freezing point in normal
living tissues varied from - 1.2° to - 2.0°, the second from
- 1.45° to - 2.74° ; that of dead tissue was higher than both
and varied from -0.5° to -0.75°. 4. The first freezing
point was raised by soaking; in some soaked tissues it
reached -0.05°. The second was not influenced by the
imbibed water. 5. When congelation was stopped at the
passage from the first freezing point to the second, the
tissue was uninjured and the first freezing point could be
obtained repeatedly in subsequent experiments. 6. The
position of the first freezing point was not affected by a
change in the cooling velocity, while that of the second
moved from - 1.45° to - 2.3° for velocities varying from 2.5
to 5.1 degrees per minute.

In the light of these findings the authors discuss the fol-
lowing four theories on the doubling of the freezing point
in living tissues :

1. The "Wound-Sa])" theory, proposed by Maximov,
according to whicli, as said above, the two i)oints would
represent, res})ectively, the freezing of the sap extruded
from the cells through the wound caused by the insertion
of the thermocouple and the freezing of tlie water of the

127

tissiio itself. An objection to this theory is that the quan-
tity of water which congeals at the first freezing point is
often considera])ly greater than that whicli can possibly be
extruded in the wounding.

2. The "Thermal Gradient" theory, suggested to Luyet
and Gehenio by the fact that the wave of congelation which
moves from the external surface of the object inward and
wiiich liberates heat gives rise to a wave of heat whicli
travels toward the thermometer. This stops or retards
the cooling curve, thus producing an apparent freezing-
point. But cases were observed in which the temperature
stayed constant at the first freezing point for nearly 10
minutes which was evidently more than the time necessary
to dissipate a wave of heat, in the conditions of the experi-
ments.

3. The theory of the "Eutectic Point" (Mez and Voigt-
lander), which considers the two points as, respectively,
the freezing and the eutectic point of the tissue. The ex-
tremely high cooling velocities used by these authors in
their determinations (c/. Curve 5, Fig. 7) render their
results the most uncertain. (For a more detailed discus-
sion see below, under Freezing Curves.)

4. The theory of the "Double Freezing Point," accord-
ing to which the 2 freezings are not only apparent and
attributable to the procedure or to the apparatus but real,
one being the congelation of intercellular, the other of
intracellular water (the latter w^ould freeze after extrac-
tion from the cells by osmosis or within the larger vacu-
oles). Luyet and Gehenio, after proposing the theory,
mention its agreement with most of the facts observed : the
occurrence of the double freezing in living tissues only,
the innocuous effect of a congelation below the first (but
above the second) freezing point, the absence of a first
freezing in dried tissues, its more frequent occurrence in
soaked tissues, the rise of the first freezing point when
more water is imbibed, etc. They point out, however, that
the existence of two different freezing points, one for the
intercellular and the other for the intracellular fiuids would

V2S

involve a pormanont osmotic disequilibrium between the
cell content and the intercellular spaces.

II. PROGRESS OF CRYSTALLIZATION
A. THE GROWTH OF CRYSTALS

AVhen a crystallization center is formed, it <»rows with
a speed which depends jirimarily on the temperature.
Tnder the same conditions, the dit^'erent faces of a given
crystal grow with different velocities, each set of faces
having a velocity coefficient. To simplify the study of the
crystallization velocity, the physicists consider separately
the linear growth of each face of the crystal, that is, the
growth in a direction perpendicular to that face. The
same general laws of growth, with various coefficients,
apply to all faces.

It is generally thought that the velocity of crystalliza-
tion increases when the temperature decreases (below the
freezing point), in other words, that the colder a body is,
the faster it freezes. Several experimenters who studied
this relation have obtained data which confirm the com-
monly accepted view and which follow a curve of the type
represented in CDEF, Figure 8. But Tammann, amongst
others, pointed out that an increase in velocity for a de-
crease in temperature would be contrary to the general
laws governing physical and chemical tranformations {cf.
Tammann, op. cit., 1925, p. 251). He then showed that the
direct experimental determination of the temperature of
crystallization always involved an error. The temperature
measured by the experimenter is that of the liquid in
which crystallization occurs and not that of the growing
surface of the crystal. When the thermometer or thermo-
couple is placed as near as practically possible to the
crystal, it is still relatively too far from the active surface
to give any adecpuite measure of the temperature of this
surface itself. The heat continuously produced by crys-
tallization on the growing surface maintains the local
temperature there higher than that of the surrounding
medium where the thermometer is placed. Even the finest

121)

Fig. 8. Relation between crystallization velocity and temperature. (After
Tammann, 1925.) The crystallization velocities are plotted in ordinates,
increasing from the origin ; the temperatures are plotted in abscissae, decreas-
ing from the origin, the latter being the freezing point. The curve CDEF
represents experimental data (it is a composite curve drawn from several of
Tammann's observations). The curve AB represents the data that one
should obtain if one could avoid the experimental errors inherent in the
procedure.

available thermoneedles have too high a heat capacity to
register this local temperature with any accuracy.

Another source of error pointed out by Tammann is
that the heat liberated during crystallization at the grow-
ing surface of the crystals causes interruptions in the
crystallization process, the latter being resumed only after
the dissipation of the heat liberated in the preceding con-
gelation ; consequently, the duration of crystallization ex-
perimentally determined consists of a succession of crystal-
lization periods and interruption periods and the resulting
figure for velocity is too low.

Taking these facts into consideration, Tammann con-
cluded that the relation between the velocity of crystalliza-
tion and the temperature should be of the type represented
by the curve AB, Figure 8. (hystallization would then
proceed more slowly at lower temperatures. Indirect
temperature determinations furnished an experimental
verification of this relation.

It is of interest to notice that the physicists who, in gen-
eral, are better trained than other experimenters in the use

M]0

of tlu'iniometric* (Icxiccs, were induced iiilo error by a mis-
iiilerj)re1ati()ii of llie I'eadiii.ns of llierniometeis. Their at-
tempt to determine directly tlie tem})erature at the surface
of a growing crystal by the finest thermocouple placed the
nearest possible lo the surface, is comparable to an attempt
at measuring' the 1em])erature of a l)iiniiii,i>' match with a
giant thermometer (iiaviiig a bulb several inches in
diameter) separated from the match by a distance of a
centimeter or so. In the last analysis, the error is due to
the fact that too many experimenters believe in ther-
mometers; they take for granted that a thermometer is an
apparatus which gives the temperature at a point in space,
while a thermometer is a complicated system of bodies in
contact, through which heat flows, usually in an uncon-
trolled manner, and from the behavior of which we try to
guess (or to use a euphemism, to calculate) what the tem-
perature is at a point in the neighborhood of the system.
Since such errors probably escape the attention of the
investigators more often than is usually thought, we
deemed it justifiable to discuss the fundamentals of
thermometry, that is, heat conduction, in a preliminary
chapter of the present work.

Strange as it may seem, then, the fact is that the lower
the temperature is, the slower is the freezing (crystalliza-
tion) and when the temperature reaches a certain mini-
mum, freezing becomes impossible because of the too
intense cold.

Since the maximum of the curve of the numlier of crystal-
lization centers (Fig. 5) is at a lower temperature than
the maximum of the curve for the velocity of crystalliza-
tion (Fig. 8, CDEF), it is clear that at temperatures at
which the number of centers is high and the velocity low,
a relatively large numl)er of small crystals will be formed,
while at temperatures at which the number of centers is
low and the velocity high, there will be a small number
of large crystals. The latter condition is realized at higher
temperatures near the freezing point, the former at lower
temperatures.

131

The growth velocity coefficients foi' the different faces
of a crystal do not vary at the same rate when the tem-
perature is changed, as we indicated above; hence, some
faces become prominent at certain temperatures and the
crystal develops more in the direction of these faces. The
temperature of crystallization determines, therefore, the
shape of the crystals. In general when the velocity of
crystallization is very low for one face it is low for all the
faces and the crystal has the shape of a spherulite.

Walton and Judd (1914) studied the rate of growth of
ice in a long glass tube previously filled with distilled
water and subcooled to various temperatures. They
initiated crystallization by ice-seeding at one end of the
tube and measured the velocity of the congelation-wave as
it moved toward the other end. They found a rate of 65
mm. per second at - 8°.

Hartmann (1914), in experiments of the same kind, ob-
tained 46.6 mm. per second at - 7°.

Tammann and Biichner (1935a), who succeeded in keep-
ing w^ater subcooled to - 13.4°, found 41.3 mm. per second
at - 7.2° and 96.8 mm. at - 13.4°. They also measured the
crystallization velocity when ice was formed from heavy
water and obtained almost the same values as with ordinary
water, at the same degrees of subcooling (that is, for tem-
peratures calculated from + 3.8° as the freezing point
of heavy water and from 0° as the freezing point of ordi-
nary water).

The same authors (1935b) determined the lowering of
the crystallization velocity of water when substances such
as sodium chloride, sulphuric acid, glycerine, alcohol and
sugar, were dissolved in it. They found that, at 5 degrees
below the freezing point, 0.85 mole of sodium chloride in
1000 gr. of water slows the rate of congelation to about 6
mm. per second; while 1.1 and 2.9 moles of sugar, in the
same conditions, slow it to about 0.3 and 0.02 mm. respec-
tively. Sugar, it was remarked, has a particularly strong
retarding effect. It might be of interest to notice that in
very dilute concentrations (about 0.01 mole per liter) the

VV2

crystallization velocity was slig-litly increased by the sub-
stances dissolved.

Callow (1925) stndied the action of various concentra-
tions of gelatin on the growth of ice crystals in gels of that
substance. He found that 1% gelatin reduces the rate of
crystallization of water to about half its value, IV/c renders
the velocity 45 times lowei-, and 3% renders it 350 times
lower.

The velocity of crystallization in biological material is
entirely unknown.

B. THE PHASE SEPARATION

1. Solutiojis and Sii.speusions. It is \\q\\ known that, in
the freezing of a solution, w^ater separates from the solute
and freezes alone, while the solution becomes more con-
centrated. Some biologists have attempted to observe this
separation under the microscope. Molisch (1897) says
that, by mounting drops of 10 /'r solutions of sodium
chloride, potassium nitrate, magnesium sulfate, cobalt
chloride, etc., on microscope slides and exposing them to
low temperatures, he could see the water freeze out in
several crystallization centers and form ice masses which
wedged betw^een them the concentrated salt solution; the
latter, in its turn, crystallized in a different form of crystal.
This separation of w^ater w^as seen in the same manner in
solutions of dyes.

According to Goeppert (1830), when plants which con-
tain a milky sap, for example, RJius, Euphorbia, Papaver,
Ficus, are frozen, the sap solidifies into transparent ice.
However, the yellow^ sap of Chelidonium gave yellow ice.

]\Iolisch (1897) reported that, wOien he put to freeze at
a temperature of - 6°, on the stage of the microscope, the
milky sap of the fig tree, Ficus elastica (a substance which
consists of an aqueous solution and of droplets of rubber),
he could see the w'ater freeze out into ice crystals wiiich
separated row's of particles of rubber. On thawing, the
two phases mixed again. Similar experiments were made
with suspensions of carmine, indigo and gum and the same

133

results were obtained. With carmine the network of par-
ticles stayed after thawini>', which fact the author attributes
to the adhesion caused by the pressure exerted by the ice.
The Brownian movement of the carmine particles had
ceased.

2. Colloids. Bruni (1909) described colloidal isingiass
and colloidal silicic acid in the frozen state as consisting of
colloidal particles interspersed with ice crystals. In silicic
acid the two phases, one of which was pure water, remained
separated after thawing.

]\Ioran (1925) noticed that, after a congelation, followed
by melting of the ice, tlie liquid portion of the white of egg
had increased and the colloidal portion had decreased. He
compares this phenomenon with the exudation of water
from gelatin, from jellies and from the muscle, after thaw-
ing.

According to Molisch (1897), a layer of a gelatin gel
frozen on a slide, under the microscope, allows one to ob-
serve the ice separated from the gel. There remains a
gelatinous network with ice particles in the meshes (Fig.
9). After thawing, the meshwork can be preserved for

Fig. 9. Network left after the thawing of a frozen gelatin solution.
(From Moliseh, 1897.)

several days if the water content of the gel is low ; it can
also be fixed as a tissue, by formol, and kept indefinitely.
With higher water contents, a reimbibition takes place and
the meshwork disappears.

Bobertag, Feist and Fischer (1908) observed that, on
thawing solutions of gelatin, carrageen, agar agar, isin-

glass, soaj), t'tc, llic waliT wliicli liad separated from llif
gelatinous mass during freezing was almost free from the
dissolved sn])stanc'e. After complete thawing, one could
still see the inhomogeneous mass to consist of clumps of the
jelly and of a dilute solution.

The freezing of gelatin gels has been the object of
extended studies by Moran (1926) and Hardy (1926).
They observed three types of freezing: the compact sur-
face freezing, the intermittent freezing and the dis-
seminated freezing.

Compact S u r face F r e e z i n g. ]\Ioran exposed
to sub-zero temperatures discs of gelatin gels of various
concentrations, measuring 3 mm. in thickness and 15 mm.
in diameter. With water contents higher than 34^ and
temperatures from - 3° to - 19°, ice formed around the
discs, while the inside gelatin core became more concen-
trated. The authors removed the ice shells and determined
the amount of water so frozen and the maximal gelatin
concentrations reached by the core. They found that these
concentrations varied from 54.3yr to 65.2% when the tem-
peratures varied from -3° to -19°. If a preparation
frozen at - 3° was put consecutively at lower temperatures
(even in liquid air) and brought back to -3°, the concen-
tration was 54.37^ , that is, the same as in gelatin frozen
at -3°.

With very high water contents and low freezing tem-
peratures, for example 88% and - 11°, there were, in ad-
dition to the surface freezing, some centers of crystalliza-
tion formed inside of the gelatin core where one could find
places containing a "sponge of gel" (Fig. 10, A).

Briefly, when freezing was slow, water separated from
the gel and came to the surface to solidify in a compact
mass of ice ; when freezing was rapid or when there was
great abundance of water, crystallization took place inside
the gel.

Intermittent F r e e z i n g. ^loi-an, studying the
congelation at - 11° of gels containing 62''/ water, observed
granulations, 2 to 3 mm. in diameter, of congealed material.

135

Fig. 10. Photomiciograplis of sections tluougli frozen gelatin gels. (From
Moran, 1926.)

irregularly distributed in a non-frozen gelatin matrix (Fig.
10, B). Sections made through these granulations at - 11°,
with instruments maintained at that temperature, or at
higher temperatures, after fixation with f ormol, revealed a
structure consisting' of alternating concentric layers (Fig.
10, C ) . ]\[oran considers these layers as made respectively
of ice, and of dehydrated gelatin.

Hardy (1926) studied Moran 's spheres in greater detail.
Layers of gelatin gels 0.5 mm. thick, containing the proper
amount of water, Avere mounted between slide and cover-
slip; they were, then, frozen at a convenient temperature
and observed in a low temperature room. The concentric
shells were seen to consist of two sorts of rings, one of

136

^vlli(•ll ronncd "lut'inhriiiU's" ahoiil ()..") micron in thickness,
\vliil(' llic other whicli srciiicd ('nch)s('(l within these mem-
])r;nies, consisted of much broader layers. Outside of the
rings, solidificntion continued in the form of rays (Fig. 11,
A), the rings l)ulgin,<!: into the l)ase of tlie rays. The rays
themselves were dixidcd transversely into com])ai'tnieiits
l)y tlie curved nienihi'aiies. At the ti]) of ;i ray, or at tlie

Fig. 11. Formation of "rays" at tlu' in'riidiory of crvstalliiK' discs in
frozen gelatin gels. (From Hardy, 1926.) An entire ray is shown in A, and
the terminal portion of a ray, at higher magnification, in B.

outer edge of a circle, one could see a series of tine etched
lines (Fig. 11, B).

Moran explains the concentric structure by assuming
that the heat developed in the formation of ice in a center
of crystallization keeps the local temperature higher at
that point for a time and prevents a further freezing. The
gelatin in the immediate neighborhood becomes more con-
centrated on account of the withdrawal of the water trans-
formed into ice and it cannot freeze. When the local tem-
perature is lowered again, a new layer of ice will be formed.
The concentric shells will result from such an alternation
of warmings and coolings.

Hardy claims that, while the '^ membrane" phase con-
sists of dehydrated gel, the layers between the membranes
are not made of ice, as Moran thought, but of a solid solu-
tion of ice and gelatin. He bases his conclusions on the
optical properties of the various phases of the system (in
polarized light) and on the behavior of the material on
thawing.

Disseminated F r e e z i n g. With weaker con-
centrations or lower temperatures, for example, with 88%
watei- at -19° or in liquid air, a third type of freezing oc-
curred. Moran described it as follows: "The interior of

137

tlic gel was now occuijied by a large iinmbcr of clear sijlieri-
eal spaces each about 3 micra in diameter and arranged in
rows." {Cf. Fig. 10, D).

Hardy called this kind of freezing "disseminated freez-
ing. ' ' The spherical masses that he observed had a diam-
eter as large as 20 micra. He showed, furthermore, that
the spheres consisted of pure ice. To explain their forma-
tion, he suggested that they had started as crystallization
centers, but that, owing to the rapid cooling, the viscosity
of the material was soon high enough to prevent their
further grow^th.

As it was said above, with high w^ater contents and very
slow freezing, some centers of crystallization are formed
within the gelatin gel and there result irregularly dis-
tributed crystalline spots wdthin the remaining non-crystal-
line gelatin. It seems that this type of freezing differs
from the one just described under the name of disseminated
freezing only by the shape of the crystals, w^hich were
prismatic in the first instance and spherical in the second.
In both cases, the crystals are of pure ice, by opposition to
what happens in the case of intermittent crystallization.
So, w^e shall distinguish two types of disseminated freezing
according to the shape of the crystals, and we shall call
"Granular disseminated" the type in which the crystals
are spherical and "Irregular disseminated" the other.

The researches of ^loran and Hardy can then be sum-
marized in the following points : 1. The irregular dis-
seminated freezing was observed mostly in gels of high
water content (88^^ w^ater) but also wdth more concen-
trated gels if freezing was moderately rapid ; 2. The gran-
ular disseminated freezing occurred with very rapid cool-
ing (in liquid air and at -19°) ; 3. Surface freezing took
place over a large range of w^ater contents but it required a
slow cooling; 4. Intermittent freezing extended also over
a large range of water content and it occurred at intermedi-
ate cooling velocities (with an external temperature from
-6° to -13.5°).

]\[oran pointed out another important fact, namely, that

a gelatin gol of wliich tlic water content is 'M.') '/( or less
never freezes at aii\' Icnipcrature.

The followini^- table «;ives, in a com})r('lH'iisive form, the
conclusions of ^loran and Hardy :

Xdlnre of

Wahr Content

Cooling Vcl

oci

ly

Type of Freezing

C

rijstallizing
Pliaxe

Flifjli, Medium

Low

Surface, Coini):i(t

Ice

Hijili. MiMlium

High

Internal, DisscniiiKited,
Irregular

Ice

Hi>,^li. .Mciliuiii

W'vy Ili^rh

Internal, Di-ssciiiiiiatcd,
Granular

Ice

Mcilium

Mcdiuin, II

ioh

Internal, Interniilteiit

Ice-gelatin

Low

Any

No freezing

3. Coagulated Material. Prillieux (1869b) described an
exudation of ice from the white and from the yolk of a
boiled egi>,- when the latter was put to freeze under a cover,
after the shell was removed. There was a layer of ice 1
mm. thick outside the white, and another of about the same
thickness between the white and the yolk. The white was
itself divided into several concentric layers of unfrozen,
soft albumin, separated by shells of ice, some of whicli were
about 1 mm. thick. The ice shells consisted of small ice
columns with their axis perpendicular to the surface of
the shells. There were tiny air bubbles along the axis of
the columns.

4. Porous uiafcrlal. If the water which freezes out of a
liciuid is hindered in its withdrawal not only by the forces
which hold it in solution or in suspension l)ut also ])y capil-
lary or osmotic forces, we should expect some dilference
in the form of the resulting crystals, their state of aggre-
gation and their velocity of formation.

Rigaud {Londou-Ed'tuh. Phil. Mag., 2, UK), 1853) de-
scribed a particular type of exudation of ice from the
mortar of a stone wall. A plate of ice covered the wall.
It consisted of juxtaposed ice columns which grew parallel
to eacli othci- and pcrjjeiidicular lo the surface from which
they arose. The columns were oi' all sizes u]) to several
centimeters in length.

139

Leslie {'^Eiwyclopcdia Britannica," vol, 3, art. Cold,
p. 258, Supplement) observed a protrusion of ice filaments,
like bundles of spun-glass, from a porous earthenware pan
into which he put water and which he maintained in the
presence of sulphuric acid in reduced air pressure. The
water was conveyed through the pores and crystallized out-
side in ice shoots, perpendicular to the surface of the
container.

Le Conte ( 1852 ) obtained a similar effect by soaking ' ' the
smaller portions of soft and spongy roots ' ' of the cypress
in potassium nitrate and letting them dry in the air.
Crystalline fibers of the salt emanated at right angles to the
surface. The author compares these fibers to the filaments
of zinc sulphate which form at the surface of the earthen-
ware cups used in batteries.

5. Tissues. Surface Freezing. The astronomer Her-
schel (the son of the famous William Herschel), in 1833,
observed, on some decaying thistles and on the stumps of
living heliotropes, ice ribands with a fibrous structure, a
silky surface and a frilled wavy shape (Fig. 12). The
ribands were formed longitudinally on the stem and per-

FlG. 12. Foiniatidii of iee rilibons on plant stvinips. (From Horscliel,
1833.)

140

pi'iidiciihii' lo il. 'I'licy orii^iiuilcd on the wood, hclow tlio
corh'X, and horcd llicii- \\;i>' llii'onuii ci'acks in the latter.
Tlic ice lihcrs were ix'rpendiculai- lo tlie stem. The at-
tac'limeiit of the riband to the stem ^vas very light and did
not correspond to any crack in the stem.

Dnnal (1848) described on the square stems of some
hd)iates, fonr ril)bons of ice comin,<>' out from the sap wood,
through the torn cortex.

Simihir ice blades on plant stems were observed, two
years later, in Georgia, by Le Conte wlio depicts the same
details as the previous observers.

Sachs (18(50) attempted to induce ice formations of the
kind described, by exposing sections of beets to the frost,
under a cover to avoid an excessive evaporation. He ob-
tained plates of ice, of velvety appearance, made of ice
columns perpendicular to the cut surface of the tissue.
The author observed these phenomena on several types of
vegetables. He noticed, furthermore, that the process
could be more easily induced in succulent plants and when
freezing was not too rapid.

Several attempts have been made to explain this exuda-
tion of ice in plant stems. Herschel notes that the water
comes from within the plant and that it must finally come
from the ground. The plant would work as a "chimney,"
moisture being exuded from the earth by "every open
spiracle, ' '

According to de Mohl ("Vermischte Schriften," Tiib-
ingen, 1845), the exudation of fluid from the tissues is due
to the contraction of the latter under the action of cold.

Le Conte holds that water will begin to freeze at the
external end of each capillary pore, where the contact
with tlie air maintains a low^er temperature. When the
water freezes into ice columns, a lateral pressure would
be exerted between tlie lattei', due to the ex])ansion which
accompanies freezing. The ice plate constituted by the
columns would then separate from the substratum in a
direction perpendicular to its surface. AVhen the ice
columns have moved away from the broader open end of

141

the c'a])illaries, water from witliiii would fill the latter
again and a new layer of ice would be formed as before.

Caspary (1854) suggests that, at freezing temperatures,
for one reason or another, an exceptionally abundant
amount of sap might ascend the plant through the vessels.
That sap would traverse the walls of the vessels to freeze
outside.

Sachs called the attention of the biologists to the fact
that a body imbibed with a liquid is always surrounded by
a film of that liquid. To show this, he covered with var-
nish a piece of a pig's bladder membrane and dipped it
into water; the varnish, which before imbibition was ad-
hering to the membrane, became loose, on account, says
the author, of the formation of a film of water between the
membrane and the varnish. Applying this principle to
the present problem he assumed that the film of w^ater
present on each w^et surface freezes first and that upon
formation of a new film, it behaves as a removable layer.

Prillieux (1869a) objected to Le Conte that the size of
the ice columns did not correspond to that of the capil-
laries. He objected to Sachs that a surface film of capil-
lary size should resist congelation instead of initiating it.
He furthermore remarked that the expansion of water
between 4° and 0° is not sufficient to explain the exudation
observed. Finally he proposed the following explanation.
Water is held in the living cells or in the boiled white of
egg by the forces of imbibition; the molecules of water
w^hich are farther away from the imbibing molecules and
are not so strongly attracted by the latter, leave them and
freeze. The crystals formed in that manner grow by
attraction of new molecules of water. So, as we under-
stand the author's interpretation, the columns of ice origi-
nate in larger intermolecular spaces and in pores.

It seems that, during the first half of this century, the
attention of the biologists has been attracted by other
questions and that the problem of the mechanism of these
particular ice formations has been left unsolved.

6. Tissues. luterceUular Freezing. In 1817, du Petit-

142

Tliouars described ice formalioiis in llic pilli and in tlic
cortical parenchyma of the stem of some plants (vine,
elderberry, etc.) The ice crystals were occasionally so
abundant that it was ])ossible to obtain a dish-full of them.
In somi' instances thei-e was a com})lete cylinder of ice
below the cortical layer. On meltin^u', tlie ice coHected in
tlie tissues i^ave almost clear water.

Later de Mohl {op. cif.) discovered that, wlien the leaves
fall after a freezing weather, a layer of ice can be seen at
the base of the petiole, separating the leaf from the branch.
The mechanism of the formation of this ice layer is prob-
ably the same as that of surface freezing of stems dis-
cussed above.

Caspary {Bof. Zeituug, 1854, p. 665) who had observed
surface freezing on various plants, in the Schoeneberg
garden, near Berlin, described also the formation of
masses of ice in the interior of fresh stems, in particular,
below the epidermis. He attempted an anatomical study
of the tissues from which the ice originated. But he says
that he could not ascertain whether the separation of the
cortex from the wood and of the wood from the medulla
was accom])anied by a tearing of the cells or if the latter
Avere only pushed apart.

Several observers, however, after Goeppert (1830), have
pointed out that the cells, in frozen tissues, are not torn by
ice crystals. Such an observation, together with that of
the presence of ice in the intercellular spaces, contributed
to estal)lish the notion that water does not freeze in the
cells but that it is withdrawn from them during the freezing
process.

Sachs (1860, vf. also his "Textl)ook of Botany," Book
HI, Ch. Ill, Sect. 7), by cross-sectioning leaf stalks of a
frozen artichoke, observed that the epidermis ^vas separ-
ated from the parenchyma by a layer of ice, and tliat the
parenchyma itself had split into several ])ortions separ-
ated from each other by ice (Fig. 13). From pieces of
leaf stalk weighing 396 grams he picked out as much as
99 grams of ice. This ice was almost pure water; after
evaporation it left only 0.1 'r of solid substance.

143

Fig. 13. Section tlirough the leaf-stalk of a frozen artichoke. (From
Sachs, 1860.) Tlie hatched spaces represent the ice masses, the black spaces,
the cavities of the ruptured tissue.

Prillieux (1869a) insisted on the fact that, in frozen
tissues, ice was found between the cells and not in them.
This was observed in petioles, in buds, in stems, etc. Some
stems presented, in a cross section, four masses of ice
radially distributed, some three, some five, depending on
their structural symmetry. A layer of ice always isolated
the epidermis; the pith often seemed filled with ice crys-
tals; the parenchyma was usually split into several por-
tions separated by ice masses. Since the cell walls were
not broken, Prillieux remarked that they could not have
been traversed by ice crystals and that, therefore, it was
in the liquid form, before freezing, that water had left the
cells.

Fig. 14. Intercellular lens-shaped masses of ice in plant tissues. (From
W'iegand, Plant World, 9, 26, 1906.) Each mass consists of two layers of
ice pillars. In the middle of the tissue, (A) the pillars have the same length
in the two layers; they are of unequal length nearer the external surface of
the tissue (B) ; only one layer remains at the surface itself (C).

144

MiilK'r-Tlmriiaii (ISSli) sliidicd llic (h'posilion of ice in
llu' jiilci-ct'llulnrs hy llic following' procedure. He sec-
tioned ^vitll a eooled knife pieces of frozen tissues, snch as
beets, potatoes, dahlia tubers, etc., took out the little lens-
shaped pieces of ice located between the cells and exam-
ined them on a cooled slide. These ice masses consisted
of two layers of ice pillars {cf. Fig. 14). Each pillar was
a more or less regular six-sided ])rism. Within the indi-
vidual i)illar, bead-like air-bubl)les extended along the axis.
The ice masses were of enormous size as coni])ai'ed with
that of the cells. The cross section of the individual crys-
tals was itself larger than that of the cells. The length of
the columns was about the same in the two adjacent layers
except if the ice mass had been formed near the external
surface of the tissue, then the layer on the external side
was thinner.

The surface freezing in compact ice crusts, as described
before, is considered by this author as the limit in the
series of forms that the ice masses take when they origi-
nate at points gradually nearer the outside surface. The
formation of ice between the base of the petioles and the
stem, described by de Mohl in his study of the fall of the
leaves after a frost, is also considered a particular case
of the same phenomenon.

As to the question of intercellular or intracellular freez-
ing, Miiller-Thurgau noted that a direct examination of
sections of frozen tissues under the microscope shows the
crystals between the cells, not in them.

Repeating an experiment previously made ])y Sachs, he
determined the residue left after evaporation of 21.08
grams of ice collected inside of a cow-beet, and found 0.04
g. It was, therefore, practically pure water which tiltered
out through the cell walls during freezing.

^^liiller-Thurgau, described also ice masses of columnar
structure in animal tissues, for example, around the ali-
mentary canal of an earthworm left to freeze in the ground,
at -6°.^

7. Cells. Surface Freezing. Moliscli (1897), exposing

145

yeast suspensions to a freezing tempei'alure of -9°, states
thai a witlulrawal of watei- was evidenced by a shrinking
of the cells (l)y a])Out 10'/<' of the original volume) and by
the fact that the vacuoles became indistinct. The cells
themselves, he thinks, nevei- froze.

The same author described also an exudation of water
from various filamentous algae: Spirogyra, CladopJiora,
Derbesia, etc., observed under the microscope during the
congelation of their medium. In a Spirogyra the diameter
decreased by 62 9^ . Then he froze these algae in olive oil
and noticed that the extruded water formed a cylinder of
ice around the plants. In some forms the ice exuded pre-
sented a characteristic filamentous pattern, with the fila-
ments sometimes twisted like in a screw, a pattern that he
also observed in moss leaves and in fern prothalia.

In staminal hairs of Tradescantia, Matruchot and Mol-
liard (1902) observed, during freezing, a loss of turgor, a
decrease in cell volume comparable to that which occurs
in plasmolysis, and a decrease or disappearance of the
large vacuoles. They consider all these features as result-
ing directly from the withdrawal of water.

8. Cellular ('o)istituents. AVliile the preceding investi-
gations refer to the exudation of water from freezing cells,
Matruchot and Molliard studied the evidence of with-
drawal of water from the cell constituents, in particular
from the nucleus. Plant tissues of various types w^ere left
to freeze for several hours (15 hours mentioned in one
case) at temperatures from -4° to -7°; then they w^ere
thawed, fixed and stained. The authors used cells with a
somewhat abundant vacuolar content taken from the lacu-
nar parenchyma of the leaves {Narcissus), from the cortex
of the hypocotyl axis {Phaseolus), from the stem {Lupi-
nus), from the parenchyma of the roots {Hyacinth us),
from the floral peduncles {Clivia, Tulipa), and also cells
of more compact cytoplasm, such as those found in the
tissues of the nucelle {Leucoium) and of the ovary {Tu-
lipa). The essentials of their results are represented in
Figure 15. The nucleus, homogeneously granular in un-

i4(;

Fig. 15. JMiase separation in tlie nncleus of frozen plant cells. (After
Matruc'hot and Molliard, 1902.) The chromatin is represented in solid black
masses.

frozen controls (Fig-. 15, A), shows vacuolization in frozen
cells. The chromatin forms a network with the meshes
elongated in the direction of the vacuoles (Fig. 15, B). If
the nncleus is between two vacuoles, the pattern is then
bipolar (Fig. 15, B), if there is only one vacuole in the
neighborhood of the nucleus, the pattern is monopolar
(Fig. 15, C). The meshes fuse into larger masses at the
equator of the nucleus in bipolar systems and at the pole
opposed to the vacuole in monopolar ones. These masses
of cliromatin become more and more compact, the fila-
ments projecting from them thin out gradually (Fig. 15,
D) and finally there results (in bipolar systems), a crown
of chromatin at the equator of the nucleus, separating two
nuclear vacuoles which bulge out on each side (Fig. 15, E),
III the cells with more condensed contents (from micelles
and ovaries), the chromatin meshes are thicker, the vacu-
oles within the meshes occupy a smaller volume, and the
entire pattern is of a different type (Fig. 1-3, F). As to
llie cytoplasm, which is described as gramilai- in the nor-
mal condition, it l)ecomes spumous or spongy by vacuoli-
zation, after freezing. In general, in the cells studied by
Matruchot and Alolliard there was, on freezing, a separa-

147

lion of water from the chromatin or from the cytoplasm
and an accumnhition of tliat water in the vacuoles.

Lnyet and Gibbs (1937) made a detailed description of
the progress of freezing in the epidermal cells of onion.
After the congelation of the moisture present at the sur-
face of the mounted epidermis, some subcooled cells froze
suddenly, becoming opaque. Two separated phases could
then be seen in the frozen cells : ice and concentrated vacuo-
lar sap. The opacity decreased during the few seconds
subsequent to the sudden freezing, a phenomenon that the
authors attributed to a rapid growth of crystals. There
followed a slow transformation and growth of ice masses
(Fig. 26) which lasted for hours.

Bugaevsky (1939), evidently unaware of the last-men-
tioned work, described the same process in epidermal and
subepidermal cells of the "underground part" of Avheat
plants. The ice crystals are said to grow within the proto-
plasm itself ; no mention is made of vacuoles.

Buck (unpublished work; cf. abstract in Auaf. Rec, 72,
SuppL, 125, 1938) obtained the curious picture represented
in Figure 16 by freezing salivary gland chromosomes of
Ch'uouomus tentans. The nuclei, dissected out of the
gland and contained in a drop of water, were let fall into
a tube of petroleum ether cooled to about - 70°. The con-
tent of the tube was then evaporated in a high vacuum at
a temperature lower than - 30°. One of the nuclei so ob-
tained, photographed in fofo,i^ shown in the Figure. One
can distinguish several lobes of the coiled chromosomes.
The parallel lines seen across the nucleus seem to bear
no relation to the orientation of the chromosomes. The
author suggests that they represent regions of compressed
nuclear material, while the clearer stripes would represent
regions of reduced chromosomal content previously occu-
pied by ice (personal communication).

C. THE FREEZING CURVES

^[any experimental investigations on death or injury by
low temperature involve the study of freezing curves, that

14S

Fig. 1(3. Frozen nuck'iis from .salivary gland of Cliironomioi tentan.s.
(Original, Buck.)

is, of curves expressing the course of tlie temperature of
a freezing object in terms of time. We shall analyse here
briefly the essential characters of freezing curves and, to
begin with, those of the freezing curve of water.

1. Freezing Curve of Water. If a mass of distilled
water, perfectly stirred, at a temperature T (above zero),
is exposed to a cooling bath at a constant temperature t
(below zero), the temperature of the water will drop, in
terms of time, according to a curve of the type repre-
sented ill Figure 17. This composite curve consists of
three limbs : AB, BC, and CD, which represent the course
of the temperature before, during, and after freezing,
respectively.

The limbs AB and CD are eooVmg curves. Under ideal
conditions, as has been stated in the third section of the
Preliminary Chapter (r/. in particular the formula 5),
such curves are exponential.

AB and CD nie not, liowever, identical. The curve AB

149

B'

Fig. 17. Freezing curve of a liquid under ideal conditions. Abscissae:
time ; ordinates : temperature.

represents the cooling of water, and the curve CD the
cooling of ice, that is, of a solid which cannot be stirred,
and which possesses a heat conductivity about 4 times
higher and a heat capacity about twice lower than that of
water. The impossibility of stirring results in a non-
uniform distribution of temperature ; in a solid body which
is being cooled the temperature decreases gradually from
the center to the periphery. The high heat conductivity
and the low specific heat of ice cause a more rapid cooling
than in the case of water; consequently, the slope of the
curve CD is steeper than that of AB.

To construct exponential curves one needs only two ex-
perimental points. Let us assume, for example, that a
mass of water at + 10° is exposed to a bath at - 5°, and that
the cooling rate is such that the temperature drops during
the first minute from 10° to 8°, that is, if - 5° is taken as the
origin, from 15° to 13° (from 10 + 5° to 8 + 5°). The two
experimental values, 13 and 15, give the ratio of the geo-

150

nu'lrii' iti-(»>;-rc'ssi()ii i-('i)r('s('iitiii,u- llif ('(tursc of tempera-
ture: 13: 15 = ().S()(). Willi lliis latio one ealeulates 1lie
temjH'rntiire after ii minutes, wliieli is 15 X 0.86()". hi
general, there is a uood agi^eemeiit hdwceii the ex})eri-
mental and the calcnhited eooiiiiu' eui'ves.

As to the portion Bf (Figure 17), it is a straight line in
the ideal conditions assumed. When the decreasing tem-
perature reaches 0°, water begins to freeze and, by liberat-
ing heat, it ])revents a further dro]) of the temperature.
Theoretieally, if the entii'e mass of watei- were at 0° and if
the heat liberated could be eliminated instantly, freezing
would be completed at once. But a portion only of water is
at 0° and freezes, the remnant is at a slightly higher tem-
perature and stays liquid. The heat liberated by the part
which freezes contributes to maintain the temperature of
the non-frozen i)ortion above 0° and delays the complete
freezing of the mass. The curve then stays horizontal,
there being an exact balance between the quantity of heat
which is withdrawn by the bath and which would bring the
temperature down, and the quantity of heat which is pro-
duced by f i-eezing and which would bring the temperature
up.

The length of the horizontal portion of the curve depends
on two main factors : the velocity of withdrawal of heat, and
the mass of liquid to be frozen. Concerning the rate of
withdrawal of heat from the freezing mass one should
remember that it does not remain constant during the
process of freezing, even if the temperature of the cooling-
bath stays constant. While the material is being solidified,
there is a gradual increase in heat conductivity and conse-
quently the rate of withdrawal of heat increases. It is
evidently necessary to take into account this change when
one calculates the length of BC. The change in specific
heat can be neglected in as much as the system consisting
of the substances separating the freezing mass from the
cooling bath can be compared to a wall limited by two
surfaces at constant tem])eratures (see, above, the treat-
ment of the "Problem of the wall")- iVnother factor

151

which considerably disturbs tlic process of withdrawal of
heat from a freezing mass and which I'enders partially
inai)i)licable the laws established under ideal conditions, is
the gi-adually increasing inetficacy of stirring during the
progress of crystallization.

The area BCC'B' is sometimes used as a measure of the
quantity of ice produced during the time interval B'C. If
the heat Q produced by freezing is entirely transmitted to
the bath, one can write that it is equal to the heat Q' dis-
sipated :

Q = Q' (A)

But the number Q of calories produced by crystallization
is equal to 80 times the number I of grams of ice formed :

Q = 80I (B)

On the other hand, the heat dissipated Q' (see, above, the
"Problem of the wall") is proportional to the difference
y of temperature between the freezing water and the cool-
ing bath, to the thickness d of the material separating
them, to the heat conductivity c of that material and to the
time t

Q' = kytcd (C)

where k is a constant of proportionality. From the equa-
tions (A), (B) and (0) one deduces

l=^ytcd (D)

that is, the amount of ice I is proportional to the area yt of
the rectangle BBT'C (when the heat conductivity c and
the thickness d are constant).

The numerical value of the product kycd, that is, the
number of calories lost by water in one unit of time, in the
conditions of the experiment, can be determined by measur-
ing, on the cooling curve, at the point B, the nnml)er of
degrees by which the temperature drops in one nnit of time
and multiplying that value by the number of grams of
water present (each gram requiring 1 calorie to lower the
temperature by one degree).

As we said, the formula D is established on the assump-
tion that the factors c and d are constant. But c, the heat

152

f()iuliu'li\"il\' of t'rccziiii;' water, docs not sta\' coiislaiil ; il
increases as more and more ice is formed. The formula I),
therefore, sliouhl l)e considered correct only in a first a])-
})roxiniati()n. The other factoi's tliat we mentioned a])Ove
as al'fcclinii,' tlie h'li^th ot" llic cui-n-c should also ])e taken
into account in the study of the area limited by the curve.

Theoretically, at the points of junction of the three curvx's
AB, BC and CD, there should ])e sharp angles; practically,
the angles ai-e rounded on account, mostly, of the lag in
the conduction of heat. As we shall see later, with bio-
logical material the j^assage from one section of the curve
into another is sometimes so gradual that it cannot be
assigned to any definite point.

We said nothing in this discussion on the heat capacity
of the thermometer, of the stirrer and of the containei-.
The presence of these objects might sometimes have an
important disturbing influence on the shape of the curve
and it is not enough simply to correct the results by intro-
ducing their water-equivalent in the calculations.

2. Freezing Curves of Solutions. When freezing bio-
logical matei'ial we are always concerned with solutions
or suspensions and not with pure liquids. The freezing
curve of an aqueous solution in ideal conditions is repre-
sented in Figure 18. Such a curve consists of 4 limbs AB,
BC, CD, DE.

The parts AB and DE are simple cooling curves of the
exponential type studied in the preceding section.

The part BC requires a detailed analysis. When the
temperature has dropped to a certain point below zero,
water starts to freeze out of the solution. This point (B
in the figure) is called, though improperly, the freezing-
point of the solution. The portion of water which freezes
at B liberates heat and the drop in temperature is retarded.
However, the curve cannot stay horizontal for any length
of time ; as soon as some of the water is solidified, the solu-
tion becomes more concentrated and its freezing point is
lowered. When the temperature is down to this lower
freezing point, moie water freezes, more heat is liberated,

153

Fig. 18. Freezing curve of a solution under ideal conditions. Abscissae:
time; ordinates: temperature.

a new delay results in the rate of cooling and the solution
becomes again more concentrated. The curve slowly fol-
lows a downward path BC.

Such a curve is of the hyperbolic type, as is shown by
the following considerations. A 0.01 weight-molar (molal)
solution begins to freeze at - 0.0186°. To lower the freez-
ing temperature by a further 0.0186 degree, that is, to bring
the freezing point to - 0.0372°, the concentration should be
doubled; for another lowering of the freezing point by
0.0186 degree the concentration should be tripled, and so
on. To double, treble, etc. a weight-molar concentration,
the weight of water in the solution should be reduced
respectively to ^, h h etc., its original value. To reduce
the weight of water to h one should crystallize ^l of it ; to

154

i-('(liu't' it to .1 one has lo cryslalli/c llic ollici- H, dc. In
llu'st' suc'c'i'ssix'c operations the (juaiit it ics ol' watci- s('))a-
ratrd l)y crystallization I'roiii the original (iuantity ])rt'seiil
would he 1 - !>, then 1 - ',, tiien 1-1, etc. Assnming that
the lieat pi-odncod by the crystallization of these (piantities
of solvent is dissipated pro|)<)rtionally to the time one lias
the following values for the frei'zing temperatures in tei-ms
ot" time (the original concentration of the solution is sup-
posed to be 0.01 weight-molar, its freezing point, therefore
- 0.0186°, and the unit of time is the time that it would take
to freeze all the water of the solution) :

Freezing rr- Freezing rr-

n ■ t lime r> :, i lime

Point Point

-0.0186° XI 1-1 = 0 - 0.0186° X 4 l-i = 0.75

- 0.0186° X 2 1-Jy = 0.5 - 0.0186° X 5 1-1=0.80

- 0.0186° X 3 1-i = 0-666

The freezing point F decreases as an arithmetic progres-
sion; the time t increases as a harmonic sequence; it is
known that the relation between two such quantities is
hyperbolic. The formula of this relation is

F = ^ (E)

where k is the freezing point depression of a weight-molar
solution.

In the establishment of this formula many factors have
l)een left behind, which should be taken into consideration
in most of the quantitative studies on freezing curves: 1.
The law of the freezing point depression holds only in dilute
solutions of non-electrolytes ; 2. The heat produced is not
withdrawn proportionally to the time, its withdrawal de-
pends on the difference between the temperature of the
bath and that of the material, a difference which is con-
tinuously decreasing; 3. By the gradual formation of ice,
the specific heat and the heat conductivity of the system
are changing and this also affects the rate of heat with-
drawal ; 4. There is some heat of solution involved in the
])hase sepai-ation. The hyperbolic relation, therefore,
should be considered only as a first approximation law.

155

One can see, by the preceding analj^sis, that, if the solu-
tion is dihite, the curve will stay practically horizontal for
a long time since a relatively large quantity of water has
to freeze before the freezing point is lowered noticeably.
With very concentrated solutions, on the contrary, the
liorizontal portion of the curve might be so small that a
sharp determination of the freezing point would be dif-
ficult.

At the temperature of congelation of the saturated solu-
tion, that is, at the eutectic point (C in the Figure), the two
phases, solvent and solute, crystallize. The curve becomes
parallel to the time axis (portion CD). For the analysis
of this portion which is a freezing curve, we refer to what
has been said in the preceding section on the freezing curve
of water. A slight complication results, however, from the
fact that there are 3 phases present, the liquid solution, the
solid solvent and the solid solute, the latter two crystalliz-
ing separately, though at the same time.

It should be mentioned also that, for dilute solutions, the
quantity of eutectic mixture is so little as compared to the
quantity of ice present that the eutectic plateau almost
vanishes while the freezing plateau is large. On the con-
trary, for concentrated solutions, in which, as we said
above, the freezing plateau disappears, the eutectic plateau
becomes considerably broader.

3. Freezing Curves of Colloids. Little is known on the
forces which have to be overcome to freeze water out of
colloids. The following investigation is one of the few
that we found in the literature, on this subject.

Fischer and Bobertag (1909) described the curious fact
that, when gelatin was dissolved in water by a previous
heating, the freezing curve so obtained had a longer hori-
zontal plateau than the freezing curve of a suspension of
the same quantity of gelatin (about 9%) dispersed in
flakes in the water (Fig. 19). A further investigation into
the cause of this phenomenon might yield valuable in-
formation on the mechanism responsible for the anomalies
of similar nature so often met with in the freezing of
tissues.

156

w»H»'>»>'«x»X»X «. «

12

16 Mm.

0 2 4 6 8 10

Fig. 19. Coininiiativc freezing curves of a solution (x x x) and of a sus-
pension (. . .) of gelatin. (Curves drawn according to the data of Fischer
and Bobertag, 1909.)

4. Fn'cziug Curves of Tissues. For a description and a
discussion of the freezing curves of plant tissues, we refer
the readers to ^^liiller-Thurgau (1881 and 1886), Voigt-
lander (1909), Maximov (1914), Walter and Weismaim
(1935), and Luyet and Gehenio (1937). Some freezing
curves of animal tissues were studied by Jensen and
Fischer (1910) and some of entire animals by Cameron and
Brownlee (1913) and by Weigmann (1936). These curves
were established for the information that they furnish on
such problems as the position of the freezing point, the
quantity of water withdrawn at a given temperature, the
mechanism of death ])y freezing, the relations between the
death i^oint, the freezing point and the quantity of ice
present, the range of subcooling, and the existence of a
eutectic point. We treated above the problems concern-
ing the freezing points ; those related to subcooling will be
reviewed in llie next chapter; the other jn-oblems men-
tioned will be studied here.

Living and Dead Tissues. The following dif-
ferences were reported in the freezing curves of living and
of dead tissues, stndii'd under otherwise comparable con-

157

ditioiis: 1. The liorizoiilal plateau was found higlier in
dead than in living tissues (for l)ibliographical references,
see above under "Freezing Points") ; 2. In dead tissues,
the plateau was maintained horizontal for a longer time and
then the curve dropped more rapidly than in living tissues
(Fig. 20) ; 3. The freezing curves of living material often

0 5 10 15 20 MIN

Fig. 20. Comparative freezing curves of living (A) and dead (B) plant
tissue. (From Luyet and Gehenio, 1937.)

showed jerks or irregularities while those of the dead were
smoother ; 4. In dead material, the position of the horizon-
tal plateau, that is, of the freezing point, was little affected
by the cooling velocities used in the experiments, except
at the very high velocities of 5 degrees per minute ; in liv-
ing material, the horizontal part of the curve was lowered
by cooling velocities of some 3 degrees per minute.

Several of the authors mentioned above have attempted
to explain these differences. In general, they attribute
most of the results to the resistance offered by the living
cell membranes and by the living protoplasm to the with-
drawal of water. The dead tissues, in which the mem-
branes have become permeable, and in which the proto-
plasmic structure has been altered, would behave as
aqueous solutions ; their water would be free. The living
tissues in which it is thought, water is held more firmly,
would release the latter only on the application of more
force. The jerks are attributed by Luyet and Gehenio to
the sudden release of various amounts of water or to the

i:)S

occasional t'rccziiin' of several vacuoles al a lime, ice-seod-
iiiii' tliroiiuli llie ineiiil)i"aiies of llie iix'iii^- cells beiuii' often
hindered.

The resulls of .Jensen and Fischer (1910), on muscle, are
quite different from the results obtained by the authors that
we just mentioned, on plant matcM'ial. The curves of dead
muscles dropi)ed more slowly after the horizontal plateau
than those of living muscle (Fig. 21). The authors attrib-
ute this to a firnirr hiufl'nic] of water in dead material.

-11.2

-22.4

-33. 6

\

r=3»^

""^^-

>N

'\\

., \

^\.

1
, 1

"n

y

0

8

»2

/6 MIN.

Fig. 21. Comparative freezing curves of living and dead muscle (From

Jensen and Fischer, 1910: ( ), fresh tissue; ( ), tissue killed by

freezing; (-■-■—), tissue killed hy heating at 100°; (• • • •), tissue killed
b_v lieating at 115°.

The freezing curve obtained by Cameron and Brownlee
(1913) on an entire frog exposed to -10° with a ther-
mometer in its stomach, seems to agree with the results
generally reported rather than with those of Jensen and
Fischer. The drop of the curve after the horizontal
Ijlateau is slower and does not present the relatively sharp
turn exhibited by the hyperbolic curve of a saline solution
frozen at the same time (Fig. 22).

E u t e c t i c Point. Mez (1905) assumed that plant
tissues should present a eutectic point just as true solutions
do. He claimed, furthermore, that this point was always
above -6°.

i:)9

-12

Fig. 22. Comparative freezing curves of an entire frog (x x x) and of
a saline solution (o o o). (After Cameron and Brownlee, 1913.)

On Mez' suggestion, Voigtlander (1909) established a
large number of freezing curves of various plant tissues.
He observed that in most of them the cooling velocity
showed a decrease at two points along the curves (Fig. 7,
Curve 5, Points F and E), and he considered these retarda-
tions in the cooling rates as representing, respectively, the
freezing and the eutectic points.

According to Fischer (1911), it is quite daring to speak
of the eutectic point of the cell constituents which are mix-
tures of colloidal substances, some of wiiich perhaps never
crystallize. Jensen and Fischer (1910), who made a com-
parative study of the freezing curves of muscles and of
saline could observe a faint trace of eutectic in the latter
but their curves show no evidence of any eutectic in the
muscle.

Maximov (1914) criticized Mez and Voigtlander 's results
on the ground that, in such a dilute solution as the cell sap,
the retardation in the drop of the curve caused by a

icn

oulc'clic I'rtM'/iiii;', should Imi'dl}' l»c noticed ;nid could iiol l)o
as lar^e as was (»l)sci\-cd. lie cxpressctl his siir])riso at
Mez' c'oiiloulioii thai the ciiU'ctic was always above -fi°,
wliiMi it is known that solutions of several snl)stances found
ill the cells have mncli lowci- eutectic points. He em-
phasized the faet that, in the numei'ous curves tliat lie
estal)lished with tissues or with extracted juices, he never
observed any indication of a eutectic freezing. He also
pointed out that, with the high cooling velocities used by
Voigtljindei', any retardation in the slant of the curve
would be so attenuated that even the freezing points would
be obscured. He finally suggested that the two retarda-
tions observed by that author were due, respectively, to
a freezing of the cell sap extruded around the thermocouple
by the insertion of the latter, and to the freezing of the
tissue. This interpretation was confirmed by the fact,
meniioned by Voigtliinder himself, that extracted juice
gave but one retardation.

Luyet and Gehenio (1937) showed that a living tissue
which gives a curve with two long horizontal plateaus (Fig.
23, A), at a low cooling rate of 2.5 degrees per minute,

0 5 10 15 20 MIN

Fig. 2o. Effect of i-ooliiig vulucity on freezing curves. (From Ijuvt't and
Gehenio, 1937.) Cooling velocity at 0°: in curve A, 2.5° per minute; in
curve B, 5.2" per minute.

gives a curve in which the plateaus almost vanish at a cool-
ing velocity of about 5 degrees per minute (Fig. 23, B),
and would be replaced by two slight retardations at the

161

cooling velocity of 10 to 14 degrees per minute used by
Voigtlander. This contirms Maximov's view that Voigt-
lander's curves present a double freezing point but no
eutectic.

Moran (1935), who defines the eutectic point as "the tem-
perature at which all free water is frozen," determined this
temperature in a concentrated filtrate of muscle juice. For
that purpose he measured the electric resistance of the
material when the temperature was lowered, there being
quite a definite change in resistance at the eutectic. He
obtained the value -37.5°, which he considers as the
' ' eutectic temperature of muscle. ' '

One must confess that there is little experimental evi-
dence for the existence of a eutectic freezing in tissues,
despite the fact that' a considerable portion of plant juices
and body fluids are true solutions, from which one would
expect a eutectic freezing. The problem of the eutectic,
therefore, remains almost entirely to be investigated.

Q u a n t i t y o f I c e F o r m e d. In the study of death
by low temperatures, biologists have attempted to find,
from an analysis of the freezing curves, the proportion of
water frozen at a given temperature, in order to determine
if death by freezing is conditioned essentially by the quan-
tity of water withdrawn from protoplasm. In doing so,
they have generally observed that the descending portion
of the freezing curve which follows the horizontal plateau
is maintained for a long time at a level higher than one
should expect if all the water were frozen.

Miiller-Thurgau (1886), by recording the time necessary
for the freezing curve to fall from the freezing point to
- 1°, from - 1° to - 2°, etc., concluded that, in a tissue, the
amount of ice formed during the first part of the freezing
period is considerably greater than that formed later ; but
there was still some crystallization when the curve had
dropped several degrees below the freezing point. This
Avas confirmed by the observation that the ice masses
formed in the intercellulars of a tissue exposed for several
hours to -10° were larger than those formed after an
exposure to -4°.

1(12

Jensen and Fisclici- (If)lO) calculak'd, from llie areas
limited l)y tlie freezing- cui'ves, tiie comparative (iiianlities
ot" iee formed at vai'ious tem]X'ratures, in living-, in pre-
\ionsly t'l-ozeii and in ))revionsly heated muscles, as also in
pliysiolo^'ical saline. The essentials of tlieii- results ^vere
discussed above.

Maximov (11)14), usiiii;' the method of the freezin,i;-curve-
area, noticed, amonj»' other interesting facts, that the quan-
tity of ice formed in a tissue of given water content does not
depend oidy on the lowest temperature reached but also
on the cooling velocity, that is, on the temperature of the
cooling bath (investigations on potato and beet).

Luyet and Condon (1938) found that the quantity of ice
formed in small pieces of potato tissue weighing 2.25 gr.
was 34.5, 45.5, 55.7 and 65.99c of the weight of the object,
after, respectively, 15, 20, 25 and 31 minutes of freezing.
The temperature had dropped during these periods of time
to -1.9°, - 2.8°, - 4.6° and - 8°, respectively. There was
still evidence that some ice was formed between the last
two temperatures.

In most of the investigations described in this last sec-
tion, no attempt has been made to bring the temperature to
very low values and to study the behavior of the curves at
these temperatures. The last portions of the freezing
curves have hardly been investigated.

III. COMPLETION OF CRYSTALLIZATION

Most of the authors who have studied the completion of
freezing and its relation to temperature and time have
done it by the calorimetric or the dilatometric method.
We refer the readers who desire more complete informa-
tion on this subject to reviews on ''Bound Water" and we
summarize here only a few papers more intimately related
to our topic.

1. Siisprnsious and Cnlloids. Foote and Saxton (1916)
determined with a dilatometer the formation of ice, at
various low temperatures, in mixtures of sand and water,
of lampblaelv and water, in moist calcium hydroxide and in

163

hydrogels of alumina, of silica and of ferric oxide. While
freozino- was completed at -4° in the sand mixture, it
required a temperature of -28° to be terminated in the
lampblack mixture. In most of the other substances
studied, it was observed that at - 20° some water was still
crystallizing out.

Moran (1926), who had previously observed that, when
gelatin gels in form of discs are frozen slowly, ice forms
at the surface, determined the quantity of water left in
the core of the discs, that is, the quantity of non-frozen
water, after exposure to various low temperatures. The
curve of Figure 24, constructed from his data, represents
the amount of water found in the cores of discs exposed to
temperatures from - 3° to - 19°. At this last temperature,

/o

45

40

35

0° -5° -10° -15" -20

o

Fig. 24. Proportion of water which remains unfrozen in gelatin gels ex-
posed to various temperatures. (Calculated from Moran 's data, 1926.) Ab-
scissae: temperatures; ordinates: unfrozen water in percent of the total
weight. The horizontal dotted line represents the quantity of water which
does not freeze at any temperature.

34.8',f (>r tlic weight of llic core was walci'. I'lil lliis Avas
almosl (,'utiri'ly ii(>ii-t'r('('zal)k' water, since ,<;els ('()iilaiiiiiij»"
34.5% waler did not t'l-eeze at any temperature, even after
immei'sion in li(iuid air. CoiiscMinently, almost all the
freezable watei- was frozen at -19°.

Lloyd and Moran {Proc. Roy. Soc. 147, 392, 1934) ex-
tended these determinations to a temperature of -45°.
They found no significant diffei-enee in tlie (piantity of ice
formed at - 20° and at - 45%

Moran (1935), using the metliod of surface freezing ob-
served that in sohitions of agar, myogen and albumin (in
collodion bags) there was still some freezing at - 20° ; how-
ever, at this temi)erature the curves representing the pro-
portion of water frozen approached the asymptotic
position. In egg white, cessation of freezing would take
place at - 31° as determined by the change in electric resis-
tance of the freezing material.

2. Tissues. Aliiller-Thurgau (1886) measured, by the
calorimetric method, the quantity of water not yet con-
gealed at about - 15° in frozen apples. The apples were
brought, after congelation, into a water-calorimeter at 16°
and the resulting drop in the temperature of the water was
recorded. The amount of ice was found to vary from
53.13 to 66.0 gr. in 100 gr. of apple, when the freezing tem-
peratures varied from - 4.5° to - 15.2°. The water content
of the apples was about 83%.

Jensen and Fischer (1910) noticed that some ice was still
being formed in frog's muscle at more than 20 degrees
below the freezing point. Their criterium is the slope of
the freezing curves.

Rubner {Abh. Preuss. Aka(L Wiss., 1922), using the
calorimetric method, found that only 75% of the water con-
tent was frozen in beef muscle at - 20°.

According to Plank {/. Ges. Kalteind., 32, 141, 1925),
the amount of ice present in ox muscle at - 20°, determined
calorimetrically, was 91% of the total water content.

]\[oran (1930), by the dilatometric method, found that
947. <'t' the water was frozen at - 10° and 98.2% at - 20°, in
muscles from beef, mutton and pork.

165

The same author (1935) studied the completion of con-
gelation in amphibian muscles. He embedded the latter
in discs of gelatin gels, exposed them to various low tem-
peratures, and measured the amount of ice formed at the
surface of the discs. He found that most of the freezable
water froze between 0° and -5°, that about lO^r of the
total water froze between - 5° and - 20° and that less than
1% froze between -20° and -37.5°, at which temperature
freezing ceased.

The duration of the freezing process at a given tempera-
ture is shown by an experiment of ^loran (1926) on a 43.7%
gelatin gel put to freeze at - 11°, the volume of which was
determined with a dilatometer. The increase in volume,
rapid during the first few days, soon became very slow, but
it ceased only after 26 days (Fig. 25).

i

1

^55

0 10 20 30 40

Duration of freezing, in days

Fig. 25. Increase in the quantity of ice formed in a gelatin gel exposed
for 26 days to - 11°. (From Moran, 1926.) The ordinates represent dila-
tometer readings.

In all these investigations it was assumed, as a principle,
and this was subsequently observed as a fact, that con-
gelation is more complete when the temperature is lower.
But, it is also a principle and an established fact, as we said
above, that the velocity of formation of nuclei and of
growth of crystals is gradually reduced to zero when one
lowers the temperature. These two principles are in evi-
dent contradiction and one of them has to be sacrificed.

]()()

.\s to the t'acliKiI ()l)s('i'\';i1 ions llicy can |)i'ol)al)]y he recon-
ciled i r 1 lie teiiiix'i'al ui'es at which crystalli/at ion was found
to 1)0 conij)lete, according' to the first principle, are ]jre-
C'isely the teniix'ratures at which the crystals cease to .i>i'OAv,
accordiiii*' to the second. The first i)riiiciple sliould then
be abandoned since the facts alleg-edly ex])laiiied by it are
really consequences of the other ])i-inciple.

IV. THE FROZEN STATE
PROPERTIES OF ICE AND OF FROZEN SYSTEMS

A historical and critical review of the investigations on
the physical constants of ice, and a large bibliography, Avill
be found in H. T. Barnes' Book "Ice Engineering" (1928).
The subject having thus been reviewed, we shall merely
mention the essential established data, indicate the use that
the biologists have made of them and suggest some other
applications. Most of our information is obtained from
Barnes' work.

1. Mechanical Properties of Ice. The plasticity of ice
is well exemplified in the movement of the glaciers; ice
really flows. The velocity of flow depends on the tempera-
ture and on the pressure exerted and it is relatively high
in large masses of ice, such as the glaciers (for figures, see
H. Hess: "Die Gletscher" 1904).

Andrews (1885) studied ice deformability at different
temperatures by measuring the degree of penetration of a
steel rod applied to blocks of ice. He found that the resis-
tance to penetration varied little when the temperature
was raised from - 40° to - 9° but decreased rapidly from
-9° to 0°. This considerable plasticity at a few degrees
below the melting point is probably the cause of the trans-
formation of the ice pattern observed by Luyet and Gibbs
(1937) in frozen epidermal cells of plants (Fig. 26).

The flow of ice does not seem to result, as it is usually
thought, from a partial melting followed l)y recrystalliza-
tion, but it consists probably in a sliding of the crystalline
planes one over the other (McOonnell, 1891). In some
other crystalline materials, gliding lines can occasionally

167

Fig. 26. Drawings 1 to 7 : Transformations in epidermal plant cells while
in the frozen state. (From Luvet and Gibbs, 1937.) Drawing 8: thawing
cell. In all the drawings, the fluid sap is represented in black and the ice
in white.

be observed and for some metals, the degree of ductility
and malleability can be related to the number of gliding-
planes ((■/. Tammann, 1925, p. 192, sqq.). Even the classi-
cal experiments known as "regelation" in which a piece
of ice gives way under the pressure of a wire applied on it,
should perhaps be explained, at least partly, by the gliding-
motion of the crystalline planes.

According to McConnell, single ice crystals are "per-
fectly brittle." However, since, in general, the fluidity
of a crystal depends on the temperature, some crystals be-
ing so fluid near the freezing point that their faces bulge
out or that their edges bend in a curve line under the action
of gravity {cf. Tammann, 1925, p. 291), it seems evident
that the individual ice crvstals should also show some

168

lluidity at li'iiiix-'raturcs iicai" thi' meltiiij;- jK)iiit. A study
of tliis property, namely, of tlie degree of fluidity of the
iee needles in formation, near 0°, would lielp in solving the
problem of the mcclianieal injury whieh ean be produced in
protoplasm by ice crystals.

Some physicists discuss the viscosity of ice and, in gen-
eral, of crystalline substances. They assume that in a
crystalline body there are layers of molecules in the licpiid
state which give to the system the consistency of a '^viscous
solid." Hess (1902) observed that the presence of sand
and dust increases the viscosity of ice. This observation
might have some importance for the biologists since, in
biological material, a number of foreign substances
admixed with ice can modify its viscosity.

Among the other mechanical properties of ice, let us
mention the tensile strength and the compressibility which,
according to Hess (1904), amount respectively to 7-8 kg.
and 25 kg. per sq. cm., and the crushing strength which
was found to vary from 23 kg. to 70 kg. per sq. cm. {cf.
Barnes, 1928).

The strength of a layer of ice 18 inches thick would be
such that it could support a railroad train.

2. Specific Gravity. Bunsen, in 1870, obtained 0.91676
as the specific gravity of ice at 0°. Barnes considers this
value as a **good average of all the latest and best measure-
ments." The specific gravity of water at 0° was found by
Chappuis (1897) to be 0.9998674. Due to the high pre-
cision of density determination, the method of utilizing the
difference in the specific gravity of ice and of water in the
study of such problems as that of the quantity of ice formed
in biological material at a given temperature, might prove
valuable.

3. Thermal Coustants. Maass and Barnes (1927) give
0.4873 as the specific heat of ice at 0°. Callendar (1912)
found 1.00934 for water at 0°.

The biologist working with low temperatures occasion-
ally needs to know the specific heat of ice from 0° C. to the
absolute zero. We represented in Figure 27 the values

169

■150

-200

250

•27 3

0.5 0.4 0.3 0.2 0.1

Fig. 27. Specific heat of ice in terms of temperatui-e. (Curve drawn from
the data of Maass and Barnes, 1927, and of Xernst, 1910.)

obtained, from 0° to -80°, by Maass and Barnes (1927)
and, from - 100° to - 200°, by Nernst (1910) . As an aver-
age between - 188° and - 252.5°, Dieterici (1903) gives the
value 0.146°. Since the curve is practically a straight line
at the temperatures plotted, one can, in calculations involv-
ing the amount of heat withdrawn for cooling some mate-
rial between two points within this range of temperatures,
take the average specific heat between the two points.

The heat conductivity of ice at 0° is 0.00573, according
to Neumann (1862) ; that of water at 0° is 0.00120, accord-
ing to H. F. Weber (1880).

The difference in conductivity along the axis of the
crystals and in a perpendicular direction was found by
Trouton (1898) to be practically negligible, the ratio of the
two being 22 : 21.

We did not find any data on the heat conductivity of ice
at very low temperatures. Lees (1908) has investigated

170

the licnl coiKliictlxil}" ol" st'V('i-aI metals a1 KJO . Ilis
rt'sulls iiulicatc thai in sonic ol' tlicni llic conduclivity is
lowiT at low tlian at lii*>Ii temperatures while in others it is
liiglier.

According- to Dickinson, Harper and ( )sl)orn (1914), the
heat of fusion of ice is 79.68 calories.

It is to l)e noticed that the heat of fusion decreases by
0.59 calories for every degree of lowering of the fi-eeziiig
point.

Barnes aud Vipond (1909) found that the heat of evapo-
ration of ice varies from about 600 calories in rapid evapo-
ration to about 700 in slow evaporation. It is not the sum
of the heat of fusion of ice (79 cal.) and of the heat of
vaporisation of water (598 cal.), but it tends, in rapid
evaporation, to become identical with the latter. This
renders probable the assumption that ice evaporates with-
out passing through the liquid phase, the molecules escap-
ing from the ice in the state of a polymeric vapor.

According to Washburn (1925) and others, the vapor
pressure of ice varies from 0° to - 90° as follows :

T.

V.P.

T.

V.P.

T.

V.P.

0°

4.579

-30°

0.286

-70°

0.002

- 5°

3.013

-40°

0.097

-80°

0.0004

-10°

1.950

-50°

0.030

-90°

0.00007

-20°

0.776

-60°

0.008

The coefficient of linear expansion of ice, as determined
by Petterson (1893) is 0.000053 between -2° and -10°.
The coefficient of cubical expansion is 0.0001620 according
to Nichols (1899).

4. Electrical Properties. It is known that ice is a good
insulator; its specific resistance at 0° is given by Johnson
(1912) as 0.367 X 10\ That of distilled water at the same
temperature is 0.5 X 10'".

We mentioned above that the difference in the electric
resistance of ice and water has been used by Moran (1935)
for the determination of the eutectic point of the muscle.

The specific inductive capacity of ice at - 5° w^as found
by Thwing (1894) to be 2.85 (wave-length of measuring

171

current: 1200 cm.); that of water at 18° is 81.07 (wave-
length practically infinite) as determined by Turner
(1900). Ice at the temperature of liquid air has a specific
inductive capacity of 1.76 to 1.88 (wave-length : 75 cm.) ac-
cording- to Behn-Kiebitz (1904). (For quotations see the
Smithsonian Tables.)

5. Optical Properfies. Ice is birefringent ; the refrac-
tive indices, as given by Reusch (1864) are 1.30598 for the
ordinary ray and 1.30734 for the extraordinary ray (red
light). ■ '

V. MELTING AND MELTED MATERIAL
A. THE PROCESS OF MELTING

Although there is an abundant biological literature on
the possible injurious and lethal etTects of thawing, the
process of fusion itself has been little investigated, far
less than the process of freezing.

Miiller-Thurgau (1881) attempted to study the course
of thawing in a few tissues. He established, though with
rather crude methods, some thawing curves. These, when
compared with the corresponding freezing curves, pre-
sented some characteristic ditTerences which later investi-
gators discussed in more detail.

Fischer (1911), after discussing some of the results
which he obtained with Bobertag (1909) in freezing and
thawing various carbon compounds, analyzed the afore-
mentioned results of Miiller-Thurgau. He concluded that
the amount of heat liberated in freezing is not the same as
that absorbed in thawing and he attributes this dit¥erence
to some endo- and exothermic processes concomitant with
freezing and thawing.

Maximow (1914) compared freezing and melting curves
of potato tissues and noticed, in particular, that the freez-
ing point was more than half a degree lower than the
melting point. As to the difference observed by Fischer
between the heat liberated in freezing and the heat ab-
sorbed in thawing, he showed that it is a question of pro-
cedure ; the difference is reversed if one changes the
cooling velocity in the freezino- curve.

172

One slioiild naturally expect that the course of melting
be diiforent t'l'om tliat of freezing;', since, in frozen tissue,
the two constituent phases are se])arated, one of tlicni,
water, hciu^' in tlic iiilci'cclhilai's, the ol hei', partly dehy-
drated i)r()t()plasm, remaining- within the cell; a frozen
tissue to be melted is, therefore, a quite different system
from an intact tissue to be frozen.

More investigation on melting curves of tissues, in par-
ticuhir of tissues wliich survive a ])artial freezing and still
possess semipermeable membranes, would throw some
light on several questions related to osmosis during freez-
ing and thawing and, in general, on the forces binding-
water in living protoplasm. A comparison between melt-
ing curves of living and of dead tissues might also prove
illuminating.

B. ALTERATIONS OBSERVED IN THAWED MATERIAL

The fundamental alteration caused in aqueous solutions
and suspensions, in aqueous colloids and in protoplasm by
freezing is the separation of ice. If, when ice is thawed,
the system can be restored to the condition in which it was
before freezing, in other words, if the phase separation is
reversible, the material might be unaltered. In this sense,
the true solutions are, at least on first approximation, un-
altered by freezing. Whether or not in suspensions, in
colloids and in protoplasm the phase separation, or any
other alteration, is reversible upon thawing, is the subject
of the following inquiry.

1. Suspensions and Colloids. Vogel (1820) observed
that starch paste loses its adhesive properties after being
frozen. This is one of the oldest reports concerning a
permanent change induced by freezing in a colloid.

According to Goeppert (1830), when the milky plants
Euphorhia and Ficiis are frozen, the milk is irreversibly
transformed into a watery fluid.

Ljubavin (J. Buss. Phys.-Chem. Soc, 21, 397, 1889) was
the first, according to Lottermoser (1907), to show that a
hydrosol can be converted into a gel by freezing. He also
pointed out tlie influence of electrolytes on that conversion.

173

Bredig {Ztsch. f. angeiv. (Item., 954, 1898) described a
precipitation of platinum and of othci- metals fi-om col-
loidal solutions, after melting'.

Lottermoser (1907 and 1908) confirmed these results
and, in particular, the fact that the lower the electrolyte
content of a sol is, the easier it acquires the property of
precipitating after thawing (although there are excep-
tions). Under ordinary conditions, hydrosols with high
electrolyte content, such as silicic acid, undergo no change
by being frozen and thawed; but when their electrolyte
content has been reduced by dialysis they can be precipi-
tated. The changes in consistency were concomitant with
changes in electric conductivity. The author considers
precipitation a consequence of the withdrawal of water
during freezing. The fine structure of the gel, he suggests,
is destroyed by the increase in volume of solidifying water.
The particles of the colloid set free in that breaking down
of the structure are wedged between the expanding ice
crystals where they are transformed into leaflets which,
after thawing, precipitate to the bottom of the vessel.

Bobertag, Feist and Fischer (1908) studied the action
of congelation at -10°, -70° and -180° on a large number
of substances belonging to various groups : true solutions
of dyes, such as eosin, safranin, etc. ; partly colloidal solu-
tions, fuchsin, methyl violet, etc., colloidal solutions of
Congo red, benzopurpurin, etc.; colloidal solutions of
tannin, gums, starch, agar-agar, gelatin, albumin and
hemoglobin; colloidal solutions of metal and metal com-
pounds. Some dyes were partly decolorized after thaw-
ing, but usually the previous color was resumed later,
although in some cases it took 2 days for the restitution of
the original condition. Tannin solutions precipitated
above the freezing point; on thawing, the precipitate fell
to the bottom of the container and, on rewarming, it dis-
solved again ; this is the ordinary behaviour of a solute of
which the solubility decreases at lower temperatures.
Gum, starch and hemoglobin solutions were turbid immedi-
ately after thawing, but cleared up entirely afterwards.

174

Agar-agar and golatiii solutions sliowcd, for ([uilc a long
time after thawing, a donhlc i)liase sti-ncturc consisting
of t'lunij)s of jelly and of a dilute li(iuid. Albumin solu-
tions stayed jH'i-mancntly lurhid aftci' treatment at -70°
and -ISO but not after being frozen at -10 . Platinum
and gold were precipitated from their colloidal solutions
by a freezing at -70°. Solutions of arsenic trisulfide and
of antimony trisultide also ))i-ecipitated. A dilute solu-
tion of iron hydroxide {li(iu<)r fcrri (Ualifsafi) did not pre-
cipitate, but pi-esented an inci'eased Tyndall etfect. A
solution of sodium silicate remained entii-ely unaltered.
In silver preiKirations, such as protargol, kollargol and
lysargin, the silver agglomerated into clumps, irregularly
distributed in the ice; but, after thawing, the preparations
were just the same as before freezing (a protective effect
of albumin is thought to be resi)onsible for the reversible
action in these preparations).

The authors think that, in all the above solutions, solute
particles are brought together during congelation and
that, after thawing, they may or may not be dispersed
again, depending on the nature of the substance. Be-
sides, some colloids would be irreversibly dehydrated l)y
freezing.

According to Bruni (1909), colloidal isinglass, frozen at
-20° or at -80°, formed, after thawing, a gel which could
not be distinguished from the original one. But colloidal
silicic acid, treated in the same manner, consisted, after
thawing, of two phases, one of which was pure water and
the other, a precipitate of amorphous leaflets of hydrated
silicic acid.

In 1911, Fischer reviewed the investigations of most of
the previous workers, including those which he had made
himself with various collaborators, and he concluded that
colloids show an extreme variation in resistance to cold.
In general, the changes which they undergo on freezing
are reversible but, for some colloids, at some detinite tem-
])ei'atures, iirevei'sible changes take ])lace. The tempera-
tures at which these iri-evei'sible pi-ocesses occur are some-

175

times determined by the age and previous history of the
material.

Bottazi and Bergami (1924) studied coagulation by
freezing in ox and dog sera and in Octopus blood. The
material, in glass containers with parallel faces 15 mm.
apart, was left for a given time in a freezing mixture, then
allowed to thaw and its turbidity was judged by the degree
to which it obscured a series of dark lines drawn against
the back wall of the container. They found that a con-
gelation of ox serum at -20° gave, after thawing, a liquid
as clear as before, except sometimes for a trace of opales-
cence. So, temperatures wiiich solidify all the water of
the proteins do not denaturate and coagulate them irre-
versibly. At higher, non-freezing temperatures, for ex-
ample, at -0.9°, the sera would coagulate partly if they
were dialyzed for 10 days and so deprived of the major
part of their electrolytes, but the coagulation process was
entirely reversible on rewarming. This partial coagula-
tion was easier to obtain if the sera were diluted with
water. The addition of NaCl to coagulable sera pre-
vented coagulation. The addition of hydrochloric acid
caused the sera to become turbid when the isoelectric point
was approached ; coagulation was, however, always rever-
sible. The serum, it is concluded, is a rather stable col-
loidal system, which becomes labile when it loses its electro-
lytes and when it is acidified near the isoelectric point.
The hemocyanin of the blood of Octopus macropus was
found still more stable. It did not coagulate at -3° or -4°
even after a month of dialysis.

According to Callow (1925), repeated freezing irrever-
sibly destroys the structure of gelatin gels.

Moran (1925) observed that frozen egg yolk loses its
fluidity on thawing and takes on a stiff pasty consistency.
This change, however, occurs only if the following con-
ditions are fulfilled: 1. The yolk must have been cooled
below -6° (called by Moran the ''critical temperature of
freezing"); a stay of several months above -6° did not
change the volk consistencv ; 2. Congelation must actually

17(i

lia\'(' taken {jlace; «'i;i;s siibcoolcd and niaiiilaiiicd at -11°
tor 7 days were fluid on thawing'; .'>. Tlie malcrial must
stay a certain leug'th of time in the frozen condition; tlie
degree of stiffness increases with tli«d lengtli of time up to
about 20 hours and then it stays constant ; 4. Thawing
must not be too rapid; yoli-: immei-sed in licjuid air (24
hrs.) and then thawed at room temperature was pasty,
while it was fluid wiien thawed in mercury at + 30°.

Moran explains these facts by assuming that the pro-
tein of the yollv, vitellin associated with lecitliin, wliich is
known to be soluble in lO''/ NaCl, gets dissolved in tlie
salt solution of the yolk wiien this solution is concentrated
by freezing. The "critical temperature of freezing," -6°,
would be the freezing point of that solution. On thawing,
the protein would irreversibly precipitate out. In an at-
tempt to verify this theory, he froze a dilute solution of
NaC^l containing some lecitho-vitellin and observed, on
thawing, a persistent cloud.

During cooling and freezing, the yolk underwent some
unexpected changes in volume, which were measured with
a dilatometer. These changes can be summarized in the
4 following points : 1, The volume of the yolk exposed for
3 days at -11° and thawed decreased by 0.0084 of its origi-
nal value (measured at 0°) ; 2. The coefficient of expansion
of the thawed yolk was higher than that of the unfrozen
yolk; 3. The contraction of the normal unfrozen yolk,
which was linear with the temperature down to 0°, showed
a higher rate in the subcooled condition from 0° to -7° ;
4. The volume of the frozen yolk, measured at -7°, in-
creased by about 0.00015 of its original value when the
material stayed for 3 days at -11°, but it decreased below
the original value when the material stayed 9 days at -11°.

Moran ascribes the decrease in volume resulting from a
long stay in the frozen state at -11° (under 4, above) to
the dissolving of the lecitho-vitellin of the yolk in the salt
solution; dissolving proteins has been observed often to
result in a decrease in volume. The long time involved
would !)(' a1ti-il)ntable to the breaking down of the struc-

177

tare (hreakiiig down which results in the liberation of the
proteins) rather than to the duration of the solving pro-
cess. The increase in volume after a relatively short
time in the frozen state (under 4, above) is attributed
tentatively to the destruction of the capillary structure and
to a reduction of capillary pressure. The decrease in vol-
ume on thawing- (under 1 above) is more puzzling since a
precipitation of the dissolved protein would increase the
volume. Moran suggests that a process comparable to
the swelling of proteins, which is known to result in a final
shrinkage of the entire mass in which they are held, takes
place in the precipitated flakes of the yolk. The decrease
in volume during subcooling (under 3 above) is ascribed to
an increase in the quantity of bound water, the degree of
hydration of stable hydrates tending, in general, to in-
crease at lower temperatures. The suggestion is made,
finally, that this decrease in volume, observed in the sub-
cooled state, might be the beginning of the same decrease
which was observed after thawing, and which could even-
tually become an irreversible change if subcooling were
maintained long enough.

Moran reported also that the irreversible changes just
mentioned do not take place when the yolk is frozen in
presence of a lOYc sucrose solution (at -11°). He com-
pares this phenomenon to what happens in plants which
are rendered winter-hardy by an increase in their sugar
content. (Such protective action, according to Shrivas-
tava {J. Phijs. Chem. 29, 166, 1925), would be due to an
adsorption of sugar on colloidal particles.)

The permanent modifications produced in yolk by
freezing w^ere studied microscopically. Normal untreated
yolk, fixed in Miiller's fluid, consists of polygonal masses
(the yolk particles). Frozen yolk, fixed in the same man-
ner, presents a horny appearance. The author explains
this by a rupture of the yolk particles on freezing, a
rupture which can be obtained also by stirring.

Nord (1927-1938) and his collaborators investigated
the effect of freezing, for various lengths of time, at tern-

17S

l)i'raliir('s cxlciuliiin' t'roin 0 lo -180°, on tlic ijliysical (miii-
stants of colloidal solulioiis of a laru'o inimbor of siib-
staiic'cs, such as, zymase, (\u<»' al])umiii, uclaliiic, ii,iim
ai'al)ic, saponin, mclacliolcstcrin, myosin, sodium olcalc,
casein, polyaci\\lic acid and its esters, j)oly\-iiiyl alcoliol,
etc. In geiier<d, dilute solutions (0,001 lo l.OV' ) of these
sul)stances exhibited, aftei' liaviui^' ])een frozen and thawed,
liii»her diffusion coefficient, interferometer value, viscosity,
catai)lioretic mii'i-ation velocity, specific conductivity and
also an increased absorption of snch ii,ases as ethylene and
acetylene. More concent lated solutions (2'/) showed,
after freezing and thawing, lower values of these constants
than did the nnfi'ozen controls. The authoi-s conclude
that, in dilute solutions, freezing effects a disaggregation
of the individual particles, whereas, at higher concentra-
tions, it i)i'oduces an aggregation of these primary
particles.

In a study of the surface tension of frozen and thawed
egg albumin and gelatin solutions, Nord (1934) found that
this property increases with the duration of freezing but
not with its repetition nor with the exposure to very low
temperatures.

Heller (1934) studied coagulation by freezing in sols of
FeCL, Fe(NO.O* and FeOOH, in terms of hydrogen-ion
concentration, of duration of dialysis and of the length of the
time of exposure to low temperatures. He found : 1. That
coagulation never occurred when the pH was lower than
6.1; 2. That a dialysis of 69 days induced coagulation in
a case in which a dialysis of 3 days was ineffective ; 3. That
the length of time the material was maintained in the
congealed state, often increased the degree of coagulation.
Comparing these results Avith those obtained in coagula-
tion by stirring and noticing similar conditions in the two
cases, he concluded that coagulation by freezing must re-
sult from a fusion of the colloidal particles under the com-
pression exerted by ice. In freezing, as in stirring,
coagulation would be possible only when the '* double
laver" at the surface of the micelles is weakened; such a

179

condition is obtained l)y dialysis. He attributed Ihe in-
fluence of tlie length of time in the frozen state to a re-
sulting- slow increase in the size of the particles; in
larger particles peptization after thawing would become
impossible.

2. Tissues. The alterations caused by freezing in tis-
sues consist in changes in the general appearance, color
and consistency of the material, in a release of enzymes
and other substances, in some chemical transformations, in
a modification of the protoplasmic structure and in some
cytological changes. A few examples of these various
alterations will be described shortly, in the order given.

Some externally observable injurious effects of freezing
in plants, and particularly in leaves, are well known by the
agriculturists. Following Goeppert (1830), who is one of
the earliest observers, we shall mention a blackening of
the surface or of the entire organ, a more fleshy consis-
tency, a somewhat transparent, shiny and glassy appear-
ance, a loss of turgor, an exudation of water and a rapid
desiccation. Flowers become, in general, dark browai.

Goeppert remarked a change of color of another type in
some white orchids which, after freezing, become pale blue
and finally dark blue. This property has been used as a
criterion of death by several investigators in their studies
of injury by cold.

Other changes in color have been described by Kylin
(1917) in algae.

Molisch (1897) noted that some plants which contain
coumarin release that odoriferous substance on being
frozen.

The release of enzymes by freezing and the consequent
increased enzymatic activity, in some tissues, is a commonly
observed fact.

Formation of sugar under the action of cold has been
reported by many investigators. The literature on ^^in-
ter hardening in plants contains abundant data on this
subject.

Luyet and Grell (1936), in centrifuging onion root tips

isn

whicli were ]tr('\iously frozen ;»1 about -80° and thawed,
found that tlu' colhihir constituents couhl ])e stratified
without ditlfic'iilty wliih' in tissues killed by heat no dis-
placement was possible. Accordingly, they think that
freezing does not induce protoplasmic coagulation as read-
ily as do other lethal agents, such as, heat, toxic substan-
ces, high pressure, radiations, mechanical injury, etc. This
is confirmed by the absence, in frozen tissue, of the char-
acteristic opacity observed in tissue killed by the other
methods.

However, Luyet and (libbs (1937) published photomicro-
graphs sliowing tiie ])rotoplasm coagulated in frozen and
thawed plant epidermal cells. The pattern of this coagu-
lum was different than that obtained in death by heat.
The authors remarked a difference in the consistency of
the two types of coagula, which might explain why the com-
l)onents of one type could be separated by centrifugation
while the components of the other could not. The coagu-
lated material was found in a thin layer adhering to the
wall of the cell in the same position as in the living state.

Bugaevsky (1939) also observed coagulated flakes in the
cells of wheat after freezing and thawing.

The tearing of cell membranes by ice has been assumed
by the earliest biologists. Goeppert (1830) contended that
he could never observe any broken membrane. After him
several authors have contributed to establish the fact that
there is never a perforation of the membranes by ice crys-
tals (for quotations see above, under the title "Phase
Separation" in tissues). But, if the membranes are not
broken, it is cei'tain that they are damaged and that they
lose their semipermeability, as is evidenced by the fact
that the cellular fluids cannot be maintained any more
within the cells and that the penetration of dyes becomes
considerably easier. Prillieux (1869a) however, con-
tended that there is more evidence for an injury to proto-
plasm than to its membranes.

Matruchot and Molliard (1902) reported the particular
phenomenon of the vanishing of the chromosomes in a

181

terminal cell of the staminal hair of Tradescantia put to
freeze at -12°. Some 18 hours after thawino-, the chromo-
somes had reappeared and cellular division had pro-
gressed. The cell was apparently not killed.

Lenoir (1931) describes, in the cells of the ovary and of
the nucelle of Frit ill aria imperialis, after freezing, swollen
chromosomes or spiremes and a disorganized linin network.

According to Detmer (1886), chloroplasts are disaggre-
gated after freezing in the same way as when plants are
killed by heat or by acids.

Several experiments on tissues described above under
''Phase Separation" contain information on the subject
discussed here.

SUMMARY

1. The formation of crystalline nuclei in a liquid is gen-
erally assumed to be initiated by collisions of molecules in
some definite conditions of orientation. 2. Crystallization
of water is sometimes considered as a precipitation of tri-
hydrol dissolved in dihydrol. 3. There is some evidence
that the formation of ice is preceded by a colloidal state.
4. It has been suggested that liquid water possesses a
quasi-crystalline structure and that little molecular rear-
rangement is required for obtaining the crystalline state.

5. The freezing point of culture media varies from
- 0.01° to about - 2.5°. 6. That of plant juices was found
to lie between -0.3° and -2.5° for most species. 7. That
of the blood of aquatic animals is, in general, about the
same as that of their medium. 8. The individual differ-
ences in the freezing point of blood can be as much as 0.3
degree (in birds). 9. The freezing point of eggs follows
a cycle, being nearly the same as that of the mother's blood
during the period of their formation, then decreasing con-
siderably and rising again during embryonic development.
10. Living protoplasm (myxomycetes) was found to freeze
at the relatively high temperature of -0.17°. 11. Animal
and plant tissues congeal at temperatures extending from
about - 0.4° to - 3°. 12. The freezing point of living tis-

^s'2

sm's is, ill iicncrnl, lower lliaii llial ot" dead inalcrial. l.'\
Living- tissues sometinics pii'sciil two t'rcczini!,' jioiiils, one
of wliich is proliably that ot" tlu' iiiterccllulai', the other,
that of the exuded iiiti'aeelhihir fluids.

14. Till' uTowlli of crystals is less ra])i(l when the tem-
IX'i-ature is lower. 15. The iioii-i)a]"allel variations in tlie
velocity of formation of nuclei and in the velocity of
growtli of crystals, in terms of temperature, result in a
dependence of number, size and form of crystals on tem-
])eratiire. 1(). The velocity of growth of ice crystals is
about 65 mm. per second at -8"; the addition of solutes
reduces this velocity (39''^ gelatine reduces it 350 times).

17. The separation of water during the congelation of
solutions, suspensions and colloids can often be observed
under the microscope. 18. In a gel cooled slowly, w^ater
comes out to the surface where it freezes in a compact
mass, while, on a more rapid cooling, internal congelation
takes place. 19. Internal congelation of gels sometimes
results in the formation of spherulites within which one
can observe two separated phases, ice and dehydrated gel,
in alternating concentric layers. 20. In coagulated mate-
rial, in porous structures and in tissues, water exudes to
the surface during freezing and forms tiny ice columns
which fuse laterally into ribbons or plates. 21. (^avities
within frozen tissues are found filled with ice which has
separated from the surrounding cells ; intercellular spaces,
in compact tissues, play the role of cavities and lens-shaped
masses of ice accumulate in them. 22. In the case of sin-
gle cells exposed to slow freezing, water exudes and con-
geals at the surface. 23. The Avithdrawal of water during
freezing has been observed also in cellular constituents, in
particular in nuclei, possibly in chromosomes.

24. The freezing curve of an ideal liquid consists of a
linear and of two exponential portions. 25. The freezing
curve of a solution consists of a hyperbolic, a linear and
two exponential portions. 26. The plateaus of the freez-
ing curves of most living tissues occupy a lower position,
are shorter, less uniform and more inflneiiced by cooling

183

velocities lluui lliosc obtained with dead matci-ial. 27.
There is little evidence of a eiitectic freezing- in tissues.
28. The quantity of ice formed per unit of time decreases
gradually, in the course of freezing.

29. In colloids and in tissues some ice formation has
still been observed below -20°. 30. It took 26 days, at
- 11°, for the completion of crystallization in a gelatin gel.

31. The following properties of ice are discussed : i^las-
ticity, fluidity of single crystals, viscosity, tensile strength,
compressibility, suppoi'ting force ; specific gravity ; specific
heat, heat conductivity, heat of fusion, heat of evaporation,
vapor pressure, coefficient of expansion; electrical resis-
tance, specific inductive capacity ; birefringence, refractive
indices.

32. The melting curves of tissues differ from the freez-
ing curves in particular by the position of the melting point
and by the "curve-area" representing the quantity of heat
involved in each of the two processes.

33. After freezing and thawing, some colloids stay unal-
tered while others are irreversibly precipitated. 34. The
causes of precipitation assumed by various investigators
are: a compression between the ice masses, a change in
electric charge during freezing or thawing, a change in
chemical structure and the reaching of the saturation tem-
perature. 35. Alterations of tissues by freezing include:
changes in external appearance, release of enzymes, inter-
nal chemical changes, disaggregation of cellular constitu-
ents, and protoplasmic precipitation. 36. The latter seems
to be of different nature than coagulation by other lethal
agents.

CHAITKR II

SUPERCOOLING AND THE SUPERCOOLED STATE

A liquid is said to ])(' sii])orcoolod (or snbcooled, or
luidorcooied) wiicii it is still in the liquid slate at a tem-
perature lower tliaii the fi'eezing point.

The fiindameiital character of the snbcooled state is to
be unstable. The circumstances which control the passage
of a snbcooled liquid into the crystalline state are very
incompletely known. The following are often mentioned
in textbooks of ])hysics: stirring, shaking, jarring, touch-
ing with a solid body, keeping in presence of impurities,
maintaining in contact with the air, mostly with dust-con-
taining air, etc. On the other hand, crystallization is said
to be prevented when the liquid is heated before being-
cooled, when it is reduced to small droplets, when it is
enclosed in capillary tubes, etc. It is difficult to say to
what extent some of these various ways of inducing or
preventing crystallization, wiiich are sometimes effective
and at other times not, have really been observed, and how
frequently they entered the physical literature through the
back door of a too conservative teaching of insufficiently
controlled observations. A consideration of their mecha-
nism of action might help one to correlate the data
concerning their efficacy.

It is generally thought that the first crystallization
nuclei originate from the collisions of the molecules and
that any factor liable to bring the molecules in closer prox-
imity should facilitate congelation while any factor render-
ing more difficult the collision of the molecules should delay
congelation. Initiation of crystallization should, then,
depend on the number of molecules present, on the time
allowed for the molecules to collide and on the speed of
motion, that is, on temperature. The presence of all-
formed crystals, which need only to grow, is expected
to induce congelation. A mechanical disturbance might,
by a sudden change in the orientation of the molecules,
increase the chances of successful collisions. On the

184

185

other hand, the destruction, by previous heating, of nuclei
in formation, should render crystallization more difficult.
The hindrance of molecular motion by capillary forces,
in thin tubes or in small droplets, should delay the for-
mation of centers. Finally, if the formation of nuclei is
a random process subject to failure according to the laws
of probability, one should expect some irregularity in the
success of experiments designed to induce crystallization,
under apparently identical conditions. We shall discuss
below, after an introductory analysis of a typical sub-
cooling curve, the most important of the factors just men-
tioned, which either cause or prevent crystallization in a
subcooled liquid.

Subcooling is generally considered an exception to the
rule that a substance crystallizes at a definite temperature.
It seems more logical, however, to consider subcooling as
the general condition, and crystallization at a fixed tem-
perature as a particular case, since the fixed freezing tem-
perature is only one, the highest, of the several tempera-
tures at which a fluid can start crystallizing, and since
congelation will not take place at the freezing point except
in the particular case w^here a crystallization center, that
is, an infinitesimal crystal, is present. Accordingly, it
would be preferable, from this point of view, to define the
freezing point not as the temperature of solidification but
as the highest temperature of solidification.

The problems which concern the biologist in the study
of subcooling are : 1. Whether, to what extent and under
what conditions the fluids present in living animals and
plants, in tissues, in cells and in protoplasm, are brought
into, or barred from the subcooled state. 2. How life is
affected by this state and, in particular, whether injury
and death result from it or from its cessation? Only the
first of these two problems belongs to the program of the
present chapter, the other will be investigated elsewhere.

I. THE SUBCOOLING CURATE
When a liquid is subcooled, the curve representing the
drop of temperature in terms of time, AB, (Fig. 28) con-

186

B"B'

Fig. 28. SulK-ooling curvo of a liquid under ideal conditions. Abscissae:
time; ordinates: teni])eraturi'.

tiiiiies its course, past the line of the freezing point BB',
down to the suhcooling point C. It then rises abruptly
to the freezini*' point B'. The subcoolino' curve consists of
2 limbs AC and CB'. The temperature interval CB" is
the degree of suhcooling.

AC is an ordinary exponential cooling curve.

Concerning the curve (/B' we shall discuss briefly two
questions : that of the maximum reached by the curve and
that of the calculation of the freezing-curve-areas (see
above, under "Freezing Curves") in cases in whicli there
is some sub-cooling.

The problem of the maximum reached by the rising tem-
perature can be treated as follows. Three factors influ-
ence the temijerature, one of which tends to raise the
maximum whik' the other two tend to lower it. These
three factors are: 1. The amount of heat Qi liberated by
freezing, during the time B"B' ; 2. The amount of heat Q-
withdrawn by the cooling bath, during the same time. 3.
The amount of heat Q-, used for bringing up the tempera-
ture of the material from the suhcooling point. Qi is
either equal to the sum Q- - Q:., or it is smaller or larger

187

than this sum. In tlie first instance, the temperature will
just reach the freezing point ; in the second, the tempera-
ture will rise to a maximum somewhat below the freezing-
point ; if the third were possible, the temperature would
rise above the freezing point ; but this cannot happen since
the heat produced by the advancing head of a crystal in
formation will first raise the temperature of the fluid im-
mediately in contact w^ith it, and when this local temper-
ature, precisely in the area where the crystal is to grow,
reaches the melting point, all crystal growth will stop. So,
after subcooling, the temperature never rises above the
freezing point, it reaches the freezing point in two of the
three cases given and remains below the freezing point
in the third.

When the freezing point cannot be reached, it is almost
always because Q.. is too high, in other words because
cooling is too rapid. The following example gives an idea
of the order of magnitude of the cooling velocity required
to prevent the ascent of temperature to the freezing point.
Let us suppose that, out of a total mass of 10 grams of
water supercooled at -4°, 1 gram freezes out, and that one
half of the heat liberated, that is, 40 calories, are with-
drawn by the cooling bath during freezing (during the
time B"B'), there is still enough heat left (40 calories)
to bring all the water from -4° to 0°. To prevent the rise
of temperature to 0°, the cooling velocity should then be
such that more than 40 calories be withdrawn during the
time B"B' (a fraction of a minute). This is a very high
cooling velocity.

Some investigators, having simply admitted (perhaps
from a too docile belief in classical statements) that, at
crystallization after subcooling, the temperature reaches
the freezing point, have made valueless determinations of
the latter. With such cooling velocities as 10 degrees per
minute, the temperature might not have the time to rise
from the subcooling point to a maximum and it will simply
be retarded in its drop (cf. Fig. 7, Curve 5) ; or if it
reaches a maximum, there is no evidence that this is the
freezing point.

188

111 li\iiig- tissues one has to consider tlie fact that water
is \villi(li';nvii from the cells before it freezes, that crystal
••rowlli is llicii i-etarded by this slow osmotic ])rocess and
that, tlicrcroi-c, less heat of crystallizniioii is lilx'i'ated
duriiii!,' the time B"B'.

In the application of the method of the freezin<i;-cnrve-
areas to cases in which there is some snbcooling-, one
should remember that this method is based on the assump-
tion that the heat withdrawn by the bath is equal to the
heat produced by freezing. When there is a snbcooling,
the heat produced is (Hjual to the sum of the heat with-
drawn and of the heat necessary to bring the temperature
from the snbcooling to the freezing point. The heat with-
drawn is proportional to the area limited by the curve
CB'D, (Fig. 28) the time axis and two abscissas, one at
the beginning and the other at the end of the freezing-
period considered. To obtain the heat produced one
should add to the number of calories found in the determi-
nation of this area the number of calories necessary to
warm the material up to the freezing point.

II. FACTORS AFFECTING CRYSTALLIZATION
IN A SUBCOOLED LIQUID

1. Temperature. By bringing the temperature far
enough below the freezing point it is almost always pos-
sible to induce crystallization. The lowest temperature
required for that pui'pose, that is, the maximal degree of
snbcooling, varies greatly with the nature of the substance
studied. Some substances are classically known to pre-
sent a very high degree of snbcooling. Phosphorus and
sulfur, which are often used in demonstration experiments,
can be maintained liquid, respectively, at about 35 and 100
degrees below the freezing point ; iron and nickel have also
been observed sui)ercooled by 100 degrees. According to
Tammann (1925, p. 231), a supercooling of 20 degrees is
quite common in organic compounds. Water is often still
liquid at -3° or -4°, more rarely at -6° or -7°, and
exceptionally, for short times, at -10°.

189

It is generally tlioiight that biological material and, in
particular, living matter can be snbcooled to a larger ex-
tent than water and aqueous solution, and several biolo-
gists attribute to that property the high resistance offered
by some plants to death by congelation. But systematic
investigations on that subject are lacking.

AVhile lowering the temperature induces crystallization
in a subcooled liquid, warming the liquid is supposed to
prevent freezing in a subsequent undercooling. The
crystallization centers, it is assumed, are not quite com-
pletely destroyed when the temperature is raised above
the freezing point, some molecular orientation would be
maintained; but, if the material is warmed to a tempera-
ture far above the freezing point, the nuclei would be en-
tirely destroyed and a high degree of subcooling or a
longer duration of the subcooled state should be possible
at a given temperature.

De Coppet (1907), in experiments on salol heated at
temperatures up to 40 degrees above the freezing point
(which is 42.6°), showed that the higher the temperature
of the previous warming the longer the material stayed in
the subcooled state at a given temperature. However,
there were a number of exceptions.

Othmer (Z. anorg. n. aUg. Chem., 91, 235, 1915) studied,
in a series of experiments on piperonal the number of
crystallization nuclei formed at 2 degrees below the freez-
ing point w^hen the material had previously been heated to
3 and to 33 degrees above the freezing point. He found 228
and 33 nuclei respectively.

Concerning the time that the material is left at a tem-
perature above the freezing point for destroying the crys-
tallization centers, some physicists have expressed the
opinion that it plays no significant role and that the tem-
perature reached is the only factor to be considered. De
Coppet (1907) conducted a few experiments on this ques-
tion and he pointed out, in particular, the results of one
experiment in which, after having maintained salol for
one hour at 68 to 78 degrees above the freezing point, he
could keep it in the subcooled condition for 6 years.

1!)0

Liiyet and Hodap]) (193S1)) iiivi'slioatcd the dcslruclioii
ot" crystallization centers l)y tenii)eratiii'es above the I'reez-
inu' ])oint in potato tissue. They determined tiie pro])Oi--
tion of sul)coolin«>'s and of spontaneous freezings in
material ])reviously heated for two minutes at various
temperatures. After exposure to 95°, 50° or 20°-24°,
the percentage of subcoolings was resj)ectively 100, 95 and
85; after a previous freezing followed by exposure to 20°-
22°, 9°-10° or 1.8°-3° the percentage of subcoolings
dropped to 75, 35 and 0 respectively (about 20 experiments
at each temperature).

2. luoculatioti. Crystallization of a supercooled liquid
can be initiated by bringing the liquid in contact with a
crystal of the same substance. This is called seeding or
inoculatiug. The procedure is used as a routine laboratory
method.

The fact that congelation starts when a crystal is pres-
ent shows that the type of modification that a subcooled
liquid undergoes to produce the first crystallite is an or-
ganization and orientation of molecules. When the first
crystallite is already formed, no such modification is re-
quired, the molecules are simply attracted to the faces of
the crystal on which they deposit.

Inoculation can be performed not only with crystals of
the same substance as the liquid but also with isomorphous
crystals, that is, with crystals in which the directions of
growth are the same. This confirms the view that the
mechanism of construction of a crystal, and evidently also
of a crystallization center, consists essentially in control-
ling the directions taken by the molecules when they enter
the crystalline structure.

Besides isomorphous crystals, some substances are said
to be specific in starting the crystallization of some partic-
ular melts ; for instance, traces of potassium hydroxide or
of nitric acid are used for inducing crystallization of
phosphorus. Assuming that this is an observed fact, one
might attempt to explain it by the theory that certain sub-
stances contribute toward orienting the molecules of other

191

substances on account of some similarity of structure, even
if that similarity is far from that presented by isomor-
plious crystals.

The biologists have tried to inoculate tissues or other
biological materials by touching them with ice crystals.
In general, the method is described as successful although,
here and there, some authors express a doubt as to its
efficacy.

Several plant physiologists used, for inoculating tissues
or organs such as leaves, a method which consisted in put-
ting a drop of water at the surface of the material, with
the idea that water would freeze first and seed the adja-
cent tissue. Others who wanted to prevent crystallization,
took precautions against any moisture on the surface.

Mtiller-Thurgau (1881) claims that potatoes cut into
pieces subcool less than intact potatoes, because of the
easier formation of centers on the cut surfaces.

According to Mez (1905), when a tissue was in contact,
at one point, with the wall of a cold glass container, in-
stead of being isolated from it by air, subcooling did not
occur because of seeding by the ice formed at the cooler
point. This author went so far as to say that he could
guarantee a strong subcooling if a plant tissue was cooled
in oil (he used castor oil) while there was no or little
subcooling if the tissue was cooled in water.

Voigtlander (1909), to obtain subcooling in plant tissues,
removed with absolute alcohol all traces of moisture on
the glassware to come in contact with the tissues and on
the thermo-needle to be inserted in them.

Harvey (1919), for preventing seeding by the extruded
sap on the cut surfaces of leaves and petioles, dried them
and covered them with vaseline. He also reported that
leaves, stems and petioles covered with wax, bloom, or a
thick mat of epidermal hair can be subcooled to lower tem-
peratures than organs not so protected, or than plants in
which the coverings were removed or broken. Such a
protection is attributed to a prevention of seeding. A
drop of water laid on the heavily covered samples did not
seed the tissue below, as it did in cases of nude epidermis.

V.)'2

Wright and Taylor (1921) remarked thai water or sap
from ])riiises on the surfaee of a ])()tato ])i'eveiited that
material from reaching' the degree of undercooling that it
would attain without tiie presence of such moisture.

On the other hand, Luyet and Hodapp (1938b) repeat-
edly obtained subcooling, and often to a considerable de-
gree, when cylindrical i)ieces of potato tissue were exposed
to low temperatures in glass vials previously filled with
water. Moisture, in this case, did not facilitate freezing.
One must notice, however, that this moisture might have
lost its ordinary properties due to the fact that it was held,
in the form of a capillary layer, between the material and
the vial.

Concerning the efficacy of seeding by surface moisture,
it seems that if such moisture can be brought to a lower
temperature than the tissue itself (as, for example, by
contact with a cooler object), or if it has a higher freezing-
point than cell sap, inoculation becomes understandable;
but one cannot see why a film of moisture of capillary size,
mixed with sap at the surface of a cut tissue, would not
subcool as much as the tissue itself.

Luyet and Gibbs (1937) remarked that seeding through
cellular membranes must be impossible in living tissues
like onion epidermis, in which one can observe the sub-
cooled cells freeze one by one, there being sometimes half-
hour intervals between the congelation of neighbouring-
cells, as was recorded previously by Molisch (1897).

3. The Time Factor. Some authors claim that, if a
liquid is maintained long enough in the subcooled state,
the molecules will unfailingly have a chance of colliding
in the conditions of orientation necessary for crystalliza-
tion. This view is based on thermodynamic considera-
tions, but what is somewhat puzzling is that the time
required for the formation of nuclei at low and at high
subcooling temperatures does not seem to vary in proj:»or-
tion to the change in thermodynamic activity of the mole-
cules. While at low temperatures one can maintain the
subcooled state for but a few seconds, at slightly higher
temperatures one can sometimes maintain it for years.

193

Tlie geologists contend that some rock inclusions are in
the subcooled state and that they have been in that condi-
tion for millions of years. If this could be established as
a fact it would serve the purpose of the longest imaginable
experiment to check the theory according to which crystal-
lization always occurs if enough time is allowed for it ; and
it would speak against this theory.

Among observations carried on over definite periods of
time, let us mention that of de Coppet (1907) who main-
tained 16 soliTtions of sodium sulfate in the supersaturated
state, far below the precipitation point, for 33 years.

Moran (1925) kept an egg yolk subcooled for a week at
-11° (that is, more than 10 degrees below the freezing-
point), while, in almost all the other cases observed, sub-
cooling of but a few degrees lasted only several hours.

These few facts, and many others, seem to indicate that
the fundamental theories proposed for explaining the ac-
tion of the time factor in initiating crystallization are none
too reliable. More investigations on the relation between
time and temperature and on the influence of the nature
of the substance on that relation could pave the way to a
deeper understanding of the problem.

4. Mechanical Disturbance. The types of disturbance
usually mentioned as inducing crystallization are : shocks
on the container of the subcooled liquid, vibrations of the
building, the room or the table where the experiments are
made, and shaking or stirring the liquid. The mechanism
of action of the disturbance consists evidently in giving to
the system which is in an instable equilibrium, the last im-
pulse necessary to destroy that equilibrium. It is thought
that this is done by some local compression on the path of
the vibratory waves created by the disturbance.

Despretz, already in 1837, pointed out the frequent in-
efficacy of repeated shaking. Several investigators after
him reported frequent negative results in attempts to
induce crystallization by shocks or by stirring.

De Coppet (1907) could not obtain the congelation of
salol by vigorous and repeated stirring, at a temperature

11)4

of .'>0 to .').') (Ici-rccs below llic fi-ccziiii;' |)oiiit. Willi su])('r-
saliiraleil solutions of sodium sulfate, maiutaiut'd at about
S di'iii-('('s below their saturation temperature, he succeeded
ill 10 out of 101 cases, in causing- crystallization by a con-
tinued stirring of several minutes. In another series of
experiments, in which the temperalui'e was about 9 degrees
below the saturation ])<)iiit, he obtained one crystallization
out of o7 trials. When small scaled tubes containing the
supersaturated solutions were dropped on a Avooden table
from a height of 10 cm,, no congelation occurred.

Bachmetjew (1907) was not able to cause the freezing
of a subcooled butterfly by hammering on the table on
which the insect was lying, or by tapping the wire of the
thermoneedle inserted in its body.

On the other hand, according to AVi-ight and Harvey
(1921), undercooling of potatoes maintained at -4° could
be terminated at any time by a shock.

Wright and Taylor (1921) studied the effect of jarring
resulting from rough handling or incidental to hauling, in
causing the undercooled potatoes to freeze. They found
that dropping them on a hard floor or even tapping them
with a pencil was generally elTective.

Luyet and Hodapp (1938a) determined the proportion
of efficient mechanical shocks on the congelation of potato
tissue. A cylindrical shell of tissue was fastened around
the bulb of a thermometer, and a slight tap was given to
the upper end of the thermometer with a wooden lover,
when the tissue was subcooled to temperatures from - 1 °
to -5°. In 63 experiments out of 122, crystallization
occurred on tapping. The percentage of successful taps
increased at lower temperatures, but to express numeri-
cally the efficacy of tapping, one must eliminate from this
percentage that of possible coincident s])oiitaneous crystal-
lizations, which also increases at lower temperatures.
Unfortunately, the latter percentage is unknown. Sum-
marizing their results, the authors conclude that no doubt
is left as to the efficacy of tapping in some experiments,
and as to its inefficacv in others. Thev remark that such

195

a ])oliavior is consistent with the theory that crystalliza-
tion occurs as an event which follows the law of prob-
ability.

As to the degree of force necessary to induce crystal-
lization, several observers have noted that it varies with
the degree of snbcooling. Fahrenheit, who is considered
the discoverer of the snbcooled state, remarked, already
in 1724, that strongly subcooled water could be made to
crystallize by a shock, while slightly subcooled water could
not.

According to Mousson (1858), water can be maintained
subcooled to - 12° or - 15° in a vacuum but then the
slightest disturbance causes solidification. The same au-
thor could obtain a high degree of snbcooling in tiny water
droplets laid on a cold plate, but touching them with the
point of a pin caused them to freeze at once.

De Coppet (1907) observed that a strong stirring could
not cause congelation of salol subcooled 30 to 35° below
the freezing point, while 10 degrees low^er, crystallization
took place readily, on the slightest disturbance. The same
author said that when he gave to sealed tubes which con-
tained supersaturated solutions of sodium sulfate, the
slight inclination necessary to bring an air bubble present
in them from one end to the other, he sometimes observed
solidification.

A study of the minimal disturbance (friction) which is
effective in causing the precipitation of supersaturated
solutions, has been made by Young (1911).

Sensitivity to mechanical disturbance, as well as sensi-
tivity to several other factors, depends on the nature of
the material. Wright and Taylor (1921) observed that,
while subcooled potatoes can be induced to freeze by a
slight tap, berries subcooled to the same degree did not
congeal under vigorous tapping.

Mez (1905) claims to have observed a rather peculiar
behavior in the cell sap of Impaileus, pressed out, filtered,
boiled, subcooled and subjected to shaking. A slight dis-
turbance started crystallization at temperatures immedi-

ifu;

alcly ])('l()w llic t'rccziiin' point and at the cxti-cnic lower
limit of suhcoolini;-, Avliile a vigorous shaking was iiicffoc-
livo in the internu'diato zone of temperatures. He con-
tends also that spontaneous erystallization occurred most
of the time between -0.72° and -2.9° and l)eiween -5.01°
and - S.17° and less frecpiently in the intermediate region.
To establish the existence of such zones of stability, more
statistical data would be necessary.

We shall mention, to finish this section, a curious but
too concisely described observation of Wartman (1860).
Some water that he had left during an entire night, at
about - 4° in a glass container 31 cm. high and 16 cm. wide
(diameter ?), had stayed liquid, but Avhen he took the con-
tainer to empty it, in the morning, three walls of ice sud-
denly formed, making 60° angles ( f ) with each other,
adhering at one side to the wall of the container and
oblique to the axis of the latter.

5. Capillarity. Some of the oldest investigations on
subcooling were made by Gay Lussac (1836) who observed
that water can be subcooled to - 12° when it is enclosed in
small tubes.

Later Despretz (1837) reported that the congelation of
a liquid is "retarded" by ten or twelve degrees if the liquid
is enclosed in a thermometric tube or even in a tube hav-
ing a diameter of one centimeter.

Mousson (1858) exposed to temperatures from -5° to
- 7°, 8 tubes which varied in diameter from 0.187 to 2.503
mm. and which were tilled with water and sealed with seal-
ing wax. After a night at the temperatures indicated,
water was frozen in all the tubes which had a diameter
larger than 0.9 mm. while it was liquid in the smaller ones.
In other experiments he sprayed droplets of water less
than 0.5 mm. in diameter on a dry surface and observed
that the smaller the drops the longer they stayed subcooled.
The fact that, in clouds, water remains liquid at rather low
temperatures is attributed to the very small size of the
droplets.

Dufoni- (1861) studied the subcooling properties of small

197

drops. He made suspensions of water in mixtures of
eliloroform or petroleum and sweet almond oil (mixtures
wliieli have the same specific gravity as water), and he
observed that the smallest drops stayed liquid at -20°.

Bigelow and Ryckenboer (1917) attempted to determine
the degree of subcooling in capillary tubes in terms of the
diameter of the latter. They encountered difficulties with
water which presents such a narrow range of subcooling
temperatures and they decided to use other substances, in
particular, sulphur. In a previous determination the same
degree of subcooling was obtained in tubes 4 millimeters in
diameter and in larger ones. It was then decided to use
tubes of about four millimeters as standards of comparison
and tubes of smaller size. Definite differences in the
degree of subcooling were sometimes observed in tubes of
different diameter; for example, a tube of 4.1 mm. gave
an average subcooling point of 59.5° while a tube of 0.164
mm. gave 53.5°. But in other cases, the results were so
inconsistent, and sometimes two tubes of the same size fur-
nished such different average data that the authors ques-
tioned the significance of the experiment. The inconsis-
tency of the results, especially in systematic investigations
like those of Bigelow and Ryckenboer, renders doubtful
the commonly accepted assumption that the degree of sub-
cooling increases gradually with the decreasing size of the
capillary spaces. There is some evidence that capillary
forces exert an action in rendering congelation more diffi-
cult, but the data so far obtained are not sufficient to say
anything on that relation.

The biologists have generally admitted without discus-
sion that capillarity increases the degree of subcooling and
they attributed to capillary forces in the intercellulars or
within the cells the fact that plant and animal tissues sub-
cool to a greater degree than water.

According to Mliller-Thurgau (1886) living plant tissues
undercool more than the extruded cell sap. The hindrance
of molecular motion in living protoplasm is considered
responsible for the maintenance of the subcooled condi-
tion.

198

I^ac'liincl jrw (liK)l) claimed to liavc oljscrvcd a vclalioii
between tlie size of insect pnpae and the decree of siil)eool-
ini»-, the hitter being less in larger pupae, and he ex])lains
his lindings by the ea])illary ])roperties of the tissues ^vhich
he assumes lo he relaled to llie si/e of tlie ])upa('.

^Fez (1905) stated that considerable snbcooling is possi-
ble only in plants with very tine intercellulars, and that
there is no or only a slight snbcooling with larger intercel-
kilar spaces.

Voigtliinder (1909) undertook to check Mez' statement.
For that purjjose he measured, on camera lucida drawings,
the areas re])resenting the intercellulars and compared
them with the degrees of subcooling observed in 16 different
plant species. For areas varying from 2.4 mm.- {St relit zia
augusta) to 25 yr {Biciuus comminns) he obtained an in-
crease in the range of subcooling from 0 to 11 degrees.
The increase was, in general, regular, although there were
some inconsistencies. In the case of the presence of
tracheae, these observations could not be confirmed. The
same author studied the relation between cell size and de-
gree of subcooling in tissues of 11 sorts of plants, using
fifty or more specimens of each. His conclusion is that
there is no relation between these two quantities.

Altogether considered, it seems that neither the physi-
cists nor the biologists have obtained convincing evidence
of the effect generally attributed to capillarity in maintain-
ing the subcooled state.

6. Impurities. Among impurities assumed to be capa-
ble of starting the solidification of a subcooled liquid, that
most often mentioned is air. We shall also describe a few^
experiments in which droplets of oil and colloidal particles
were considered the inoculating agents.

According to one of the early investigators of the
changes of state, Dufour (1861), one should, for obtaining
a good subcooling of water, free it from air and maintain
it in an atmosphere at reduced pressure.

Mez (1905) reports that, while he could not obtain sub-
cooling invariabK' noi- to anv considerable degree with

199

pressod-oiit plant sa|), even when, afler repeated l)oiling
and lilt('riiii>', the licjiiid was clear and transparent, he did
()l)1ain subeooling at will, when he had previously fi-ozen
and thawed that clear liquid several times and let the air
bubbles enclosed in the ice escape during thawing. To
prevent the dissolving of air bubbles anew, he maintained
a layer of oil at the surface of the material. The difficulty
of subeooling tissues which contain tracheae is attributed
by him to the abundance of air in them. He explains in
the same manner the fact that the intercellulars freeze
before the cell contents. Concerning the mechanism of
action of the air bubbles, Mez suggests that their separa-
tion under the action of cold consumes heat and that the
resulting local cooling favors the formation of crystal
nuclei.

Some authors think that the action of air in inducing
crystallization is indirect and that it should be attributed
to dust particles or infinitesimal crystals contained in the
air. But against this contention one has the observation
of Moran (1925) that chicken eggs show an increase in the
tendency to subcool when their shell is coated with vaseline.

Cases of crystallization of a subcooled liquid in a sealed
glass container, at the breaking of the latter, are not un-
frequently mentioned in the literature. Some authors
attribute crystallization to a disturbance at breaking,
others, to the sudden contact with the air.

Mez (1905) could not readily obtain subeooling in cell-
sap wdiich had been heated wdiile it was covered by a layer
of oil. He thinks that some emulsification took place and
that the oil droplets in the sap play the same role as air
bubbles in preventing subeooling.

The efficacy of colloidal micelles in inducing crystalliza-
tion is suggested by Flichtbauer (1904) who expresses the
opinion that some dust particles which prevent subeooling
are of colloidal nature.

Mez (1905) considers the difficulty in subeooling turl)id
cell-sap as due partly to the action of mucous and gummy
substances. He thinks that colloidal particles might give
the start to the formation of nuclei.

200

Voigtliiiidci' ( 1!»();») atlcinptcd to iii\-('st inalc this sni»'ges-
tioii. Hi' used .Malvaeeat' wliicli are known to ])v ricli in
mucin. Out of 71 specimens, 52 did not suhcool and tlie
others furnished but a very slij>ht subcoolin*;-.

It is of interest to noliee that, accordinj^' to tlie hist men-
tioned authors, eoUolds favor crystallization, while niosl
of the physicists and biologists hold that anything which
delays molecular motion should prevent the formation of
nuclei.

J)esi)retz (1837), assuming the possibility of inoculation
by the presence of a foreign body, remarked that the delay
in the congelation of water (subcooling) takes place as wtU
in a copper or in a lead container as in glass.

The notion of impurity being conventional, the entire
question of inoculation by an impurity resolves itself, in
the last analysis, to that of inoculation by a body other tlian
the subcooled liquid itself.

7. Other Factors. Among other factors which might
have an influence in inducing crystallization we shall men-
tion the cooling velocity and the concentration of the super-
cooled or supersaturated solutions.

Some data on the first of these two factors will be found
in the work of Fiichtbauer (1904) who concludes that the
cooling velocity does not influence subcooling.

According to Bakhmetieff (1901) the relation between
the degree of subcooling and the cooling velocity is a rather
complicated one. At some cooling rates there would be a
maximal subcooling, and at higher and lower cooling rates,
subcooling would be less. The author observed many ex-
ceptions to that "law," and he attributes them to individ-
ual differences or to various degrees of development of the
organisms investigated (insects). It seems that Bakh-
metieff's notion of the method of establishing a law of
nature was different from that commonly accepted.

Voigtlander (1909), from numerous experiments on
plant tissues, concluded that there is no relation between
velocity of cooling and degree of subcooling.

Jones, Miller and Bailey (1919), working with potato,

201

reported that wlicii llic lempci-ature was lowered slowly
to -5° the subcooling- point of the material a])proached
that temperature, while if cooling- to - 5° was rajjid, sub-
cooling ceased near -3°.

Wright and Taylor (1921) confirmed the observations
of the last mentioned authors by showing that with a cool-
ing temperature of -9° a subcooling point of -6.5° was
obtained (on potato), while with a cooling temperature of
-12.9° subcooling ceased at -5°.

As to the factor, solute concentration, a work of Jatfe
(1903) would indicate that more concentrated solutions
would be more sensitive to crystallization by shock.

Voigtliinder (1909) attempted to investigate the influ-
ence of the osmotic concentration on the degree of sub-
cooling, in plants. He worked with 20 different species
and made at least fifty determinations on each. No rela-
tion was found; the subcooling point varied at random
from -4.19° to -11.07°, and the maximum subcooling was
about the same in plants isotonic with 1% and in plants
isotonic with 4.6 y^ KNO3. Unfortunately, in the same
work, strange results were obtained for the freezing points,
there being no relation between the latter and osmotic
pressure; for example, materials isotonic with 2.6yc and
4A% KNO3 were found to have the same freezing point.
However, since at subcooling there is a reversal of the
direction of the freezing curve, which can be more accu-
rately determined than the simple slowing of the curve at
freezing {Cf. Fig. 7, curve 5), Voigtlander's subcooling
maxima might be sufficiently well observed to be taken in
consideration.

That a higher concentration favors subcooling would
result from an observation of Moran (1925) who exposed
chicken eggs to evaporation until they lost about l^f of
their weight and noticed, then, that subcooling took place
more readily.

Weigman (1936) says that he obtained deep subcoolings
with snails {helix) possessing an operculum, while uncov-
ered snails presented only slight subcoolings. Since it is

202

known llial, in llic antninn, the snails lose a considerable
amoiinl of water before enteriii*;' into the dormant state,
the increased tendency to suIk'ooI siioiild, jx'rhaps, l)e
atti-il)iited to the concentration of body fluids.

SUMMARY

1. Some fundamental principles on the nature of sub-
cooling- and the mechanism of crystallization in a subeooled
liquid are outlined.

2. In an analysis of the sulx'ooling curve, two problems
are discussed: a. The maximal temperature reached by
the ascending curve; b. The quantity of heat liberated
as calculated by the method of the freezing-curve-area.

3. The factors inducing or preventing cystallization, as
studied by the physicists and the biologists, are reviewed ;
the following topics are considered: a. Temperature, spe-
cific response to temperature, destruction of centers by
previous warming, duration of previous warming; b. In-
oculation, inoculation by isomorphous crystals and other
bodies, inoculation of tissues through cell membranes
mostly by surface moisture ; c. Time factor, maintenance
of the subeooled state for long periods; d. ^lechanical dis-
turbance, its frequent inefficacy, its undeniable efficacy in
other cases, the magnitude of the disturbance required, the
specific sensitivity to mechanical disturbance; e. Capillar-
ity, the inconsistency of the experimental data; f. Impuri-
ties, air, oil droplets, colloidal particles.

CHAPTER III

THE VITREOUS STATE, VITRIFICATION,
DEVITRIFICATION AND VITROMELTING

The subject of this chapter is new in biology. The first
experiments on '* Vitrification of Protoplasm" were re-
ported by Luyet in 1937. Since the data are still scarce,
instead of following our usual procedure which consisted
in selecting the essentials in the literature, we shall at-
tempt to give, in compact form, a complete account of the
experiments made up to the present (May 1939) and shall
supplement it with some observations and remarks sug-
gested by our own experience with the subject.

The first part of this chapter will be devoted to a study
of the principles, facts and methods relating to the vitre-
ous state in physical systems, and the second wdll treat of
the application of these principles and methods to biologi-
cal material.

I. PHYSICAL SYSTEMS

1. The Vitreous State. The vitreous state has been
known in silicates for hundreds of years, but it was gen-
erally assumed that the possibility of becoming vitreous
was an exceptional property of these bodies. At the end
of the last century (1898), Tammann pointed out that a
large number of substances can be obtained as glasses and
suggested that this property might be universal. Out of
a series of 153 carbon compounds investigated, 59, that is,
38 per cent, could be vitrified.

The production of the vitreous state is conditioned by
temperature in the manner illustrated in the diagram. Fig.
29. If the temperatures are plotted on a horizontal line,
from the absolute zero up, the states, gas, liquid, crystal-
line and vitreous, are represented by the zones 0, L, C, V,
and the changes of state by the zone D and the points ]\[
and B. Upon a lowering of temperature, a body in the
gas state becomes liquid at the liquefaction point B, it
thereafter crystallizes at the freezing point M and stays

203

204

crystalliiu' down lo llic absolute zero. \\\\\ if, 1)\- alti'ii])!
cooliiiu', one can brinj;- a iKiiiid 1 lii'on,i;ii llic /one (' before
it lias the time to crystallize, it takes the vitreous state and
stays vitreous at h)\ver temperatures. If a l)ody iu tlic
viti-eous state at a low temperature is warmed up slowly,
it devitrifies, that is, it becomes crystalliue when it reaches
the devitrification temperatures 1), it becomes liquid at the
melting ])oiut M and it is transformed into a gas at the
boiling point H.

V C L G

A.Z. DM B

Fig. 29. Diiifji.-nii rc'incsi'iithig, on a teiiiperaturo scale of which the ori-
gin is the absolute zero, A. Z., tlie four states of matter: gas G, liquid L,
ervstalliue C and vitreous V, and the three changes of state: boiling B, melt-
ing M and devitrification D. The upper arrow indicates tlie change of tem-
perature for i)assing from the liquid to the vitreous state, and the lower arrow
the change of temi)eiature involved in subcooling.

A glass is amorphous, that is, its molecules are dis-
tributed at random, while, in a crystal, there is an ordered
arrangement of the atoms. A glass is isotropic, its physi-
cal properties being the same in all directions, w^hile most
of the crystals are anisotropic, their physical properties
having higher coefficients in some directions than in others.
In polarized light, between crossed nicols, a glass is opaqne,
while most crystals are brightly illuminated. A glass dif-
fers from a li(iuid in that it is hard and breakable, while
a liquid is fluid and deformable.

In the passage from one state to another, an intermedi-
ate state can, as we said above, be skipped over. A crystal
can become a gas w^ithout taking the intermediate liquid
state, a process wiiich is known as sublimation. Similarly,
when the crystalline state is skipped ovei- by rapid cooling,
the body passes from the liquid to the vitreous condition,
it is said to vitrify. If the crystalline state is avoided by
rapid warming, the vitreous body becomes liquid, a pi'ocess
which we call vitromelting or vitrofusion.

The two transition processes, melting and boiling, are

205

not ciitirely eomparable to devitrificatioii. Melting and
boiling- are reversible while devitrification is not. A gas
becomes a liqnid on cooling and a liqnid becomes a gas on
warming; a liquid crystallizes when one lowers its tem-
perature and a crystal melts when its temperature is
raised ; while a glass crystallizes on warming but a crystal
does not vitrify on cooling.

Furthermore, melting and boiling take place at tempera-
tures which are practically points, while devitrification
occurs over a larger range of temperatures. To illustrate
this diiference, we indicated in the diagram (Fig. 29) the
boiling and melting points by the lines of separation B and
M, and the range of devitrification temperatures by the
zone D.

According to these principles, the behavior of silicates is
not exceptional. Their vitreous zone (V in Fig. 29) ex-
tends from the absolute zero to about 1000° C. Since the
atmospheric temperature, which prevails about us, is
within this zone, we are familiar with the vitreous state of
silicates. A silicate glass heated to the devitrification
temperatures (below the melting point) becomes opaque
by crystallization. At a higher temperature, the crystals
melt.

From observations made on some silicates which devit-
rify very slowly, becoming opaque in the course of several
years, it has been concluded that the vitreous state is un-
stable and that if enough time, i.e., centuries or thousands
of years, were allowed, devitrification would always take
place. The fact that natural flint has an opalescent ap-
pearance, due probably to the presence of minute crystals,
is sometimes considered an indication of the instability of
the vitreous state, it being assumed that crystallization is
still being completed. But there is no evidence that the
opacit}^ of flint has developed during the millions of years
which have followed its vitrification and that the crystals
noM^ observed were not formed at the same time as flint
itself.

It has been mentioned above that glasses are regarded

by many as suprrcoolcd li([iii(ls. This dctiiiit ion is uscrul,
in a sense, sinee it reminds one of tlie faet tliat i»lassi's re-
semble liquids in that they ])iesent a random arrauiivment
of theii" moleenles. But sueh a eoneeption ii>nores the faet
that, while a snpereooled litiuid is in so nnstabli' a state of
etinilibrium that the eontaet with a erystal invariably i)ro-
duees its erystallization, a ^lass ean be put in eontaet with
a erystal without losing* its stability. A snpereooled liquid
has been bronght from the zone L (Fig. l2J)) into the zone
(' of erystallization tem])eratures, where it is highly un-
stable; a glass has been brought, from the zone L, below
the zone of erystallization temperatui-es, into the zone V
where it is praetieally stable. The ehange in temperature
whieh leads to the formation of a glass is represented dia-
grammatieally by the upper arrow in Fig. 29, that whieh
leads to the formation of a snpereooled liquitl is re])rt'-
sented by the lower arrow.

From what has been said it follows that a body ean erys-
tallize (/.('., freeze, to use the common term) only within
a limited range of temperatures (C and D, Fig, 29). In
the zone of the vitreous state (V, P^ig. 29) freezing is im-
l)ossible, the temj)erature being too low. Since the crystal-
lization range can be reached from above and from below,
there are two ways of freezing a body: one is to cool it
down from the liquid state, the other to warm it up from
the vitreous state. We are so accustomed to the idea that
freezing is more intense at lowei- temperatures, that the
statement which jirecedes appears paradoxical.

As we shall see later, for aqueous colloids, the zone of
erystallization temperatures extends over some tens of
degrees below zero ; consequently these colloids cannot be
frozen (crystallized) if the tem])erature within them is
below this range.

The reason why plants or animals freeze in nature, when
the atmos]iheric temperature drops to - 30*^ or -40", is
that the dro]) is too slow. The objects which are being
cooled remain for a few minutes at the dangerous (freez-
ing) temperatures. To avoid freezing, the temperature

207

should drop at a rate of some linndred degrees per second,
within the objects themselves.

2. Vitrification. As a consequence of what has been
said on the position occupied by the vitreous state at the
lower end of the temperature scale and the impossibility
of passing from the crystalline to the vitreous state, the
only method of vitrifying a substance is to take it in the
liquid or gas state and cool it rapidly so as to skip over the
zone of crystallization temperatures in less time than is
necessary for the material to freeze.

The ease with which an intermediate state can be skipped
over depends on the range of temperatures at which that
state obtains. The liquid state of CO:.., for example, is
easily avoided when solid C0_, is warmed up, on account
of the fact that the melting and the boiling point almost
coincide.

The range of freezing temperatures, although large in
some substances under ordinary conditions, can become
narrower when such processes as subcooling occur. For
example, in a gel of which the freezing zone extends from
-2° to -12°, a subcooling to -7° will reduce this zone to
one half its normal size, that is, to a range of from - 7° to
-12°.

Another factor of considerable import in the vitrifica-
tion of a substance is its crystallization velocity. It is
evident that when the crystals grow faster one must
traverse the crystallization zone more rapidly if one wants
to avoid crystallization.

The essential point in the vitrification technique being
to overcome the velocity of formation of crystals, the main
problem is to secure a high cooling velocity. Practically
the only method used to cool a liquid or a solid body (for a
gas, it is different) is to bring that body in contact with
another at a lower temperature. The cooling velocity will
be higher Avhen the temperature of the cooling medium is
lower and when the contact is better. Liquid air (- 190°)
is appropriate for the vitrification of aqueous colloids,
aqueous solutions and protoplasm. Liquid hydrogen or

•JOS

li(Hii(l Iicliiini would Ix' still Ix'Hci', tlicii- hoilitii;- points
Ix'iiiii' rrspcct i\cly ;il»oiit (iO niid SO (l(\i>it'('s lower. A liijuid
l)alli cooled to about -7.") with solid ('( ). also allows vitri-
lication of most aqueous colloids, although ^\•itll a lower
efficiency.

It is ini])ortaiit that tlie cooling l)ath ho li(iuid; a liquid
insures a bettei- contact with the substance to be vitritied
than eilliei- a solid or a gas. Attempts at vitrifying thin
layers of gelatine gels by application of smooth plates of
solid COj on both sides gave ])ooi'er results than immersion
in a liquid bath at the same temperature.

The fact that liquid air, when evaporating, forms a pro-
tective mantle around the object to be cooled has led the
histologists who use rapid freezing as a method of fixation
to look for another cooling liquid. They have adopted
isopentane cooled in liquid air. Isopentane has a very low
freezing point (-159°), it can be subcooled to -200°, and
it has a relatively high boiling point (28°) ; because of this
last property it does not boil when it comes in contact with
the object to be cooled. Nevertheless, experimentation
has convinced us that isopentane is not so satisfactory for
the dissipation of heat as one might expect. Small quan-
tities of a gelatine gel were placed in glass tubes about 1
millimeter in inside diameter, closed at one end. The tubes
were immersed in a bath at - 10° where their contents
froze. Some of them were then immersed in water at 20°,
the rest in isopentane at the same temperature, both warm-
ing media being well stirred. It was found that the time
necessary for melting the gelatine was considerably longer
in isopentane than in water. This is doubtless due, to a
large extent, to the difference in heat conductivity of the
two liquids (inclusive of the difference in contact con-
ductivity).

The velocity of cooling also depends on the mass to be
frozen and on its surface. One will obtain rapid elimina-
tion of heat by reducing the material to sheets with the
smallest possible thickness and the largest possible area.
Calculation shows thai when a glass strip, 0.1 mm. thick,

209

is transferred from air at 20° to a liquid bath at - 200°, the
temperature drops about 200 degrees duriii<>' the first sec-
ond. It is generally admitted that, with liquid air as a
cooling- bath, the drop in temperature is somewhat slower
because of the formation, around the object, of the pro-
tective air mantle mentioned above ; however, the cooling
velocity obtainable w4th liquid air amply suffices to vitrify
objects whose thickness is of the order of 0.1 millimeter.

When the mass of material to be cooled is too large, only
the most external layer vitrifies. The inner parts, wdiich
lose their heat too slowly, freeze.

Whenever crystallization begins at some point on an
object, the conditions for vitrification of the rest are im-
paired. The p;ortion which crystallizes liberates heat ;
this spreads to the surrounding parts and maintains their
temperature at the freezing point. Hence, the whole mass
might freeze.

In our research on the vitrification of colloids we have
found, moreover, that their water content determines the
possibility or impossibility of vitrification. In general,
with 50^ gelatin solutions, w^e have been able to vitrify
layers 0.3 mm. in thickness (by the method of immersion
in liquid air), while, wdth solutions containing 90% water,
W'C could vitrify only smears a few micra thick.

Attempts at vitrifying pure w^ater have been made by
a few investigators. Burton and Oliver (1935) obtained,
from steam, some solid water in wdiich X-ray analysis did
not reveal any crystalline structure.

Previously, Haw^kes (1929) had mentioned an experi-
ment in which a drop of solid amorphous water was ob-
tained, by chance, during rapid cooling.

The difficulty experienced in vitrifying pure water has
been attributed, in general, to the high velocity of crystal-
lization of that substance. Walton and Judd (1914) who
measured this velocity, found 65 mm. per second (an
exceptionally high velocity).

According to Callow (1925), the addition of 3^/( gelatine
to w^ater reduces its velocity of crystallization to 1 350 of
its value.

1210

'Phis ]»i()i)i'rly lias rriidcrcd j)()ssil)l(' our ('X])('iiiii('ii1s on
llic xitrificalioii of colloids (Ijuyct, 1!).")7). We ])ro('('('d('d
ill llu' foUowin,!'' inaniicr: A drop of a ^)i)' /> i»-('larni solulioii
was ))iit, when still hoi, on a thin i;-lass support and s])r('ad
out so as to form a layer about 0.2 mm. thick. Tliis prepa-
ration was then immersed in liquid air. AVhen we took it
out, the u'elatin was vitrilied, as shown 1)\' its li-ansparency
when viewi'd ai>'ainst ordinary liiiht and its ()])acity Ix'tweon
crossed uicols. After ahout 10 seconds of exposure to
room temperature, the gelatin became opaque under ordi-
nary illumination and reestablished light l)etween crossed
iiicnls (devitrilication, r/. Fig. ,')0). This fundamental ex-
periment has been the starting point of all the investiga-
tions which we made on the vitrification of protoplasm.

Fig. .'}(!. Vitrified ;iii(i dcvitiitied gelatin gels on glass sui)i)oits, plioto-
giaplied against a dark baekground. The figure on the left shows the vitri-
fied gelatin almost transparent; the figure on the right shows the same prepa-
ration after it has been warmed up and has become opacpie l).v devitrification
(crystallization). (Original, Luyet.)

To sum up, the method of vitrification consists essen-
tially in immersing a thin film of the liquid to be treated
in a bath at a very low temperature.

Vitrification is a process of quite a different nature than
that used in the refrigeration industry as ** rapid freez-
ing." When some hundred ])ounds of food are fi'ozen in
10 or 15 minutes, small ciystals are formed which cause
less damage within the tissues than larger crystals, as
those found in slower freezing, would. In vitrification one
aims at avoiding crystallization altogether and, to achieve
this, the cooling velocity should be about a thousand times
higher than in the industrial process.

211

3. Dcrifri/ifafioji: When one raises the temperature of
a vitreous substance, it crystallizes. In the ^lass industry,
this process is called devitrification or recrystallization.

Among the various methods of ascertaining the passage
from the vitreous to the crystalline state, the simplest is
that of observing the decrease in transparency. When a
large number of small crystals are formed in a body in the
vitreous state, light is scattered in all directions, the trans-
parency is attenuated and, in some cases, the body becomes
completely opaque.

This method, however, is not always infallible. In de-
vitrifying concentrated solutions of magnesium chloride,
we observed that the transparency remained altogether
unaltered, although the material reestablished light be-
tween crossed nicols. The method of analysis by polarized
light is, therefore, necessary in certain cases.

The best method of diagnosing the commencement of
devitrification would be the analysis by X-rays. This
might allow one to perceive crystallites of very small
dimensions in most of the substances which we consider
glasses.

The possible existence, in a body in the vitreous state,
of these crystallites or of what we previously called nuclei
of crystallization is suggested by the fact that, during de-
vitrification, a glass passes from the condition of trans-
parency to that of opacity by a continuous and uniform
darkening. One cannot distinguish separate centers of
crystallization in this case as one can when one induces
crystallization from the liquid state. If the opacity of a
devitrified body is due to the presence of crystals, the
gradual darkening ought to be explained by an increase in
the dimensions of the crystals already formed or by the
formation of new crystals. So, when a preparation begins
to lose its transparency, it does so either because nuclei of
crystallization are forming, or because the already pre-
formed nuclei, at first too small to cause the obscuration of
the vitreous substance when it is observed with ordinary
light, are growing in size.

'2V2

When the tomporatiire is sufficioiitly liii;h, devitrification
])rocoods witli an easily measnrahle velocity. For exam-
ple, a IM solntion of sncrose, vitrified in a thin layer, de-
vitrities within 10 seconds when exposed to a temperature
of -26^'; it devitrities in a minute at about - oO% while it
does not devitrify at all, even within an hour, at -35^.
We call - 26°, - 30°, etc., ''temperatures of devitrification,"
but it is evident that one must indicate the time required
for devitrification at each temperature if one wishes to
give to the notion of devitrification temperatures a precise
meaning.

We have undertaken to establish the devitrification tem-
peratures, or better, the time-temperature curves, for
aqueous solutions of various organic (Luyet, 1939) and
inorganic substances. The method employed was as fol-
lows: A small drop of the solution to be vitrified was
placed between two glass strips, each about 0.1 mm. thick,
kept apart by two bits of glass 0.1 mm. in thickness. This
preparation was first immersed in liquid air ; thereafter it
was placed in an isopentane bath maintained at a constant
temperature, and the time necessary for complete crystal-
lization was determined. The opacity of a frozen prepa-
ration served as a term of comparison to show when the
devitrification was complete.

The curves obtained seem to indicate that, contrary to
the general belief, devitrification does not take place at all
at very low temperatures. If one extrapolates the curve
of Figure 31, for example, it becomes parallel to the time
axis at a temperature a little below that at which devitri-
fication occurs within a minute. The hypothesis of a very
slow rate of devitrification, requiring hundreds of years
for producing an observable result, seems, therefore, to be
out of the question, at least for certain substances like
those studied here.

AVithin a rather large range of concentrations, for ex-
ample, from 0.9M to 2.2M for a sucrose solution, the de-
vitrification temperatures change but little. Thus, lAL and
2M sucrose solutions have devitrification temperatures

213

time: in minutes
_^ ^.

2 10

Fig. 31.

"1

I 2 10 20 30

Time-temperature curves of devitrification of a 20% gelatin
solution. The dots indicate a complete devitrification; the circles signify that
devitrification was not completed at the time and temperatures recorded.

which differ by only 0.4 degree (- 31.4^ and - 31.8° respec-
tively; devitrification in 5 minutes). For determining
these temperatures, two drops, one of each solution, were
mounted on the same preparation and the increase in
opacity of the two was observed simultaneously.

This lowering of the devitrification temperatures with
increase in concentration reminds one of the freezing point
lowering of aqueous solutions. Nevertheless, we cannot
say at the present time to what extent these two processes
are referable to the same cause.

•J 14

At coiicciit I'atidiis hiii'lu'r tliaii tliosc (if tlic al)()\'(' iiicii-
lioiK'd raiiiiv, a devil rificalioii ot" a (lilTci'dil !>"])(' (»l)taiiis,
wliicli wi' sliall describe farther on.

Siiiee devitrification is a clianj^c of stale c()m])aral)le, in
many respects to other cliani'cs of state like melting and
boilini>', it is i)ro])a])le that, like theni, it takes ])lace at a
temperature which is a function of the chemical structure
of the substance under investigation. One should expect
a rise in tlu' devitrification temperatures as one passes,
within the sami' sei'ies, from compounds of simple molec-
uhir structure to those with a more c()mi)lex one, as is the
case with the boiling point, which rises when one passes,
for example, from methane to pentane. We could estab-
lish the existence of such a relation. In a series of sugars
we obtained (duration of devitrification, 5 minutes)' :

Glucose 2M C, H,,0„ -40.6°

Sucrose 2M Ci,H,,(),, -31.8°

Kaffinose IM CisH.s.O,, 5H,0 - 27.2°

Dextrin 2M x {C, H,„0.-,). - 9.4°

The other high-molecular-weight substances which we have
studied, such as, gelatine, albumin, gums, dextrin, have
high devitritication temperatures (about -10°). Sub-
stances whose chemical composition is more or less similar
to that of the sugars, but which have much lower molecular
weights, such as glycerine, ethylene glycol and formalde-
hyde, have been found, in preliminary experiments, to
have devitrification temperatures in the neighborhood of
-60° to -70°.

However, the relation which we just noted cannot be
simply compared to that between the molecular complexity
of a substance and its melting or boiling point. In the one
case, it is question of solutions, in the other of definite
compounds.

It is to be expected that certain molecular groupings in
the solute have a specific effect on the devitrification tem-

1 Liiyt't, R. J., ii:iiKM- in jircss in ./. P/(.(/.v. Chrm.

215

peratuix's of llic solution. In prelimiiiaiy investigations
on this problem' we have observed that dextrose and levu-
lose, wliich have the same empirical formula but differ in
the structural arrangement of their atoms, have the same
devitrification temperatures, while sucrose and lactose,
which are likewise isomers, exhibit a slight difference
therein.

There also exists a relation between devitrification tem-
peratures and water of hydration or, more generally,
between devitrification temperatures and the wat<'r-binding
capacity of the solute (Luyet, 1939). With sucrose solu-
tions we found that, when the concentration w^as higher
than that corresponding to ten molecules of water per one
of sucrose, devitrification did not take place at all, no
matter what the temperature was. With lower concentra-
tions, corresponding to one molecule of sugar for every
11, 12 or 13 of \vater, devitrification occurred at tempera-
tures of about - 50°, and the frozen mass exhibited a par-
ticular type of crystallization which we designate by
''crystallization in tufts" there being tufts of crystalline
needles in the vitreous mass. With lesser concentrations,
a different type of devitrification set in : the preparation
assumed an amber color which gradually darkened to com-
plete opacity. The devitrification temperatures were
about 15 degrees higher in this case. It is this type of
devitrification which we have studied particularly and
which has furnished the numerical data given above. One
can, it seems, interpret the results observed in these three
ranges of concentration by supposing that the first 10
molecules of water which come in contact with a sugar
molecule attach themselves so firmly to it that they cannot
be torn loose by the forces of crystallization. The next
three molecules would be bound by a different type of
bond, and, when more than 13 molecules of water are
present per molecule of sugar, this excess water would be
held only by the forces of solution and would be free to
solidify. (In making this hypothesis, we are supposing

1 In collaboration with Dr. C. and Miss M. .Jordan.

21(1

tliat only water cryslnlli/A's duriiii'' llic successive devitri-
fications; l)iit tliis i)oint is to be investigated.)

The existence of three ranges of concentration witli the
properties just described has been observed in a great
number of solutions. Thus, solutions of gum arabic^ do
not devitrify if their concentration exceeds 68''/. They
devitrify in the form of tufts between 6S'/( and G-^Vc , and
in the amber-color form below 64%.

Devitrifications at more than two ranges of tempera-
ture were noticed with solutions of substances such as
urea.-

Witli sohitions of sodium chloride, two devitrifications
of the anilxM-color type occur at different temperatures,
one of them in the neighborhood of -28°. When the de-
vitrified material is warmed up, a partial melting takes
place at about -21°, the well-known freezing temperature
of the eutectic mixture.

We have also applied the method of devitrification to
the study of the mode of binding of water in substances
which set as gels. A preliminary work'' has shown that a
sugar solution to which pectin has been added and which
has been allowed to set, devitrifies at temperatures slightly
below those at which an identical but alkaline solution,
which does not set, devitrifies.

From what we have said on the higher devitrification
temperatures of solutions of substances such as gelatine,
dextrin, the gums, etc., it follows that these solutions can
freeze only from about zero to 10 or 12 degrees below zero.

4. Yitrofusion. The direct passage from the vitreous
to the liquid state can be effected by a rapid warming.
The conditions foi- assuring this ])assage and avoiding the
intermediate crystalline state are fundamentally the same
as those required for vitrification : 1. The greatest possible
temperature difference between the warming bath and the
object ; 2. The smallest possible mass and the greatest pos-

1 Eesearch made in collaboration with Mr. J. Fnltoii.

2 Studied in collaboration with Mr. H. Noe.
a In collalioratioii witli Mr. W. Sclimiesing.

217

sible surface area of tlie latter ; 3. A high heat coiidiictivity
of the warming bath and, in particular, a good contact con-
ductivity (as it has been said in the previous section, a
liquid bath is far superior in this respect to all others).

II. LIVING MATTER

1. General Proeedure. The principle that the solidifica-
tion of a liquid into a glass requires less molecular rear-
rangement than the transformation of that liquid into a
crystal suggests that vitrification might not injure proto-
plasm in conditions in which crystallization kills it.

Our first attempts at vitrifying living matter have con-
sisted in applying to it the methods of vitrification and
vitrofusion and then testing for its vitality. Only a few
experiments, and these on but a few types of material,
have been carried out to ascertain whether or not the
treated protoplasm had actually been brought into the
vitreous state. In the following account, therefore, the
vitality of living matter treated by the vitrification meth-
ods is taken as indicative of a probability that the material
was actually vitrified. Vitality after treatment does not,
indeed, constitute a proof of vitrification, but, if masses of
protoplasm, of small size and not too high water content,
are still alive after rapid cooling and rapid warming, while
they are killed in larger quantity, or when they have a
higher water content, or when they are cooled or warmed
slowly, vitality does afford at least indirect evidence that
vitrification has been achieved.

In as far as living beings are comparable to the gelatin
solutions which we studied previously, we can expect to
succeed in vitrifying organisms whose thickness is about
one third of a mm., if their water content is about 50^/(
and smaller organisms of greater water content, provided
the latter does not exceed 90 ^c.

2. Methods. For rapid cooling, we used the method of
immersion in liquid air. For rapid warming, we ordi-
narily immersed the material in water at a temperature
of about 20°. Occasionally we employed water heated to

1>1S

,")() or (id , <>i" cx't'ii hoiliii^' walci', hut in the last case, the
object remaiiK'd in the bath I'oi" U'ss Uiaii one tiflli of a
second, w]u'rcii))on it was iniiru'(bately pbiiiged into cold
water. Immersion in mercury lieated to 40° gave good
]-esubs ill tlie case of moss. Isopentane recommends itself
])articnlarly in exi)eriments on })r()tozoa because of its
immiscil)ility with water. One can place on a thin cover-
glass a small drop of water containing- the protozoa, im-
merse tliis preparation in licinid air, then in isopentane,
and repeat the operation several times; the drop is still
intact with the organisms within it. But in spite of this
notable advantage, it seems, on the basis of experiments
reported above, that isopentane is too slow as a warming-
medium.

In order to reduce the heat capacity of the preparation,
one must use only very thin supports. Microscope cover-
glasses are often too thick. Frequently w^e substituted
for them sheets of mica ; since these cleave easily, one can
get sheets which are only some ten micra in thickness.
Metallic foil presents the inconvenience of not being trans-
parent and so not allowing of microscopic observation.

Instead of thin supports, we sometimes used, with advan-
tage, a ring about two millimeters in diameter, made of
as thin a metal wire as possible, and fastened to a light,
rigid rod. One simply dips this loop into the culture, and
thus obtains, in the thin film within the ring, quite a con-
siderable number of organisms. With the diameter of
loop indicated, it is only seldom that the tilm breaks either
when immersed in liquid air or when immersed in the
warming medium. We often found it advantageous to
reduce the thickness of the wire still further by flattening
it with a hammer.

With protozoa we also employed another method which
consisted in placing- the culture in an atomizer and spray-
ing it thence into liquid air. But the freezing of the w^ater
which encloses the organisms liberates heat and retards
cooling.

We likewise tried to emulsify the culture with oil. When

219

this emulsion was placed on a tliin support, each organism
could be seen to be enclosed in a cell consisting of a droplet
of nutritive medium surrounded by oil. The objection
mentioned for the previous method applies to this one also.

Perhaps one might have success by spraying the cultures
in thin sheets into liquid air by means of a syringe with a
flattened and adjustable jet.

As for the methods of drying, we used, besides that which
consists in allowing the preparation to evaporate in the
air or in desiccators containing various concentrations of
sulfuric acid, also the plasmolytic method of immersing
the preparation into a solution of salt or of sugar.

3. Experiments and Results. We undertook a first series
of experiments with euglenae. The organisms were first
concentrated by centrifugation. A small drop of the con-
centrated culture was thereupon placed on a glass slide
and left to evaporate in the air till only a swarming mass
of animals remained. The preparation was then dipped
into liquid air and after this into water at 20° or at 40°.
No euglena ever came back alive from the ordeal. Think-
ing that the organisms contained too much water, we tried
to carry the evaporation still farther or to reduce the water
content by adding to the droplet concentrated solutions of
sugar. But concentrations which killed the euglenae
within one minute, did not suffice to produce the desired
result. The use of strips of mica instead of glass supports,
to lessen the heat capacity, was likewise ineffectual. On
the whole, all our attempts to revive euglenae in the vege-
tative state were unsuccessful. The organisms were ordi-
narily not deformed, but they showed no sign of life.
Whether they had actually been vitrified or frozen is
uncertain.

We next repeated with paramecia all the experiments
made with euglenae. The results were completely nega-
tive. But, while the euglenae did not suffer any deforma-
tion by the treatment, the paramecia always did, and often
they were found completely broken in pieces when taken
out of the liquid air.

220

Exporimonts \vitli ciliates smalU'r than paramocia, that
is, with oolpoda, in tlio vrgotativr state, likewise uavo
negative rosnlts.

The same is true also ot' experiments with amoebae.

(hi the whole, onr investigations on the three principal
groups of protozoa, the rhizopods, the eiliates and the
tlagellates, did not allow us to revive a single organism.
It seems probable that these animals oould not really be
vitritied on account of their too high water content.

However, revival was obtained in some experiments with
myxamoebae (Gehenio and Liiyet, unpublished). Out of
thirty attempts made to vitrify these organisms by immer-
sion in liquid air on the wire loop, live gave living
myxamoebae whose contractile vacuoles resumed their
function and maintained it for several lionrs. Though the
percentage of animals revived is small, we consider the
fact as highly signiticant.

Next we tried the spermatozoa of the frog (Luyet and
Hodapp, 1938c). A smear on a thin cover-glass gave nega-
tive results. A second attempt on a sheet of mica was
no more successful. A third series of experiments in
which the spermatozoa were previously immersed in a
20% sucrose solution for being dehydrated before immer-
sion in liquid air, yielded some motile organisms, less than
l^c of the number treated. By incri'asing the concentra-
tion of the sucrose solution to 40' < or 507^, we could
increase the percentage of motile or non-disorganized
forms to 20/( or more. To sum up, by employing with
the spermatozoa of the frog the method of mica sheets, of
dehydration in concentrated sugar solutions and of rapid
warming in water at 20°, we obtained some living forms
in each preparation.

Studies on the duration of immersion in liquid air which
the spermatozoa can support have shown that the number
of survivors and their activity are the same after live days
as after three seconds. This finding is in good agreement
with the assumption that the material is vitrified and stays
unaltered at low temperatures.

221

Experiments like those just described were also carried
out with the spermatozoa of tlie ral, but not a single one
could be revived.

Goetz and Scott-Goetz (1938) described experiments in
which yeast previously treated by the vitrification methods
was killed when exposed, in monocellular layers, in a wire
loop, to temperatures of some few degrees below zero,
while it was intact when exposed, under the same condi-
tions, to 150° below zero. This seems to indicate that the
very low temperatures, at which the vitreous state can be
maintained, do not kill, while the higher temperatures
which cause devitrification are lethal.

The epidermis of the onion, a classical subject in plant
cytology, seemed to be particularly appropriate for our
researches, especially because of the ease with which one
can obtain very thin monocellular layers (Luyet and
Thoennes, 1938b). A piece of epidermis, held on a small
metal fork, was immersed into liquid air and then into
water at 20°. The vitality of the cells was tested by plas-
molysis. A first series of observations furnished only
dead cells. Thinking that the quantity of water present
in the large vacuoles of the epidermal cells rendered vitri-
fication impossible, we tried to reduce this quantity of
water by plasmolysis in salt solutions. No cell could be
revived. But we had not taken account of the fact, re-
ported by many investigators, that a direct immersion in
water after plasmolysis in a concentrated salt solution is
often fatal. The invasion of the strongly plasmolysed cells
by water produces a too violent expansion which causes the
bursting of the protoplast. We therefore tried to warm
tlie cells rapidly by immersing them not directly into water,
but into a saline solution at 20°. This time we found a
considerable number of cells capable of being deplas-
molysed or plasmolysed to a further extent.

A study, with polarized light, of the plasmolysed onion
epidermis, in liquid air, showed that the cellular proto-
plasm, concentrated by plasmolysis, was isotropic, while
the space which surrounded it and which contained only

salt solution, was anisotropic (Luyct and Thoennes,
193Sa).

Plant loaves can be vitrified, at least partially, when their
water content is not too hi<ih. We have shown this in the
followinii' mannei'. A leaf which, wlieii examined against
a source of light (an electric lamp) exhibits a certain
degree of translncidity, is dipped into liquid air. It be-
comes hard and ])reakable but its degree of transparency
has hardly changed when one takes the leaf out and again
examines it against the lamp. After a few seconds of
exposure to room temperature one sees the leaf become
considerably oi)a(iue and, a few seconds later, it again
acquires its original translncidity. It seems evident that
all the water of the leaf was not frozen at the temperature
of liquid air, that it froze only when the temperature rose
to the zone of devitrification, and that it melted at a still
higher temperature. The leaves treated in the manner
described showed little or no vitality. Though this mate-
rial allows of an easy observation of vitrification and devit-
rification, we think that it could not be subjected to vitro-
fusion (the transition from the vitreous to the liquid state
without passing through the crystalline state), even when
boiling water was used as the warming bath.

Moss gave results in perfect agreement with our antici-
pations (Luyet and Gehenio, 1938). Specimens of the
genus M)iiiiw were placed in containers in which solutions
of sulfuric acid of known concentration maintained an
atmosphere having a given degree of humidity. After a
stay of less than 24 hours in one of these containers, a state
of equilibrium was established. A portion of the moss was
then taken out for a determination of the water content,
while the remainder was immersed in liquid air and in
water at 20°. The vitality of the cells was tested by plas-
molv«is. The results can be summarized as follows:

223

Water Content MetJiod Results

More than 65% Slow devitrification Dead

More than 65% Rapid warming Survival

65% to 30% Slow devitrification Partly dead

65% to 30% Rapid warming Survival

Less than 30% Slow devitrification Survival

Less than 30% Rapid warming Survival

One sees that when the moss is sufficiently dried, it always
survives treatment by low temperatures, whatever be the
rapidity of cooling or of warming. When the water content
is high, the plants die if one lets them freeze, but survive
the vitrification if one prevents freezing by a rapid
warming.

Finally we tried to vitrify muscle fibers (Luyet and
Thoennes, 1938c). Bundles of 6 to 10 fibers, taken from a
chloroformed frog and mounted on a metal frame, were
immersed in liquid air and then into Ringer's solution at
20°. The fibers so treated were thereupon placed on the
microscope and subjected to an electric shock. Some fibers,
often most of the fibers present, contracted. However,
they ceased to respond sooner than normal fibers to re-
peated electric shocks, and, for the same reaction, they
required an induction current of higher potential. But
whether the period of immersion in liquid air lasted several
hours or only a few seconds seemed to make no dilTerence.

In a series of researches which we have just begun, we
intend to study the extent of the zone of crystallization in
various organic fluids such as muscle plasma, moss juice,
yeast extract, the protoplasmic fluid of the protozoa, etc.
Perhaps these researches will reveal the reason why cer-
tain living beings are very resistant to low temperatures
while others are not. The extent of the temperature inter-
val within which they freeze might be the determining-
factor in their sensitivity. Our first results on muscle
fibers seem to indicate that the temperatures at which the
muscle dies during devitrification are the same as the tem-
peratures of devitrification of muscle plasma, determined
by the method previously described for aqueous solutions.

224

Altliougli IIktc ai"(' several unexplained exeepi ions, llic
results obtained in the \it riticalion of pr<)1()i)lasm, and in
l)artic'iilar those fiii'nisiied l)y moss, e])idermal cells and
muscle fibers, seem lo conlirui llie view thai a s;ood vitri-
ficatiou is not injurious, there Ix'iui;' no molecular dis-
turbance, while an incomi)lele vitrification or devitrifica-
tion and, (I forfiori, cryslallizaliou, are injurious to the
extent tliat they disrupt the living structure.

SimiMARY

1. At different temperatures, matter can exist in four
physical states: as a gas, as a liquid, as a crystal and as
a glass. The last-named state occupies the lowest zone in
the scale of temperatures. 2. Crystallization is possible
only within a limited range of temperatures. Below this
range, matter is too inert to crystallize (in other words,
too cold to freeze). 3. The vitreous state is obtained by
cooling a liciuid very rapidly. The rapidity has for its
object to make the liquid traverse the zone of crystalliza-
tion temperatures before it has the time to become crystal-
line. 4. By using, as a method of rapid cooling, the immer-
sion in liquid air of material previously reduced to thin
layers, we vitrified solutions of various organic and inor-
ganic substances, such as gelatine, albumin, amino acids,
agar, gums, dextrin, sugars, glycerine, formaldehyde,
sodium chloride, sodium hydroxide, etc. 5. The more
dilute a solution is, the more difficult it is to vitrify it.
Pure water could not be vitrified by our methods. 6. A
vitreous substance devitrifies, that is, crystallizes, when
one raises its temperature. One can therefore cause the
crystallization of a body by warming it up from the vitre-
ous state. 7. The temperatures of devitrification of solu-
tions rise when one passes from a solute of simple molecu-
lar composition to a more complex one, in the same series.
8. At high concentrations of a given solute (for example,
one molecule of sucrose for 9 or 10 of water), devitrifica-
tion does not take i)lace. At intermediate concentrations
(one molecule of sucrose for 10 to 14 of water), devitri-

225

ficatioii occurs in a definite range of temperatures and the
devitrifying mass acquires a tufty structure. When one
passes to lower concentrations, the ranges of devitrifica-
tion temperatures move suddenly higher and the devitrify-
ing material appears as a transparent mass of amber color
which gradually darkens to complete opacity. The beha-
vior of these three groups of concentrations is attributed
to three modes of binding of water. 9. Within the range
of concentrations of the last group mentioned, the devitri-
fication temperatures decrease slightly with increasing-
concentration, a phenomenon perhaps comparable to the
depression of the freezing point of solutions. 10. By a
rapid warming (immersion in warm water), one can avoid
devitrification (crystallization) and pass directly from the
vitreous to the liquid state; we call this change of state
"vitrofusion."

11. Assuming as a working hypothesis that it is usually
the formation of ice which kills protoplasm at low tem-
peratures, we studied the vitality of organisms subjected
to the vitrification and vitrofusion procedure. 12. The
protozoa : eugiena, Paramecium and amoeba did not sur-
vive the treatment, but w^e have no guarantee that they
have not been frozen. Some myxamoebae survived. 13.
Some frog spermatozoa resumed their motility after solidi-
fication in liquid air ; rat spermatozoa never did. 14. The
cells of the epidermis of the onion were capable of plasmo-
lysis after vitrification and vitrofusion. 15. Plant leaves
could be vitrified, but we are not sure that their vitrofusion
has succeeded. In general, they appeared severely in-
jured after being brought back to room temperature. 16.
Moss leaves have given results in perfect accord with the
anticipations relative to the innocuousness of vitrification
and the fatal action of freezing. The cells were always
alive after rapid cooling and rapid warming and always
dead after slow treatment. 17. Frog muscle fibers re-
sponded to electric stimuli after the vitrification and vitro-
fusion procedure.

BIBLIOGRAPHY

ANDREWS, S. T., rroc. h'oi/. So,-.. /'^ 544, 1885.
ATKINS. \V. R., Sc. rroc. Jfoi/. Duhlin Sac, 1;.\ ll2.'.. li»OSta.
, Bioch. J.. 4. 480, l!)(ti»l).

BACHMETJEW, P., ExiKTimfiitcllf Kntdinoldjiiscln' Studicu voiii j.liysi-

kalisch-c'hoinisclicii Staiidiiniikt aiis. Vol. I. 'ri'niporaturverhaltni.ssc lici

Insekten. Loipzijj, JlMH; X'ul. II. l-;iiifiii.ss dcr jiusseren Faktoron aiif

Insekten. Sophia, 1907.
BACKMAN, E. L., and Kr\NSTHo:\I, .1., Jich. i/i.s. riij/sioL, 144, 287,

1912.
HAKIIMETIEFF, P., Arch. Sc. biol. St.-IVIcr.shoiir(/. S, 242, 1901.
HAKNHS, II. T., Ice Eiigiiu'ering. Montreal, 1928.
BARNES, H. T., and VIPONl), W. S., Thjisicdl Hcv., 2S, 453, 1909.
BEEN and KIEBITZ, Boltzinann Ft'stsehrift, p. 010, 1904.
BERXAL, J. D., and FOWLER, R. H., J. Chcm. Phy.^., 1, 515, 1933.
BIALASZEWICZ, K., Arch. f. Entw.-Mrch., 34, 489-540, 1912.
BIGELOW. S. L., and RYCKENBOER, E. A., J. Phy.^. Chem., 31, 474, 1917.
BOBERTAG, O., FEIST, K., and FISCHER, II. W., Ber. d. deiitsch. Chcm.

Gc.s., 47, 3675, 1908.
BOTTAZZI, F., e BERGAMI, G., Arch. Scienzc Biol., 6, 74, 1924.
BRFNI, G., Ber. d. deut.-ich. Chem. Ge.s., 42, 563, 1909.
BUGAEVSKY, M. F., C. r. Ac. Sci. U.F.S.S., JJ, 131, 1939.
BUNSEN, R., Pogg. A7in., 141, 1, 1870.
BURTON, E. F., and OLIVER, W. F., Proc Jiotj. Soc. London, A, 1.53, 166,

1935.
CALLENDAR, H. L., Proc. Boij. Soc. London, A, 86, 254, 1912.
CALLOW, E. H., Proc. Boy. Soc. London, A, 108, 307, 1925.
CAMERON, A. T., and BROWNLEE, T. I., Proc. Boy. Soc. Canada, Sect. 4,

7, 107, 1913.
CASPARY, R., Bot. Zeifg., 665-674; 681-690; and 697-706, 1854.
CHAMBERS, R., and HALE, H. P., Proc. Boy. Soc. London, B, 110, 336,

1932.
CHAPPUIS, Ann. d. Physil, 63, 202, 1897.
COLLIP, J. B., J. Biol. Chem., 42, 207, 1920a.

■ , J. Biol. Chem., 42, 221, 1920b.

LE CONTE, J., London-Edinh.-Diiblin Philo.s. Mug. and Join: of Sc, 36, 329,

1850.

■ , .(/((. ./. Sci., 63, 84, 195, 1852.

de COPPET, L. C, Ann. Chim. Phy.'i., 10, 457, 1907.

DESPRETZ, C. r. Acad. Sci., 5, 19, 1837.

DKTMER, W., Bot. Zcif., 44, 513, 1886.

DICKINSON, HARPER and OSBORNE, Bur. of Stand. Bull, 10, 235, 1914.

DIETERICI, C, Ann. d. Phy.^ik, 12, 160, 1903.

DIXON, H. H., and ATKINS, W. R., Sc. Proc. Boy. Dublin Soc, 12, 275,

1910.
DUFOUR, L., Pogg. Ann., 114, 530, 1861.
DUNAL, F., Mem. Ac Montpellier, (Secf. Sc), 7. 153, 1848.
FAHRENHEIT, G., Phil. Tran.s., 39, 78, 1724.
FISCHER, H. W., Bcitr. z. Biol. d. Pfl., 10, 133, 1911.

227

FISCHER, H. W., and BOBERTAG, O., Bioch. Ztschr., 18, 58, 1909.
FOOTE, H. W., and SAXTON, B., J. Am. Chem. Soc, 38, 588, 1916.
FrCHTBAUER, Ztschr. f. physik. Chem.. 48, 558, 1904.
GARREY, W. E., Biol. Bull., 8, 257, 1905.

, Biol. Bull., 28, 77, 1915.

GAY LUS8AC, Ann. de Chimie {2), 58, 36,3, 1836.

GOEPPERT, Ueber die Warmeentwickelung in den Pflanzen, deren Gefiieren,

und die Scliutzniittel gegen dasselbe. Breslau, 1830.
GOETZ, A., and 8C0TT GOETZ, S., Biodyn., No. 43, 1, 1938.

, , Naturwiss., 26, 427, 1938.

, , Proc. Am. Philos. Soc, 79, 361, 1938.

HALE, H. P., Proc. Boy. Soc. London. B, 112, 473, 1935.

HARDY, W., Pmc. Boy. Soc. London, A, 112, 47, 1926.

HARRIS, J. A., GORTNER, R. A., HOFMAN, W. F., and VALENTINE,

A. T., Proc. Soc. E.rp. Biol, and Med., 18, 106, 1921.
HARRIS, J. A., GORTNER, R. A., and LAWRENCE, J. V., Torrey Bot.

Club Bull, 44, 267, 1917.
HARTMANN, R., Ztschr. f. anorg. u. allg. Chem., SS, 128, 1914.
HARVEY, R. B., Bot. Gaz., 67, 441, 1919.
HAWKES, L., Nature, 123, 244, 1929.
HELLER, W., C. r. Acad. Sci., 199, 354, 1934.

HERSCHEL, J., London-Edinb. Philo.<<. Mag. and Jour, of Sc. 2, 110, 1833.
HESS, H., Ann. d. Physik, 8, 405, 1902.

, Die Gletscher, 1904.

JACCARD, P., u. FREY-WYSSLING, A., Jahrb. f. wi.s.s. Bot., 79, 655, 1934.

JAFFe, G., Ztschr. f. physik. Chem., 43, 567, 1903.

JENSEN, P., u. FISCHER, H. W., Ztschr. f. allg. Phy.nol., 11, 23, 1910.

JOHNSON, J. H. L., .V. S. Inst, of Sci., 13, 126, 1912.

JONES, MILLER and BAILEY, Wise. Agr. Exp. Sta. Bes. Bull., 46, 1, 1919.

KYLIN, H., Ber. d. deutsch. Bot. Ges., 3.5, 370, 1917.

LEES, C. H., Proc. Boy. Soc. London, A, 80, 143, 1908.

LENOIR, M., C. r. Soc. Biol, 107, 1151, 1931.

LOTTERMOSER, A., Ztsclir. f. physik. Chem.. 60, 451, 1907.

, Ber. d. deutsch. chem. Ges., 41, 3976, 1908.

LUYET, B. J., Biodyn., No. 29, 1, 1937.

, /. Phys. Chem. (in press), 1939.

LUYET, B. J., and CONDON, H. M., Biodyn., No. 37, 1, 1938.
LUYET, B. J., and GEHENIO, P. M., Biodyn., No. 30, 1, 1937.

■ , Biodyn., No. 42, 1, 1938.

LUYET, B. J., and GIBBS, M. C, Biodyn., No. 25, 1, 1937.
LUYET, B. J., and GRELL, S. M., Biodyn., No. 23, 1, 1936.
LUYET, B. J., and HODAPP, E. L., Protoplasma, 30, 254, 1938a.

■ , C. r. Soc. Biol., 127, 786, 1938b.

, Proc. Soc. Exp. Biol, and Med., 39, 433, 1938e.

LUYET, B. J., et THOENNES, G., C. r. Acad. Sci., 206, 2002, 1938a.

, Science, 88, 284, 1938b.

, C. r. Acad. Sci., 207, 1256, 1938c.

MAASS, O., and BARNES, W. H., Private comnumication McGill Univer-
sity, 1927. (Quoted from H. T. Barnes, "Ice Engineering," 1928.)

228

MATRrCHOT. L.. ot MOLLTAKD, M.. h'rr. Gn\. Bat.. //. 4(il ; 4(i;'. ct -)-2'2,

1902.
^[AXIMOV, N. A., Jahrb. f. n-i.ss. Bot., J,>', :V2:, l!tl4.
ilKZ, C, Flora, 94, 89, 1905.

^lOLlSCII, TI., Untersuchungoii iihcr das Kifiicrcii (Ut I'tlanzi'ii. .Inia, 1897.
MORAN, T., Proc. Boy. Soc. London, B, i)8, 436, 1925.

, Proc. Soy. Soc. London, A, 1J3, 30, 1926.

, Proc. Boy. Soc. London, B, 107, 182, 1930.

, Proc. Boy. Soc. London, B, 118, 548, 1935.

MOl^SSOX, A., Ann. d. Phy.sik u. Chemie, 105, 161, 1858.
Mi'LLER-THURGAU, H., Landw. Jahrb., 9, 133, 1880.

, Landw. Jahrb., 15, 453, 1886.

:\rrCONNELL, J. C, Proc. Boy. Soc, 49, 323, 1891.
XERNST, W., Deutsch. Phys. Ges., 12, 14, 565, 1910.
XEUMAXX, F. E., Ann. de Chimie (3), 66, 183, 1862.
XICHOLS, E. L., Physical Bcv., 8, 21, 1899.
XORD, F. F., Protoplasnia, Jl, 116, 1934.
PKTTERSOX, O., Vega Expedition, Vol. 2, 1883.
PRILLIEUX, E., Ann. Sci. Nat. 5e Ser. Bot., U, 125, 18(59a.

, Bidl. Soc. Bot. France, 16, 140, 1869b.

REUSCH, E., Pogg. Ann., 121, 573, 1864.

SACHS, Ber. u. VerJi. d. Kiin. Sachs. Ges. d. Wiss. su Leipzig, Nat.-wiss.

Klas.^e, 12, 1, 1860.
SCOTT, G. G., Annals X. T. Acad. Sci., 23, 1, 1913.

, Am. Naturalist, 50, 641, 1916.

TAMMANN, G., Ztschr. f. phys. Chem., 25, 472, 1898.
TAMMANX, G., The States of Aggregation, New York, 1925.
TAMMANX, G., and BuCHNEE, A., Ztschr. f. anorg. u. allg. Chem., 222,

12, 1935a.

, Ztschr. f. anorg. ii. allg. Chem., 222, 371, 1935b.

duPETIT-THOUARS, Le verger francais, 1817.

THWTXG, Ztschr. f. physil: Chem., 14, 286, 1894.

TROUTOX, J. J., Proc. Boy. Soc. Dublin, 8, 691, 1898.

TURXER, Ztschr. f. physik. Chem., 35, 385, 1900.

VOGEL, A., Gilbert's Ann. d. Physik, 64, 167, 1820.

VOIGTLaNDEE, H., Cohns Beitr. z. Biol. d. Pfl., 9, 359, 1909.

WALTER, H., und WEISMANX, 0., Jahrb. f. wiss. Bot., 82, 273, 1935.

WALTOX, J. H., and JUDD, R. C, J. Phys. Chem., 18, 722, 1914.

WARTMAX, Arch. Sc. Phys. Nat. Geneve, Nouv. per. 7, 277, 1860.

WASHBURX, E. W., Science, 61, 54, 1925.

WEI5ER, H. F., Wied. Ann., 10, 103, 304, 1880.

WEIGMANN, R., Biol. Zbl., 58, 301, 1936.

WIEGAXD, K. M., Plant World, 9, 26, 1906.

WRIGHT, R. C, and HARVEY, R. B., V. S. Depl. Agr. Bull., 895, 1, 1921.

WRIGHT, R. C, and TAYLOR, G. F., U. S. Dept. Agr. Bull., 016, 1. 1921.

YOUXG, S. W., J. Am. Chem. Soc, 33, 148, 1911.

ZACHAROWA, T. ^f.. Jahr}>. f. wiss. Bnl., 65, 61, 1926.

PART III

THE MECHANISM OF INJURY AND DEATH RY
LOW TEMPERATURE

Some confusion in the study of death arises from the
faihire to distinguish between organismal, systemic, cel-
lular and protoplasmic death. It is evident that a theory
which hokls that the death of a frog results from the de-
struction of the red cells in the frozen blood refers to
organismal death; a theory, according to which the leaves
of a tree die after a layer of ice has been formed at their
point of attachment and severed their connection with
the stem, considers systemic death ; a theory stating that
the death of a tissue results from a tearing of the cellu-
lar membranes by ice crystals is concerned with cellular
death ; finally, a theory which considers the fundamental
physico-chemical processes involved in the destruction of
living matter, such as the precipitation of colloids by con-
gelation, regards protoplasmic death. In this work we
are not concerned with organismal nor with systemic
death by cold, though occasionally reference will be made
to these; we are concerned primarily with protoplasmic
death. But the investigators, in general, do not distin-
guish between cellular and protoplasmic death, their
theories and their experiments concerning the two phe-
nomena are so intimately related that we shall treat these
two subjects together.

There is some evidence that the mechanism of injury
and death b}^ cold is different when death is accompanied
by ice formation and when it is not. These two cases
will, therefore, be discussed in separate chapters,

229

(:iiapti:k i

ACTl()^ Ol COLD wniioLT
ICK KOHMAIION

( )lisci-\;iti(>iis ,-ni(l theories on llie aclioii ol' low leiiiper-
ature without ice i'oriii;ilit)ii can he classitied into two
g-roups: ]. Those concerned with orgaiiisiiis which die or
are injured in the proximity of tlieir freezing point; 2.
Those which refer to tiie action of extreme cokl, at tem-
peratures at which the material solidities without form-
ing ice crystals, that is, vitrities. The study of the first
group will he sulxlivided into two sections: one, con-
cerning the effects of chilling ahove the fi-eezing i)oint;
the other, the action of cold in the snhcooled state.

1. A<TI()X or cold) IX TIIIO NEIGHBORHOOD OF
Till-: FIMvEZING POINT OF PKOTOPLASM

A. ACTION OF COLD ABOVE THE FREEZING POINT

This subject has been competently reviewed by Bel-
ehradek (1935) in the chapter ''Chilling, Chilling-Coma
and Death by Chilling" of his monograi)li ''Temperature
and Living Matter". The reader will find in that chap-
ter, presented in a condensed tabular form, the data of
numerous investigators on the temperatures of cessa-
tion of protoplasmic streaming, ciliary movement, cellu-
lar multi])lication, gi'owth, metabolic activity and irri-
tability. There follows a compendium and discussion of
the observations and theories concerning injury and death
by cold at temperatures above the freezing point. Nu-
merous bibliograi^hical references are given. Since the
subject has been already reviewed, we shall jjresent here
only a brief outline of the essential points of interest.
A few ([uestions which have not been discussed by
Belehradek will be considered in detail. For these only
will the l)ibliographical references be given.

Practically all the authors cited by this reviewer have
found that chilling l)ecomes injni'ions or lethal only

230

231

after liaviiii!;- exerted its action on protoplasm for a
relatively long time. We encountered in the literature
a few reports of rapid injury by cold. The mechanism
of injury is apparently different in these two cases; a
slow injury suggests a disturbance in the interplay of
physiological functions while a rapid injury is more likely
to be due to some sudden structural changes such as
precipitation, solidification, etc. The two cases will be
treated here separately.

1. Sloir IiiJiirioKS Action. A. Observations. Ho-
moiotherms enter into a state of coma when their body
is cooled to some temperature above zero and they are
killed on further cooling.

A state of rigor, induced by chilling, has been de-
scribed in homoiotherms and poikilotherms, in particular
in fishes.

The numerous observations made on insects, either in
the adult or in the larval stages, are contradictory. Both
a high resistance and a high sensitivity to cold have been
reported (cf. Bachmetjew, 1901 and 1907, and Uvarov,
1931).

Prolonged exposure of some plants to near-zero tem-
peratures is sometimes fatal.

In general, organisms adapted to high temperatures
are more easily affected by cooling. Among these one
should mention, besides the tropical plants, some poikilo-
therms, in particular reptiles.

While the plants and animals just mentioned are higher
forms, less differentiated or undifferentiated protoplasm
has also been reported to be injured or killed by cold
without freezing. Eggs are, according to some authors,
very sensitive: ant eggs ( Pictet, 1893), eggs of the Med-
iterranean fruit fly (Bach and Pemberton, J. Agr. Res.,
5, 657, 1916), bedbug eggs (Hase, Ztschr. f. Parasitenk.,
2, 368, 1930), chicken eggs (Moran, 1925). However,
there are also reports claiming a high degree of resis-
tance to cold in eggs.

O'M

/). I) i s (' u s s i () 11. "Cold aiicsllu'sia", "cold riii^or"
and death by chilling-, in lionioiothcrnis and, in several
cases, in ]ioikilotheniis, have been atlril)nted to a specific
injury to the nervous system, but nothing is known of
the physico-chemical mechanism of this injury. Camer-
on and Brownlee (191.'-)) consitler the fact that an excised
frog's heart is more resistant to cold than the entire
frog, as indicating that cold must exert a special action
on the nervous system; the latter is intact in llie entire
frog and absent in the excised heart.

Death at temperatures above the freezing point is in
plants probably due to a disequilibrium resulting from a
change in the rate of the various physiological functions,
in jiai'ticular, of water absorjjtion from the ground and of
transpiration.

In organisms adapted to high temperatures it is in-
teresting to note that injury and death are due to the
same factor which has produced adaptation, namely a
change in the temperature. While high temperature, act-
ing gradually for a long time, modified the organism so
as to make it fit the new conditions, low temperature,
later acting al)ru]itly for a short time, exerted an inju-
rious or lethal action.

While these interpretations deal almost exclusively
with organisinal death, the following structural changes
have been invoked to explain the mechanism of proto-
plasmic death :

1. Chemical changes (very few specific suggestions
have been made) ;

2. Changes in the velocities of interrelated chemical
reactions ;

3. Impairment of the functions of elimination and con-
sequent accumulation of toxic products;

4. Impairment of the functions of osmosis and permea-
bility ;

5. Impairment of the functions controlling ]jrotoplas-
mic water relations and resulting delivdration ;

233

6. Changes in the viscosity of protoplasm;

7. Changes in the adsorptive properties of some pro-
topUisniic constituents;

8. Solidification of protoplasmic fats;

9. Precipitation of some of the components of the liv-
ing structure;

10. Coagulation processes.

Belehradek discusses the interrelationship between sev-
eral of these mechanisms. Many of them might be in-
volved at the same time in causing death.

The observations concerning deJiydration as a result of
the action of cold without ice formation call for a few
notes and remarks.

According to Molisch (1897), when Tradescantia hair
cells are exposed to cold for a long time the protoplast
separates in some places from the cell wall, indicating
that water has been extruded,

Greeley (1901) described the same phenomenon in
Spirogijra filaments cooled from 20 "^ to +1° and kept at
that temperature for 3 hours. When the filaments were
cooled while immersed in olive oil he could see water
droplets exuding from the cells into the oil.

Klemm (1895) looked upon such a protoplasmic con-
traction as an initiation of a disorganization which grad-
ually will result in death.

Chambers and Hale (1932), however, noted that main-
taining onion epidermis at - 10 in the unfrozen condi-
tion for several hours does not induce plasmolysis. The
apparent contradiction between this last observation and
those previously recorded might be due to the fact that
plasmolysis by cold is exhibited only by some types of
protoplasm and only under some particular conditions.

Concerning the cause of dehydration, we wish to point
out that the rounding up of protoplasm in Tradescantia
hair, such as occurs under the action of cold, can also be
produced by various forms of more or less violent stim-

•2:\4

Illation. A comparison of this pliciioiiiciioii wilh llic
t'oiitrac'tioii of iimscular 1 issue and of oIIrt forms of
eontrat'tile protoi)lasm 1)\ cold mii;lit not l)c out of
place.

At first it appears somewhat surprising that the with-
drawal of water involved in this ])h('nomenon be at all
injurious and if it is injurious one would hardly expect
it to lead to death.

2. l\apid Lijurious Aclhni. (a) In the ciliate Sff^utor
coendeus, Greeley (1901 and 11)U2) observed that while a
slow cooling- caused encystment or sporulation, a sudden
cooling was lethal. (The absence of ice formation within
the organisms themselves is evidently implied in the au-
thor's account.) In the last case, that is, after sudden
cooling, the animals were deformed, their pigments were
released and complete disorganization followed. In the
former case, when the rate of cooling was about 10 de-
grees per hour, the following sequence of phenomena
which is partly the same as that which often precedes
death was observed: 1. A cessation of body movements
and of ciliary motion; 2. The formation of vacuoles
which gradually increased in size; 3. A change in the
form of the cell which gradually became spherical; 4.
The separation of the nuclear material; 5. A transforma-
tion of the protoplasm into a granular mass; 6. The
throwing off of the ectosarc; 7. The transformation of
the cell into a cyst. The animal resumed its normal
activity upon an elevation of temperature.

The fact that the final result of the action of low
temperature was death or encystment depending on the
velocity of cooling postulates that some similar mechan-
ism must underlie these tw^o processes.

The reduction of the water content, Greeley suggests,
might be responsible for l)oth, cystogenic and lethal ac-
tion, as indicated by fnrtlicr cxpcrinicnts in which an
immersion of Sfoitor in a strongly hypertonic M/10
solution of sucrose, produced the same etfects as a

235

sudden lowering' of the teniix'i'Mtui'e, and an imniersion
in M/lOO sucrose the same effects as a slow cooling.

In cooling- experiments of the same type with Monas,
Greeley (1902) described a typical sporulati(m caused
by low temperature. At +4 resting- cells were formed
which, when exposed for 5 to 7 days to +1% divided into
several spherical spores (from 2 to 25 in a cell). So the
physiologT,cal processes involved in reproduction by
spores seem to bear some similarity with those which
lead to injury and death.

(})) Gehenio and Luyet (1939) found that the Plasmo-
dium of the myxomycete Physarmn polycephalum w^as
killed by sudden or moderately rapid exposure to cold at
temperatures above the freezing point. They described
the following phenomena as stages in the process of
death, when the temperature of the organisms was low-
ered gradually from room temperature at the rate of one
degree per minute: 1. At +5° to 0°, movement ceased; 2.
At +3° and below, hyaline vesicles formed; 3. At 0' or
below, the protoplasm underwent disorganization; 4. At
the same temperature the pigment granules broke down.
A sudden cooling to a given low temperature w^as consid-
erably more injurious than a slow cooling to the same
temperature.

■ The authors propose the following interpretation of
their observations. Under the action of the lowering of
temperature the protoplasmic sol would set gradually to
a gel. They relate this hypothetical gelation to the in-
crease in viscosity wdiich was noted by Heilbrunn (192-1:)
in protoplasm cooled to a temperature slightly above zero
and to the cessation of protoplasmic streaming. But the
gelation ' 'would be a reversible process preceding death
and not constituting death. . . .After complete setting,
the gel would undergo a syncretic breakdown, squeezing
out the dispersion medium enmeshed within it, and this
would be the death process itself. At the periphery of
the i)lasmodiuni the locally expressed fluid would appear

in the lonii ol" vesicles or blisters. Within the interior
of the ])lnsiu()<liuni, syneresis would manifest itself in the
phase separation or disorganization."

This view, the authors state, lits in with the fact that
the changes induced by cold are, in general, the same as
those produced by otlier lethal agents; the approximation
of the molecules wliieii causes gehition would be a lower-
ing of temperatuie in the present case, while it would be
an electrostatic discharge or a dehydration or an adsorp-
tion, etc., in the case of otlier killing agents. The influence
of the time factor (death })roduced in 5 seconds at -1°),
the greater elTect of abrupt cooling and the percentage of
recovery after exposure to cold at a given temperature
and for a given time, are also, the authors point out, con-
sonant with the theory.

B. ACTION OF COLD IN THE SUBCOOLED STATE

1. Observations. The literature on the action of cold in
the subcooled state is summarized in Table 1.

2. Discussion. A. M e c h a n i s m of Injury and
Death. It readily appears that an injurious action of
cold in the subcooled state, though definitely observed, is
rather unfrequent. Subcooling probably affects some
types of protoplasm more than others but it is, at present,
impossible to say which types.

As in the case of injury above the freezing point, the
time factor plays an important role. This and other sim-
ilarities make it quite certain that, in the last analysis,
when no ice is formed, the mechanism of action of cold on
protoplasm is the same above and below the freezing
point. What has been said in the preceding section would
then adequately apply here.

On first consideration one might think that, in the cases
in which cold injures protoplasm, subcooling must cause
more damage than chilling, for the simple reason that the
subcooling temperature is lower. But if injury is due to
a disequilibrium of functions, the latte7% in general, are

237

slower at lower temperatures (as is the case for osmosis,
for example) and less damage should be jjroduced during
a given time in the subeooled state than during the same
time at temperatures above the freezing point. If, on the
other hand, injury results from a process akin to precipi-
tation in a saturated solution, the lower the temperature
the greater would be the chances of its happening. A
comparative study of the degree of damage done in the
same time in the subeooled state and above freezing might
allow one to decide between these two possible mechan-
isms of injury.

TABLE 1

Action of Cold in the Subcooi.ed State

(a) Injury by subcooling

Temp, and

Organisms

Time of
Exposure

Results

Investigators

B. coli

-0.5 or

50% killed

Hilliard, Torossian

-6.0°

& Stone, 1915;

(In glu-

Hilliard & Davis,

cose solu-

1918

tion)

Paramaecium

Below -4°

Marked swell-

Efimoff, 1924

-

30 min.

ing & death

-16°

Swelling,

Wolfson, 1935

Long

visibility of

time (In

nucleus &

capillary

death

tubes)

Colpidmm colpoda and

Below -4°

Shrivelling &

Efimoff, 1924

Spirostomiini ambi-

30 min.

death

guum

White blood cells of

-2° to -3°

Death

Schenk, 1870

poikilotherms

8 hrs.

White blood cells

-3°

Death

Schenk, 1870

of homoiotherms

>15 min.

Mycelia and hyphae of

-13°

Death

Lindner, 1915

Aspergillus and Pen-

24 hrs.

icillium

Germinating tubes

-4.2° to

75% killed

Bartetzko, 1910

of Aspergillus niger

-13°
9 days
(In 50%
glucose)

Cells of red cabbage

-3.9°

Death

Iljin, 1934

leaves

>1 day

238

(b) Innocuousness of subcooling

Organisms

Temp, and
Time of
Exposure

Criterion of
Vitality

Investigators

Streptococci of

-17° to

Citovicz, 1928

scarlet fever

-18°
2 wks.

Amoeba

-5°

Chambers & Hale,
1932

Paraviaeciuni

-9°

Short

time
-14.2° to

Efimoff, 1924

-16°

Wolf son, 1935

Short

time

Colpidinm and

-9°

Efimoff, 1924

Si)irostomu))i

Short
time

Etiglena

-0.2°
1 hr.

Jahn, 1933

Leucocytes of

-7°

Schenk, 1870

poikilotherms

Short
time

Leucocytes of the

-3°

Schenk, 1870

rabbit

15 min.

Chicken eggs

-4.6°
47 hrs.

Moran, 1925

-2.9°

Moran, 1925

118 hrs.

Marine algae

-1.8°

Some physio-

Kjellmann (Quoted

logical

from Bot. Ztg.,

activity

33, 771, 1875)

l^^itella

-2°

Protoplasmic
streaming

Kylin, 1917

Germinating spores of

-14°

Bartetzko, 1910

AnperyiUus, Penicil-

2 hrs.

liu7n, Botrytis, and

Phycomyces

Aspergillus

-6° to
-11°
4 days

Bartetzko, 1910

Various fungi

-3°

Growth

Horowitz-Wlassowa
& Grinberg, 1933

-7.8°

Growth

Brooks & Hansford,
(Food Inv. Bd.,
Spec. Rpt. No. 17,
1923)

-8.9°

Growth

Smart, 1935

-6° and

-10°

Growth

Bidault, 1921

-10°

Growth

Haines, 1930

239

Temp, and

Organisms

Time of
Exposure

-10° to

Criterion of
Vitality

Investigators

Hyphae of

No stiffening

Molisch. 1897

Phy corny ces

-120

of protopl.
fluids

Submerged fungal

-13°

Lindner, 1915

mycelia

8 hrs.

Aerial hyphae

-11°
41/2 hrs.

Lindner. 1915

Hair cells of Trianea

-13°

Klemm. 1895

and Momordica

<15 min.

Hair cells of Trades-

-50 to -9°

Molisch. 1897

canticK Episcia and

6 hrs.

Pelargonium

Plant tissues

-13°

Ability to

Voigtlaender, 1909

(various)

plasmolyse

Rye seedlings

-11.1°

Zacharowa, 1926

Embryonic mamma-

-5°

Growth

Simonin, 1931

lian tissue

5 days

Frog gastrocnemius

-4°

No loss of

Moran, 1929

2 days

irritability

Frog muscles

-15°

No loss of

Chambers & Hale,

3 hrs.

contractility

1932

-18°

Kodis, 1898

Bees, bumble-bees,

-2.9° to

Kalabuchov. 1934

wasps, and larvae

-17.1°

of beetles

48 hrs.

Frogs

-10°

Kodis, 1898

Salamanders

-2.2°

Jecklin. 1935

Toads

-1°

Kalabuchov, 1934

Tortoises

-5.3°

Kalabuchov, 1934

Bleaks and Stickle-

-3.06°

Schmidt, Platanov,

backs

& Person, 1936

Carps

-30

Regnard, 1895

-5°

Kalabuchov, 1934

CiteUus suslica and

-0.5° and

Murigin, 1937

Citellus pigtnaeus

-1°

(ground squirrels)

Xyctalus noctula

-5.9"

Kalabuchov, 1934

(bat)

L>4()

//. 1\ () 1 (' r 1 ;i y (' (I I) y S u h c o o 1 i ii <•• i ii
X a 1 II r e. Conceriiiiig Iho role j)lay('<l by subcooliiiji: in
the preservation or in the injni-y of j)hints and animals
in natnre, a review of the literature I'eveals that the most
diverse views have been held. These views depend on the
theory accepted by each individual author for the mechan-
ism of death by cold. They are, too often, rather theo-
retical and lacking in ex])erimental evidence.

Since the generality of investigators have found that
protoplasm is not injured in the subcooled state, they nat-
urally concluded that subcooled animals or plants are
"protected" against the damage of frost. The resistance
of plants in winter to temperatures of several degrees
below zero is often attributed to a tendency to subcool.
The fact that trees in northern forests and poikilotliermic
animals in cold climates are not killed by long and severe
winters is also thought by many to be due to a particular
ability to undergo deep subcooling (Kalabnchov, per-
sonal communication).

Weigman (1936) pointed out that hibernating snails
have a freezing point as high as -0.39° ; their survival to
low temperatures therefore cannot be attributed to a low^-
ering of the freezing point at the onset of the state of
dormant life, it must then be due to the ability to sub-
cool,

Harvey (1919) suggested another indirect protective
role of subcooling, namely, that plants left for some
length of time in the subcooled state might undergo adap-
tative changes and become hardened against frost.

The authors who held the theory of the specific mini-
nmni (see below) according to which death occurs at a
certain given temperature, no matter whether ice is
formed or not, took sides against the view of ''protective"
subcooling. For them, only that which prevents an organ-
ism from reaching the specific minimum is protective.
Subcooling itself does not, of course, prevent the drop
of temperature, while freezing, by liberating heat, does.

241

So they conclude, paradoxical as it may seem, that sub-
cooling is dangerous while freezing is protective. A few
of the arguments of the main proponent of this theory,
]\Iez (11)05), will suffice to illustrate it and to show how
it has been applied to the most varied plK'iiomeua of
nature.

According to this author, the tissues of the stem of
Impatiois were killed sooner when freezing was preceded
by subcooling than when it was not. But in these experi-
ments the quantities of ice formed in the two cases were
not compared, a factor which has been shown to be of
fundamental importance in causing death.

Mez then applies his theory to the supposedly observed
fact that trees and shrubs are more injured in "stagnant"
air than in air agitated by winds. Shaking would, in the
latter case, prevent subcooling which is assumed to be
the damaging agent.

The frost resistance acquired by ''hardened" plants is
attributed by this author to the prevention of subcooling
by oils formed during hardening.

The fact mentioned by Sachs and Molisch that a sprout
of Tradescautia, exposed to 5°, half in water and half
in air, had the latter part killed and the former un-
harmed, is explained by Mez on the assumption that the
part frozen in air was previously subcooled to a greater
extent.

II. ACTION OF EXTREME COLD

While injury and death can result from cold without
ice formation at near-zero temperatures, it seems that, in
general, extremely low temperatures are harmless if ice is
not formed. So, instead of discussing the mechanism of
injury by cold in this section, we shall consider the cause
of the innocuousness of xevy low temperatures.

1. Observations. The organisms which resist the tem-
peratures of liquefaction of "permanent" gases can be di-
vided into two groups: 1. Those which support drying

242

niul which, in ihc dry stale, an- ii(»l atVcrtcd l)y the ht\v
temperatures in question; '2. Thox- which, either with
tlieir full water content or after luniiii;- been only slio-htly
dehydrated, are innnune against cxtrenu' cold. This sec-
ond class, in its turn, includes two subdivisions: 1. There
are organisms which survive liquid air temperatures only
when special precautions are taken, such as extremely
rapid cooling- and re warming; '2. There are others which,
without any of these precautions, can be immersed in
liiiuid air, liquid hydrogen or iiiiuid helium without in-
jury. The experimental data concerning these three
groups are presented in tables 2, 3 and 4.

2. Discussitni. A. C a u s e o f the K e s i s t a n c e
t o K X t r e ni e C o 1 d. The fact that all the organ-
isms mentioned in the tirst grou]) (Table 2) resist very
low temperatures when they art' dried suggests that it is
the absence of freezable water which innnunizes them.
The type of precautions which save the organisms of
the third group (Table 4) from death, that is, rapid cool-
ing and rewarming through the temperatures at which
freezing of water would be i)Ossible (from 0 to some tens
of degrees below 0 ), supports the assumption that, if
ice formation is prevented, danger is avoided. This as-
sumption has naturally been applied to the organisms of
the second group (Table 3), in other words, it has been
thought that when bacteria, yeasts, fungi, algae, ]irotozoa
and germ cells in the vegetative state or nematodes, ro-
tifers and tardigrades, with their full water content, re-
sist innnersion in Tuiuid air, it is because their water does
not freeze.

The cause of the assumed absence of ice in the organ-
isms of the second group has been sought in the capillary
forces within the intermicellar spaces. Some types of
protoplasm (the bacteria, in particular) w^ould be of a
denser structure and the capillary spaces in them would
be so small that '"water Avill not be changed into ice at
any temperature" (Lipman, 1939). Concerning this view

243

we wish to point out that it is not established experimen-
tally that the degree of subcooling- of water becomes in-
creasingly greater when the size of the capillary spaces
reaches the order of a micron or less. The generally
assumed relation between degree of subcooling and ca-
pillary size already ceases to be consistent when the
capillary sizes are of the order of 0.1 mm. (cf. our re-
view, ''The Physical States of Protoplasm at Low Tem-

T ABLE 2
OuciA.MSMs Which, When Di:y. Resist Extuemely Low Te.mperatures

Organisms

Temp.

j Time of Exposure to

Cold & Other Conditions Iiivestigators

1. Bacteria and Fungi

Bacteria :

Staph, pyogenes
aureus

Common bacilli
(Spores)

Myxomycetes:
Pliysarum

polycephaluni

L. air

L. helium
4.2° to
I.350K

L. air

125 days

Paul & Prall,
1907

6 hrs.; previously des- Lipman,
iccated for 2 weeks 1936a

12 hrs.

Gehenio &
Luyet (un-
published)

2.

Algae

Pleurococcus vulgaris

-|

Chlorella vulgaris

Stichococcus

hacillaris

Hantzschia

480 hrs. at -190° and

amphioxys

71/2 hrs. at -269° to

Pinnularia viridis

-190° and

-271°; previously

Chlorococcum

-269° to

desiccated over bari-

Becquerel,

huniicohim

-271°

um oxide for 3 mos.

1932d,1936

Palmella niiniata

and then sealed into

Oscillatoria

tubes evacuated to

Glaeotila

lO-'mm. of mercury

Hormidium

Siphonema

Pediastrum

Tribonema elegans

4° to

Several hours; previ-

Becquerel,

1.84°K

ously dried and kept
in high vacuum for
22 years

1932b

244

Organisms

Temp.

' Time of Exposure to i

Cold & Other Conditions Investigators

3. Protozoa

Colpoda cmuUus
(Cysts)

Amoeba proteus
Aiitocha lh)i(i.r
Amoeba dartylifera
Aetinophri/s sol
Paramaeviion

bursaria
Euglena riridi.s

L. air

L. air

-190° and
-269° to
-271°

13V2 iirs.; previously
air-dried

12i.> days; previously
subjected to a vac-
uum of about 10-^
mm. of mercury for
2 to 3 days

480 hrs. at -190° and
71/2 hrs. at -269° to
-271°; in soils pre-
viously dried in a
vacuum over barium
oxide for 3 months,
then sealed in glass
tubes evacuated to
lO-'mm. of mercury

Taylor &

Strickland,

1936
Taylor &

Strickland,

1936

Becquerel,
1936

4. Spores and Seeds

Spores of the fungi,

L. hydr.

77 hrs.; previously

Becquerel,

Mucor,

dried and sealed in

1910

Rhizopus,

tubes evacuated to

Sterigmatoeystis

lO'cm. of mercury.

and Aspergillus

Left for 2 years in
vacuum after expo-
sure to low tempera-
ture

Spores of the mosses.

L. nitr.

10 days; air-dried

Becquerel,

Dicranella,

1932a

Atrichum,

Hypnum,

Leucobryum,

Funaria and

BracJiythecium

Spores of 2 genera of

L. helium

9 hrs. at 4° and 1 hr.

Becquerel,

mosses

4° to

at 1.84°K; previous-

1932a

1.84°K

ly desiccated and
sealed in tubes evac-
uated to 10 "mm. of
mercury

Spores of the fern.

L. helium

6 hrs. at 5° to 3° and

Becquerel,

Polysticfiitm filix

5° to 3°K

5 hrs. at 3°K; previ-

1930

mas

ously desiccated and
sealed in the highest
obtainable vacuum

Pollen grains of

L. helium

7 hrs.; previously thor-

Becquerel,

Antirrhi7ium and

Minimum

oughly dried and

1929

Nicotiana

1.3°K

sealed in tubes evac-
uated to 10 "mm. of
mercury

245

Organisms

Temp.

Time of Exposure to
Cold & Other Conditions

Investigators

Seeds (various)

-180°

1 hr.

Dewar &
McKen-
drick, 189l'

-200°

Pictet, 1893

L. air

110 hrs.; previously

Brown & Es-

air-dried (with still

combe, 1897

10 to 12% water)

L. hydr.

Dewar. 1899

L. hydr.

6 his.; previously air-

Thiselton-

dried

Dyer. 1899

L. air

24 hrs.

Adams, 1905

L. air

132 hrs.; previously

Becquerel,

vacuum-dried

1909

L. air and

3 weeks in liquid air

Becquerel,

L. liydr.

and 77 hrs. in liquid
Ho; previously desic-
cated over barium
oxide for 2 wks. and
sealed in tubes evac-
uated to lO-'mm. of
mercury; kept sealed
for 1 yr. after treat-
ment

1905

L. helium

101/4 hrs.; previously

Becquerel,

3.8°K

desiccated over bari-
um oxide for 2 wks.
and sealed in tubes
evacuated to lO'^mm.
of mercury

1925

L. air

60 days; previously

Lipman &

dried over CaCL

Lewis, 1931

L. helium

40 hrs. at 4.2°, 2 hrs.

Lipman.

4.2° to

at 1.35° and 2 hrs. at

1936a

1.35°K

1.35° to 4.2°K; pre-
viously desiccated for
2 wks. over HoSO^
in a partial vacuum

5. Metaphyta

Lichens:

Parnielia. Xantlioria
and Cladonia

L. nitr.

18 days; previously air-
dried

Becquerel,
1932d

Moss:

Hypnum

L. nitr.

18 days; previously air-
dried

Becquerel.
1932d

Protonemata of 8
genera of mosses

L. air

50 hrs. ; previously vac-
uum-dried over
H,SO,

Lipman,
1936b

Mn i u m

L. air

30 seconds

Luyet & Ge-
henio.1938

246

Organisms

Pteridophytes:

Spermatophytes:
Tuberous roots of
lianuncnlaceae

Sprouting seeds of
wheat, rye. lucern
and Hcliauthus

Temp.

Time of Exposure to
Cold & Other Conditions Investigators

L. air

L. nitr,

L. nitr

2 hrs.

IS days; previously air
and vacuum-dried

Luyet & Har-
tuug, 1939
(unpub-
lished)

Becquerel,
1932b

IS days; previously air- Becquerel,
and vacuum-dried , 1932c

(('. Mf'tazoa

Nematodes :

Fleet IIS. Tylenehus

L. helium

Air-dried

Rahm, 1920,

and Dori/hiimus

1921, 1923

Rotifers:

Callidina and

L. air

125 hrs.; previously air-

Rahm, 1920,

Adineta

dried for IS days to
14 months

1921. 1923

L. hydr.

26 hrs.; previously air-

Rahm. 1920.

dried

1921. 1923

L. helium

7% hrs.; previously

Rahm. 1920.

-269° to

air-dried for IS days

1921. 1923

-271.88°

to 14 months

Adineta gracilis, Ro-

L. nitr.

480 hrs. to liquid N.

Becquerel,

tifer vulgaris and

and

and 71/^ hrs. to liquid

1936

Callidina angusti-

L. helium

He; previously vac-

coUis

uum-dried for 3 mos.

Tardigrades:

Maerobiotus.

L. air

125 hrs.; air-dried for

Rahm, 1920,

Eeliiniseus and

18 days to 14 months

1921. 1923

Miliiesiimi

L. hydr.

26 hrs.; air-dried for

Rahm. 1920.

18 days to 14 months

1921, 1923

L. helium

7% hrs.; air-dried for

Rahm, 1920,

-269° to

18 days to 14 months

1921, 1923

-271.88°

Maerobiotus

-269° to

7y2 hrs.; vacuum-dried

Becquerel,

Hufelandi

-271°

for 3 months

1936

Arthropods:

Art em ill salina

L. air

Air-dried

Gilchrist,

(eggs)

1939 (un-
published)

247

TABLE 3

OuGAMSjis Which, ix Tin; Wkt Condition, Resist
Extremely Low Te.mpekatvues

Organisms

Temp.

Time of Exposure to
Cold & Other Conditions

Investigators

1. Bacteria, Yeasts and Other Fungi

Bacteria:

Bacteria (various)

-200°

Pictet. 1893

-192°

11/2 hrs.

White, 1899

L. air

20 hrs.; in broth or on

Macfadyen,

solid media; veg. and

1900

spore forms

L. nitr.

10 hrs.

Macfadyen
& Rowland,
1900b

L. hydr.

Beijerinck &
Jacobsen,
1908

-180°

8 days

Moussu, 1912

L. air

48 hrs.; veg. forms on
agar slants

Lipman.1937

B. typhosus

L. air

6 months

Macfadyen
& Rowland,
1900a

-253°

DeJong, 1922

B. coli

L. air

6 months

Macfadyen
& Rowland,
1900a, 1902

-253°

DeJong, 1922

L. hydr.

3 hrs.; in 0.85% NaCl
solution

Kadisch,1931

Staph, pyogenes

L. air

6 months

Macfadyen

aureus

& Rowland,
1900a, 1902

L. hydr.

50 hrs.; suspended in

Kadisch,1931

-252°

0.85% NaCI solution

B. anthracis

L. air

15 hrs.; bouillon sus-
pensions of veg. form

Belli. 1902

Spirochaeta pallida

L. nitr.

14 days

Jahnel, 1937

Bacillus of Chicken

L. air

15 hrs.; bouillon sus-

Belli. 1902

Cholera

pensions of veg. form

Sodoku spirilla

L. nitr.

14 days

Jahnel, 1937

G07WC0CCllS

L. nitr.

24 hours

Lumiere &

-195°

Chevrotier,
1914

B. faecalis

-253°

DeJong, 1922

alcaligenes

B. lactis aerogenes

-253°

DeJong, 1922

248

Time of Exposure to

Organisms

Temp.

Cold & Other Conditions

Investigators

Bacteria of

-253°

DeJong, 1922

Paratliyphoid A

and F^ and of

Enteritis

Tubercle i)acillus

L. air

6 weeks

Swithin-
bank, 1901

L. hydr.

50 hrs.; suspended in
.85% NaCl solution

Kadisch,1931

L. air

20 alternate freezings

Weinzirl &

and thawings

Weiser,
1934

Avian Tubercle

L. nitr.

200 alternate freezings

Kyes & Pot-

bacillus

and thawings

ter, 1939

Luminescent

L. helium

Several hrs.; fresh veg.

Zirpolo, 1932

Bacteria

state

Yeasts:

Beer yeast

L. air

6 min.

Doemens,
1897

Yeasts (various)

L. air

6 months; washed and

Macfadyen

pressed

& Rowland,
1902

Pathogenic yeasts

-252°

3 hrs.; suspended in
0.85% NaCl solution

Kadisch,1931

-268.8°

2 hrs.; suspended in
0.85% NaCl solution

Kadisch,1931

-272°

11/^ hrs.; suspended in
0.85% NaCl solution

Kadisch,1931

Saccharomyces

L. air

13 hrs.; cultured on

Kaercher,

cerevisiae

-183° to
-192°

agar slants

1931

Fungi :

Mycelia of

-185°

Vz hr. ; on agar slants

Heldmaier,

Schizophyllum and

1929

Collybia

L. air

13 hrs.; on agar slants

Kaercher,
1931

Mycelia of

L. air

13 hrs.; on agar slants

Kaercher,

Aspergillus and

1931

Armillaria

L. air

48 hrs.; on agar media

Lipman, 1937

Mycelia of

L. air

13 hrs.; on agar slants

Kaercher,

Hypholoma,

1931

Clitocybe,

Placodes,

Xylaria and

Phycomyces

Mycelia of various

L. air

48 hrs.; on agar media

Lipman, 1937

fungi

249

Organisms

Temp.

Time of Exposure to
Cold & Other Conditions

Investigators

Diatoms

ChloreUa

Sticliococcus
bacillaris

Trypanosomes
(various)

Trypanosoma
gam biense

Trypanosom,a Lewisi

Trypanosoma brucei
Trypanosoma

venezuelense
Trypanosoma

equiperdum

Dourine

Trypanosome

Eggs of Macrohiotus
(Tardigrad)

Spores of various
fungi

Spores of
Melanconium,
Coniothyrium,
Euroiium and
Cystospora

2. Monocellular Algae
-200°

L. air

L. air
-183° to
-192°

3. Protozoa

Frozen in water

Pictet, 1893

cultures

1 hr.; suspended in

Warburg,

Knopp's solution

1919

13 hrs.; cultured on

Kaercher,

agar slants

1931

L. air

L. air
L. air

L. air

•L. air
L. nitr.

5 to 25 min.

20 min.
75 min.

1 hr.

21 days
20 hrs.

4. Germ Cells and Spores
L. air and

L. hydr.

-252°

-268° to
-272°
L. air

50 hrs.; suspended in
0.85% NaCl solution
71/2 hrs.

1 hr. ; suspended in
water

Laveran &

Mesnil,

1904
Gaylord,1908

Quoted from

Doflein,

1911
Laveran &

Mesnil,

1904

DeJong, 1922

Jahnel, 1937

Rahm, 1920,
1923

Kadisch,1931

Kadisch,1931

Alexopoulos
& Drum-
mond,1934

Rotifers (various)

5. Metazoa

L. hydr.
-253°

2 days in L. air and 1 1 Rahm, 1920,
day in L. hydrogen 1921, 1923

250

TABLE 4

ORtiAMSMs Which, in thk Wi:t Condition. Sikvivk Im.mkrsion in Liquid
Aiu, Pkovidki) Tiiky Akk Cooi.kd and Rkwakmi:!) Suddenly

( VlTKlI l( ATION Fl{0( KDUKi; )

Organisms

Conditions of Exposure

Myxamoebae

Moss Leaves (with full

water content)
Onion Epidermis

Fros Spermatozoa

Frog Muscle Fibers

Investigators

Suspended in films of, Gehenio and Luyet,
water in a thin wire 1939

loop

Previously plasmolysed
in NaCl solution

Previously plasmolysed
in sucrose solution

(unpublished)
Luyet and Gehenio,

1938
Luyet andThoennes,

1938a
Luyet and Hodapp,

1938
Luyet andThoennes,

1938b

peratures"). However, the absence of experimental evi-
dence is not an argument against the possibility of the
theory.

Another reason which might be suggested for the re-
sistance to extreme cold of the organisms of the second
group is that their small size provides them with such a
large surface area, in comparison with their volume,
that they can lose water by exosmosis in the short time
during which their culture medium freezes. The cells,
being then dehydrated as a result of the congelation of
the water around them, would naturally become resis-
tant. An objection to this interpretation is that osmosis
is known to be a comparatively slow process which would
hardly account for the dehydration of even a small speck
of protoplasm during the few seconds necessary for freez-
ing a small drop of culture medium in li(|uid air.

Luyet and Gehenio (19.'^9, p. 123) proposed a third ex-
planation for the prevention of congelation in microor-
ganisms which survive the lowest temperatures in the
vegetative state. According to Luyet ( VXW)) water solu-
tions of substances which have a hii>h mok'cnlar weig-ht

251

have a narrow freezing range. For example, a concen-
trated solution of dextrin freezes only between about - 1°
and -9. Similarly the substance of some types of pro-
toplasm might have a very narrow freezing range. If,
then, the degree of subcooling is such that the freezing
range can easily be traversed, the formation of ice is
avoided.

As to the organisms of the first group, those which
can be dried, they resist not only cold but almost any
injurious agent w^hen they are in the dry condition. Such
a general resistance is again attributed to the absence
of water. But evidently the ability to support without
injury the removal of water is due to some intrinsic
property characteristic of some given types of proto-
plasm.

A priori it seems that the resistance to cold could be
attributed directly to this intrinsic property rather than
to the actual absence of water and consequent absence of
ice. But experiments have shown that most of the desic-
cable organisms are killed when frozen without being
previously dried. To mention one instance, Adams (1905)
found that seeds which contain more than 12% water may
be killed by freezing, while if they are dried to a fur-
ther extent they remain uninjured. These experiments
clearly speak in favor of the theory of the actual ab-
sence of freezable water as the cause of the resistance to
extreme cold in desiccable organisms.

In the instance given, 12% of the weight of the seeds
would then be unfreezable water. This proportion, it
might be remarked, is low when compared to the 34.5%
water content which has been found by Moran (1926) to
stay unfrozen at any low temperature in gelatin gels. The
quantity of water which, in several colloids, cannot be
unbound by crystallization forces is considerably higher
than is usually thought.

Coming now to the explanation of the survival of pro-
toplasm treated by the rapid cooling and rewarming

•^.yj.

method (tliird .uioup aboNc) we li;i\(' cxidciice from all
angles that it' cooling niid rcwai'iniiii;- are sufficiently
rapid to prevent the formation of ice and to really vitrify
protoplasm, life is preserved, while if a good vitrification
cannot be achieved, the damage is in proportion to the
degree of crystallization (for experimental data cf. our
review: ''The Physical kStates of Proto])lasm at Low
Temperatures"). Death then seems to result from the
disruption of the units which constitute living matter
when the molecules of water are torn away from these
units by the forces of crystallization, and the cause of the
innocuousness of low temperature seems to be the absence
of f reezable water.

In this discussion of the three groups of cases in which
extreme cold is innocuous, we assumed, on the basis of
circumstantial evidence, that no ice was formed in the
protoplasm. We know, how^ever, only one direct obser-
vation of the actual absence of ice in protoplasm at very
low temperatures: Luyet and Thoennes (1938a) reported
that the plasmolyzed protoplasts of the cells of onion epi-
dermis do not lose their isotropic properties when im-
mersed in liquid air.

B. Resistance to Extreme Cold and
the Structure of Living Matter. The
experiments on vitrification show that living matter can
be hardened into a solid without being killed. The passage
from the liquid to the solid state is not lethal. On the
other hand, the passage from the liquid to the crystalline
state at near-zero temperatures and the passage from
the solid amorphous to the crystalline state in devitri-
fication experiments is lethal. Now, the change involved
in the transformation of a liquid into an amorphous solid
is simply an increase in cohesion connected with a closer
approximation of the molecules, while crystallization in-
volves a rearrangement of the molecules. The struc-
ture of living matter is, therefore, such that life is com-
patible with the increase in compactness and density

etno

M-liieli occurs in vitrification l)ut that it is destroyed by
the molecular rearrangement which takes place in crys-
tallization.

Furthermore, a considerable decrease in molecular
motion has no injurious effect on living matter (cf. Lip-
man and Lewis, 1934). In the present state of our knowl-
edge the influence of temperature on life can be repre-
sented diagrammatically as in the accompanying figure.

Upper

Lethal
Zone

Optimal
Zone

Dangerous
Zone

Safe
Zone

(No Lower
Absolute

Limit

-"W
- Coagu-
ation

- Active

Life
1-0°

Freezinf

-100

Latent
I- Life

-200°

Limit )
Zero

Fig. 32. Diagram iUustrating the influence of temperature on vital
processes.

While apparently one can always kill protoplasm by rais-
ing its temperature, that is, by increasing its molecular

iiiotii>ii, one oaiiiiot kill il. it sih'ius. by loworinu" its tom-
poraturo oven to near tlio absolute y.ovo, that is, by iu>arly
stoppiiiii" its moleeular motion. In the last analysis, how
ever, the nieehanisni o\' action of hi^h and that o^ low
temperature mi^ht not be so ditVerent as it appears. The
theories ot" injury by hiiih temperature let'. Belehradek,
llK>r)) attribute death to some indireet et'feet of heat, sueh
as protoplasmic coaiiulation. tlu' destruction oi' some en-
zyme, the \apori/,atit>n o[' lipoids, etc., in the same way as
death by cold is attributed to some indirect elVect of
temperature such as the freeziuii' ol' water. It seems
then that it is not temperature itself, hiiili or low, in (»ther
words, it is not the ra])id or the slow molecular m(»tion,
which disturbs the structural architecture o\' livinu' mat-
ter, but the indirect chanues caused by certain particular
rates of molecular motion.

Water api)ears to pla>' a role o\' fundamental impor-
tance not only, as is well known, in the fi(>ictii>ns o( liviuii*
nuitter at the temperatures of active life but also in the
sfntctiirt' o^ the livinu' units, as is shown by the destruc-
Xiou o[' proto]ilasm when the molecules of water arc torn
away, in the process of devitrilication, for example.

Adams 1^1905) points out that at extremely low tem-
peratures not only water should be soliditied in proto-
plasm but also carbon ilioxide. and that oxyuen and nitro-
jjon should litpiefy or soliilify tO(>. The se]iaratiim of
these substances from protoplasm and the mechanical
injury possible as a result of the reduced lucssure caused
by these changes of state, might be expected t(^ cause
damage. However, the fact seems to be that the sep-
aration of these substances is hannless, while the separa-
tion of water is highly injurious. The importance of
the crystallization of water in death by low tempera-
ture is emphasized in the diagram above where one of
the main lethal zones is at the freezing point of water.

The observatitui that living matter can stay for a long
time near the absolute zero without showinu- anv measnr-

255

able amount of activity ))ut, witlioul loosiii<^- ils ability to
become active again when ))roug'lit back to higher tem-
peratures, has led some thinkers (de Caiidolle, 1895; Cho-
dat, 1H%; Brown and Kscombe, 1897; etc.) to consider
the static aspect of life, w liich is usually overhwked in the
classical definitions. An organism which resists extreme
cold behaves like a watch which, though well wound, is
stopped by some braking meclianism. This watch is in
perfect condition as to its constructional features and it
will start of its own accord as soon as the brake is re-
moved. In a similar manner, the activities of living mat-
ter can be stopped entii-ely without destruction of the
mechanism which conditions them. This state of affairs
is consistent with the hypothesis that the force which con-
trols the vital activities requires a special .structure of
matter, and that, when that structure is destroyed, the
organism is dead, while, when the structure is maintained,
the protoplasm is alive, though it might not be active. To
use the comparison of de Candolle, an organism in the
state of latent life is like an explosive which does not
show any evidence of its tremendous potential energy as
long as it is not fired.

SUMMARY

1. An injurious action of cold, above the freezing point
of protoplasm, has been reported in all homoiotherms,
in some poikilotherms, in some higher plants and in some
undifferentiated living forms. 2. This action, in which
the time factor is rather important, has been attributed to
a disturbance of physiological functions, to chemical
changes, to the accumulation of toxic products, to an al-
teration of permeability, to dehydration, to changes in
viscosity and in adsorptive properties and to processes
of solidification, precipitation and coagulation. .3. A
rapid lethal action of cold, above zero, has been reported
in a few cases; it has been attributed to syneresis pre-
ceded by gelation.

256

4. The majority of investigators have found subcooling
innocuous. 5. AVlien subcooling is injurious, the impor-
tance of the time factor suggests tliat the mechanism of
action is the same as that of cold above zero. 6. In nature
the subcooled state represents probably a condition of
safetj^ against injury, though there are some arguments
in favor of the o])])osite view.

7. The organisms Avhicli resist extreme cold belong to
three groups : a) some resist only in the dry state, b) some
with their full water content, c) of the latter, some sur-
vive only if si)ecial precautions of rapid cooling and re-
warming are taken. 8. In all these, the main cause of the
resistance seems to be the impossibility of freezing. The
formation of ice might be prevented by the bound state
of the water in the partially dried organisms, by capillary
forces, by a possible rapid exosmosis of water in the small
living forms or by a narrow range of freezing tempera-
tures.

9. Studies on the resistance of living matter to extreme
cold indicate that: a) molecular rearrangements such
as take place in crystallization are lethal, while solidifi-
cation into the amorphous state is not; b) cold alone, that
is, a decrease in molecular motion, is innocuous ; c) water
plays an important role not only in the functional activity
of living matter but also in the structure of the living
units; d) life is probably conditioned by some special
structure which, at low temperatures, allows for a state
of latent life and at higher temperatures furnishes the
basic mechanism for vital activities; the destruction of
this structure would induce death.

CHAPTER II

ACTION OF COLD ACCOMPANIED BY
ICE FORMATION

The theories on the mechanism of death by freezing
attribute the lethal injury to the following various causes
which will serve as a basis for our classification :

1. A mere withdrawal of energy ;

2. The attainment of a minimal temperature ;

3. Mechanical injury ;

4. Too rapid thawing;

5. Dehydration;

6. Various physiological, physical and chemical
changes.

Often, when the experimental data are too few or too
inconsistent to justify a pertinent discussion, we shall
limit ourselves to a mere presentation of the theories and
of the facts recorded in the literature.

I. THEOKV ATTKIBUTING DEATH TO A MERE
WITHDRAWAL OF ENERGY

In the last analysis, all the theories to be reviewed
hereafter attribute injury and death to the withdrawal
of energy, that is, to cooling, but the present theory con-
siders the withdrawal of energy as the immediate and
final lethal mechanism, while the others assume that cool-
ing causes some intermediate action, such as the forma-
tion of ice, which is then considered the immediate lethal
factor.

The idea that death might result from a decrease in
the energy content of an organism is perhaps the first to
come to one's mind when one considers death by cold in
the warm-blooded animals. These creatures constantly
produce energy so as to maintain their body at a tem-
perature higher than that of their milieu. They compete
with the milieu and, if they fail in this competition, death

257

258

follows, it is then iialur.-il to lliiiik tlint dcalh results
from the inability to jjroduec eiion^li ciicrnv to compen-
sate for that whieli is withdi-awii.

This theory has been aj)|)rKMl to cold -hlooch-d animals
which, it has been claimed for a lon<i- time, succnmb to
cold when they lose more heat than they can supply. As
a typical representative of the many authors who held
this view, during the last century, we shall mention Pla-
teau (1872). He claims to have observed that aquatic
arthropoda immersed in freezing- watei- die when they
are caught in the ice in such a way that they cannot make
any more movement and consequently cannot produce
any more heat.

While the production of energy by cold-])looded ani-
mals seems to have been universally recognized by the
earliest physiologists, the production of heat by plants
was still a subject of controversy a century ago. Goep-
pert, in 1830, in his book entitled: "Ueber die Warme-
Entwicklung in den Pflanzen, deren Gefrieren und die
Schutzmittel gegen dasselbe", states (p. 228) that the
plants "do not possess the ability to produce their own
heat" ("eigene Warme"). His conclusion is that, since
plants cannot resist cold by producing heat themselves
i}or by receiving heat from the ground, "their vital force
("Lebenskraft") is the first and most important and per-
haps the only source from which the resistance to the
harmful influence of cold arises" (p. 225). Each species
would possess a certain vital force which requires a given
withdrawal of energy for its destruction.

It is interesting to mention, in connection with this con-
cept of specific energy content of living matter, an idea
which was quite generally accepted during the last cen-
tury, namely, that freezing could not take place in living
plants and animals, but that life had to be destroyed be-
fore congelation could occur. The destruction of life, it
was thought, was caused by cold ahme, that is, by the
withdrawal of energy.

259

The theory of the impossibility for an organism to
freeze as long' as it is alive was still accepted some fifty
years ago since Miiller-Thurgau (1886) deemed it neces-
sary to point out that his own experiments, in which
plants were found alive after freezing and thawing, spoke
against such a view.

There is some resemblance between this apparently
antiquated theory and some modern forms of the "bound
water" hypothesis, according to which living matter
would bind a certain quantity of M^ater in such a way that
the crystallization forces would have to tear away this
water and, in doing so, kill the protoplasm, before freez-
ing can occur.

II. THEORY ATTRIBUTING DEATH TO THE ATTAIN-
MENT OF A MINIMAL TEMPERATURE

The theory that death is due to the removal of a given
quantity of energy leads naturally to the idea that there
is a definite death temperature for each species of ani-
mal or plant. In this form, the theory has been known
as that of the "specific minimum". It was developed
mostly by Mez (1905) and his pupils Apelt (1907), Rein
(1908) and Voigtlander (1909).

Mez (1905), considering that some plants are killed
by cold at temperatures above zero while others resist
hard freezing, and that some seeds present a high cold
resistance (Avena, Triticum) while others are rather
sensitive to cold {Lobelia), developed the theory that for
each plant species there is a temperature minimum be-
low which the plant cannot live. This minimum, or death
point would be characteristic of each species, under a
given set of conditions, such as, water content, age, stage
of development, etc., but it would change when any one
of these conditions changes. Drying, for example, would
lower the specific minimum. The development of a seed
into embryonic tissues would raise the minimum.

260

Mez applies his theory to llic ex])l;niation of various
observations or assumptions on the Ix'havior of phmts
at low teniix'ratures. Adaplalioii 1<» cliniatG would con-
sist in a change of the specific mi 11111111111. The resistance
of plants which stay alive in ice would be partly attrib-
utable to the fact that the heat developed during ice for-
mation protects them and retards the drop of the tem-
perature to the specific mininnini. Subcooling might then
be rather harmful, as has been said in the preceding cha])-
ter. The injurious effect of rapid thawing is explained on
the assumption that if the heat necessary to melt the ice
be withdrawn too suddenly from the regions surrounding
the thawing spot, the temperature of the internal por-
tions of the material may be lowered to the specific min-
imum. To confirm this interpretation Mez says that, 5
mm. below the surface of a frozen apple thaw^ed by the
warmth of the hand, he could measure a lowering of tem-
perature of 1.8 degree.

The idea that each animal or plant species is charac-
terized by a given death temperature appeals to those
biologists who are in quest of shai'])ly defined, typical
physical processes. It suggests that death might l)e cor-
related w^itli some physical change, which would take
place at a definite temperature, as is the case for melt-
ing. Apelt (1907) seems to have followed this line of
thought. He evidently assumed that the death tempera-
ture of a plant species could be measured with the same
precision as a melting point when he claimed to have
established the range of death temperatures in some
types of potato within 0.007 degree. His procedure con-
sisted in exposing potato to a given temperature and diag-
nosing death by the change in color of the tissue and
by the failure of the cells previously stained with methy-
lene blue to plasmolyse.

While Apelt 's precision has not been taken seriously
by all biologists, several points in his argumentation and
experiments deserve mention. He established, for ex-

261

ample, a clear disliiiction hetweou the theory of death
by withdrawal of energy and that of death at a tem-
perature minimum and he rejected the former as con-
tradicted by the following facts. Cells of potato tubers
were not killed in one hour at a temperature 0.3 to 0.4
degree higher than that which was instantly lethal,
neither were they killed in two hours at a temperature
one degree higher than the death point. Death, then,
is not attributable to a withdrawal of energy, since the
same amount of energy could be withdrawn in a longer
time at a higher temi)erature as in a shorter time at a
lower temperature. Retaining the theory of the specific
minimum, the author thinks that a protoplasmic disor-
ganization when the temperature reaches that minimum
constitutes the mechanism of death.

III. THEOKV ATTRIHUTING DEATH TO MECHANICAL

INJURY

Death has been considered as resulting in various ways
from a mechanical injury inflicted during freezing or
thawing. A. Some authors have thought that the expan-
sion of ice during its formation bursts the cells, as it
bursts a bottle filled with water ; B. Others have assumed
that the ice crystals in the cells or in the intercellulars
damage the protoplasm or the cell parts by piercing or
tearing them; C. Some investigators have invoked the
pressure exerted by the expanding ice as the cause of
death, its action being not to burst the cells, but to
compress the protoplasm; D. A few biologists have at-
tributed death to a mechanical injury inflicted, not by the
ice itself, but by the jarring of the protoplast during
plasmolysis and deplasmolysis, as a result of which the
cytoplasmic mass would be torn off from the cell walls
where it adhered.

A. Most of the reviewers have attributed to Duhamel
and Buffon the idea that death results from a mechanical
injury caused by the expanding ice in living tissues. We

262

could iKtt liiid aii\- statement to that elTect in the authors
mentioned. They say that hij;- eiaeks can be produced
in trees as a eouseiiuence ol" tlie foiiuation of ice in tlieir
intei'ior l)ut their theory of death is entirely dilTerent and
will be ex])laine(l later.

Accordinu" to Senebier (ISOO) ice miulit cause death
l)y its ex))ansioii.

But soon several investigators showed that tlie ctdls
are not broken by freeziu^-. Goepjx'il (18l>0) says that
the large cells of Calla ctJiiopica do uot show, w^heu their
sap is couuoaled, any expansion of the cell wall observ-
able under the niicroscoi)e. lie furthermore states that
in the lim)) tissue, after thawiui-', the cellular structure
remains intact and that the cell walls are never torn.

]\Iorren (1853) confirmed the obs<'rvation that there is
no evidence of ruptured walls.

Xageli (1860) calculated what should be the expansion
of a cell during the formation of ice in it and showe<l that
this expansion is never great enough to break the cells,
the membranes of which are always sufficiently expan-
sible. Besides, he observed that often the cells in con-
tact with the intercellular masses of ice are actually not
damaged.

Schacht ( 1854) noted that the juice exuded from pota-
toes after freezing does not contain starch and he con-
cluded from that observation that the cells could not be
lacerated.

Prillieux (18()5)), after describing the formation of ice
in the intercellulars, states that ice may tear the tissues
but never the cells and that there is no necessity of as-
suming that the membranes are torn to explain that the
juices come out of the cells.

To summarize : The opinion that ice bursts the cells as
it would a bottle has been advanced by some early biolo-
gists. Between 18,')() and 1870 several authors demon-
strated that it is not based on exj)erimental and ob-
servational ('\i(leiice. From the Ix'giiiniiig <>f the ])res-

263

eiit century this idea has generally been aljancloned by
the physiologists, though occasionally some authors won-
der it' the reaction against it has been exaggerated. The
extrusion of water from the cells during freezing and the
absence of ice in the cells after the congelation of a tissue
have generally been observed after sloiv freezing as it
usually occurs in nature. With rapid freezing or when
the water content of the cells is very high, intracellular
congelation takes place (Molisch, 1897; and, more re-
cently, Stuckey and Curtis, 1938). The mechanical injury
in intracellular freezing may be quite different from that
of the generally observed extracellular formation of ice.

Stuckey and Curtis {loc. cif.), who reported to have ob-
served ice formation within the cytoplasm itself in the
cells of the prothallia of Polypodium aureitm, claim that
death always resulted from such intracellular freezing.
They consider death as due, according to all evidence, to
a mechanical injury by ice.

B. While the authors cited above have established that
ice does not destroy the cells by bursting them or by tear-
ing their membranes, more recent investigators have sup-
posed that tiny ice crystals tear the protoplasm itself.
Maximov (1914) for example, attributes death partly to
the destruction of the fine structure of the protoplasm as
a result of mechanical injury.

Stiles (1930) thinks that the formation of a new phase,
namely ice, constitutes a mechanical disturbance result-
ing in a breaking down of the colloidal system. The types
of protoplasm in which the separation of the materials on
freezing is followed after thawing by their restitution to
the former state, would not be killed, the other types
would. He assumes that, if the crystals be smaller, the
mechanical injury would be less, and several types of
protoplasm might resist freezing. As a means of induc-
ing the formation of smaller crystals he proposes the use
of lower freezing temperatures, according to the finding
of Tammann (1898) on the relation between the temper-

2G4

atui't', tlic imiiihci" ol' (•rys1;iHr/.;it ion cciilcrs t'oi'iiird and
llit'ii- \('l()t'it>- of i;i-()\vtli. Bui llic pari of this suggestion
concerning tlie i)ossibility of avoiding injury ])y causing
the formation of sniallei- crystals could not l)e conlirnied
ex])erinientally, as the following observations show.

lljin ( IJ'.'U), after comparing exix'riiiiciits on rapid
and slow cooling of the cells of red cabbage leaves, con-
cludes that sudden freezing is more injurious than slow
freezing.

The abrupt iunuersiou of sticklebacks in li(|uid air by
Weigman (IK.'Ui) killed the animals readily.

Identical experiments by Luyet (1938) on gold lish gave
the same results.

Luyet and Thoennes (1938a) found that monocellu-
lar layers of plant ej)! dermis presented only dead cells
after rapid freezing in li(]uid air.

As is evident, these experiments which do not confirm
the idea of a lesser injurious effect of smaller crystals,
do not invalidate the theory of a possible mechanical
injury on protoplasmic structure by ice.

Some have thought that a mechanical contact with ice
might result in a coagulation of protoplasm, since it is
well known that touching or piercing wdth a needle
(Chambers) or i)ricking a cell may result in its coagula-
tion. Lepeschkin (1936) apparently follow^s this trend of
thought, when he speaks of "mechanical coagulation" by
freezing.

C. The killing of protoplasm under the action of the
pressure exerted by the expanding ice is often referred to
in the biological literature. Among the authors who dis-
cuss this theory more extensively let us mention Plateau
(1872) who attempts to show that there is no pressure
within the ice in formation. It is a known physical prin-
ciple, according to him, that the cavities in a solid body
expand like the body itself. Therefore the cell contents
cannot be crushed by the freezing of the tissue around
them. He claims to liaxc shown Ihe al)sence of pressure

265

experimentally with an apparatus consisting of a glass
tube on the end of which was a rubber bulb tilled with
a li(iuid and iinniersed vertically, the open end up, in a
tiask containing water. When the latter froze in the
flask, the level of the fluid in the tube stayed the same,
indicating 'that there was no pressure exerted on the rub-
ber bulb. Plateau, it seems, did not notice that the re-
sults of his experiment disagreed with the principle that
he invoked, he should have observed a lowering of the
level of the fluid in the manometric tube, if 'the cavity
around the bulb were expanding.

lljin (1936), discussing the case of plant cells frozen
in water, conceives the mechanism of pressure by ice as
follows: The water in which the material is immersed
freezes first and forms a wall around the protoplast of
each cell. When, later, the sap of the vacuole freezes, the
protoplast is wedged between two masses of ice. On these
assumptions he calculates, for different forms of cells,
how much the volume of the protoplasm should give so as
not to be crushed by the ice formed in the vacuole. His
figures indicate that there is definitely a possibility of
some crushing action. If the cells are frozen in air, it is
assumed that the pressure might be exerted against the
frozen cell walls. The fact that the congelation of the
vacuolar sap is always followed by death is in agreement
with the concept of an injurious pressure effect of the
frozen sap.

As a whole, both the problem of the existence of a
pressure in a frozen tissue and the problem of the effi-
cacy of pressure in causing death call for more experi-
mental evidence.

A consideration of the enormous pressure required to
kill protoplasm makes one doubt the possibility of the
existence of such pressures in cells. Most of the tis-
sues of metazoa resist a hydrostatic pressure of sev-
eral hundred atmospheres. Protozoa and bacteria are
killed only at pressures of the order of 1,000 atmospheres

266

(cf. Cattell's review, ID.'Ui). Yeast takes more than four
thousand atmosi)heres (Liiyol, 1<>:'7). TnchT the action
of pressures of this nia^iiituck', ice sliould melt several
degrees l)eh)W zero. Besides, I'oi- ohtaiiiiii,^- sueli ])res-
sures it would be necessary to prevent the exi)ansion of
ice by holding it in some material more resistant than
the wjdls of an animal or plant tissue.

The ex])eriiiients of Melseiis ( ISTO) who roiiiKJ that,
in a culture of yeast ex])osed in a steel boml) calculated
to burst at 8000 atmospheres, there were living cells after
the temperature was lowered until the bomb burst, are
also significant in the discussion of this problem.

How enormous hydrostatic pressures have no action
on protoplasm while pricking with a glass needle may,
in some instances, start coagulation, is entirely unknown.
The answer to this question might throw some light on the
tyi)e of mechanical injury caused by ice.

D. lljin (li)34) presented a new theory of death l)y me-
chanical injury, applicable to typical i)lant cells which
consist of a protoplast adhering to cell walls and filled
with cell sap. He distinguishes two general cases: that
in which death occurs during freezing and that in which
it occurs during thawing, and he subdistinguishes two
cases of death during freezing, that in which ice is formed
only in the intercellular spaces and that in which there
is ice also in the vacuoles. When death is caused by
thawing, the too rapid invasion of the protoplasm by
water would damage the living structure which is not
capable of expanding rapidly enough and is torn by
being pulled about. When death occurs during freez-
ing but without congelation of the vacuolar content, the
w^ithdrawal of water from the vacuole would cause the
latter to shrink and the protoplasmic layer, still attached
on one side to the cell walls, would be stretched between
these cell walls and the vacuole whose contraction it has
to follow; this stretching woud be injurious. In the ca§e
of formation of ice, both around the cell and in the vac-

267

uole, llio protoplast would Ix' killed by being squeezed
between two masses of ice, when the vacuole expands on
freezing, lljin's experiments in which he succeeded
in keeping alive, by cautious slow freezing and thawing,
cells which otherwise would have l)een destroye«l give
much weight to his theory.

IV. THEORY OF DEATH BY TOO RAriD THAWING

For the investigators who maintain that cold, when
not accompanied by ice formation, is not generally lethal
and that freezing is usually a necessary condition for
death, the question arose as to whether death occurs dur-
ing freezing itself or during thawing. Almost all w^ho
favor the latter assumption think that it is the rapidity of
the thawing which renders it dangerous. So the theory
of ''death by thawing" and that of ''death by too rapid
thawing" will be treated together.

The origin of the theory of "death by too rapid thaw-
ing" seems to be the old popular idea that, when a per-
son has frozen limbs, he should be warmed gradually.
This notion, frequent in the medical literature, is found
here and there also in the biological literature. For ex-
ample, Duhamel and Buff on (1737) say that when ani-
mals are frozen one puts them in snow, in water or in
dung to \varm them slowly. These authors also give as
a well-known fact that frozen fruit decays if thawed too
rapidly. They claim, furthermore, that they could save
plants (orange trees and geraniums) which w^ere coated
wath ice, by covering them so as to prevent a too rapid
thawing by the sun, or by exposing them to a slight
rain which also would cause a slow thawing. After refut-
ing the idea that the injurious action of the sun on frozen
trees might be due to a condensation of the rays by the
lenses constituted by the droplets of melting water, as
some have maintained, Duhamel and Buffon present the
following tentative hypothesis for explaining the mechan-
ism of action of rapid thawing : the vessels distended by

2(^8

the increase in volume of llic I'rozcii s;ip cannot, in fast
thawing, resume their normal size smoothly enough
("avec assez de douceur"), then they break, the sap
evaporates and the plant dries up.

A similar idea is held by Pichel (1816, quoted by Miil-
ler-Tliurgau, 1886). Injury in twigs would result from a
tearing of vessels or of essential structures in the too
rapid thawing of the unequally expanded outer and inner
layers of the twigs. The free course of the sap would
thus be disturbed.

Goeppert (1830) called into doubt the view of many
of his predecessors that slow thawing saves frozen plants
from death. He thawed slowly, at about 0°, in snow^, frozen
bulbs of onions, tulips, etc., and observed that they were
killed.

The experiments of Sachs (1860; see also Sachs' Hand-
buch der exp. Physiol, der Pflanzen, Leipzig, 1865 and
later editions) again revived the older theory that slow
thawing can forestall the death of frozen plants. He
froze pieces of beet and of pumpkin and leaves of beet,
cabbage, bean, etc., at - 4° to - 6°R (- 5° to - 7.5°C) and
thawed them in water at 0^, in air at 2' to 3°R or in
water at 6° to 10°R. In the first case, wdth slow thawing
at 0% the plants were alive, in the last two cases they
w^ere killed. Sachs' interpretation is that when thawing
is slow the molecules of water pulled loose from the pro-
t()l)lasm during crystallization can again take up their
former position and reestablish the conditions existing
before freezing, while if thawing is rapid some of the
water may flow away and not be reabsorbed and thus the
previous conditions of concentration and imbibition can-
not be restored and death may result. As to the fact that
a lower water content decreases the sensitivity to cold,
Sachs explains it by assuming that when there is less
water to freeze it can more readily be reabsorbed after
thawinij;.

269

Goeppert (1871), more than forty years after his first
observations, undertook a new series of experiments which
led him again to the conchision that it is during freezing
and not during thawing that the phints are killed. He put
to freeze orchids which contain indican and turn blue at
death {Calauthe) ; he found that the blue color appeared
in the frozen state before thawing.

Prillieux (1872) repeated these experiments and came
to the conclusion that the indican plants became blue only
after thawing. It seems that the ditferent conclusions
reached by these two authors are due partly to a dis-
agreement on what is called blue. Before thawing the
plants are of a steel blue color (Stahlblau) and after
thawing they are of a deep dark blue.

Kunisch (1880), instead of utilizing the change of color
of indican plants at death, tried to revive them and to put
them to grow after freezing and slow thawing but he
registered only negative results.

Miiller-Thurgau (1880) also experimented with indican
plants. Using the petals of PJiajus, he could observe by
a slow lowering of the temperature that the change in
color took place during freezing, not however at the freez-
ing point but when, after some ice formation, the temper-
ature dropped to a lower level. Any attempt to keep
alive by slow thawing petals which had turned blue on
freezing failed. The same author (1886) summarizing
the results of other experiments in which he subjected
"several hundreds" of frozen plants to rapid or slow
thawing at various temperatures (for example, potatoes
thawed in sand at 45" and at 0") concludes that there is
no evidence that slow thawing ever saved these plants
from death. However, a few years later (1894), he found
that frozen pears and apples show considerable injury aft-
er thawing in water at 0° or in luke-warm water, whereas
they show only slight or no injury if thawed more slowly
in air at 0° or at 20°. (One might mention here that Miil-
ler-Thurgau pointed out the error made by previous in-

270

vestigators who tlion^lil tli;i1 lliawiiii;' in wator should be
slowiM- tliaii tliawiii^' in air, at the same UMnporatiire).

Molisc'h ( lSi)7), who reviowed the ])i-ol)k'iii of death by
rapid thawing-, himself made a large number of experi-
ments on this ])oint. He use<l mostly leaves of various
higher phints, in i)artienhir, of Af/rrdhnu in wiiich deatli
can be diagnosed by the o(h)r of couniarin released from
the cells. He also performed some experiments with
algae of the class Florideae, which change their color at
death. Ka])id tliawing was carried out in water at 30° ;
for slow thawing the material remained 5 hours at -1°,
5 hours at 0 and then it was brought to 2 \ The leaves
were cut longitudinally in two, one half being thawed
ra])idly, the other slowly. The author concludes that the
rapidity of thawing had no effect on survival except in
one case, namely with the plant Agave.

According to Sorauer (Handbuch der Pfianzenkrank-
heiten, 1909) frozen leaves of Cineraria presented dead
spots only at places where they had been thawed too
rapidly between the warm fingers.

Hedlund (1912, quoted by Akerman, 1919), also found
that plants frozen at moderately low temperatures can
be saved by slow thawing.

Winkler (1913) on the contrary, came to the conclu-
sion that survival is not controllc^d l)y the rapidity of
the thawing.

Chandler (1913), experimenting on fruit and vegeta-
bles, found in exceptional cases some evidence of a more
injurious action of rapid thawdng; in general, however,
the rapidity of thawing made no ditference on the re-
sults.

Akermann reviewed this subject (1913) and himself
made a systematic series of observations on the effect
of rapid and slow^ thawing on various plants (red cab-
bage leaves. Viburnum, Aucuha, potato, Tradescantia).
He concludes that slow^ thawing might play a role in
saving from deatli some species of frozen ])lants ])nt

271

only if they had not been exposed to too low a tem-
perature.

Iljin (11)34) revived the old theory of injury and
death by too rapid thawing and established it on a new
basis. He observed that when, during freezing, water
is extruded by osmosis from the vacuoles of plant cells,
the protoplast contracts and the opposite sides of the
cytoplasmic sac approach each other and come in con-
tact in the center of the cells, while they are still sepa-
rated by cell sap in the outer portions. On thawing,
water suddenly invades the contracted protoplast and
tears it, if it cannot pull apart the adhering sides. The
author says that he observed the same phenomenon,
namely that the protoplast tears open, in too rapid
deplasmolysis. In the case of death by desiccation he
claims to have evidence that injury results from the too
rapid invasion of the protoplasm by water on remoist-
ening. In his experiments on low temperature effects
he succeeded in saving from death cells of red cabbage
frozen at the temperature of solid carbon dioxide. His
method consisted in slowly restoring the water to the
cells by letting cooled hypertonic solutions of sugar, gly-
cerine or hiorganic salts of gradually decreasing con-
centrations fall dropwise on the frozen sections of the
tissue.

Of the few authors who found slow thawing more in-
jurious than rapid thawing we shall mention here a re-
cent work of Turner and Brayton (1939) on the spiro-
chetes of relapsing fever. When rewarming and thaw-
ing took place in 30 seconds, (in water at 37°) or in 25
to 35 minutes (at room temperature), there were living
spirochetes. As a test of vitality the authors used
motility and pathogenicity for mice.

The numerous data recorded in this section do not
allow one to draw any general conclusion. It is prob-
able that the etfects of slow thawing are different in
different organisms, as a comparison of the results on

spirtx'lictcs, for cxniiiplc .-iikI those on the lissut'S of
higher plants, seems to indicnte. Px'sides there are I'Vi-
(lently otlier factors in the piolth-ni which haxc not yet
been analysed.

\-. 'n!i:<H:v of diiatii r.v 1)i:iivi>i:ati()N

After it had been observed that watei" eoiiios out of
the cells ilurin.n- fi-eezin<'- and i)asses into the intercellu-
lar sj^aces, the theory was proposed that death results
from the fact that congelation deprives the ])rotoi)lasni
of its moisture. Death by freezing- would then ])e ideu-
tical, in the last analxsis, with death by drou,i;lit. Sev-
eral eminent jilant pli> siolo,i;-ists towai'd the end of
the last and the be^innin^- of the i»i-esent century took
sides on this (juestion. We shall lu're sunnnarize, iu
ehrouolo,i;ical order, the most im])ortant works which
have conti-ibnted to the (leveloi)meiit of this theory.

Sachs (18G0) is usually not considered an advocate
of the dehydration theory of death l)y freezing, though
some of his statements place him among the pioneers
who jtoinled onl tiie important alteration which I'e-
sults when water molecules are disengagi'd from a living
structure. He says that the molecules of water belong to
the structural organization of protoplasm and of the cell
walls, and are in a certain state of equilibrium wdtli the
otlier constituent molecules. By freezing and thawing,
the water molecules are i)ulled away from the structure
and a new^ state of equilibrium is established in w^hich the
other molecules have a stronger attraction for each other
than for water. This is ])recisely the mechanism which
several later investigatoi-s who hold the water with-
drawal theory assumed to explain injur>- and death by
freezing.

MiilJcr-TJiinfidH (ISSd), who is ordinarily held as the
founder of this theory, ])ro])osed it in a I'ather hesitating
phraseology. He says that death is usually considered
as resulting fi'om a destrnction of the ordered arrange-

273

iiu'iit of the eoiistitiu'iit parts of pi'otoi)lasiii, including
water, and that sneh destruction has been attributed to
one of the three following factors : temperature alone,
the withdrawal of water during freezing, some processes
which take place during thawing (three theories which,
according to him, can be traced liack to the previous
century.) After discussing the pros and cons for the
first and the third possibilities he gives his preference to
the second, saying that, since water withdrawal is the
most essential change which occurs during freezing, it
could very well be the cause of death by cold.

He then goes a stejj further and gives a body to the
dehydration theory by suggesting a mechanism by which
death might result from the withdrawal of water. He
considers that the solid constituents of protoplasm, /. e.,
the micelles, are separated by the water phase, being far-
ther apart in i)rotoplasni which has a higher water con-
tent: the withdrawal of water will result in displacing
these micelles from the positions that they occupied in
the ordered arrangement of living protoplasm. How a
molecular structure can be altered by freezing is exem-
plified by starch paste which, after congelation, loses its
property of holding water of imbibition.

Miiller-Thurgau also holds that : 1. The sudden with-
drawal of water during the rapid freezing which follows
subcooling is particularly dangerous; 2. Often the dis-
turbance produced by dehydration would .be reversible
and the original structure could be restored if the water
witlldra^^^l by freezing would not evaporate after thawing
before it can be returned to the cells. Conseijuently, he
claims that a means of keeping alive frozen material is
to prevent evaporation during and after thawing.

Several observed facts are interpreted by Miiller-
Thurgau as being in good fitting with his dehydration
theory. The greater sensitivity to cold in plants with
higher moisture content, as exhibited, for example, by
soaked seeds, and the relativelv higher resistance of less

274

h>(li-a1o(l uiatcrinl as in llic uikIcxcIoixmI huds, are ex-
plained on tlie assninj)! ion that when tlic micelles are
closer together, as in drier tissue, less damage is done
since there is less water to be withdrawn.

The fact that many of the plants which resist cold are
also those which resist drought, in i)ai-ticular, moss and
lichens, is adduced as evidence for the theory.

Sachs' experiments in which plants thawed in w^ater
recovered while ])lants thawed in the air died, are in-
terpreted as favoring the dehydration theory, since re-
covery would be possible when enough water is furnished
to rehydrate the cells, while death would occur when too
nnich water evaporates, as in the case of thawing in the
air.

To the objection that some plants would be killed by
a relatively slight dehydration in freezing while they
resist considerable drying under other circumstances, he
answers that, in freezing, the withdrawal of water is
particularly sudden and acts by its suddenness.

Moliscli (IHin) contributed important experimental
data to the theory of Miiller-Thurgau which he accepted
almost entirely. He first pointed out that the theory
of a disturbance of the functional harmony cannot ex-
plain the cases in which death takes place innnediately
upon congelation. He remarked that the theory of a
lethal action of temperature alone without ice forma-
tion is contradicted by the numerous observations of the
innocuousness of subcooling. Finally, he showed that
the theory of death by too rapid thawing does not apply
in a large number of cases that he studied. Though each
of these three theories, he concludes, might explain death
in some particular instances, death is, in general, coin-
cident with the formation of ice and with its attendant
withdrawal of water.

The following points, several of which had already
been indicated by Miiller-Thurgau, are emphasized by
Molisch: 1. A relatively large projxntion of ice is formed

275

at the beginning- of freezing at near-zero temperatures;
2. A further withdrawal of water at lower temperatures
causes death, o. Cold resistance is increased by dehydra-
tion (wilted tobacco leaves were found to resist freezing
more than fresh ones) ; 4. The high concentrations which
result from delwdration exert a toxic effect ; 5. The differ-
ences in resistance to both freezing and drying in various
plants are specific characters. Molisch furthermore at-
tributes the resistance of bacteria, spores, seeds, moss,
etc., to the fact that these organisms can be exposed to
low temperatures without releasing their water.

According to Matruchot and Molliard (1902), who had
observed that, in frozen plant cells, Avater had been sep-
arated from the cytoplasm and from the nucleus, the de-
hydration theory explains niosl: of the facts known on
the action of low temperature. They discuss, in partic-
ular, the following ones : 1. Cytoplasmic streaming de-
creases and finally stops, when a cell is cooled; this
would be due to the more solid consistency acquired by
protoplasm on the withdrawal of water. 2. Numerous
plants and animals revive after freezing or after dry-
ing when water is supplied to them; in both cases, the
organism would come back to life if water could be re-
imbibed by the protoplasm and the conditions which
existed previously reestablished, while the organisms
would die if the separation of water went so far as to
constitute an irreversible process. 3. The injurious ef-
fect of too rapid thawing would be due to the sudden
invasion of the dehydrated tissues by water, a too rapid
imbibition rendering impossible the reestablishment of
the previous state. 4. The various degrees of resistance
oiTered by different species to both desiccation and freez-
ing would be explainable by different specific water-hold-
ing capacities. 5. The high resistance to cold of plants
with a thick or heavily cutinized epidermis could be at-
tributed to the ability of these plants to retain water.

276

The lirst of those processes, namely the eessalioii of
proto])hisniic streainiiiji:, does not seem to l^e satisfac-
torily exi)huned by dehydration. Protophisniic stream-
ing- has been observed to l)econie sh)wer and to stop
under the action of h)W tenii)ei-ature ^vitllont ice for-
mation, that is, without (h'h>(li'ation to any noticeable
extent.

Ill an attem))t to analyse the mechanism itself of death
])y dehydration, Matruchot and Molliard, following some
views previously held by Dastre, distin,i>uish 3 sorts of
water in living matter: 1. External water, that is, the wa-
ter of the cell sap, which does not enter into the make-up
of living protoplasm; 2. Iiiterpos<(1 water, the molecules
of which move freely in the capillary spaces between the
micelles, that is, within the meshes which constitute
protoplasm; 3. Constituent water which is either a part
of the protoplasmic molecules or is attached to them by
adhesion forces. The withdraw^al of external Avater
would be harmless and would leave a still liquid proto-
plasm. The withdrawal of interposed water would not
be usually lethal and Avould result in the production of
that more solid sort of protoplasm found in seeds or
spores. As to the constituent water, its separation from
protoplasm would induce death.

Pfeffer (IdO'i) formulated several objections to the wa-
ter withdrawal theory. The essential points in his ob-
jections are the following: 1. There is a contradiction
between the fact that desiccation of a plant increases its
resistance to cold and the theory that desiccation by cold
causes death. 2. More water can be removed without in-
jury by transpiration and by plasmolysis than by freez-
ing. 3. Death by cold is in several cases independent of
the water content: some plants survive cold when they
are in the turgescent state while others, like seeds, sur-
vive in the dry state. 4. To account for the death of
some plants at temperatures far below the freezing
point one would have to assume that some moisture does
not freeze until these low temperatures are reached, an

277

assumption which is ' not in asreemont with what is
known on the rate of format ion of ice in terms of tem-
perature.

Some of these fundamental objections were resumed by
Mes f 190.5). This author w^onders why dehydration should
be harmful when caused by freezing and harmless oth-
erwise. Concerning- the formation of ice at tempera-
tures far below^ the freezing point, he quotes his experi-
ments with ImpaUens stems in which he claims to have
shown by the shape of the freezing curves that congel-
ation is completed at -6-. Cooling at a low^er tempera-
ture, therefore, w^ould not induce any further desicca-
tion and one does not see how it w^ould be injurious,
except if one admits the theory of the ''specific mini-
mum ' '.

According to Mez, several experiments in which death
was attributed by Molisch to freezing and interpreted
as identical wdth death by desiccation (water algae,
staminal hair of Tradescantia, potato) represent really
cases of death by desiccation and not of death by freez-
ing.

Apelt (W07), supporting the argument of Mez, says
that in his experiments on potato tubers he ahvays found
that the death point was definitely belo^v the freezing
point. This fact is interpreted as signifying that death
is not due to a desiccation during freezine-

According to the same author, the dehydration theory
has against it that, in one experiment on potato, re-
peated freezing (more than 4 or 5 times) at a temperature
slightly above the death temperature resulted in death.
Since it is assumed that the amount of water congealed
in each freezing at the same temperature is the same,
one does not see how^ the repetition of the experiment
could become injurious.

Gorke (1907) brought forth a new argument in favor
of the dehydration theory by showing that proteins can
be precipitated by freezing when the salt concentration

278

iiK'i-cascs as a rcsiill of the iciiioxal of watci" (the de-
tails of tliis llicory will he i;i\('ii hclow).

Vit'ifitUnidir / IHO'.i), rcsniiiiii.u' the x'icws of Mcz, claims
that the ('I'll sap usually ))r('S('ii1s a cutcctic ])()int at
around - (i . lie. thcrcfoi'c, denies that thei'e can ])e any
further desiccation at the tenijx'iatui-e of '.]() at which
some ])lants die.

The same author argues that if death is due to some
watei- withdrawal, oi- to the salting' out of some protein,
or to the eutectic fi'eezin.n' of some complex mixture, some
thermal effect should ajjpear. Xone, however, has ever
been observed.

One of the most natural explanations of the fact ob-
served so many times that a tissue survives the forma-
tion of ice in the neii^hborhood of its freezing- point and is
killed on further freezing at a loAver temperature is
that, as long- as there is free water to freeze, no damage
is done, and that injury begins when more strongly
bound water freezes. Jensen and Fischer (1910), found,
in a comparison of the freezing curve of frog's muscle
with that of an isotonic salt solution, that only about 3,9%
of the water of the muscle might be more firmly bound.
Besides, the dead muscles also seemed to contain bound
water. Death, therefore, could hardly be explained by the
unl)in(ling of that water.

According to Irmscher (1912), the similarity pointed
out by other investigators in the resistance of plants to
injury by drying and l)y freezing can also be observed
in moss. In general, the species which are more resis-
tant to cold are also more resistant to drought. (There
are, however, several exceptions.)

Another similarity between the mechanism of action of
desiccation and that of freezing, pointed out by the same
author, is that cold hardiness can be induced by expo-
sure to drought as well as by exposure to cold. Moss
which had been dried and which regained its full turgor
bv innnersion in water was found more resistant to cold

279

than normal moss. The natural conclusion from this
observation is that the protoplasmic changes induced by
desiccation must be the same as those induced by cold.

Irmscher, furthermore, repeated with moss the exjjeri-
ments of Molisch on the difference between material
frozen in water and that frozen in air. Moss partially
immersed in water was exposed for 3 days to — 5° to
-6\ The submerged portion, all surrounded by ice,
remained alive, while the tips which projected out were
killed. The more complete dehydration in air is as-
sumed to be responsible for the injurious effect.

Cameron and Broivulee (1913), discussing experiments
in which frogs were killed after having been frozen by
an exposure of several hours at or below the freezing
point, attribute death to the excessive concentration of
the cell contents caused by dehydration. (They note that
such concentrated cell contents should not freeze.)

In order to clarify the problem of the correlation
between ice formation and death in plants, Maximov
( Wl'f) undertook to study: 1. The death temperature as
related to the freezing point ; 2. The infiuence on death of
the time the material is left at a given freezing temper-
ature; 3. The relation between the quantity of ice formed
at a given time and temperature and the occurrence of
death. Using potato tissue in which the vitality of the
cells was judged by the plasmolysis test, he found that
the tissues were completely killed in a short time at
-2.66 while almost all the cells were alive at -1.96°
and -2.11°. Furthermore, a piece of potato left to freeze
for 10 minutes between 0° and - 1.82° was alive, wdiile
one left for about 3 hours at the same temperatures was
practically all dead.

These and other experiments confirmed his views that
death progresses step by step with ice formation and,
consequently, with water withdrawal. So this author
accepts, in general, the dehydration theory of ^liiller-
Thurgau and Molisch, but modifies it by suggesting that

280

the (Inui.'inc by freezing' I'csulls not only from llic water
witlulrawal itsclt" but also t'loiii a pri'cijjilatioii ol" tlic col-
loids of the protoplasm. Sudi a i)recipitalioii would be
caused by ])otli an increased concentration of the colloids
subsequent to ficcziiii;- and by a mechanical compression
between the k'v masses. The connnon action of these two
factors would be to brin^' the colloidal i)ai-ticles of pro-
toplasm in too close a proximity so tliat coagulation
would result.

Having admitted a mechanical action of ice on the
precipitation of colloids, Maximov considers as answered
the objection of Pfetfer and others to the dehydration
theory, namely, that some plants withstand desiccation
while they are killed by freezing. The factor pressure,
present in freezing, is absent in desiccation.

The fact pointed out by Maximov himself (1912) that
immersion in a concentrated solution protects a tissue
against the action of cold except if the temperature of
the solution is below the cryohydrate point, is brought
to supi)ort the theory; above the cryohydrate point the
etTect of pressure by ice is lessened by the fact that a
part of the solution is liquid and gives way under
pressure.

Maximov departs in some minor ])oints from ]\luller-
Thurgau's views; for example, he denies that the rapid
water withdrawal after subcooling is the cause of death
since he observed that the death temperature was about
the same when freezing w^as preceded by a considerable
subcooling and when there was no subcooling.

He criticizes Fischer's theory that death by cold con-
sists in the loss by the protoplasmic colloids of the ca-
pacity for adsori)tion, saying that only a small portion
of water is adsorbed in protoplasm, the rest is "free"
water, held in the cell by osmotic forces, not by adsorp-
tion. The release of water after death is explainable,
according to Maximov, who here quotes Niigeli (ISGl),

281

by an increase of permeability and it does not involve a
loss of the adsorption capacity.

Twenty-five years later, Maximov (1938) held about
the same fundamental views. The withdrawal of water
during- freezing would cause an alteration in the proto-
plasmic colloids; in particular, it w^ould increase per-
meability. He could, in leaves, determine an increase in
cell permeability which paralleled the degree of wilting
and, consequently, he considers freezing and wilting as
similar in their injurious action.

KyJ'ni (1!U7) reported that various algae, for which
the temperature of death by freezing in sea water was
previously recorded, were killed at temperatures above
freezing when the increasing concentration of the water
in w^hich they were immersed was high enough to have
the same freezing point as the water in which the algae
were killed by freezing. Accordingly death of the or-
ganisms is attributed to the concentration resulting from
freezing and not to the congelation itself.

Moran (1929), who worked mostly with isolated frog's
muscles, remarks that neither cold alone kills, as is
shown by the well known innocuousness of subcooling,
nor does the formation of a relatively large quantity of
ice in a tissue, since muscles left for -1-8 hours in the
frozen state at -1.5° (freezing point -0.42-) were ir-
ritable after thawing. Having rejected these two in-
terpretations, he points out that it is the removal by
freezing of more than a certain critical quantity of water
wiiich seems to be fatal, as is shown by the fact that a
muscle loses its power to react to an electric stimulus
when frozen at a temperature lower than - 1.9°.

Comparing these results with those obtained in his
study of death by desiccation, namely, that it was im-
possible to revive a muscle in which 78% or more of the
water content had been removed, he pointed out that the
temperature of death by freezing was precisely the
freezing point of a solution of sodium chloride which was

282

coiicoiit rated by evaporation from its ])oint of isotonicity
with the normal nniscle until it had lost 78'/ of its water.
Death by freezing- would then take ])lace when more than
this |ti-()i)()rtioii of watci- freezes out from the already
concentrated })rotoi)lasm. (A determimition of the freez-
in«^ point of a solution containini;- the salts of the nmscle
at the concentration reached after drying to the critical
degree gave 1.6".)

The similarity of action of freezing and drying was
further evidenced by the similarity in the changes of the
electric resistance observed in tissues which were killed
by either of the two injurious agents.

Moran, however, remarks that the action of freezing
and that of drying are not altogether identical on all
sorts of materials. Drying is not known ever to pro-
duce an irreversible change in gelatin gels, while freez-
ing does.

As to the mechanism by which a removal of water be-
comes injurious, he makes the following suggestions : 1.
Dehydration may result in a destruction of "an interface,
a catalyst, or a structure holding catalysts"; 2. It may
upset the ionic balance and cause a change in the vis-
cosity and permeability of protoplasm; 3. It might pro-
duce some internal strains of the type observed in gels
which become anisotropic by being frozen and thawed.

Com])aring death to the irreversible change brought
about at - 6 in frozen egg yolk and probably due to the
action of concentrated electrolytes on the protein-lipid
complex, Moran sees ''closely similar relations" in the
two cases; they would ditfer, however, in the fact that the
irreversible change in yolk requires some time so that
one can avoid it by rapid cooling, while "no sensible time
appears to be needed for permanent destruction" of the
living system and it is impossible, by rapid freezing, to
avoid death.

Some of the authors who hold the dehydi-ation theory
of death bv cold assume that if the water withdrawn can

283

be reabsorbed after Ihawiiio-, death can be prevented.
Concerning- this (lueslion, a remark of Stik'S (quoted by
Jones and Gortner, 1932) is of interest. Hydrated gela-
tin h)ses 4 to 6 times more water on thawing when it
has been frozen slowly than when it has been frozen
rapidly. Jones and Gortner (1932) mention, as a natural
explanation of this observation, the fact described by
Moran (1926) and by Hardy (1926) that, in rapid freez-
ing, a large number of small crystals are formed within
the gelatin mass and a reabsorption of that water after
thawing is easy and rapid, while, in slow freezing, the
presence of larger quantities of ice at the same place
and mostly at the external surface impairs or slows
reimbibition after thawing.

A determination by Luyet and Condon (J9S8) of the
time at which the cells of potato tubers are killed when
gradually increasing proportions of water are with-
drawn by freezing, led these authors to the conclusion
that injury begins after the withdrawal of about 35% of
the water content, at a temperature of 0.2 to 0.3 degree
below the freezing point (between B and C in the graph)

Fiu. 33. Freezing curve of a piece of potato. Abscissae: time in min-
utes from the beginning of freezing; Ordinates: temperature. During
the periods of time indicated by A and B no ceH was kiUed; death oc-
curred during the period C; freezing continued when all the cells were
killed, during the period D. (From Luyet and Condon, 1938.)

and that all the cells are killed when about 70' < of the
water has been removed at 3.5 degrees below the freezing

284

point (botweoii V and 1> in the titriiro). At'tor death, more
water freezes mit wlieii the temperature is hnvered ( rei!:ioii
D in tlie figure). Tf death, in thesi' ex])erinients, took
])hiee while the material was I'l-ozeii ( and not on thawinir),
it did not correspond to the removal of the most tightly
bound water, since some was still unfrozen after death.

This observation led Lnifet (1U-11>) to distinguish be-
tween fitdl and hound (unfreezable) water. There woidd
be 5 kinds of water in protophism : 1. Excess water which
can be removed without atlt'ecting the activity of ])roto-
plasm; for examiile, without decreasing the res])iratory
rate. '2. Mifuholic water which iiithiences the ratt' of liv-
ing processes but can be withdrawn without inducing
death. 3. Vital water, tlie separation of which is lethal.
4. Boinunit frcczabic water which, in some organisms,
stays unfrozen during death by congelation but can ])e
frozen after death. 5. U)ifrrezahlc water which never
freezes at any temperature. The author accounts for the
speeilic differences in cold resistance by assuming that, in
some types of protoplasm, the vital water is the most
tightly bound, unfreezable water, \\ hiU', in others, it is the
freer, freezable water.

Becquerel (1939) claims that cells killed by freezing do
not show any evidence of having been dehydrated by
plasmolysis. He ex])osed e])idermal stri])s of onion or of
the petals of red hyacinth to temjieratures extending
from -150" to -25"^ and examined individual cells under
the inicrosco])e before, during and after freezing and
thawing. He observed that neither the cytojilasni, nor the
vacuole nor the nucleus undergoes any change which
might be interpreted as resulting from ])lasmolysis. He
concludes that ''almost all the physiologists so far have
made the mistake" of considering as plasmolysed a coag-
ulated c^'toplasm slightly separated from the cell wall.
It seems that the condition reported by Becquerel is due
to the fact that he used a rapid method of freezing which
did not allow plasmolysis, which is usually a slow proc-
ess, to take place.

285

Levitt (1939), in a study of cold hardiness in cabbage,
brings a quite new argument against the theory that re-
sistance to injur}" by cold is simply resistance to dehydra-
tion. He determined by calorimetric methods the propor-
tion of frozen and unfrozen water in hardened and in un-
hardened plants at their critical freezing temperatures,
that is, at temperatures at which 50-75 per cent of the
plants are killed (-5.6° for hardened, -2.1° for unhard-
ened). The "unhardened tissue retained 3.5 times as
much water in the liquid state per gram dry matter as did
hardened (6.30 and 1.75 gm. respectively)". Hardiness,
that is, resistance to injury by cold, ''is therefore not de-
termined simply by resistance to dehydration."

It is quite evident that as yet one cannot draw any
general conclusion from the data here presented.

VI. THEOEY OF DEATH BY VARIOUS PHYSIOLOGICAL,
PHYSICAL OR CHEMICAL ALTERATIONS

Various jjliysiological changes which accompany freez-
ing have been assumed to cause injury and death. Though,
in the last analysis, the mechanism of their lethal action
might be physical or chemical, they will be treated here
as physiological changes, as they were presented by
their authors. The physical alterations, other than crys-
tallization of water, which have been observed to result
from freezing and thawing in aqueous colloids are : pre-
cipitation, agglomeration or dispersion of particles, coag-
ulation, gelation, changes in transparency (turbidity,
opalescence) and changes in consistency (cf. our previous
review of this subject). Any of these physical alterations,
as well as a number of chemical changes, might consti-
tute the mechanism of injury and death by freezing. In
the literature one finds such a general suggestion repeat-
ed again and again. But specific suggestions as to wiiich
protoplasmic constituents might be altered, and in what
manner, are few and still fewer the experimental attempts
to test these possibilities. We shall mention here the
more specific suggestions and those which are backed by
some experimental evidence.

28G

/. Plii/sioJdfiicdJ (li(nif/es. A weaken iiii;- of vitaliiy by
tlio action of colil (Ui ])roto])lasm lias l)een assumed by
Irnischor (1912) to oxi)lain liow a ])rolon<;-ed or repeated
exposui'e causes a ,ni'a<luall\' inci-casini^' (bunage. He had
observed that some moss species whicli resisted one freez-
ing at "20 were killed by 4 fi'eezings at 15' and that
moss which was not entii-ely killed l)y one freezing at
— 1") , this temperature being maintained for IS hours,
died entirely after an ex])osure of 4 days to - 10 . (Sim-
ilar experiments were reported by numerous other au-
thors, in particular by Goeppert, 1830, on Lamiiim and
by Ai)elt, 11)07, on ])otat()).

Schaffnit and Liidtke ( 1932), who studied the influence
of low temperature on the metabolism of plants subjected
to various diets, came also to the conclusion that before
death there is evidenc«' of a gradual weakening under the
action of cold. Death would then be due, finally, to dis-
turbed metabolism.

To assume that low temperature weakens protoplasm
and renders it more vulnerable to cold is in direct oppo-
sition with the observed fact that low temperature hard-
ens against cold.

According to Krebs (1931), the muscles would lose
their ability to synthesize glycogen and phosphagen
after being frozen. Death would then occur when the
energy demands exceed the supply.

Cold injury might be related to physiological changes
which take place at some particular temperature. Moran
(1925) cites, as an example of a change of the kind
which might be invoked here, the observation of Vernon
(,//. of Physiol, /7, 277, 1894) and of Castle {Jl Gen.
Physiol., 7, 189, 1924) that respiration in the frog and
in other animals and plants undergoes a critical change
at around +15 . It is possible that, in a similar man-
ner, some unknown activity, ])erhaps some enzymatic
process, is critically altered at the temperature at which

287

the water phase is separated by freezing- from the proto-
phisniic substance.

Some authors, after Klemm (1895), have observed that
freezing causes a granulation of protoplasm. The mech-
anism of this alteration, which is probably physiological
in nature, is unknown.

Freezing might be more injurious to some vital activi-
ties than to others and destroy one particular function
before it destroys them all at death. This assumption
w^ould explain the observation of Pictet (1893) that a
wound made by freezing takes a much longer time to heal
than a wound inflicted by other injurious agents. The
subepidermal tissues would have lost their powder of re-
geueratlug the dead portion.

2. Physical and Chouical Changvs. Most of the phy-
sical changes assumed to take place at freezing tempera-
tures are phase separations. The most important phase
separation is evidently that of water; but other phases
might separate either directly when a saturation temper-
ature is reached or indirectly as a result of dehydration.

Gorke (1907) assumed that the proteins dissolved in
the cell sap of plants might precipitate at death, such a
precipitation being due to the gradually increasing con-
centration of the sap subsequent to the withdrawal of
water by freezing. He attempted an experimental study
of his theory by determining the amount of precipitable
protein in the sap extracted from previously frozen and
from unfrozen plants. The proteins w^ere salted out and
the nitrogen content of the precipitate was determined by
the Kjeldahl method. The sap from frozen material
contained only 2/3 the quantity of nitrogen found in the
sap from living plants. Unfortunately Gorke 's proce-
dure is subject to criticism (as Voigtlander, 1909, pointed
out) in the fact that he extracted the sap by a slight
pressure in frozen tissue, in which the fluid came out
readily, wdiile he crushed the living tissue; this differ-
ence in treatment might well account for the difference

288

ill ])r()t('iii coiitciil observed, l^iit this criticism does not
ai)i)ly to a second .uroup of exi)ei-iineiits in wliicli Gorke
measured the amount of ])roteiu wliich precipitated in
the freezing of previously extruded sap. lie found a
noticeable quantity of nitrogen in the precipitate of the
frozen specimens. From these and other experiments
in which it was found that the precipitation of proteins
in i)hints of varying cold resistance (for example, mus-
tard leaves and spruce needles) requires either a dif-
ferent low temperature (-3° and -40° respectively) or
a different salt concentration (2.7 N and over 5.4 N NaCl
respectively), Gorkc^ concludes that the specificity ob-
served in the behavior of plants exposed to cold de-
pends on the ease of precipitation of the proteins.

Xord (1934) remarks that if death is characterized by
the transition from a clear, homogeneous protoplasm to
a coarse, microscopically heterogeneous material, it is
natural to think that such a transformation corresponds
to the freezing point of some aqueous solutions and to
the coagulation point of some proteins. Since his inves-
tigations on the action of freezing on various suspensions,
colloidal solutions, and enzymes have resulted in the
conclusion that the size of the colloid particles is modi-
fied by crystallization of water, he correlates death with
changes of this kind.

Mirsky (1937a and b), who observed that extracted
myosin coagulates by freezing or drying and that myosin
within the nniscle coagulates on being rehydrated after
having been dried, postulates that death by freezing, in
the muscle, results from a coagulation or aggregation of
the myosin molecules caused by some change during
freezing or during the reimbibition of water.

Harvey (1918) found a higher hydrogen ion concen-
tration in the sap of frozen than of unfrozen plants.
Whether this ionic dissociation is an immediate effect
of freezing or whether it results from various interme-
diate transformations, it is im])ossible to say.

289

A liberation of ions on freezing has also been reported
by Dexter, Tottingliam and Graber (1930) who observed
a higher electric conductivity and a higher electrolyte con-
tent in frozen than in unfrozen plant sap,

Fischer (1911) suggests that one of the modifications
caused by freezing in colloids might be a loss of their
power to absorb water. He attributes to this change the
ditference observed by some biologists in the quantity
of heat liberated during the freezing of a tissue and the
quantity of heat reabsorbed on thawing.

Becquerel (1937, 1938, 1939)', considering the resem-
blance between a tissue killed by freezing and no longer
capable of holding its water and a gel which loses its
water when its structure breaks down (syneresis), pro-
poses the theory that death is a structural disorganiza-
tion identical with that occurring in such gels. He ob-
served in cells of the epidermis of the onion, immersed in
liquid air and thawed, a decrease of the volume of the
nuclei which could be interpreted as comparable with the
contraction of gels which lose their water by syneresis.
Becquerel then pictures death as follows : During freez-
ing (he speaks in particular of freezing in liquid air),
syncretic phenomena take place which consist in a sep-
aration of water from the rest of the colloidal system and
in an irreversible chemical change of the colloidal par-
ticles; on thawing, the particles so modified aggregate
into larger masses and form a coagulum. The fluid ex-
truded after thawing consists of the syncretic w^ater
and of the cell sap which easily traverse the now dis-
rupted protoplasm.

Concerning the water-holding properties of living tis-
sues, it is interesting to compare them Avith those of
aqueous gels. Though these two systems might be quite
similar in many respects, they differ, among other fea-
tures, in their behaviour on freezing. As Jones and
Gortner (1932) have pointed out, when one freezes (rapid
freezing) a gelatin gel several times in succession, the

saiiu' ninouiit of water crystallizes each time, at the same
initial freezing; point. This shows that no irreversible
alteration takes place from one freezinj;- to the other.
In the case of livin.ii' tissues, on the contrary, the freezing
l)oinl is raised 1>\' a lirst lethal freezing and the ([uantity
of water which crystallizes at a gi\-en temperature, in
a second congelation, is different from the (juantity
solidified in the first.

Several authors have assumed that, at death, pro-
teins are denatured in a salt solution which became con-
centrated by freezing. Concerning this theory, as applied
to nmscle proteins, Moran (1929) remarks that all at-
tempts to produce synthetically a solution of the same
composition as the fluid of the muscle have failed and he
attributes this failure to the fact that the calcium is not
simply in solution in the living muscle but that it is
associated with the proteins. This remark suggests the
hypotliesis that the destruction of the complex, protein-
calcium, might constitute death by cold.

SUMMARY

1. Death in frozen animals or plants has been attributed
by some pioneer authors to a withdrawal of energy and
the resulting impossibility on the part of the cooled or-
ganism to supply the energy necessary for vital activities.
2. According to this theory freezing is impossible as
long as the organism is alive; life has to be destroyed to
allow the formation of ice.

3. Other investigators have attributed death of frozen
plants to the attainment of a given minimal temperature,
characteristic of each species. 4. Accordingly, when this
'^specific minimal temperature" is l)elow the freezing
point, the formation of ice protects against injury and
death, while subcooling is dangerous.

5. Death has been attributed to a mechanical injury
consisting in a bursting of the cells by the expansion of
the ice in formation; but the ru])turing of cells after slow

291

freezing" in iiaturo could never be observed. 6. A mechan-
ical injury resulting in the destruction of the fine struc-
ture of protoplasm by ice crystals has been invoked as
the cause of death. 7. Some investigators have thought of
a damage by pressure between growing ice masses. 8.
Others have proposed that the protoplasm might be in-
jured by the jarring involved in freezing and thawing.

9. A large number of investigators have studied exper-
imentally the theory that the so-called death by freez-
ing is caused by a too rapid thawing. Xo general con-
clusion can be derived from their results which seem
largely to disagree.

10. Most plant physiologists have attributed death by
freezing to a dehydration of protoplasm. Some consider
dehydration as a step toward a precipitation or a coagu-
lation; others think of a destruction of the structure of
living molecules, perhaps of catalysts, by the withdrawal
of their constituent water; for others dehydration might
cause such essential changes as an increase in permea-
bility, an increase in viscosity, an upsetting of the ionic
balance, etc. ; finally, some authors attribute death to the
toxic concentration resulting from dehydration. 11. Ar-
guments in favor of the dehydration theory of freezing
are: the resemblance, repeatedly recognized, between the
action of freezing and that of drying and the resem-
blance in the conditions of sensitivity to these two fac-
tors. 12. The main objections against the theory are:
that in some plants more water can be removed without
injury by other means than by freezing ; that dehydration
by freezing is assumed to be lethal, while dehydration
by air-drying increases the resistance to cold.

13. Various physiological changes which accompany
freezing have been assumed to cause injury : a weaken-
ing of vitality liy cold, an impairment of the synthesizing
ability, an impairment of functions such as regeneration,
etc. 1-4. Among the physical or chemical changes which
might be responsible for death by freezing the most im-

21)2

poitaiil iiit'iitioiKMl ill tlic lilcrnlinc* arc : n i)recipitation, a
(Iciiatiiralion, or. a coanulatioii of |)i'()t('iiis, an ionic disso-
ciation, a loss of adsorjtlivc or watcr-biiidin.i;- ])roi)orlies,
a synerotic release (»t' water, the destruction of some com-
plex of llie iN'pe proleiii-calcinm.

BIBLIOGRAPHY

ADAMS, J.. Sc. Pio'-. Roil- ^f>r.. Dublin, i. 1. lIHl.-j.
AKERMANN, A., Botauiska Xotiscr. 33, 1913.

• — , Bat. yot.. .'/.'', 105, 1919.

ALEXOPOULOS. C. .1.. and J. DRUMMOND, Tnius. IUi)iois Acad. Set,

Jd. 63, l!t34.
APELT, A.. Cohiis Beitr. z. Biol. d. Pfl., 9, 215, 1907.
BACHMETJEW. P., Experimentelle Entomologische Stiulien vom physi-

kalisch-cheniischen Standpunkt aus. Vol. T. Temperaturverhalt-

nisse bei Insekten. Leipzig, 1901; Vol. II. Einfluss dev ausseren

Faktoren auf Insekten. Sophia, 1907.
BARTETZKO, H., Jahrh. f. wiss. Bot.. '/7, -jT. 1910.
BECQUEREL, P., C. r. Acad. Sci.. I'/O, 1652, 1905; /'/8, 1052, 1909; 150,

1437, 1910; /.S/, 805, 1925; 188, 1308, 1929; IHO. 1134, 1930; lO.'t, 1378,

1932a; 1!>.',, 1974, 1932b; 19',, 2158, 1932c; 2(l.i, 978, 1936; .20.',, 1267,

1937; 2(k:, 1587, 1938.

, Rapport 6c Congr. Inf. <Jii Froid. Biienos-Ayrcs. 1932d.

, Chronica Botanica, J, 10, 1939.

BEIJERINCK, M. W., and H. C. JACOBSEN, First Internat. Congr.

Refrig.. 2, 195, 1908.
BELEHRADEK, J., Temperature and Living Matter. Berlin, 1935.
BELLI, C. M., Cbl. Bakt.. SI, 355, 1902.
BIDAULT, C, C. r. Soc. Biol.. 85, 1017, 1921.
BROWN, H. T., and F. ESCOMBE, Proc. Roy. Soc. London, 62, 160,

1897.
CAMERON, A. T., and T. I. BROWNLEE, Proc. Roy. Soc, Canada, 7,

Sect. 4, 107, 1913.
CATTELL. M.. Biol. Rev. Cambridge PJiilos. -S'or.. // (4). 441, 1936.
CHAMBERS. R., and H. P. HALE, Proc. Roy. Soc, London, B, 110, 336,

1932.
CHANDLER, W. H., Mo. Agr. Exp. Sta. Res. Bull.. 8, 143, 1913.
CHODAT, R., Bull. Herb. Boisier, J,, 890, 1896.
CITOVICZ, B., C. r. Soc. Biol.. 99, 1699, 1928.

UK CAXDOLLE, C, Arch. Sc. Phys. Nat. Oenrvc, .?r pn:. ,«, 497, 1895.
i>K .JONG, D. A., Arch. Nrerland. Physiol.. 7, 588, 1922.
DEWAR, .1., C. r. Acad. Sci.. 129, 434, 1899.
DEWAR, J., and J. G. McKENDRICK. Proc. Roy. Inst. Great Britain,

l.i, 695, 1892.
DEXTER, S. I., W. E. TOTTINGHAM and L. F. GRABER, Plant Phy-
siol.. 5, 215, 1930.
DOEMENS, Allg. Brau. u. Hopf. Ztg.. 2, 2225, 1897.
DOFLEIN, F., Lehrbuch der Protozoenkunde. 3rd Ed., i). 230, Jena, 1911.

293

DUHAMEL, H.. and G. BUFFON, Hist. Ac. Roy. Sc. Mim.. Math. Phys.,

233, 1737.
EFIMOFF, A. W., Arch. Protistenk, .',9, 433, 1924.
FISCHER, H. W., Beitr. z. Biol. d. Pfl.. 10, 133, 1911.
GAYLORD, H. R., J. Inf. Dis., 5, 443, 1908.

GEHEXIO. P. M., and B. .1. LUYET, Biodyn.. No. .')5, 1, 1939.
GOEPPERT, H. R., ITeber die Warmeentwickelnng in den Pflanzen. de-

ren Gefrieren, und die Schutzmittel gegen dasselbe. Breslaii, 1830.

, Bat. Zeit., 20, 48, 65, 1871.

GORKE, H., Lnndw. Versiichs-Stat, 0-',, 149, 1907.
GREELEY, A. W., Am. J. Physiol., 6, 122, 1901.

, Biol. Bull., 3, 165, 1902.

HAINES, R. B., J. Exp. Biol., 8, 379, 1930.

HARDY, W., Proc. Roy. Soc, A. 112, 47, 1926.

HARVEY, R. B., Bot. Gaz.. 67, 441, 1919.

HEILBRUNN, L. V., Am. J. Physiol. dS, 645, 1924.

HELDMAIER, C, Ztschr. f. Bot., 22, 161, 1929.

HILLIARD, C. M., C. TOROSSIAN and R. P. STONE, Science, i2, 770,

1915.
HILLIARD. C. M., and M. A. DAVIS, ./. Bact.. .?. 423, 1918.
HOROWITZ-WLASSOWA, L. M., and L. D. GRINBERG, Cbl. Bakt. 89.

54, 1933.
ILJIN, W. S., Protoplasma, 20, 105, 1934.
IRMSCHER, E., Jalirh. f. wiss. Bot., 50, 387, 1912.
JAHN, T. L., Arch. Protistenk, 79, 249, 1933.
JAHNEL, F., Klin. Woch., JG, 1304, 1937.
JECKLIN, L., Rev. Suisse Zool. i,2, 731, 1935.

JENSEN, P., and H. W. FISCHER, Ztschr. f. aUg. Physiol.. 11. 23, 1910.
JONES, I. D., and R. A. GORTNER, J. Phys. Chem. 36, 387, 1932.
KADISCH, E., Med. Klin., 1074, 1109, 1931.
KALABUCHOV, N., C. r. Acad. 8ci.. U.R.S.S., I, 424, 1934
KaRCHER, H., Planta, L',, 515, 1931.
KLEMM, P., Jahrh. f. uHss. Bot.. 28. 641, 1895.
KODIS, T., Cbl. f. Physiol.. 12, 592, 1898.
KREBS, E., Proc. Roy. Soc. London, B. 108. 535, 1931.
KUNISCH, H., Inaugural Diss., Breslau, 1880.
KYES, P., and T. S. POTTER. J. Inf. Dis.. 6!,, 123, 1939.
KYLIN, H., Ber. d. deutsch. hot. Ges., 35, 370, 1917.
LAVERAN, A., and F. MESNIL, Trypanosomes et trypanosomiases,

Paris, 1904.
LEPESCHKIN, W. W.. Protoplasma, 25. 300, 1936.
LEVITT, J., Plant Physiol. / '/, 93, 1939.
LINDNER, J., Jahrh. f. tciss. Bot., 55, 1, 1915.
LIPMAN, C. B., Plant Physiol.. 11, 201, 1936a.

, Bull. Torrey Bot. Club. 63, 515, 1936b, 6'/, 537, 1937.

■ , Biodyn.. \o. ',5, 1, 1939.

LIPMAN, C. B., and G. N. LEWIS, Plant Physiol. 9, 392, 1934.
LUMIfcRE, A., and J. CHEVROTIER, C. r. Acad. Sci., 158, 139, 1914.
LUYET, B., C. r. Acad. Sci., 20-'f, 1214, 1937.

2!>4

-. C. r. Soc. Biol.. 121, 788, 1938.

-, Arvhiv /. exp. /.cUforsch.. 22, 487, 19:59.

LUYET. 1?. .1.. and K. 1.. IIODAri', /'/<». Soc. E.rp. Biol, and M('(l.. ■{'■>.
LUYET, 1$. J., and I'. M. GEIfEXIO, lliinhin., Wo. ',2. 1, 19:'.S; Xo. ',S,

1, 1939.
LUYET, B. J., and K. L. IIODAPP. Pror. Nor. /•;.r/>. Biul. and .M<<1.. .{.'>,

433 1938.
LUYET, B. J., and C!. TllOEXNES. C. r. A<a<l. Sri.. 2it(]. 2002, 1938a;

201', 1256, 1938b.
MACFADYEN, A., Proc. Boii. Soc. London, (Ui. 180, and Lancet, 1, 849,

1900.
IMACFADYEN. A., and S. ROWLAND, Proc. lioy. Soc. London, (!(!, 339,

190na; CC, 488, 1900b.

, Ann. Bat.. J6, 589, 1902.

MATRUCHOT, L.. and M. MOLLTARD, Rev. dm. Bot.. 1), 401, 463, 522,

1902.
MAXLMOV, N. A., licv. dciitsch. hot. (Ics.. .W, r>2, 293, .")04, 1912.

, Jahrh. f. wiss. Bot., 5.i, 327, 1914.

, C. r. Acad. Set.. U.R.S.S., 21, 183, 1938.

MELSENS, M., C. r. Acad. Sci.. 10, 629, 1870.

.MEZ. C, Flora, '.).',, 89, 1905.

:\IIRSKY A. E., .7. Gen. Physiol.. 20, 455, 1937a; 20, 461, 1937b.

MOLISCH, H., Untersucbungen iiber das Erfrieren der Pflanzen. Jena,

1897.
MORAN, T., Proc. Boy. 8oc. London, B, 98, 436, 1925; B. 105, 177, 1929,

A, 112, 30, 1926.
MORREN, C. F., Bull. Acad. Sci.. Bruxcllcs, 20, 1853.

iMOUSSU, G., Office national des recberches scientifiques, France, 1912.
MiJLLER-THURGAU, H., Landw. Jahrh.. !>, 133, 1880; 15, 453, 1886.
■ , Schioeiz. Ztsilir. f. Oh.st u. Weinhau, .?, 316,

1894.

MURIGIN, I. I., Bull. Biol. Med. Exp., U.R.S.S., J, 100, 1937.

NaGELI, C, Sitz. d. Konig. Bayer. Akad. d. Wiss., Miinchen. 1, 1, 265,
1861.

NaGELI, K. \V.. Beitriige zur wissentscbaftlichen Botanilv. II. Leipzig,

1860.
NORD, F. F., Protoplasma. 21, 116, 1934.
PAUL, T., and F. PRALL, Arh. aus dcm Kaiscrl. Gcsundheitsarnte, 26,

73, 1907.
PFEFFER, W., Pflanzenpbysiologie, II, Leipzig, 1904.
PICTET, R., Arch. 8c Phys. Nat., 3e Srr.. 30, 293, 1893.
PLANK, R., E. EIIRENBAUM and K. REUTER, Die Konservierung

von Fischen durcb das Gefrierverfabren. Berlin, 1916.

PLATEAU, F., Bull, de I'Acad. Roy. Belgique, 2e Scr.. .V,, 291, 1872.

PRILLIEUX, E., Ann. Sci. Nat., 5e, Scr., Bot., 12, 125, 1869.

, Bull, de la Soc. Bot. de France, 19, 152, 1872.

RAHM, G., K. Akad. Wetensch.. Amsterdam, Natuiirkinidiqe Afdeclirig,
25, 235, 1920; 29, 499, 1921.

, Ztschr. /. alUi. Physiol., 20. 1, 1923.

REGNARD, P., C. r. Soc. Biol.. IQe Scr., 2, 652, 1895.

295

REIN, R., y.tschr. f. Katunciss.. 80, 1, 1908.
SACHS, J., Landic. Vcrsiichs-Stat. 2, 167, 1860.
SCHAFFNIT, E., and M. LiJDTKE, Phytopath. Z., .',, 329, 1932.
SCHEXK, S. L., Qitziingsh. Wlen. Akad.. Hit, 2.'), 1870.

SCHMIDT, P. .J., G. P., PLATOXOV, and S. A. PERSON, C. r. Acad. ScL,

U.R.S.S., 3, 30.5, 1936.
SENEBIER, J., Physiologie vegetale. ISod.
SniOXIX, C, C. r. Soc. Biol.. 101 . 1029, 1931 .
SMART, H. F., Science, S2, 52.5, 1935.
STILES. W., Protoplasma, 9, 459, 1930.

STUCKEY, I. H., and O. F. CURTIS, Plant Physiol.. 13, 815, 1938.
SWITHINBANK, H., Proc. Roy. Soc, London, 6S, 498, 1901.
TA:\hAIAXX, G., /Jschr. f. phys. Chem. .io, 441, 1898.
TAYLOR, C. v., and A. G. R. STRICKLAND, Physiol. Zool., 0, 15, 1936.
THISELTON-DYER, W., Proc. Roy. Soc, London, 6'.J. 361, 1899.
TURNER, T. B., and N. L. BRAYTON, J. Exp. Med., 70, 639, 1939.
UVAROV, B. P., Trruis. Entomol. Soc. London, 70, 1, 1931.
VOIGTLaNDER, H., Cohns Beitr. z. Biol. d. Pfl.. 0, 359, 1909.
WARBURG, O., Bioch. Ztschr.. 100, 230, 1919.
WEIGMANX, R., Biol. ZU., 5S, 301, 1936.
WEINZIRL, J., and R. S. WEISER, Tubercle. 15, 210, 1934.
WHITE, A. C, Med. Rec. -56, 109, 1899.
WIXKLER, A., Jahrl). f. iciss. Bot.. 52, 467, 1913.
WOLFSON, C, Ecology, 16, 630, 1935.
ZACHAROWA, T. M., Jahrb. f. iciss. Bot., 65, 61, 1926.

ZIRPOLO, G., Proc. Sixth Int. Congr. Cryoscopy. Buenos Aires, p. 450.
1932.

gkm:p>al luiujodrxAiMiv of tiik litkha-
Tiwi: ox TiiK iM\i:si:rxVATioN and

TllK DKSTIU'CTIOX Ol' LIl'i: AT

i.ow ri:Mi>i:i\ATrrxi:s^

ill! ( 'lirniKihiiiicdl Order)

1 I'M]
i^EAr.Mri;, li.A.I*\, Mriiioircs poiii- sci-xir n I'liisloirc dcs
insoetos. I*;ii-Is; iSliulies on low Icinpcralure'S in Vol, 2,
pp. 141-147).

1737
DiiiAMKL, ir. AND G. BuFFON, Observation dos diftorents
c'lTcls (pic pro(]nisont snr los voovtanx Ics i>'i'andes gcloes
d'lii\('r ct Ics pctitcs ti;-clccs du pi-inlcni[)S. I/isf. Ac.Roy.
Sc.,Meu,. Mdlh.rJiijs., 233-298.

1776
Si'ALLANZAXi, L., Opuscoll (U fislca aniniale c vegetabile.
Modena ; (Experiments on the action of low temperatures
in Opusc. 1, Chapters V and VI).

1778
HuxTKi;, J., Of the Heat of Animals and Vegetables.
PJiilos. Trans., GS, 7-49.

*This bibliography is not a list of the references utilized in the pres-
ent monograph — such a list has been printed, in sections, at the end of
each Part — it is intended to be a compilation, as complete as possible,
of the researches published up to 1940 in the field of "The Preserva-
tion and the Destruction of Life at Low Temperatures." The expres-
sion "complete bibliography" has become, at present, practically mean-
ingless for a large numl)er of topics; we present, therefore, this bil)li()-
graphical list as a nucleus of the essential references, which is to l)e
completed in future editions. The elaboration of monographic l)il)lio-
graphies, and the organization of some means of providing a periodical
republication and completion of such works is probably to become an
important task for the next generation of scientists (perhaps as im-
portant as the organization of research laboratories has been during
the first half of this century). To emphasize this particular character
of the present bibliography, and also to show the historical develop-
ment of the research on the sul)ject, we arranged the references in the
chronological order,

296

297

1817
Du Petit-Tiiouaks. a., Snr Ics effets de la gelee sur les
plaiites. Vcrncr Frai/rals, I'aris.

1820
VoGEL, A., Ueber die Veraenderungen, die einige Stoffe
des orgaiiischeii Reielies beim Gefrieren erloiden. Cfil-
hert's Ann. d. Physik, ()4, 167.

1829
Xeuffer, W., I'ntersuchmigen iiber die Temperatnr-
Verandei'uiigou der Vegetabilieii inid vcrsehiedeiie damit
in Beziehuiig steliende Gegenstande. Iiiaug. Diss., Tii-
bingen.

1830
GoEPPERT, H.R., Ueber die AYarmeentwiclvelung in den
Pflanzen, deren Gefrieren, und die Schutzmittel gegen das-
selbe. 1-272. Breslau.

1833
Herschel, J., Xotice of a Remarkable Deposition of Ice
round the Decaying Stems of Vegetables during Frost.
London-Ediuh. PhilosMag., 2, 110-111; (On p. 190 there
is a note by S. P. Pigaud on the same subject).

1838
AuDoiis^, Influence du froid chez les insectes. Ann. Soc.
Entom. dc France, 7, Bull. 39,

Among subjects which have been deliberately omitted, though they
concern rather directly our topic, we shall mention: cohl liardiuess in
plants, preservation of food by refrigeration (and, as such, cold injuri/
to edible plants, fruit, etc.). plii/siological effect of cold on germination,
growth, morphogenesis, habitat, etc. (and. as such, snoic vegetation,
arctic vegetation, etc.), properties of phi/sical systems at low tempera-
tures (the properties of colloids, however, are included). These sub-
jects either have been investigated enough to constitute separate units,
or the accepted division of the sciences has assigned them to specialists
in other branches, or they have already been or are reviewed by com-
petent authors whose work it would be useless to duplicate.

When the title of a paper does not indicate what relation its content
bears to low temperature, we added a note in parentheses to that effect.

1840

l*i:KV()sr. lu'clicrclics sur Ics ;iirnii;il('iil('S spcniiiil i(iiu's.
C.r. Acdtl. Sci., II, I)(I7~!H)S; ( l-'i^cc/iiii;- of fro,;;' spci-iiinlo-
zoa).

1848

DixAL. F., Dos effots de la geU'o sur Ics plnntes. Mem.
Ac. MouipcU'wr. Sect. Sci., 1, 15:1.

1850
Le Coxtk, J., Obsoi'vatioiis on a Iiciiiaikablc Exudaliou
of Ice from llio Stems of Vegetables and on a Singular
Protrusiou of ley Colunms from CV'rlain Kinds of Kartli
during Frosty Weather. Loiidon-Ediiih. Phllos. Mag., '30,
329-342.

1852
Le Conte, J., Observations on the Freezing of Vegeta-
bles and the Causes Which Enable Some Plants to En-
dure the Action of Extreme Cold. Aw. J. Sci., 63, 84-92,
195-206.

1853

Goeppert, H.R., Ueber das Verhalten der Pflanzen bei
niederer Temperatur. Bot. Ztfj., 11, 123-124.

DE QuATREFAGES, A,, Reclierclies sur la vitalive de quel-
ques poissons d'eau douee. Ami. Sci. Xdf.. ( ."!), /.'', 341-3(19;
(Freezing of tish spermatozoa).

1854
Caspary, R., Auft'allende Eisl)ildung auf Pflanzen. Bot.
Ztg., 665-674, 681-690 and 697-706.

1855
CASPAitv, P., Ueber l^^i-ostspalten. Bol. Zlg., 449-462,
473-482 and 489-500.

1860
Sachs. .J., Untersuchungen iiber das Erfrieren der
Pflanzen. Luinhr. Vcrsiiclis-Shil .. Xo. 5, 167-201.

299

AVartmax, ]■•]., Xolc relative a riiiHueiice cle t'roids ex-
cossifs sur les graincs. Arch. Sc. Pliys. Nat. Geneve,
Nouv. per., 7, 277-279.

1861
Nageli, Ueber die Wirkuiig des Frostes auf die Pflaii-
zenzelleii. Sifsiuif/shrr.l-fjJ.-lHit/cr. Alad. Wiss. su Miiii-
chen, 264-271.

1862
Walther, a., Beitrage ziir Lehre voii der tliierisclien
Warme. Yircliow's Arch., 2.')^ 414:-4:11 ; (Observations on
death of rabbits by cold).

1864
KuHNE, W., Untersuchungen iiber das Protoplasma
und die Contractilitat. Leipzig; (Action of low tempera-
ture: pp. 3-6, 46-47, 68, SS, 100-102).

1866

PoucHET, F.A., Eecherclies experimentales sur la con-
gelation des animaux. J. Anat. et Physiol., 3, 1-36.

RoTW, M., Ueber einige Bezieliungen des Flinnnerepi-
thels zum contractilen Protoplasma, Arch. path. Anat. u.
Physiol., 37; (Action of low temperature: pp. 188-189).

1868
Sachs, J., Lehrbucli der Botanik. Leipzig; (Action of
low temperature: p. 562).

1869

Prtllieux, E., Sur les proprietes endosmotiques des cel-
lules gelees. Bull. Soc. Bot. de France, 1(>, 91-100.

— , Etfet de la gelee sur les plantes. Forma-
tion de glaQons dans les tissus des plantes. Bull. Soc. Bot.
dc France, 16, 140-152.

, Sur la formation de gla(^ons ti I'interieur

des ])lante.s. Aidi.ScL Nat., rf Her. Bot., 12, 125.

1S70

Mklskns. M., Note sur In vit;ill1(' dc l;i Icxurc dc
hiri-c. C.r.Acdd. Sci., 1(1, ()L*i)-()."!2 ; ( ( 'oiiililiicd nclioii of
l)ressure and cold).

ScHENK. S.L., iiber den Kiiilluss iilcdcrci' 'rciiipcra-
tui'i;rnd(' aid' ('inii;-(' Filciiiciilai-oi'.uaiiisiiicii. Sil :iii/(/slt.
Win). Akdil., (iO, 25-;U).

1871

CoHX, F., Das Gefricrcii dor Zcllcii von Xifrlla si/ii-
carpa. Bot. Zffi., ^'^ 7l\''..

GoEPPEKT, I1.I\., Ilolic (k'L- Kaltegradc, wclclic d'k' \'('ii'('-
tation iibeiiiaupt ertriij;!. Bot. ZUj., 2!), 48-58, G5-7G.

Hermann, L., Die Erstarruiig- in Foli>-o starker Kalte-
grade. Arch. rjes. Phi/sioL, .'/, 189-192.

Verson", E., Ucber den Einfluss niedrigen Teniperatu-
reii aiif di^e Lebensfaliigkeit der Eier des genieinen Seid-
enspinners. Ost. Scidenhaii Zig., A, 57-59, 65-6G.

1872

DoENHOFF, Beitriige 7Air Physiologie. I. Ueber das Ver-
halten kaltbliitiger Thiere gegen Frosttemperatur. An]\.
Aiiaf. u. Physiol., 724-727.

^[i'LLER, W., Ueber die Widerstaiidsfahigkeit des
Frosches gegen holie iind niedere Teni])eraturen. Arch.
Aiiaf. n. Physiol., 754-759.

Plateau, F., Reclierches physieo-eliiini(|nes snr les arti-
ciiles aquatiiiues. Bull. Acad. roy. dc Bclyiqiic, J'' scr., A'l;
(Action of low temperature: pp. 291-809).

PRn^LiEiTx, E., De 1 'influence de la congelation sur le
])oids des tissus vegetaux. C.r. Acad. Sci., 7'/, l.')44-l.')4().

, CV)loration en bleu des (leurs de (|uel(j[ues

orchidees sous 1 'influence de la gelee. Bull. Soc. Bot. de
France, W, 152-159.

1874
CoHN, F., AVirknng der Kiillc anf I'llanzciizellen. .laJiih.
Ayr. CIh'ui., 197-198.

301

Sciii^MACMTKK, Ft., Bciti-jige zui' M oi'] )li<)l()gio 1111(1 Biolo-
gic der Het'o. Sifzuiif/sh. Wiai. Ahail. 70, 157-188; (Some
observations on the action of cxijosiirc to low tempera-
tures).

1875

GoEPPERT, H.K., Ueber das Anfthauen gefrorener Ge-
wachse. Bof. Zif/., .,'./, ()09-612.

, Ueber die Fahigkeit krautartiger Ge-

wachse, Kalte zu ertragen. Bof. Zffi., .^.>, 613-616.

Uloth, W., Ueber die Keimnng von Pflanzensamen in
Eis. Flora: 3S, 266-268.

1876

DucLAUx, E., De Uaction physiologic pn^ (ju'exercent, sur
les graines de vers a soies, des temperatures inferieures
a zero. C.r. Acad. Sci, .S.?, 1049-1051.

Haberlandt, G., Uber den Einfluss des Frostes auf die
Chlorophyllkorner. Bot. Ztschr., 2(), 219-255.

Velten, W., Die Einwirkung der Temperatur auf die
Protoplasma-bewegung. Flora, rj<), 177-182, 193-199, 209-
217.

1877

FiRSCH, A., iJber den Eintluss niederer Temperaturen
auf die Lebensfahigkeit der Bakterien. Sitzungsh. kais.
Ahad. ir/.s,9,, Mafli.-Xdf iinriss. Klassc, 7.7, 257-269.

1878

DeVries, H., Ueber das Erfrieren der Ptlanzen. Leo-
pohlhuKi (Dresden), /'/, 103-108.

1879

DE Candolle, C. and R. Pictet, Eecherches concernant
Paction des basses temi)eratures sur la faculte germina-
tive des graines. Arch. Sc. Phi/s. Naf. Geneve, 3" per.,
2, 629-632.

isso

KiNiscii. 11., I'lx'r die t(>l!Icli(' l^iiiiwirkuii.i;' iiicdci'cr
'r('nii)('i-;ilui-('ii .•ml' dlf rilaiizcii. liinn.i;'. Diss., l-.KI,
BiH'slau.

Mi'-LLKit-'riirKCAi-. 11., rebel- das ( Jel'riei-eii niid Vh'-
friereii der Ptlaii/.eii. Lai/'lir. Jdlirh., U, l.'JIMS!).

1884

DK Caxdollk. C. AM) H. PiCTET, CoiiiiHiiii icat'ioii without
title on till' action of low temperatures on seeds. Arrli.Sc.
Phijs.Xaf. (u'ucn\ ..'' per., II, ;52r)-:',-J(i.

FoL, Coimiiunieatioii williont title on the action cold on
protozoa. Arch. Sc.riii/s. Nat. Gtnierc, ,V jx'-r., 11, 1)27.

PicTET. \\. ANi> K. Yung, De Taction dn froid siir les
microbes. (\r.. AauL Sci., 9(S, 747-74!).

SoRATKH. I\, Wirkung-en kiinstliclier Froste. Bcr.
deutscli. hnf. r/c.s., 2, XXII-XXV.

VoYLE, J., Elfcct of Cold on Eggs of Bark-lice. U.S.
Urpt. Agr. Rep., 413-414.

1885
CoLEMAX, J.J. AND J.G. McKendrick, Tlic Mcclianical
Production of Cold and the Effects of Cold \\\)(n\ ]\licro-
])hytes. (liei)i. Neifs, Ji, 61-64.

1886

Detmer, AV., Feber Zerstorung der Molekularstructur
des Protoplasma der Pllanzenzelle. Bof. Zff/. '/'/, 51,')-
524. (Action of low temperature: ])]). 520-52.')).

MC'ller-Thurgau, H., Feber das (Jefrieren und Er-
frieren der PHanzen. 11. Tlieil. LinnUr. Jahrb. /•>, 453-
610.

PoKDEL, H., Uber das vitale Temperaturmininmm wir-
belloser Tliiere. Ztselir. Xafunriss., 5.9, 183-214.

1889
Heubel. F., Die Wiederbelebung des Herzens nach dem
Einti-itt vollkonimeiu'r llerznuiskelstaiTe. I\l'lii(/ers Arcli.,
'/5, 461-5S1; (Action of low teniperat ni'e : pp. 5(53-568).

303

L.TUBAVix. X., On the Frocziiii:,' of Some (\)lloi(l;il So-
hilioiis. ,/. Iiiiss. I*lii/sic<)-Cliciu. Hoc, il, .'5!>7; ( Iviissian).

1890

JuMELLE, H., La vie des lichens peiidniit 1 'liiver. C.r.Soc.
Biol. (Mem.), 42, 115-119.

KocHs, W., Kami die Kontinuitat der Lebensvorgange
zeitweilig vollig luiterbroelieii werden ? BloL ZbJ. 10, 673-
686.

1891

Ambeoxx, H., Einige Beobaclitmigen liber das Gef rieren
der Kolloide. B< r. k.-sdchs. Ges. Wiss. zii Leipzig, I, 28.

CoLix, G., De Pact ion des froids excessifs sur les ani-
maiix. C.r. Acad. Sci., 112, 397-399.

KxAUTHE, K., Meine Erfahrungen iiber das Verhalten
von Ampbibien und Fischen gegenliber der Kalte. Zool.
Anz., V,, 104-106 and 109-115.

Muller-Erzbach, W., Die AViderstandsfahigkeit des
Froscbes gegen das Einfrieren. Zool. Anz., ///, 383-384.

1892

d'Arsoxval. Action ]jbysiob)gique des tres basses tem-
peratures. (\r.Soc.BioL, 'i'l, 808-809.

FoRSTER, J., Uber die Entwickhmg von Bakterien bei
niederen Temperaturen. Chl.BaM., 12, 431-436.

JuMELLE, H., Recbercbes pbysiologiques snr les li-
cbens. Bcv. gen. Bof., .'/; (Action of low temperature:
pp. 266-272 and 305-316).

KocHs, W., Ueber die Vorgange beim Einfrieren und
Austrocknen von Tier(^n und Pflanzen. Biol. Zhl., 12,
330-339.

1893
PicTET, R., De Pemploi metbodique des basses tempe-
ratures en biologie. Arch.Sc.Phi/s.Xaf., Ser..l, .](), 293-314.

1894
Bay, J., Crystals of Ice on Plants. Bof. Gaz., lU, 321-
326.

r>04

M iLi,i;i; 'riicKcAr. II. M., C'lici- dns I'll rricrcii dcs ()1)-
slcs. Sclncci:. Xlsilir. Ohslii. W < iiilxiii , .)', .'SKi-.'')!^! .

Win ri:i;Ni rz, 1\., \'cri;l('ic'lu'ii(l(' \'ci-sii(*1k' iihcr Ahkiili-
hiiii;- 1111(1 Finiissuiiu'. A icIi.c.rp.Pal Ii.k.PIki niuik., ->■>, 2S()-
'.]()-[; (( )ii (Icalli lt\' ('t)l(l ill lioiiioiotlicniis).

1895

DAhMKK. M., rdicr Fjisl)il(liiii,i;' in IMlaiizcii mil Kiiek-
sic'lit nut' (lie .•niatoiiiisc'lic BcsclialTciilicil (Icrsclhcii. Flora,
SO, 4oG-444.

DE Ca.\1)(»m,k. ('., Siir la \ii' lalciitc dcs i;raiiit'S. Arch.
Sc. PJn/s. Xdt. (icneve, 3' per., ri.i, 497-512 ; ( Action of low
1(>iiilK'i-atin(': ))p. 499-505).

Klkm.m. I*., Desoi-gaiiisatioiiserschcimuigcii dcr Zclle.
Jdlirh.f.irlss. Hot., 2S; (Action of low temperature: pp.
G41-G44).

Klepzoff, C, Ziir Frage iiber den Einfluss niederer
Temperaturen auf die vegetativen Formen des Bacillus
aiithracls. Chi. Bali., 11, 289-295.

KocHS, "W., Kami ein zu einem Fisklumpen gefrorenes
Tier wieder lehendig werden ? Biol. ZhI., /->, 1)72-1)77.

Regnaki), p., Actions des tres basses temperatures sur
les animaux a(|iia1 i(|iies. C.r.Hoc.B'iol., '/T, ()52-658.

1896

CiioDAT, 1\., Fxi)(''riences relatives i\ Faction des basses
temperatures sui* Mucor mucedo. Bull. IJcrh. Bois.sier, .'/,
890-897.

MoLiscH, H., Das Frfrieren von Pflanzen bei Tempera-
turen iiber dem Fispunkt. Sitzu7i(/sh.kf/l.Aka(l.]Vi.'^s. zu
W 'it'll, 10.1.

1897

Brown, TI.T. and F. Fscombk, Note on the Influence of
Ver,y Low Temperatures on the (xerminative Power of
Seeds. rroclioi/.Soc, Loiuloii, liJ, 1(50-1 (55.

DAVENi'oitr. C.B., Fx])erimeiital Morithology. New
York; (Action of low temperature: ])\). 2.'!9-249).

MoLiscH, H., Untei-snchniigpii iiber das Frfi'ieren der
Pllanzen. 1-7."'). Jena.

305

1898

d'Aksoxval, liifliuMico (le la dessicatioii siir I'action de
I'air li(ini(le sur les l)act(''rie8. C.r.Soc.BioL, .',0, 877-878.

Gladix, G.P., The Viability of Pest Bacilli under Va-
rious Physical Conditions and under the Action of Va-
rious Disinfectants. Diss., St. Petersburg'; ( Russian).

KoDis, T., Die Unterkiihlung der thierischen und pflanz-
lichen Gewebe. ChJ. Physiol, 12, 592-595.

1899

Dewak, J,, Connnunication witlioul title on the action
of liquid hydrogen on seeds. C.r.AcafLSci, 129, 434.

Kasansky, M.W. Die Einwirkung der Winterkalte auf
die Pest- und Diphtheriebacillen. Chl.Bakt., 2-7, 122-124.

Eabaud, E., De I'influence de la congelation sur le de-
veloppenient de IVruf de poule. ('./■. AcofLSci., 1.2!^, 1183-
1185.

Thiseltox-Dyee, W., On the Influence of the Temper-
ature of Li(iuid Hydrogen on the Germinal ive Power of
Seeds. Proc.Boi/.Soc, London, (>'), 361-368; also A}ui. Boi.,
/.?, 599-607.

White, A.C, Licpiid Air; Its Application in Medicine
and Surgery. Med.Bec, oO, 109-112; (Action of liquid air
on various bacteria).

1900

Chaxoz and M. Doyox, Action de basses temperatures
sur la coagulabilite du sang et du lait et sur le pouvoir
coagulant de la presure. C.r.Soc.BioL, o2, 453-454.

Chaxoz, p. Courmont and M. Doyon, Action du re-
froidissement par Pair liquide sur les serums aggluti-
nants et les cultures agglutinables. C.r.Soc.BioL, 52, 764-
765.

Macfadyen, a., On the Influence of the Temperature
of Liquid Air on Bacteria. Proc.Boy.Soc, London, 66,
180-182; also Lauccf, 1, 849.

Macfadyen, A. and S. Rowland, Further Note on the
Influence of the Temperature of Liquid Air on Bacteria.
Proc.Boy.Soc, London, iU>, 339-340.

306

Maciauvkn. a. ami S. Iaowi.a ni>. liilhiciicc of llic Tciii-
pi'int me (»r lj(iui(l 1 1 ydi'oLicii on l>;i(*l('ri<M. Pinc.Itni/.Soc.,
London, m;, 4SS-4S!).

M AiiM'ciioi'. Ij. a.\i> .M . .Moi.i.iAiMi. Sur cci-lnins plir-
iionioiK'S prrscMit^'s pai* Ics noyanx sons T.-iclion dii I'iomI.
C.r.Aca/I.Sci., I.U), TSS-TDl.

M AiKKi, AND liA(ii;ii'KE, Dotenuinnl ion d Jiclion dcs i)lus
bassos 1('nip(''i-;itni'('s conipal iblcs axcc la v\r dc la ,i;re-
iioiiillc. ( 'ompai-aisoii de ractiou dc la clialcui- ct dii I'l-oid
8iir cct animal. Cr. Sac. Hid]., .7,?, 4'.V2-4'.]').

PozKi.'sKi. Action dc (luclipics fcnncnls soluhlcs ai)rcs
i-ct'roidisscniciil x'crs -191 an nioxcn dv Tair li(|uide.
('j:S„r.h'i,>l., .'>.!, 71 4-71 G.

SKIHiWlCK. \\'/i\ AND (\E.A. WiNSI.OW. K.\ pci-illlClltcllc

iind statislisclic Sludicn iibcr dcii i^jnfhiss del' Kaltc aiif
dcii Ty])liiisl)acillus mid seine A\'rl('ilun,i''. Cbl. Bakt., 27,
G84.

1901

Bachmetjew, p., Exporimontelle Entomologische Stu-
dien vom physikaliscli-cliemischen Standpunkt aus. Vol.
I. Temperaturvcrhaltiiisse bei Iiisekleii. Leii)zig.

Bakhmetieff, v., De la toin})eratui-e \-ilfde iiiiiiiiiia eliez
les animaux dont la temperature dii sang est variable.
Arch. sc. hioI.St.-Pefershourg, S, 242-264.

Brehme, W. Ueber die Widerstaiidslaliigkeit der Oho-
leraxibrionen und Tyiiliiisbaeilleii gi'geii iiiedere Teinpe-
ratiireii. Aich.jAlijg., 'lO, ;]20-346.

Greeley, A.W., On the Analogy Between tlie Effects of
Loss of Water and Lowering of Temperature. Am., J.
Phi/sioJ., i;, 122-128.

AIatruchot, L. and M. ]\[olltard, Sur I'identite des
modiUcations de structure produites dans les cellules
vegetales par le gel, la plasmolyse, et la fanaison. 6'./-.
Acad.Sci., ]32, 495-498.

Pai;k. AV.ir., Duration of Life of Typlioid Baeilli, De-
rived from Twenty Different Sources, in Ice. Effect of

Intense Cold on Bacteria, ./. liosfoit Soc. Med. Sci, •'),
371-373.

1902

Belli, C.]\I., Der Einfliiss niederster, mil fliissio-oi- Luft
erlialtener Tempera turen aiif die Virulenz der pathogenen
Keime. Chi Bait., .?/, 355-360.

]\[acfadyen, a., On the Influence of the Prolonged Ac-
tion of the Temperature of Liquid Air on Micro-organ-
isms, and on the Effects of :\Ieclianical Trituration at the
Temperature of Liquid Air on Photogenic Bacteria. Proc.
Roy. Soc, London, 11, 76-77.

Macfadyen, a. axd S. Rowland, On the Suspension of
Life at Low Temperatures. Ann. B of., Hi, 589-590.

Matkuchot, L. axd :\r. MoLLLVRD, Modifications pro-
duites par le gel dans la structure des cellules vegetates.
Rrv.f/eu.Bof., I'l, 401-419, 463-482 and 522-538.

(JxoRATo, R., Der Widerstand des Influenzabacillus ge-
gen physische und chemische Mittel. Chi. Bait., .H, 704-
712; (Some observations on resistance to low tempera-
ture ) .

SiMPsox, S., Temperature Range in the Monkey in
Ether Anaesthesia. J. Physiol, 28, XXXVII-XL.

SoRAUEE, P. Erostblasen an Blattern. Zisclir. Pflaii-
zenkrank., 12, 44-47.

1903

Barratt, J.O.W., Centrifugalisation and Disintegra-
tion in Relation to the Virus of Rabies. ChJ. Bakf., J.
Abt., So, 769-775; (Trituration at temperature of licpiid
air).

Reyxolds, J.B., Freezing Points of Fruit Juices. Ont.-
Agy.Coll. and Exp.Farms Rep., 20, 13-15.

1904
Laverax, a. axd F. Mesxil, Trypanosomes et trypa-
nosomiases. Paris: Mason; (Action of liquid air on try-
panosomes).

oU8

T^iii i:i;. W.. Pllaiizciipliysiolo^ic, Ijcipzi.i;'; ((iciu'ral
(list'ussioii on llic erroets ol" low tcinix'iatui-e in Vol. 2, ])p.
87-96).

iDo:)

Adams. .!., Tlic l\lTct't of \'ci-\ I.ow 'Pciiipcraluic on
Moist Seeds. Sci./'rnc.Hoi/.Snr., Duhliu, J, 1 (i.

Bi:((irKi{KL, 1*., Aetioii <le Tnir lifiuidc sui- la \ie dc la
.UTaiiu'. (\r.ArafJ. Sci.. I 'I'l, 1(552- 1(354.

BicivKL. A., Xoti/ iihei- die Kesistenz des l*e|»siiis gegen
niodri.ii'e 'Peinpei-atureii. Dciif scli.ni<'il.W(i(h(iis(]i r., •* /,
loo.>.

GAiaa:v. W'.l^., 'Plie ( )siiioti(' rressui-e of Sea AVfdcr and
ol' the i^lood of Mai-iiio Aiiiiiials. BioJ. l>nll., N, 257-270;
( Freezing;' ])oiiit detcriiiiiiatioiis).

Lt"i)Kk, Weiterc Beitiiige siir lianiolyse. 11. Cbl. Baki.,
'/(>, 577-58o; (Action of low temperature: pp. 581-583).

Mez. C, Xeue rntersuclimigen iiber das Erfrieren eis-
bestandiger Pflanzen. Flora, !)',, 89-128.

, ?]inigo PHanzengeograpliische Folgernngen

ans einer neuen Theoi-ie iiber das Erfrici'en eisbestandi-
ger PHanzen. llnl JdJnh.Beihlaft., .."/, 40-42.

NoAcK, P., i'^ber Frostblasen und ilire Entsteliung.
Ztscln-.f.Pflauzcnl-rauk., 1o, 29-44.

Smith, E.E. and D.B. Swixgle, Dei- EinHuss des Ge-
frierens auf Bakterien. Chi. Bait., M, .'557-359.

AViESNER, J., t'^ber ?^rostlanbfall ncbst Benierkungen
iiber die ]\leclianik der Blattal)losnng. Bcr.d.deutsch.hof.
Ges., i.?, 49-60.

1906

LiESEGAX(;, P.E., t'^lx'i- das Erfi-ieren der Pllanzen.
Fhn-a. '.)(■>, 523-524.

AloLiscn, H., Eisl)lnnien. \' atunvlss.W uclivnschr., .^1,
204-205.

TkodoPvKsco, e.g., Observations niorphologiques et
biologi(|nes sur le genre Dnnaiiclla. Ixcr.f/ru.Bof., IS.
415-418; (Note on action of low temperature).

309

\ViK(iAM., K.M., The OcriiirciKv of Ice in I'laiil Tis-
sue, riaiif Worhl, !l, 26-:]9.

, The Passa^-e of AVator from tlic IMaiit

Cell During Freezing. Plant Worhl,'.), 107-118.

1907

Apklt, a., Xeue rntersuchungcn iiber deu Kaltetod der
Kartoffel. Beifr. z. Biol. d. Pflanzen, 9, 215-262.

Bachmetjew, p., Experimentelle Entomologisclie Stu-
(licn voni ])liysika]isc]i-clu'iiiiselien Staiidpniikt aiis. Vol.
11. Einfluss der iiusseren Faktoren auf Insekten. Sophia.

Becqup:rel, P., Recherches snr la vielatente des graines.
Auii. Sc. Nat., Ser. !), .7, 193-311; (Action of low temper-
ature : pp. 222-231.

GoRKE, H., tJber chcmische Vorgange heini Erfrieren
der Pflanzen. Laudir. VcrsiicJis-Stat., Oo, 149-160.

KovcHOFP, J., Enzymatische Eiweisszersetzung in er-
frorenen Pflanzen. Brr.deutscli.ljot.Gfs., 2J, 473-479.

LiDFORss, B., Die AVintergriine Flora. Lund. Univ. Ars-
skrift., N. F., 2, 1-76.

AIoBius, M., Die Erkaltung der Pflanzen. Ber. dcutsch.
J>»t. Ges., 2.1, 67-70.

AIoLiscH, H., tJber das Gefrieren der Kolloiden. Flora,
ni, 121-122.

Paul, T., and F. Prall,, Die AVertbcstimmung von
Desinfektionsmitteln niit Stapliylokokken, die bei der
Temperatur der fliissigen Luft aufbewahrt wurden.
Arh. h. Gesundheitsamtc , 26, 73-129.

1908

Bobertag, ()., K. Feist, and H.W. Fischer, Uber das
Ausfrieren von }i\dYoso\en. Be r.d. dcutsch. chem. Ges., '/I,
3675-3679.

Gaylord, H.R., The Resistance of Embryonic Epithe-
lium, Transplantable Mouse Cancer, and Certain Organ-
isms to Freezing with Liquid Air. J. Inf. Dis., o, 443-
448,

810

(irii-.iKi;. A. AMI 1^\ I'^LiiiN. ('her (his Aut't rclcii iiiid
Ausfru'rcii dcr Ilydiosolc. />'ry. (IchIscIi. cIiciii. (Ics., '//,

4-jr);)-42(5().

Lo r iKiiMnsKK, A., t'Mx'i' d.-is Aiisri-i('i-('ii \()ii llydroso-
Icll. />'< I. fh'illsch. cJnni. (Ics. ', I , ;VM{\-?y\)l\).

M.wi.Mow, X., ()ii llic (^)iu'sti(»ii of Death l)y Freoziii,^' in
riaiils. ,/. S,,r. Sal. ,!,■ St. rrlrrshmirf/, Bof. Sect., 82-40;
(Russian with (Jci-iiiaii siiiiiiiiary ).

Rein, I\., riitcisuc'lmni^cii iilxT dcii Kiiltctod dor
Pflanzoii. Zfschr. Xal unriss. SO, l-oS.

Salvix-]\I()()HK. J.E. AXi) J.O.AV. BAiatAiT, Note upon llic
Eltt'oct of Li(iuid Aii- upon tlio (irat'tal)l(' Cancer of Mice.
Lcnicet, /, 227.

1909

Atkixs, W.R., The Osmotic Pressures of the Bh)od
and Eggs of Birds. Sci. Proc. Boy. Duhlin Soc, 12, 123-
130; (Data on freezing points).

, The Osmotic Pressure of the Egg of the

Common Yowl and Its Changes During Inenbation.
Bioch. J . 'i, 480-484; (Data on freezing points).

Becquerel, p., Sur la suspension de la vie chez cer-
taines graines. C.r. Acad. Sri. ^ 1 '/S, 1052-1054.

Bruni, G., tJber das Ausfrieren von Gallerten. Bcr.
deutsch. chem.Ges., Ji2, 563-565.

Fischer, II.AV. and (). Bobertag, tJber das Ausfrieren
von Gelen. Biocli. Zfschr., IS, 58-94.

GuiGXARi), L., Influence de I'anesthesie et du gel sur le
dedoublement de certains glucosides chez les plantes.
C.r.Acad. Sci., I y.K 91-93

Heckel, E., Influence des anesthesiques et du gel sur
les plantes a coumarine. C.r.Acad. Sci., I'i9, 829-831.

VoiGTLANDER, H., Uiit erkiililuug und Kaltetod der Ptlan-
zen. Coins BiHr. Hiid. PjL, !), 3.59-414.

1910.
Bahiki/ko. II., rntcrsuchnngcu iil)er das Erl'ricrcn von
Scliininiclpil/cii. Jahrh. iciss. Hot., 'jl, 57-98.

311

Bfx'qukrel, r., Koclicrelies cxpri-iiiiciitales siir la vie
lalciilc (los spores des Mucorinees et des Ascomvcetes.
(\r. Av(uL Sri,, 1.10, 1437-1439.

Dixox, ir.II. AND AY.R. Atkixs, On Osmotic Pressure
ill I Mauls; and on a Thermoelectric ]\Ietliod of Deter-
mining Freezing Points. Sci. Proc. Boij. DuJ)liu Soc,
/J, 275-311.

Geoegevitch, P.M.,Uber den Einfluss von exlremen
Teniperaturen auf die Zellen der Wurzelspitze von Gal-
toiiia cmid leans. Beth. hot. ChJ., 2o (1), 127-136.

Harris, D.F., Observations on Frogs at Temperatures
below zero. J. Plnjsiol. jO, LIII-LV.

Jensen, P., and II. W. Fischer, Der Zustand des Was-
sers in der iiberlebenden und al)getoteten Muskelsub-
stanz. Ztschr. f. aUg. Physiol., 11, 23-93.

RiCHTER, A., Zur Frage iiber den Tod von Ptianzen in-
folge niedriger Temperatur. ( Kalteresistenz von Asper-
fjUhis nicjer.) Chi. Bali., 2S, 617-624.

Schaffnit, E., Studien iiber den Einfluss niederer
Teniperaturen auf die pflanzliche Zelle. Miff. Kais. Wil-
helm's lusf. Laudiv. Brombercj 3, 93-144.

WisLOucH, S.M., On the Freezing (Cold Death) of the
Alga Sfichococcus hacillaris Nag. under Various Envi-
ronmental Conditions. Bull. Jardin Bot. Sf. Pefershourg,
10, 166-180; (Russian with German sunnnary).

1911

Fischer, H.W., Gefrieren und Erfrieren, eine physico-
chemische Studie. Coluis Beifr. Biol. Pfl., 10, 133-234.

MoLiscH, H., Das Erfrieren der Pflanzen. Ver. z. Verbr.
Nafurwiss. Kewifnisse, Wien., .7/, 141-176.

1912

Backman, E.L. axu J. RiTNNSTROM, Der osmotische
Druck wahrend der Embryonalentwieklung von Ban a
friuporaria. Arch. ges. Physiol., t','i, 287-345; (Freez-
ing points of frog eggs).

312

I>i.\L.\sxi;\\ ic/.. I\., I'hcr (l;is X'crli.-illcii dcs osiiiot isclicii
Dnu'kcs wiilirciid dci- I*]ii1 w ickluii.u' ^^^'^ Wirbclt iciTiii-
bryoiuMi. Arch. Kul ir.-Mcch., .//, 4Si)-r)4(); ( Kreeziii*;'
]io"mts of I'ro^e: eg'gs).

BiMNow. II., I)('i- Kiillctod dcs isolici'lcii uiid duicli-
l)lii1('1cii l-'i-oscliiimskcls. Xlsclir. (ill/). Phi/siol., I.>, VAu-

Ikmscii i;i;. 1^]., t'lx'i' die lu'sisiciiz del' Laiihiiioosc i;'o-
gon Ausl rockimiii;- uiid Kiiltc Jahrh. iciss. Bof., .')(), 387-
4r)0.

ljii:sK(iA.N(i, l-i., J)c'r()riiiali<)ii('ii voii (lallcrtcii diircli Gc-
frioreiL KoIJoid. Zfsclir., /o, 'J-Jf).

^rAxnr()\v, X.A., Clu'iniselic Scliul/inillcl dci" l*llaii-
zcii i>vi>-ou Firl'rioreii. h'rr. (loifscli. hoi. Gcs., '>0, 52-05,
2i)3-;J05, 504-516.

Ohlweiler, W."\V., Tlio Kolation Botwocii the Den-
sity of Cell Saps and the Freezing Points of Leaves.
Missouri Bot. Gard. Ann. Rep., 23, 101-131.

Smith, E.F., Das Verhalten von ]\riki'o-organisnien
gegen niedere Temperaturen. ChL I'xiht.. .».>, .").'')5.

TioTsuDA, I., rel)ei' die Konzeiil ral ion (Ut StTuiiKiua-
liliilen dureh (lei'rieren uud iiher den Eintiuss holier
kaltegrade (fliissige Lnt't) aul' die Antikorper. Ztsclir.
Imnnin., /J, 97-116.

1913

Cameron, A.T. and T. I. Browjstlee, The Effect of Tjow
Temperature on the Frog. Proc. Boi/. Sor. Canada, 7,
Sect. 4, 107-124.

Chandler, AV.H., The Killing of r*L-nil Tissue l)y Low
Temperature. Mis.stnirl Af/r. Exj). SI a. Bcs. Bull., 8,
143-309.

Dixon, E.H., and A\'. Aikixs. ()siuotic' I'ressures in
Plants. Methods of Exiraeliiig Sap from IMaiil Oi-gans.
Cryoscopic and (Conductivity Measni-enient on Some
Vegeta])h' Saps. Proc. Boi/. DuhVni Soc., /.,', 422-434.

313

Kkittt, R.C, Factors Iiilliiciiciiij;- the Sur\i\nl of Bac-
teria at Tcinpcralurcs in tlio Vicinity of tlic Freezing
Point of Water. Science, -H, 877-879.

1914

Camp:rox, A.T., Further Experiments on the Effect of
Low Temperature on the Frog. Proc. Boy. Soc. Canada,
S, Sect. 4, 261.

CAMEROisr, A.T. AND T.I. Brownlee, The Etfect of Low
Temi)eratures on Cokl-l^h^oded Animals. Quart. J. Exp.
Phi/sioL, 7, 115-130.

LuMiEKE, A. AND J. Cheveotier, Sur la resistance du
gonocoque aux basses temperatures, C.r. Acad. Sci., loS,
139-140.

Maximov, X.A., Experimentelle und kritische L'nter-
sucliungen iiber das Gefrieren und Erfrieren der Pflanzen.
Jahrh. wiss. Bot., 33, 327-420.

PoppE, K., tJber den Einfluss niedriger Temperaturen
auf Milzbrandbazillen. Zfsclir. FJeisch- n. Milcliln/r/., 2//,
485-489.

Russell, W., De la survie des tissus vegetaux apres le
gel. C.r. Acad. Sci., /JN, 508-510.

1915

Estreicher-Kiersxowska, E., Uber die Kalteresistenz
und den Kaltetod der Samen. Diss., Freiburg (Schweiz).

Garry, W.E., Some Cryoscopic and Osmotic Data.
BioU. Bull., 28, 77-86.

Hepburx, J.S., The Influence of Low Temperatures
upon Enzymes. Bioch. Bull., '/, 136-150.

Hepburx^ J.S. and C.B. Bazzoni, The Eetention of Ac-
tivity by Lirease and by Oxidase after Exposure to the
Temperature of Liquid Air. J. Franklin Inst., 180, 603-
605.

HlLLLVRD, CM., C. TorOSSL\N AND E.P. StOXE, XotCS OU

the Factors Involved in the Germicidal Effect of Freezing
and Low Temperatures. Science, .^2, 770-771.

LixDNKK. ,1., V\h'V (Icii bjiil'luss i;iiiistiger TtMiipcralurcn
aiit' .i;-(>rrt)icii(' Scirnuiiu'lpilzc. Zur Kciiiiliiis dcr Kiiltf-
roaislc'uz \"oii ^lsi» if/iUas iti/jcr. Jalnh. iciss. I lot., ■>.»,
1-52.

Schmidt. P.J., A. I^)xoMARKn and I*\ S.wi.liki:. Sin- la
l)iolot>-ic do la Irifliiiic. (\ r. Soc. li'mL, 7N, :5()()-:5()7 ; ( Low
teinperatiire sliulies).

lOlf)

Bai;ii:a.m. 11.1^]., Effect of Xa1iii-al Low Trini)cratur(' on
Certain Kun.ui and Bacteria. ./. J//r. lies., .7, 651-655.

CoBLEXTZ, W.W., The Exndalion of Ice from Stems of
IMaiils. Sri Mniifhli/, .>, 334-:U!).

KooiK. H.AW AND B. Saxton, The Fiffcet of I^'rce/in.i;' on
(V'rlaiii hioi-,s;ani(* I I\(lroi;els. J. Am. Cliciii. .S'r>r., .>N, 588-
6(11).

SeoTT, G.G., The Evohitionary Sii;nilicance of the Os-
motic Pressure of the Bh)0(h Ain. XatKiaUst, .'lO, (i41-663;
(Data on freezini;' points),

1917

KvLix, H., Uher die Kalteresistcnz dcr Mccrcsalucii.
Bcr. deutsch. hot. Ges., 33, 370-384.

Schmidt, P.J. and F.V. Stchepki-\a, Sur I'analHose des
vers de terre. C.r. Hoc. Biol., 80, 366-368.

1918

Hilliard, C.^r. AXD ^r.A. Davis, The Germicidal Action
of Freezini>' Tcmpci-atnrcs npoii Bacteria. J. Bacf., >>, 4'23-
431.

Plaxk, B., ('"her d<'n Einfhiss der Gefriergeschwindig-
keit auf die liistoh)gischen Vera nde run g'eii tierischer
Gewebe. Zfsrhr. aJlf/. Phi/sioL, 17, 221-238.

1919
Akermax, a., ('hci- die Bcdcntnnu dcr Art des Auf-
tauens fiir die I'jrliallnng gcfroi'cnci- I'flanzcn. L'of. Not.
49-64, 105-12(i.

315

Beek, F.J., Doctor dissertation (in Dutch) on the freez-
ing- of dysentery l)acleria, Leydcn, li)l!l; ((^)n()1ed by de
Jong, 1922).

Harvey, K.B., Importance of Epidermal Coverings.
Bof. Gaz., 01, 441-444; (Prevention of ice-seeding).

Jones, L.R. and ]\I. JMiller, Frost Necrosis of Tidi])
Leaves. Phyfopafh. 0, 475-476.

Jones, L.R., M. Miller and E. Bailey, Frost Necrosis
of Potato Tubers. Wise. Agr. Exp, Sfa. Res. Bull., .',0,
1-46.

Matisse, G., Action de hi chaleur et du froid sur I'acti-
vite des etres vivants. Paris, 1-556; (Lethal low tempera-
tures: pp. 41-45).

Vass, A.F., The Influence of Low Temperature on Soil
Bacteria. Cornell Univ. Agr. Exp. Sta. Mem., 27, 1043-
1074.

Vaughan, R.E. and M. Miller, Freezing Injuries to Po-
tato Tubers. Wise. Agr. Coll. Ext. Serv. Cire., 120, 1-4.

Warburg, 0., Uber die Geschwindigkeit der photoche-
mischen Kohlensaurezersetzung in lebenden Zellen.
Bioeh. Ztselir. 100 ; (Action of li(juid air on Cliloif^lla, p.
234).

1920

CoLLip, J.B., Osmotic Pressure of Serum and Er3^thro-
cytes in Various Vertebrate Types as Determined by the
Cryoscopic Method. J. Biol. Chem., J,2, 207-212.

, Osmotic Pressure of Tissue as Deter-
mined ])y the Cryoscopic Method. J. Biol. Chem., .',2, 221-
236.

Eahm, G., Einwirkung sehr niederer Temperaturen auf
die ]\Ioosfauna. K. Akad. Wetenseh., Amsterdam, Natuur-
kuiidigr AfderliHg, 2o, 235-248; also 2.9, 499-512, 1921.

Thunberg, T., Zur Kenntnis des intermediaren Stoff-
wechsels und der dabei wirksamen Enzyme. Versuche, die
Enzyme durch die Einwirkung starker Kalte zu diffe-
renzieren. Skaiidiiiar. Areli. Physiol., '/<), 71-80.

31()

l^iDAfi.r, (\, Sur Ics iiioisissni'cs dcs \i;iii(l('S coii^clrcs.
C.r.Snr.lliol., s:,, lolT-lois.

IIaIIKIS, ,I.A., U.A. (loiMNKi:. W.K. ll(. I.MAN AND A.T.

Valentin K. M;i\iiiiiiiii WmIhcs of ()siii()li(' Coiicciil ration,
in I'laiit Tissue Kliiids. I'l oc Soc.K.rii.llinl . ,nnl Mr,i., IS,
lOG-UH); (Freezing- point data).

Harvey, R.B., ^MoIIkkIs foi- tiic Diicct Determination of
the Kreeziiin Point and i\illiii,i;' Point of Plaid Tissues.
Proc. Am. Assoc. Atl r.Sci., 7'/, (iO.

Haiivkv. P.B. and \j.(). IiK(;kimbal, Freeziiis;- I'oiids of
^laize with \'ai-ious Moisture Contents. Pioc. .iui. Assoc.
Adr. Sci.. 7',. (iO.

Wright, K.C, and R.B. Harvey, The Freezing Point of
Potatoes as Determined liy the Thei-nioeleetric ]\Iethod.
U.S.Depf.A(/r.BuU.,S!i.-,, 1-7.

AVitUiiiT. R.C. AND G.F. Taylor, Freezing Injury to Po-
tatoes When Fndercooled. U.S. Drpi.Afiy.Ilnll. U HI, 1-15.

1922

DK J()X(!, I). A., M iero-organisnies et basses lenipera-
tnres. Arc}i.N('rrLP]ii/si(>L,7, 588-591.

Duval, M. and P. Portier, Limite de resistance an froid
des chenilles de Cossiis cossus. C.r.Soc.BloJ., Sd, 2-4.

Ongkiehono, Doctor dissertation (in Dutch) on freez-
ing of intestinal bacteria, Leyden ; ((^)uoted i)y do. -Jong,
1922).

Rahm, P.G., Die Pliysiologische Kalte])rolilenie. Vcrli.
deufscJi.ZooI. Ges., 27, 85.

Stiles, W., The Preservation of Food l)y Freezing, with
Special Reference to Fish and Meat; A study in (leneral
Physiology. Foo(] hiv. Boairl, Special Be p. Xc 7. Ijondon,
ll.M. Stationary Office.

SrMMKRS, J.N., Effect of Low Temperature on the
Hatching of (Jypsy-Moth F.ggs. U.S. I)<'p/.Af/r. null., lOSO,
1-U.

317

Tait, J., Tlio Heart of nil)('riiatiii,i;- Aiiiiiials. Aut.J.
Plii/slol., ■')!), 4()7; (Resistance to cold).

Zaxdbekgkn, K,, Doetoi" dissei'tatioii (in Diitcli) on tlic
action of cold on trypaiiosonies, Leydeii; ( (.^)uoted by de
Jong, 1922).

1923

Bailey, E.M., The Cryoscopy of :\nik. J.Assoc.Off.Agr.
CheiH., G, 429-434.

Brittox, S.W., Effects of Lowering the Temperature of
Homoiotlierniic Animals. Quart J .E x p.Fh ys'i ol ., /•>, 55-68.

B-AHM, P.G., Biologisclie und pliysiologisclie Beitrage
zur Kenntnis der Moosfauna. Zfsclir. (ill//. Physiol., 2(1,
1-34.

Scott, AV., What is the Relation Between the Moisture
Content and Vial)ility of Seed Corn When Subjected to
Low Temperatures ? loiva Acad. Sci. Proc, 30, 254-257.

Tait, J. and S.W. Britton, Artificial Cooling of a Hi-
bernating Animal. Q uar f. , J. E x p. PhijsioL, 226-227 { Suppl. ) .

Wright, R.C. axd G.F. Taylor, The Freezing Tempera-
ture of Some Fruits, Vegetables, and Cut Flowers. U.S.
Depf. Afjr. Bull, llS-i, 1-8.

1924

Bottazzi, F. and G. Bkrga:mt, Azione delle basse tem-
perature sui systemi colloidali liquidi. Arch. S dense
Biol., I!, 74-93.

Brittox, S.W., The Effects of Extreme Temperatures
on Fishes. Am.J .Plnjsiol., 67, 411-421.

DiEHL, H.C. AXD R.C. Wright, Freezing Injury of Ap-
ples. J. A[/r. Pes., 29, 99-127.

Efimoff, A.W., rl)er Ausfrieren und Uberkaltung der
Protozoen. ArcJi. f. Protisfpvk.. '/H. 433-446.

1925
Begqiterel, p., La suspension de la vie des graines dans
le vide h la temperature de 1 'helium liquide. C.r. Acad. Sci.,
1S1, 805-807.

318

(\vLi.()\v. Ivll., ^riic \'<'I()('ily (.r l(v (^ryslnllisalioii
throiiiiii Sii]>('i'('t)ii!('(l (Iclatliic (ids. I'loc. Iioi/- S(><\,

KfSA, ;]u7-:52:;.

Carter, W., '^riic l^ilTccI of I^ow 'rciiipcraturcs on
/InicliHs iihli'clns Say, an Insect AlTcdini;' Seed. ,/. Afir.
Ris., .;/, lGr)-lSL>.

CoBLKNTz.AV.W., Frost Flo\v(M-s. 'V\w K'csull of Exuda-
tion of Ice b'l'om the Stems of IMants. Am. Forests, .11,
()Sl>-GS4.

AfoRAX, '1\, The Klfoct of Low Teni])eratni'e on Hen's
F^^'^^. Pror. Hofi. Hoc, !)SB, 4;'.G-4r)(5.

RuBENTSCiiiK. L., ijber die I jeheiisfahii^keit der Vvo-
])aklerieii bei eiuei- Teniperatnr iinter 0 C. Chi. Ihikf.,
(}',, 1G6-174.

Sanderson, E.S., Effect of Freezing and Thawing on
tlie Bactei'iopliag'e. Science, ()2, 377.

192G

Gertz, ()., liber die Kalteresistenz der Algenoxydasen.
Bof. Not., 263-268.

Ha^rdy, W., a Microscopic Study of the Freezing of Gel.
Proc.Roy.Sor., 112 A, 47-61.

MoRAN, T., The Freezing of (Jehitine Gel. Proc. B(yy.
Soc.,112A,?>0-AQ.

Payne, N.M., The Fft'eet of Environmental Tempera-
tures upon Insect Freezing Points. Ecolorjij, 7, 99-106.

, Freezing and Survival of Insects at Low

Temperatures. Quart. Rev. Biol., I, 270-282.

Prucha, M.J. AND J.M. Brannon, Viability of Bacte-
rium tijpliosuui in Ice Cream. J. Bact., J I, 27-29.

Stockman, S. and F.C. Minett, Researches on the Vi-
rus of Foot-and-Mouth Disease. J.Comp.PatJt. and
Therap., .>!) (1), 1-30; (Some observations on resistance
to low temperatures).

Zacmakow A. T.M., rel)ei- den l^]in(luss niedriger Tem-
pei'atni'en auf die rilaiizeii. .1 ah ili.w'iss.Rol ., (>.'>, 61-87.

319

1927

KuvsvKowsKv. K.X. AND G.X. l*A\\ij)\\, Beitmgc ziir
Biologie der Sperniatozoen. Ztschr. Tierzucht. u. Zi'ich-
fuHf/sbioL, 10, 257-283; (Some observations on the action
of low temperature).

Pawlow, (t.X., Effect of Low Temperatures on the Sper-
matozoa. Russ. J. Pln/sioL, Id, 291-300; (Russian with
German summary).

Payne, N.i\[., Measures of Insect Cold Hardiness. Blul.
Bull, r,:>, 4-1:9-457.

, Freezing and Survival of Insects at Low

Temperatures. J. Morpli., //.?, 521-546.

KivERs, T.:\[., Effect of Eepeated Freezing (-185 = C)
and Thawing on Colon Bacilli, Virus III, Vaccine Virus,
Bacteriophage, Complement and Trypsin. J. Exp. Med.,
',o, 11-21.

Wright, K.C, Some Effects of Freezing on Onions.
U.S. Drpt. Agr. Clrc. No. J,lo, 1-8.

Weight, R.C. and H.C. Diehl, Freezing Injury to Po-
tatoes. U. S. Dept. Agr. Teclin. Bull., 27, 1-23.

1928

CiTOvicz, B., La transformation des Streptocoques he-
molytiques en Streptocoques du group viridaus par re-
froidissement. C. r. Sue. Biol., 99, 1699-1700.

Payne, N.M., Cold Hardiness in the Japanese Beetle,
PopiUia Japonica Newman. Biol. Bull., JJ, 163-179.

Tanner, F.W. and B.W. Williamson, The Elfect of
Freezing on Yeasts. Proc. Soc. Exp. Biol, and Med., 2'),
377-381.

1929

Becquerel, p.. La vie latente des grains de pollen dans
le vide a 271 C. au-dessous de zero. C.r.Aead.Sei., ISS,
1308-1310.

Maximow, N.A., Internal Factors of Frost and Drought
Resistance in Plants. Profoplasina, 7, 259-291.

:]20

MnitAN. 'I'.. Crilical 'rciiipcint me of l^'i-cc/iiiii,-. rroc.
Roll. Soc. lihin, 177- 1 !>.").

Xii.ssox-Lhis.sxkk, G., Dc'jiIIi fiom l.ow Toini)oralnvo
and Resistance of Plants to Cold. Quart. Rev. Biol., '/,
113-117.

Smith. \\A\, Tlic I^'onnal ioii of Lactic Acid in Mus-
flc ill the Krozcii State. Prnc. Ifoi/. Soc. KloB. 198-207.

Wkiomann. K'.. I'lter riiterseliiede in der Kiiltel)e-
stjiiidi.ukeit \<>ii I'^roseiien, ]^]ideelist'ii uiid Alli,i>atoi"eii.
I'crli. i)lii/sil,-.~iii(il. (Ics. ]]' iiizhiiifi, ■')'/, 88-97.

, Die Wirkuiig starker Ahkiililnn.u- auf

Anipliibien und Keptilitai. Z. ZooL, Ul'i, 64:1-092.

1930

AcuaiADi, M., Sulle teniperatui'e di eoiift'elanieiito delle
pere e delle mele. Ann. Chim. AppL, 20, 188-196.

Becquerel, p., La vie latente des spores de Foiigere
dans le vide anx l)asses tenqjeratures de I'helinm li(|uide.
C.r.Anul.Sci., HID, 1134-1136.

BoLDYKEWA, N.W., Tlic Hibernation of Water Organ-
isms in Ice. Hii<h-ol)iol. Z., i), 45-84; (Russian with Ger-
man sunnnary).

BrssE, W.P., Eftect of Low Temperatures on Germina-
tioii of Impermeable Seeds. Bot. Gaz., 89, 169-179.

BUSSE, W.F. AND C.Ii. BURNHAM, SoUlC Effccts of LoW

Temperatures on Seeds. Bot. Gas., 00, 399-411.

Cameron, A.T., Temperature and Life and Death.
Trans.Boij.Soc. Canada, Sect. 5, 2//, 53-93.

Caerick, D.B., Some Cold-storage and Freezing Stud-
ies on the Fruit of the Vinifera Grape. Cornell Univ. Agr.
Exp. Sid. Mem.. I.U, }-'M.

Fischer, IML, Influence des basses temperatures sur
la vie de rildix po mafia L. C.r. Acad. Sci., lOo, 369-371.

Kruyt, H.P. axi) K.C. Winkler, iTber den Einfluss
liydralierter Koljoidc auf die (lefi-ierpuiiklseniiedrigung.
Z. aiinrf/. all (I. Clwin., /NN, 200-204.

MoitAX, T., The Frozen St<de in Mainmalian Muscle.
Proc.Boy.Soc, 101 H, 182-187.

321

Pavxk, X.^I., Some Kl'tVcts of Low Tcinpcratiirc on Tii-
teriial Structure and Function in Animals. Ecolof/i/, II,
500-504.

Sacharov, X., Studies in C^)ld-Hesistanee in Insects.
Ecoh >(/!/, II, r)()5-517.

Stiles, AV., < )n the Cold Deatli of Plants. Protophisma,
0, 459-468.

Weigmann, K., Weitere Untersucliungeu liber die
Kaltebestandi.nkeit poikilotliermer AVirbeltiere. Z. ZooJ.,
ISO, 195-209.

Weimee, J.L., Alfalfa l\oot Injuries Kesulting- from
Freezing. J. Agr. Res., .'/O, 121-143.

1931

Fernald, E.L, Freezing Point Depressions of Aspara-
gus Shoots Determined by a Thermo-electric Method.
Contr. Boyce Thompson Inst. Plant Res., 3, 483-498.

Jennison, H.M., Further Anent ''Frost Flowers". An
Explanation of Frost Crystals in Dried Plant Stems.
Toneija, 31, 111-112.

Kadisch, E., Beitrage zur Wirkung der Kalte auf patho-
gene Fadenpilze, Hefen und Bakterien. Ausdehnung die-
ser Versuche bis in die Nahe des absoluten XuUpunktes
(bis -272°C). Med. Klin., 1074-1078, and 1109-11P2.

Karcher, H., tJber die Kalteresistenz einiger Pilze und
Algen. PI ant a, 1',, 515-516.

Krebs, E., The Breakdown and Kesynthesis of Phos-
phagen in Frozen and Thawed Muscle. Proc. Roy. Soc,
lOSB, 535-552.

Lenoir, M., Action du gel sur les noyaux des cellules
de Povaire et du nucelle chez FritiUaria imperialis L.
C.r.Soc.Biol., 107, 1151-1153.

Martin, J.H., J.A. Harris and I.D. Jones, Freezing-
Point Depression and Specific Conductivity of Sorghum
Tissue Fluids. J. Ayr. Res., ',2, 57-69.

Newton, R. and W.R. Brown, Frost Precipitation of
Proteins of Plant Juice. CUniad. J. Res., 5, 87-110.

SiMoNiN, ('., lu'clicrclics sui- l;i ^ui'xic dcs lissiis ex-
poses mix hnsscs 1ciii|)t''i-;il urcs. C.r.Soc. Iliol.. Kil, lO'J!)-
1 iXV2.

'r.\NNi:i:, F.W. ANiili.l. Wai.i.ack, I'^lTcct of I'^rcc/iiii;' oil
M ic'i'o-or.n'aiiisnis in \';irious Mciistra. Pioc Sdc.E.iji.
liinl. (UUI Mr, I., >i), 32-34.

ToKKKV. I\.X., Fantastic Frosi (^i-ystals on Dried Sleins
of Diltaiiy. Tonci/n, .11, 10 TJ.

I'vAiiov. B.I'., Iiisecls and (Miniale. Trans. l\iii diiioI .
Soc, Loudon, !'.>, 1-247; ( Action of low leinperature :
])]). 7-l()).

1932

ArcusriNK, D.L., Fffects ol" (^)uick j^'ree/iiii;' upon lii-
t'ecli\-e Lai'\-ae of 'i iichhiclhi spiralis. J. Paras., li), 175.

Bkcquerel. p., La vie lateiite dcs spores des Alonsses
aux basses temperatures. CM. Acad Sci., l^'i, 1378-1380.

, L'anliydrobiose des tiibercules des

l^eiioiicules dans I'azote li(iuide. (\r. Acad.Sci., !!>'/, 1974-
li»7(;.

, La reviviscence des plaiitules des-

sechees soumises aux actions du vide et des tres basses
temperaiures. (\r. Acad. Sci., I!)'/, 2158-2159.

Chambers, K. and H.P. Hale, The Formation of Tee
ill Pi-otoplasm. Proc. Roy. Soc, IKlB, 336-352.

b'lxx, ]).B., Denaturation of Proteins in Muscle Juice
by Freezino-. Proc. Roy. Soc, UIB, 396-411.

Hampil, B., The Influence of Temperature on the l/ife
Processes and Death of Bacteria. Quart. Rev. JJioL, 7,
172-196.

ScHAFEXiT, E. AX!) M. Li'dtkk, Beilriii>e znr Keiinlnis
\-on Kaltewirkun^'eii anf die ])llaiizliclie Zelle. Pliylo-
IHifh. Z., .',., 329-3,S2; also .'>, 505-565, 193:5.

ZiHi'OLO. G., Stiidi sulla bioluminesceii/a batterica. A-
zioiie dell 'idroi^cno e dell 'elio li<|iiido. Holl . Soc. A al . A a-
/>oli, '/'/, 229-235.

323

1933

Hauvey, R.B., Pliysioloi^y of llic Ada] )1 at ion of Plants
to Low Temperature. Proc. iro//r/'.v (Irain Exldb. and
Conf., Canada, 2, 145-151.

Horowitz-Wlassowa, L.]\r. axi» L.U. Grixberg, Ziir
Frage liber ^jsychrophile Mikroben. Chi. Bakt. 89, 54-62 ;
(Growth and developmcMit of bacteria at sub-zero tem-
peratures).

ZiRPOLO, G., Eicerclie eriol)iologiclie sui batteri lumi-
nosi del Cefalopodi. Arcli. Zool. IfaL, /N, 359-405.

1934

Alexopoulos, C.J. and J. Drummoni), Resistance of
Fungous Spores to Low Temperatures. Trans. Illinois
Acad. Sci, .?(), 63.

BoRODix, X.A., The Anabiosis or Phenomenon of Re-
suscitation of Fishes after Being Frozen. Zool. Jalirh.
Abt. allg. Zool., 53, 313-342.

Deschiexs, R., Influence du froid sur les formes ve-
getatives d'amibe dysenterique. Cr.Soc.BioL, 11'), 793-
795.

Iljix, W.S., Uber den Kaltetod der Pflanzen und seine
Ursachen. Protoplasm a, 20, 105-124.

Kalabuchov, N., "Anabiosis" in Vertebrates and In-
sects at a Temperature Below Zero. C.r.Acad.Sci,
U.R.S.S., 1, 424-426.

LiPMAX, C.B. and G.N. Lewis, Tolerance of Liquid-
Air Temperatures bv Seeds of Higher Plants for Sixty
Days. Plant Physioi, 9, 392-394.

NoRD, F.F., Die Kryolyse und ilire Beziehung zur Zell-
physiologie. Protoplasma, 21, 116-128.

Weixzirl, J. AXD R.S. Weiser, Desensitization of Tu-
berculous Guinea Pigs by ]\Ieans of Natural Tuberculin
Prepared from Fractured Bacilli. Tubercle, Jo, 210-216;
(Effect of alternate freezing and thawing).

1935
Belehradek, J., Temperature and Living Matter. Pro-
toplasma Monograph, Borntraeger, Berlin; (Chapters
VIII and IX, pp. 117-158, treat of low temperature).

:V24

(li;i:.\ I iior.sK. (i.A., l' iiri'cc/aMc ;iii(l i''icf/.;il)lc W'.'ilcr
l''i|iiili!)iiuiii ill l*l;iiil ^rissiU's ;is I iilliiciiccd hy Sub-zero
'r('iiiii.T;iliii-cs. /'I, ml PlnisioL, Id, TSl^SS.

IIai.k. II.IV. The Krcc/iu.u' IN)iiil of ^'()lk and While of

K.iii;-. Pin,'. llnl/.Snc, / /.V;. 47:1-479.

II.\i;\i:v. IMk, An Aiiuotatcd Bi])rK)L>i-ni)liy of llie Low
'i'einperatui-e lu'lat ions of I*l;ni1s. l-'2'2.'5. Bui'i^css, Miiine-
ajtolis ; ( Miiiu'().iirai)li('(! ).

l\.\i,Ai;r(ii()W. X..I., Anabiose bei Wii'bell ieren iind In-
sekten bei 4'enipei-atui-en iiuler 0 . Znol. Jalirh., Ahl.
(iU<l. ZooL, .>•>, 47-04.

Kessler, W., Ubor die iiiiieren I'rsachen der Kjillcrt'-
sistenz del' !*llan/eii. Phn/hi, .J'/, '.\]'2''.]^y2.

LoziNA-LoziNSKiJ, L.K., Die Aiiabiosc bei don Raiiiien
dev Pi/i-aiisfa jnihilalis Hiibii. iiaeh (Jcfrieriin.i;-. C.r. Acad.

Smart, H.F., Gi'owth and Snrvi\al of M iero-Organisnis
at Sub-Freezing- Temperatures. Science, 8.^, 525.

A\'.\T.TER, 11., AND O. AVeismann, Uber die Gefrierinmkte
und osniotiselien Werte lebender nnd toter pflanzlieher
(Jewebe. Jdlirh. iriss. Bof., S2, 27:U\]().

Wartenberg, H., tJber ^'Frostblasen" an Blattern des
Apfelbaumes. Phj/ofpafh. Ztsclir.. N, 515-522.

AVoLFsox, C, ()l)servations on I'araniecium during Ex-
posure to Sub-Zero Temperatures. Ecology, IG, 630-639.

1936

Becquerel, p., T^a vie latente de (luelijues Algues et Ani-
maux inferieurs anx basses teniperalures et la conser-
vation de la vie dans I'univers. ('. r. Acad. Sci, 202, 978-
981.

Edlich, Y., Finwirkung von Teni])eratur und Wasser
auf aerophile Algeii. Arcli. Mikrohiol., 7, 62-109; (Action
of low temperatui-e : ])p. 92-96).

Gi('I\I.ii()i;n, .] ., (Jradienteii des I'ji'fi-iereiis \'on Lanb-
l)liitleni. rrnln/,l(ts)ii(i, 2(1. 90-9(i.

325

Kaitkukv. p., Kxpci-iiiicuts in llie Kevhalizatioii of (Jr-
^aiiisnis from llic Pcriiiaiu'iitly Frozen Subsoil. C.r.Acad.
ScL, U.R.S.S., A, 137-140.

LiPMAx, C.B., Normal Viability of Seeds and Bacterial
Spores After Exposure to Temperatures near the Abso-
lute Zero. Plant Physiol., It, 201-205.

, The Tolerance of Liquid Air Tempera-
tures by Dry Moss Protonema. Butt. Torrei/ Bot. Ctuh,
63, 515-518. '

LuYET, B.J. AND S.M. Grell, A study with the Ultra-
centrifuge of the Mechanism of Death in Frozen Cells.
Biodyu., 1, No. 23, 1-16.

Schmidt, P.J., G.P. Platoxov and S.A. Person, On the
Anabiosis of Fishes in Supercooled Water. C.r.Acad.Sci.,
r.R.S.S., 3, 305-308.

Taylor, C.V. and A.G.R. Strickland, Effects of High
Vacua and Extreme Temperatures on Cysts of Colpoda
cucidlus. Physiol. Zool., 9, 15-26.

Weigmann, R., Zur Kaltebestandigkeit poikilothermer
Tiere. Untersuchungen an Schnecken und Fischen. Biot.
Zbl., 58, 301-322.

1937

Becquerel, p., La inort par le gel de la cellule vegetale
dans Fazote liquide a -190 \ C.r.Acad.Sci., 20',, 1267-1269.

FiRsovA, M.K., Die Wirkung von tiefen Temperaturen
und von Ausfrieren auf die Keimfahigkeit der Sameii des
Schlafmohnes {Papaver somiiiferum L.). Trudy prili.
Bot., No. 2, 121-124; (Russian with English sunmiary).

Galvialo, M.L, The Influence of High and Low Tem-
peratures on the Activity of Catalase and Diastase. Rus-
sian J. Physiol, 22, 224-227.

Griffiths, H. J., Some Observations on the Overwinter-
ing of Certain Helminth Parasites of Sheep in Canada.
Canad. J. Res., tlD, 156-162.

Jahnel, F., Uber das Uberleben von Syphilis- und Re-
currensspirochaten sowie Sodokuspirillen in fliissigem

:V2(\

StickstolT ( 'rciii|)ci-;ilur P.Mi ) mid die I'Viiiwii-kuiii;- ;iii-
(Ici-cr K;ill(',ui':i<l(' auf iWcsr M iki-(.()i'i;;iiiisiii('ii. h'Hii.

1 1 'or//.. /'/, i;!()4-i;u):).

l.Ki'Kscii KIN, W.W., Zoll-Xckrohiosf unci I'roloplnsiiia-
l\u\. I'i()l()]»l;isiii;i .M()iio»>ra])li, Boriitrae^er, Px'iliii;
( Chapter !>, pp. lOd-HH), deals with death l)y h»\v tempera-
tures).

Lii'MA.N, C.B., Tolerance oi' i/niuid Air Temperatures
by Spore-free and Very Youn^- Cultures of Kun.^i and
Bacteria Growin.i;' on A,i>,ar Media. II hU. Tone// Bot.
Ch(h, iy'i, 537-546.

LuYKT. B.J., The Vitrification of Ori-anic Colloids and
of Protoplasm. Biodi/n., 1, No, 29, 1-1 4.

LuYET, B.J. AND P.M. Gehenio, The Double Freezin,<>,'
Point of Living Tissues. Biodyn., 1, No. 30, 1-23.

LuYET, B.J. AND M.C. GiBBs, On the Mechanism of Con-
gelation and of Death in the Eapid Freezing of Epider-
mal Plant Cells. Biodyn., 1, No. 25, 1-18.

^luRiGix, I.I., A Contribution to the Problem of Survi-
\al of Hibernating Mammals at Temperatures below
Zero. Bull Biol. Med. Exp., U.R.S.S., ',, 100-102.

1938

Becquerel, p., La congelation cellulaire et la synerese.
C.r.Acad.ScL, 206, 1587-1590.

BuGAEVsKY, M.F., Dynamics of Vegetable Cell Decay
Due to Low Temperature. C.r.Acad.Sci., U.R.S.S., 22,
131-134.

GoETz, A. AND S. ScoTT GoETz, Das Verglasen einzel-
liger Organismen. Naturwiss., 26, 427-429.

,Death by Devitrification

in Yeast Cells. Biodyn., 2, No. 43, 1-8.

— , Vitrification and Crys-

tallization of Organic Cells at Low Temperatures. J.
Aj>i>L Phys., .'/, 71S-729.

, Vitrification and Crys-
tallization of Protophyta at Low Temperatures. Proc.
Aw.Philos.Soc, 79, 361-388.

327

JjTn'KT, B., Siir la siir\i(' dcs poissoiis plunges dans I'air
li(|ni(le. C.r.Soc. Iliol., I.>1\ 788-790.

LuYET, B.J. Axi) ir.AF. Condon, Temperature Relation-
ships and Ice-Wfder Proportions during Death by Freez-
ing in Phuit Tissues. Biodi/u., 2, Xo. 37, 1-8.

LuYET, B.J. ANn P.j\[. Gehenio, The 8ui-vival of ^loss
Vitrified in Liquid Air and Its Kehition to Water Content.
Biodyn., 2, No. 42, 1-7.

LuYET, B.J., AND E.L. HoDApp, On the Effect of Mechan-
ical Shocks on the Congelation of Subcooled Plant Tis-
sues. Prof o plasma , -V), 25-1-257.

■ , Sur les noyaux cristal-

lins dans un tissu vegetal, prealablement chauffe ou gele.
C.r. SocBioL, 121, 786-787.

, Revival of Frog's

Spermatozoa Vitrified in Liquid Air. Proc. Soc. Exp.
Biol, and Med., S9, 433-434.

LuYET, B.J. AND G. Thoennes, Demonstration des pro-
prietes isotropiques de masses cellulaires vitrifiees a la
temperature de Pair liquide. C.r. Acad. Sci., 206, 2002.

, The Survival of Plant

Cells Lnmersed in Liquid Air. Science, <SN, 284-285.

, La reviviscence de

fibres musculaires vitrifiees dans Pair liquide. C.r. Acad.
Sci., 207, 1256.

Ramsden, W., Coagulation by Shearing and by Freez-
ing. Nature, l',2, 1120-1121.

Stuckey, I.H. AND O.F. CuETis, Icc Fomiatious and the
Death of Plant Cells by Freezing. Plant Physiol, 13, 815-
833.

Turner, T.B., The Preservation of Virulent Treponema
pallid mn and Treponema pertenue in the Frozen State;
with a Xote on the Preservation of Filtrable Viruses. J.
Exp. Med., 67, 61-78.

ZiMPFER, P., The Eifect of Certain Variables upon the
Freezing Point Depression of Plant Tissues. Ahstr. of
Doct. Diss., Ohio State Univ., No. 26, 67-73,

:V2S

I^KcgrKitKi.. 1'., lk"'l(' tic la snik'ivsc dans Ic iiu'caiiisiut'
(If la con^uvlatioii cH-lliilaiic. ('Inoiiicd liot<inic(i, •'>, 10-11.

(iKiiKXio. 1*..M., A.\i» \^.^. Lrvi:r, A Study of the .Meclian-
isiii of Death l)y Cold in llie Plasiinxliuni of the Myxomy-
cetes. I>lii/l//ii.,.,\ Xo. .")."), \-'2'2.

KvKs. 1*. AND '1\S. I'oTiKK, Tlic Hosistaiico of Avian
Tubercle! Bacilli lo Low Temperatures with FiS])ecial Ref-
erence to ^1 nil i pic Changes in Temperature. ./. Infect.
D'is.,6.',, 123-134.

Levitt, J., The Kelation of Cal)liage Hardiness to Bound
Water, Unfrozen Water, and Cell Contraction When Fro-
zen. PJcud Phiisl,,!.. I',, 93-112.

LiPMAN, C.B., On the Difference in Resistance of Va-
rious Types of Cells to Extremely Low Temjiei-atures.
2?/o(/7//?.,2, No. 45, 1-4.

LuYET, B.J., AVater and the Ultra-Structure of Proto-
plasm. Archiv. exp. Zellforsch., 22, 487-490.

Turner, T.B. and W.L. Fleming, Prolonged Mainte-
nance of Spirochetes and Filtrable Viruses in the Frozen
State. J. Exp. Med., I'O, 629-637.

Turner, T.B,, and N.L. Brayton, Factors Influencing
the Survival of Spirochetes in the Frozen State. J. Exp.
Med., 70, 639-650.

SUBJECT INDEX

Absidi'K 65

Absolute zero. 12; for tempera-
tures approaching the Abs. zero,
see liquid helium

Actinomyces. 3S, 61, 62

Actinophrys, 29, 244

AcUnetn. 72, 73, 246

AethaUum, 62

Agar solutions, 164, 174

Ageratum, 44, 270

Agglutinin, 16

Air. effect on subcooling. 198; see
liquid air

Albumin solutions, 164. 174. 178

Algae. 27, 32, 38, 66, 238, 243, 249,
277, 281

Allantoic fluid. 120

Alteniaria, 64

Amboceptor, 17

Amniotic fluid, 120

Amoeba, death point, 29; effect of
extreme cold, 244; freezing
point. 121; subcooling, 238; vit-
rification, 220

Amphibia. 33, 35, 36, 55 seq.. 82
seq.. 116, 118, 119, 120, 121, 158,
164. 223, 232, 239, 250, 278, 279,
281, 286

Amylase, 15

Anabiotic temperatures, definition,
9

Anaphylaxis, 16

Aiiixia. 62

Annelids, 73

Anodonta. 56

Antibodies, 17

Antirrhinnm. 40, 244

Ants, 76, 78

Apatococcus. 27, 28

Aphids, 76

Apples, 164, 260, 269

Argyroneta. 78

Annillaria. 65, 248

Artemia. 246

Arthropods, 33, 34. 35. 75 seq.. 147,

194. 198, 231. 239, 246, 258
Ascophyta. 64

Aspergillus. 15, 39, 62, 63, 64, 65,

237, 238, 244, 248
Atrichum. 39, 244
Atriplex, 116
Aucuba, 270
Avena, 259

Bacilli, 21, 22, 243; B. authracis.

20, 247; B. coli. 17. 21, 22, 23,
237, 247; B. faeealis alcaligenes,

21, 247; B. lactis aerogenes. 21,
247; B. pestis, 20; B. typhosus.
20, 21, 22. 23, 247; B. of chicken
cholera. 20, 247

Bacteria, 14, 19 seq.. 32, 243, 247,
265. 275; dysentery B.. 23; en-
teritis B., 21, 248; paratyphoid
B., 21, 248; typhoid B., 21

Bacteriophage, 17, 18

Barley, 40

Bat, 93, 239

Bean, 268

Bedbug, 76

Bees, 76. 77. 78, 81, 239

Beet, 50, 122, 268

Beetles, 81, 239

Bile, 116

Birds, 36, 37, 56, 116, 118. 119, 120,
176, 177, 199, 201, 238

Bleaks, 90, 239

Blood cells, 54, 55, 116, 237

Boletus, 65

Bombinator, 119

Bombyx. 78

Bothrydium. 38

Botrytis. 62. 63, 238

Bound water, 157 seq.. 259, 278,
284

Brachythecium. 39, 244

Brown tail moth, 80

Buckwheat, 51

Buds, 52

Bufo. 85

Bullhead. 89

Bumblebee, 81, 239

Butterflies, 76, 194

Bythiiiia, 75

329

c'ahhaKc t:.. :.:;. J:!T. 2tll, liOS, 270

Cactuses, 116

Caldiithr, 26!»

Calla. 262

Callidiiia. 72, 7o, 246

Cambium. '\2

Ca)iii)(initl(i. 16

rai)illarity. 1!m;. l'I2

Carassiiis. 87

Carbon dioxide (solid), 12, ir>, 17,

40, 82. 208
Carpio. 87

Carps, 87, 88, 90, 239
Cat, 55, 92, 93
Catalase, 14, 16
Caterpillars, 76, 78, 79
Crphalotheciuvi. 64
Ceramium. 67
Cerebrospinal fluid. 116
(liara. 66
CJielidonhivi, 132
Chick embryo, 56
Chilling, 230 seq.: sec Cold, Homo-

iotherms

Chilomonas, 38

Chironomus, 147

ChlorcUa, 28, 32, 243, 249

Chlorococciim, 28, 243

Chlorophyll granules, 28, 181

Choctost Ilium. 62

Cholera vibriones, 19; see also
Bacilli

Cliondrus. 67

Chromosomes, 147, 180

Chydorus, 81

Ciliates, 30, 31, 219. 220, 234, 237,

238, 244
Cineraria, 270
Citellus, 94, 239
riadouia. 68, 245
Cladopliora, 66, 67, 145
Cladosporium, 61, 62
Clitocybe, 65, 248
Clivia, 145
Closterium, 27
Vlostridinm. 16, 19
Coagulation by freezing, 180, 280
Cocci, 21
Cod, 88

('(idiiiiii. 66

Coelenterates, 71

Cold, above the freezing point, 70,
91 se(i.: 2:!n-236, 281; below the
freezing point, in the absence of
ice, 26, 224, 236-241, 242 seq.

Coleoptera, 77, 78

Colloids, 133, IC.i'-ltil, L'Ki

Colloidal ice, 114

ColhjUa, 64, 65, 248

Colpidiiim. 30, 237, 238

Colpoda. 31, 220, 237, 244

Complement, 16, 17

Congelation, definition, 13

Conifers, 52

Coniophora, 63

Coniothyrium, 249

Contact conductivity, 106, 208

Cooling, body (problem of), 108;
curves, 148; law of, 108; rate, 12,
108 seq.. 123, 138, 150, 157, 160,
187, 200, 204, 207, 208, 234, 235,
242

Coprinus, 65

Corn, 51

Cortex, 52

Cossus, 79

Cow-pox vaccine, 18

Crustacea, 77

Crystalline nuclei, 112, 113, 128

Crystallization, 101, 102, 112 seq.,
184 seq.. 203 seq.: temperatures,
206 seq.. 251; velocity, 90, 129
seq.. 134 seq.. 207, 209, 210; see
Devitrification, Freezing

Ciilex, 78

Cyclamen, 46

Cyclops. 11, 81

Cylindrosporium. 64

Cyprinus, 87

Cytnspora. 39, 249

Dahlia, 46
Dallia. 86
Daphnia. 11
Daucus, 50, 123

Death, cellular. 229; organismal,
229, 232; protoplasmic, 22!t, 2:!2;
systemic, 229; teniperatur(>s, in,
11, 19 seq.; theories, 229 seq.

Degree K, see Absolute Zero

O.J L

Dehydration and death by cold,

233, 251, 257, 272. 277
Delesseria, 14, 67
Dematium, 38
Derbesia, 66, 145
Desiccation, see Dehydration
Desmids, 27
Devitrification, 102, 2U3, 211, 213

seq.
Diastase, 16
Diatoms, 27, 249
DicraneUa, 39, 244
Diilymium, 62
Dihydrol, 114
Diphi/llobothrin))). 71
Diptera, 78
Dog, 91

DoryJaimus. 72, 246
Double freezing point, 124 seq.
Dunaliella, 38
Dysentery bacilli, 17

Earthworm, 73

Eberth bacilli, 17

Echiniscus. 82, 246

Eel, 55, 87, 88

Eelpont, 88

Egg yolk, 175, 193

Eggs, cold resistance, 231; death
point, 34; freezing point, 118
seq.; subcooling, 201, 238

Embryonic tissue, 55

Encystment due to cold, 234

Enteromorpha, 67

Enzymes, 14-16, 18

Enzymoids, 16, 17

Epidermophytes, 39

Episcia. 44, 46, 49, 239

Epithelium, 55, 56, 57

Euglena. 28, 31, 219, 238, 244

Euphorbia. 132. 172

Eurotium. 39, 249

Eutectic point, 124, 127, 158 seq.,

280

Evergreens, 52

Fennel, 41
Fern, 40, 244
Ficus. 132, 172
Fishes, 86

Flagellates, 31, 32, 38, 219, 235,

238, 244, 249
Flies, 76, 77, 78
Florideae. 270
Flounder, 88

Freezing, 112 seq.: curves, 147
seq.; definition, 13; point, 10,
11, 114 seq.: 230 seq.; (frozen)
state, 166 seq.; see Crystalliza-
tion, Ice

FritiUaria. 181

Frog, 35, 36, 55, 56, 57, 58, 59, 82,
83, 84, 85, 120, 121, 158, 220, 223,

239, 279
Fucus, 67
Funaria, 39, 244

Fungi, 15, 16, 17, 19 seq.. 38, 39,
61 seq., 121, 123, 145, 221, 235,
237, 238, 239, 242, 243, 244, 247,
248, 266

Fusarium, 64, 65

Gasocrystallization, 102

Gasovitrification, 102

Gastric juice, 116

Gelatin, 113, seq.. 155, 156, 164. 174,
175, 209, 283, 289

Geranium, 267

Germ cells, 32, 249

Glaeotila. 67, 243

Glass, properties, 203 seq. ; see Vit-
reous state

GlomereUa. 64

Gold fish, 87. 88, 90, 264

Gonococci, 20, 247

Grasshopper, 76

Ground-squirrel, 94, 239

Guinea-pig, 93

Haematococeus, 37, 38

Hair cells, 43, 277

Hantzschia. 28, 243

Heart, 55, 59

Heat, conduction, 104 seq. : of crys-
tallization, 150, 188, 209; speci-
fic, 106, 109, 150

Heavy water, 131

Hedgehog, 93

Helianthus, 54, 246

Helix, 74, 75

Helminthes, 34, 71. 72, 242, 246

Hemiptera, 77

*^•^>

Hemocyanin, 17r>

Hemolysin, !(>

HimuphilUN infliiciizar, 2?>

Herpes virus, IS

ilonioiotlienns. 1.'), 17, '.V.^. r)5, 91

scq.. ih;. It! I. 17.''. i*:'.i. 2:".s. 2;!!),

257
Hookwoiin. 71
normidiinn. (u, 70, 243
Honnodi'iKhon. G2
Hormones, Ki
ni/arinfhiis, 4t;. 11.".. 284
Ih/drachiia. 7S
Hi/pholovia. 65, 24S
Hypniini. :'.!>. <!fl, 241, 24.'.

Ice. tolloidal. 114; properties, 166,
srq.: quantity present in mate-
rial being frozen, 151, 161 seq..
281 seq.: sec Cold, Crystalliza-
tion, Devitrification. Freezing,
Water

I)ui)(ilii)is. ni5, 241, 277

Intracellulars. K!

Inoculation (Ice), 112. 121, 190
seq.

Insects, 33, 34. 35, 76 seq.. 147, 198,
231, 239

Internal medium, 117 scq.

Intestine, 55

Intestinal juice, 116

Inulase, 15

Isopentane, 208, 218

Japanese beetle, SO

Laminaria, 67

Lamium, 286

Larix. 70

Laurencin. 67

Leaves, 49, 50, 52, 121, 145, 179,

191, 222, 235, 250, 264, 270
Leech, 73
Lepiota, 65
Leucobryum, 39, 244
Leucocytes, 238
Leucoium. 145
Lichens, 68, 245
Limnaea. 74, 75
Lipase, 14

Liciuid air. 12. 1.". scq.. 207 seq.,
217 .S'7/.. 241 scq.. 2i\\

Liquid IhIjiiiii. 12, 22, 26, 28, 29,
:;2, 3;i. m. 12, 43. 54, 67, 72, 73,
S2, 208, 243 sc(i.

Litpiid hydrogen, 12, 21. 2*;. 35. :',9,
II, 72, 73, 82, 207, 2lti .s<f/.

Liver. 55

LoheUa. 41, 259

Lucern, 54, 246

Lung, 55

fjiij)iinis, 145

Mdcacus. 92

Macrobiotus. 82, 246, 249

Marauta. 46

Mechanical in.jiiry by freezing, 257,
261

Mcl(nic()iiiu))i . ;19. 349

Melting. 101, 102, 171; sec Thaw-
ing

Membranes, tearing of, 180, 261

Meruleus, 63

Metabolic Water, definition, 284

Micrococcus, 22

Microsterias, 27

Milk, freezing point, 116

Mihiesiitm. 82, 246

MisguDius. 87

Mite, 78

Mneviiopsis, 71

Mnium, 222, 245

Momordica, 43, 239

Monas, 235

Moniezia, 72

MoniJia. 38

:\Ionkey, 92

MortierclJa. 65

Mosquitoes, 76, 78

Moss, 38, 54, 68, 222, 244, 245, 250,
275, 278, 279, 286

Mouse, 55, 94

Mouth cancer virus, 18

Mucor. 38, 39, 61, 62, 64. 65, 244

Mud-minnow, 88

Mummichog, 88

Mils, 94

Muscle, death point, 57; euteetic
point, 161; freezing curve, 158,
278; freezing point. 123; mech-
anism of death by cold, 281;
physiol. changes by freezing,
286; quantity of water frozen,
164, subcooling, 239; vitrifica-
tion, 223, 250

333

Rlyogen, 164
Myosin, 288
Myotis, 93

Myxamoebae, 220, 250
Myxomyoetes, 62, 121, 123, 243

KarcissKS. 14.")
Nemalion. 67

Nematodes, 71, 72, 242. 246
Nematodinis. 72
Nerve, 55, 59
Neuropteron, 77
Nicotiana. 40, 46, 47, 244
Nitella, 66, 238
Xyctahis. 93, 239

Oak borer, 80
Oats, 40, 41
Octopus. 175
Oiflium. 38

Onion, 45, 46, 50, 147, 221, 233, 250,

268, 284

Orange tree, 267
Orchids, 179, 269
OsciUatoria, 67, 243
Ostertagia, 72
Ox, 55
Oxidase, 14, 15, 16

Pahnella. 28, 243

Pancreatic juice, 116

Papaver, 132

Paramaecium. 30, 31, 219 237
244

Parmelia, 68, 245
Pea, 51
Pears, 269
Pediastruvi, 67, 243
Pelargonium, 44, 47, 239
Penicillium. 38, 61, 62. 63 65
238

Pennaria, 71

Pepsin, 15, 16

Perch, 88

Peroxidase, 14

Phajus. 48, 269

Phase separation in freezing,
seq.

Phaseolus. 121, 145

Photobacterium, 21

238,

237,

132

Phijronnjvcs. 63, 65, 238, 239, 248

Physaruvi. 121, 123, 235, 243

Physcia, 68

Physical state, see State

Pliytophtliora, 64

Pinnularia, 28, 243

Piper, 46

Placodes. 65. 248

Plauorhis. 74

Plectus. 72, 246

Pleurococcus, 27, 28, 243

Ploivrightia, 64

Pollaclv, 88

Pollen. 40, 244

Polypodium. 263

Polystichum, 40, 244

Porphyra. 67

Potato, 14, 47, 48, 50, 121. 122,
123, 124. 125, 171, 191, 260, 262,
270. 277, 279. 283, 286
Precipitin, 16
Prirmila, 46

Problem of the Wall, 104, 107
Proteases, 14
Prothrombin. 15
Protococcus. 28
Protonemata, 54

Protophyta, 19 seq.. 38, 235, 237,
242, 243, 247, 248. 249, 265, 266
Protozoa, 29 seq., 38, 121, 218, 219
220. 234, 235, 237. 238, 242, 244,
249, 250, 265
Ptomains. 17
Pumpkin, 268
Phyhtiella. 67
Pyrausta. 81
Pythium. 65

Quercus. 52

Rabbits, 91, 92

Rabies virus, 18

Rana. 57, 60. 83, 119, 120

Ranunculaceae, 54, 246

Rat, 55

Recrystallization, sec Vitrification

Reductase, 14

Regelation, 167

Rennet, 15

Reptiles, 17, 33, 55, 86. 116, 117.
239

I{}iiz(>( toiiio, Go

Rhizopods, L'!t. 121. 220. 2nS. 211.

2r>()
Rhizopim. :'.i\ G:., 2-1 ■<
RhodcHS, 87
Hhodoiui/ces, 38
W;i»«, 132

Richmann's Law, 1 H)
Hicinus. 198
Roots, 49, r)4
Rotifers, 72, 73, 246, 249
h'otifrr vulgaris, 73, 246
Rye, r.l. .-.1. 121. L':'.!i. 216

Sactharumyrcs, 23, 26, 27, 248

SdUnnandra, 86, 239

Saline. i:>!t. 278

Saliva, 116

Saroinae. 21

Schizophi/Uuiii. 64. 6.', 248

Sclrrotinia. 64

Scolopendra. 76

Sculpin, 88

Sea raven, 88

Seeding (Ice), see Inoculation

Seeds, 32, 40, 34, 244, 245, 251,

275
^elaginrUa. 246
Serum. 116, 175

Silkworm, 76

Xiijhonevui. 67, 243

Sitophiliis, 81

Skate, 59, 88

SiHi-rintlius, 78

Snails, 74, 75, 201, 240

Sodoku spirilla, 247

Sol-gel transformation, 172

Si)ecific minimum, theory of, 240,
259

Si)ermatic fluid, 116

Spermatozoa, 32, 33, 34, 220, 250

Sj)haero})sis, 64

Spiders, 77, 78

Spirilla, 21

Hpirochaeta, 247, 271

Spirogyra, 66, 145, 233

Kpirostomum, 30, 237, 238

Si)ores, 32, 37, 243, 244, 249, 275

Spi)r(itrir)i mil . <(2

S|)()rulati<>n caused by cooling, 234
Slaithiilorociiis, 17, 20, 21, 22, 243,

L'47
State ( physical ), 101 scq.; frozen,

166 .s-cr/. ; supercooled, 184 seq..

236; vitreous, 203 scq., 252
Steady state, 104, 105
Stems, 49
mentor, 30, 234
^tcrigmatocystis, 39, 244
Htichococriis. 27, 28, 32, 243, 249
Stickle-back. 87, 90, 239. 264
Stomatal cells, 46
Strelitzia, 198
Streiitococci of scarlet fever, 19,

238
Subcooling, 102, 131, 184 srq.. 2ii6.

236 seq. : see Inoculation
Sublimation, 204
Sucrase, 15
Sucrose, 115

Sulphur dioxide, litpiid, 12
Supercooling, see Subcooling
Survival temperatures, definition,

9
Sweat, 116
Syneresis, 235, 236, 289

Tardigrades, 82, 246

Tegenaria, 78

Tenches, 87

Testudo. 86

Thawing, 171 seq.. 257. 260, 266,

267 seq.; see Melting
Thermoaseus, 38, 62
Thermoidium, 38, 62
Thermomyces, 38, 62
Tissues, 121 seq.. 124, 156, 179,

239
Toad, 55, 82, 84. 85, 239
Toncod, 88
Tortoise, 86, 239
Toxins, 18
Tnidesrantia. 43, 44, 145, 233, 239,

241, 270, 277
Trailliella. 67
Trees, 52
Triania, 43, 239
Trihonevia, 67, 243
Trichina. 71, 72
'fricJioilcnna. (!5

335

TricJioslroiigi/Ius, 72
Trihydrol, 11 :!
Tritivum. 259
Tritons, 55

Trypanosomes, ?A. ?,2. 249
Trypsin, 15, 16
Tubercle bacillus. 22, 248
Tuberous tissue, 47
Tiilipa. 145, 268
Turtle, 33, 55, 86, 117
Tussilago. 124
Tylenchus, 72, 246
Typhus bacillus, 19

riothrix, 38
Urease, 16
Urine, 116

Vaccine virus, 18

Variable state, 105. 107

Venom, cobra, 17

Venturia, 64

Viburnum, 270

Virus III, 18

Viruses, 17-18

Vital temperatures, definition, 9

Vital water, definition. 284

Vitamins, 13, 14

Vitreous state, 101 scq.. 203 seq.,
252

Vitrification, 11, 101, 201, 203 seq.,
207 seq.. 217 seq.. 250, 252; tem-
peratures, 204, 211 seq.

Vitrofusion, see Vitromelting

Vitromelting. 102, 203 scq., 216

Vitrosublimation, 102

Wasps, 81, 239

Water, colloidal, 114; (water) con-
tent, 12, 217, 223, 242, 251, 272
seq.: freezing curve. 148; freez-
ing point. 115; freezing velocity,
131; of hydration, 215; sub-
cooled. 131. 195, 196; vitrifica-
tion, 209; see Bound W., De-
hydration. Metabolic W., Vital
W.

Wlieat. 40, 41, 51, 54, 246
Wood, 52
Woodchuck, 93

Xanthoria, 68, 245
Xylaria. 65. 248

Yeasts, 15, 25 seq., 32, 145, "^21,
248. 266

Zymase, 15

Airiioii iNi)i:x

Adams, 41, !Hi. 245, 251. 2r)l, 2;)2.

S08
Assradi. 320
Akernian, 270. 292. 314
Alexopoulos, 39, 9(J. 249, 292, 323
Ambroiin, 303
Andrews, 166, 226
Apelt, 47, 48, -lO, 96, 259, 260. 277,

286, 292, 309
Atkins. 116. lis. 119. 226. 310, ;;il.

312
Aiidoin, 297
Augustine. 72. 96, 322

Bach, 35, 231

Bachmetjew, 34, 75, 78, 79, 96, US,

194, 198. 226, 231, 292, 306, 309
Backman, 115, 118, 120, 226, 311
Bahrmann. 60, 96
Bailey. 200. 227, 315, 317
Bakhmetieff, 34, 76, 96, 200, 226.

306
Barnes, 114, 106. 168. 169. 170, 226,

227
Barrat, IS, 96, 99, 307, 310
Bartetzko, 63, 96, 237, 238,

310
Bartram, 64, 96, 314
Bay, 303

Bazzoni, 16, 97, 313
Becquerel, 28, 29, 31, 32, 39, 40

46, 54, 67, 68, 69, 73, 82, 56,

244, 245, 246, 284, 289, 292,

309. 310, 311, 317, 319, 320,

324. 325, 326, 328
Beek, 21, 96, 315
Behn, 171. 226
Beijerinck, 247. 292
Belehradek, 34, 96, 230. 233.

292. 323
Belli. 20, 96, 247, 292. 307
Bergami, 175. 220, 317
Bernal, 114, 226
Berry, 25, 61, 96
Bialaszewicz, 115, 118, 119,

312
Bickel, 16, 96, 308
Bidault. 62, 238, 292, 316

292,

42,
243,
308,
322,

254,

226,

Bigelow, 197, 226

B()I)ertag, 133, 155, 171. 173, 226,

227, 309, 310
Boldyrewa, 320
Borodin, 86, 88, 96, 323
Bottazzi, 115, 175, 226, 317
Brannan, 20, 99, 318
Brayton, 271, 295, 328
Bredig, 173
Brehme. 19, 96. 306
Britton, 59. 88, 93, 96, lOO. 317
Brooks, 61, 238
Brown, 41, 97, 245, 255, 292, 304,

321
Brownlee. 59, 85, 97, 121. 150, 158.

226. 232. 279, 292. 312. 313
Bruni, 133, 174, 226, 310
Brunow, 57. 97, 312
Buchner, 15
Buck, 147

Buchner, A., 131, 228
Biihler, 59, 60, 97
Buffon. 261, 267, 293, 296
Bugaevsky, 147. 180, 226, 326
Bunsen, 168. 226
Burnham, 320
Burton, 209, 226
Busse, 320

Callendar, 168, 226

Callow, 132, 175, 209, 226, 318

Cameron, 59, 85, 97, 121, 156, 158,

226. 232, 279, 292, 312, 313, 320
Carrick, 320
Carter, 318

Caspary, 141, 142, 226, 298
Castle, 280
Cattell, 266, 292
Chambers, 29, 45, 58, 97, 121, 226.

233, 238, 239, 264, 292, 322
Chandler, 270, 292, 312
Chanoz, 15, 17, 97, 305
Chappuis, 168, 226
Chevrotier, 20, 98, 247, 293, 313
Chodat, 27. 39, 64, 97, 255, 292, 304
Citovicz, 19, 97, 238, 292, 319

336

337

Coblentz, 314. 318
Cohn, 66, 97, 300
Coleman, 91. 97. 302
Colin. 40. 91. 97, 303
Collip, 116, 118, 121, 226, 315
Condon, 48, 162, 227, 283, 327
de Coppet, 189, 193, 195, 226
Courmont, 17, 97, 305
Curtis. 263, 295, 327

Dakin. 115

Dalmer, 304

Dalton. 110

d'Arsonval, 15, 24. 97, 303. 305

Dastre, 276

Davenport, 97, 304

Davis, 22. 98. 237, 293. 314

de Candolle. 40. 41. 97, 255, 292,

301. 302, 304
de Jong. 21, 23. 32. 97. 247. 248,

249, 292. 316
de Mohl, 140, 142, 144
Deschiens, 29, 97, 323
Despretz, 193, 196, 200. 22G
Detmer, 181, 226, 302
deVries, 301
Dewar, 245, 292. 305
Dexter. 289, 292
d'Herelle, 17
Dickinson. 170. 226
Diehl, 317, 319
Dleterici, 169, 226
Dixon. 116, 226, 311, 312
Doemens, 26. 27. 24S. 292
Doenhoff. 35. 73, 77. 97, 300
Doflein. 32, 249. 292
Doyon. 15. 17. 97. 305
Drummond. 39. 96, 249. 292, 323
Duclaux, 301
Dufour, 196, 198, 226
Duhamel, 261. 267, 293, 296
Dulong, 110
Dunal, 140, 226, 298
du Petit-Thoiiars, 142, 228, 297
Duval. 79, 81, 97, 118. 316

Edlich. 27, 97, 324
Edwards, 40, 56, 97
Efimoff, 30, 97, 237, 238, 293, 317

l'^lir(>iiljauni, 294

Escombe, 41, 97, 245, 255, 292, 304

Essex, 34, 71

Estrelcher-Kiersnowska, 313

Fahrenheit, 195, 226

Feist, 133, 173, 226, 309

Fernald, 321

Finn, 322

Firsch, 301

Firsova, 325

Fischer, H.W., 57, 97, 115, 121, 123.
133. 155, 156, 158, 159, 162, 164,
171, 173, 174, 226, 227, 278, 280,
289, 293, 309, 310, 311

Fischer, P.H., 74, 97, 320

Fischer-Sigwart, 36

Fleming, 328

Flosdorf, 22, 97

Flury, 310

Fol, 29, 31, 97, 302

Foote. 162, 227, 314

Forster. 303

Fourier, 104, 108

Fowler, 114, 226

Frazer, 115

Frey-Wyssling, 123

Fiichtbauer, 199, 200, 227

Fulton. 216

Galvialo. 16, 97. 325

Carrey, 115, 117, 227, 308, 313

Gaylord, 18, 31, 55, 97, 249, 293, 309

Gay Lussac, 196, 227

Gehenio, 113, 121, 123, 124. 127,
156, 160. 220, 222, 227, 235. 243,
245, 250, 293. 284, 326, 327, 328

Georgevitch, 311

Gertz, 14. 97. 318

Gibbs, 98, 147. 166. 180, 192, 227,

326
Gicklhorn, 324
Gilchrist, 246
Gladin. 20, 97, 305
Glage, 14
Goeppert, 47, 97, 132, 142, 172, 179.

ISO, 227. 258. 262. 268. 269, 286,

293, 297, 298, 300, 301

Goetz, 221, 227, 326

Gorke, 277, 287, 288, 293, 309

Gortner, 116, 227, 283, 289, 293, 316

338

(;ral)fr. 2S0, 2!)2
Greathouse. o24
Greeley, 30, 97, 2:53, 234, 235, 293,

3(H;
Grell. 179, 227, 32.5
Griffiths, 72, 32.')
Grinbeig, Gl, 98, 238, 323
Giinther, 31
Guignard. 310
Gutbier, 310

Haberlaiult. ;no

Haines, 62, 97, 238, 293

Hale. 29. 4.5, 58, 97, 118, 121, 226,

227. 233, 238. 239, 292. 322. 324
Hampil, 24, 97, 322
Hansford, 61, 238
Hardy, 134. 135, 136. 137, 138, 227.

283, 293, 318
Harper, 170, 226
Harris, D.F., 85, 97. 311
Harris, J.A.. 116. 227, 316. 321
Hartmann, 131, 227
Hartung. 246
Harvey, 50, 97. 191, 194, 227. 228.

240, 288. 293. 315, 316. 323. 324
Hase, 35. 231
Hawkes, 209, 227
Heckel. 310
Hedluud, 270
Heilbrunn, 235, 293
Heldmaier, 64, 248, 293
Heller, 178. 227
Hepburn, 14. 16. 97, 313
Hermann. 300
Herring, 92. 93, 100
Herschel, 139, 140, 227, 297
Hess, 166, 168, 227
Heubel, 59, 97, 302
Hilliard, 22, 98. 237. 293. 313, 314
Hodapp, 190, 192, 194. 220. 227.

250. 294, 327
Hober. 117
Hoffman, 115
Hofman, 116. 227, 316
Horowitz-Wlassowa, 61, 98, 238,

293, 323
Howard, 118
Hunter, 83, 98, 296

Iljin. 53, 98. 237, 264, 265, 266, 271,

293, 323
Illanes, 36, 98
Irmscher. 38, 68, 98, 278, 279, 286.

293. 312

Jaccard, 12:!, 227
Jacol)sen, 247, 292
Jaffe, 201, 227
Jahn, 31, 98, 238, 293
Jahnel, 247, 249, 293. 325
Jecklin, 86, 98, 239, 293
Jennison, 321

Jensen, 57, 97, 115, 121, 123, 156,
158. 159. 162, 164, 227, 278, 293,

:m

.Johnson, 170, 227

Jones, 200, 227, 283, 289, 293, 315.

321
Jordan. C, 215
Jordan, M., 215
Judd. 131, 209, 228
Jumelle. 68. 98, 303

Kadisch, 21, 25, 26. 39, 98, 247, 248,

249. 293, 321
Kalabuchov, 81, 85, 88. 93, 98, 239.

240, 293. 323, 324
Kapterev, 74, 81, 98, 325
Karcher. 26, 28, 65, 67, 98, 248, 249,

293. 321
Kasansky. 20, 24, 98, 305
Keith, 23, 98, 313
Kennon, 115
Kessler, 324
Kiebitz, 171, 226
Kjava, 71
Kjellmann, 38. 238
Klebs, 31
Klemm, 43, 66. 98. 233. 239. 287.

293. 304
Klepzoff. 24. 98, 304
Knauthe, 84, 87, 98, 303
Kochs, 83, 98, 303, 304
Kodis, 84, 98, 239, 293, 305
Kol, 27

Kovchoff. 14, 98, .309
Krebs. 286, 293. 321
Krumwiede, 22, 24
Kruyt, 320
Krysykowsky, 319

339

Kiihiie. 29, 43, oT, 62, 98, 299
Kunisch, 269, 293. 302
Kyes. 248, 293, 328
Kyliu, 66, 67, 98, 179, 227. 238, 281,
293, 314

Lagriffe, 84, 98, 306

Laveran, 249, 293, 307

Lawrence, 116, 227

Le Conte, 139, 140, 141, 226, 298

Lees, 169, 227

Lenoir, 181, 227, 321

Lepeschkin, 264. 293, 326

Leslie, 139

Levitt, 285, 293, 328

Lewis, 42, 98, 245, 253, 293, 323

Lidforss, 309

Liesegang, 308, 312

Lindner, 62, 63, 98, 237. 239, 293.

314
Lipman, 21, 22, 42, 54, 65, 98, 242.

243, 245, 247, 248, 253, 293, 323,

325, 326
Lipschiitz, 36, 98
Ljubavin, 172, 303
Lloyd, 164
Loeb, 115

Lottermoser. 172. 173. 227, 310
Lozina-Lozinskij, 81, 98, 324
Ludke, 17, 98, 286, 295, 308, 322
Lumiere, 17, 20, 98, 247, 293, 313
Luyet, 11, 45, 46, 48, 90, 98, 101,

113, 121, 123, 124, 127, 147, 156,

157, 160, 162, 166, 179, 180, 190.

192, 194, 203, 210, 212. 215, 220,

221, 222, 223, 227, 235, 243, 245,

246, 250, 252, 264, 266. 283, 284,
293. 294, 325, 326, 327, 328

Maass, 168. 169. 227

Macfadyen. 15, 17, 21, 24, 26, 98.

247. 248. 294, 305, 306, 307
Magath, 34, 71

Magoon, 61. 96
Mainx, 31
Mantegazza, 34
Martin, 321
Martine, 110
Matisse, 315

Matruchot, 145, 146, 180, 228, 275,
276, 294, 306, 307

Maurel, 84, 98, 306

Maximow, 48, 50, 98, 122, 124,
156. 159, 161, 162, 171. 228,
279, 280, 281, 294. 310, 312.
319

McConnell. 166, 167, 228

McKendrick. 91, 97. 245, 292.

McLean, 19, 99

Melsens, 25, 98, 266, 294, 300

Mesnil, 249, 293, 307

Mez. 47, 51, 98, 124. 127, 158,
160, 191, 195, 198, 199, 228,
259, 260, 277, 294, 308

Michel, 14

Miller, 200, 227, 315

Minett, 18, 100, 318

Mirsky, 288. 294

Mobius, 309

Moissan, 41

Molisch, 25, 29. 44. 45, 46. 49
66, 70, 99, 132, 133, 144, 179,
228, 233, 239, 241, 263, 270,
277, 279, 294, 304, 308, 309,

Molliard, 145, 146, 180, 228,
276, 294, 306, 307

Moran, 36, 37, 56, 58, 99, 119,
134, 135, 136, 137, 138. 161,
164, 165, 170, 175, 176, 177,
199, 201, 228, 231. 238, 239,
281, 282, 283, 286, 290, 294,
320

Morren, 262, 294

Morse, 115

Mouriquand, 14

Mousson, 195, 196, 228

Moiissu. 247, 294

Mudd. 22, 97

Miiller. 83. 99, 300

Miiller-Erzbach, 303

Miiller-Thurgau. 47, 49, 99,
144. 156. 161, 164, 171, 191,
228, 259, 268. 269. 272, 273,
279, 280. 294, 302, 304
Murigin. 94, 99. 239, 294. 326

Nageli, 262, 280, 294, 299
Nernst, 169, 228
Neuffer, 297
Neumann, 169, 228
Newton, 108, 321
Nichols, 170, 228
Nicolas, 17

126,
263,
.313

302

159,
241,

63,
192,
274,
311
275,

133,
163,
193,
251,
318,

121,
197,
274,

;uo

Xilsson-LoissiuT, :!20

Noack. :?S. ci'. !l!l. :!0S

Xoe. 21(;

Xord. 177. 17S. 228. 2SS. 204. 323

Ohlvvt'iler. 312
Oliver. 71. 209. 22G
Onskiehons. 21. 99. 316
Onoiato. 23, 99, 307
Osborn, 170, 226
Othmer, 189

Park, 22, 24, 306

Paul, 20, 99, 243, 294, 309

Pawlow. 33, 99, 319

Payne. 71. 79. 81. 99. 318, 319. 321

Peclet. 106

Pemberton. 3,'), 231

Pennington. 14

Person. 90. 99, 230, 295, 325

Petit, 110

Petterson, 170. 228

Pfeffer, 276. 280, 294, 308

Pichel, 268

Pictet. 15. 17, 18. 20, 26, 27. 35,

36, 40, 41, 57, 74. 84, 87, 91, 97.

99, 231. 245, 247, 249, 287, 294

302. 303
Plank, 164, 294. 314
Platanov, 90, 99, 239, 295, 325
Plateau, 77, 99, 258, 264, 265, 294,

300

Ponomarer. 71. 99, 314

Poppe, 313

Portier, 79, 81, 97, 316

Potter, 248, 293, 328

Pouchet, 55, 87, 99, 299

Pozerski, 15, 99, 306

Prall, 20, 99, 243, 294, 309

Prevost, 33, 298

Prillieux, 138, 141, 143. 180, 228,

262. 269, 294. 299, 300
Prucha, 20, 99, 318

Quatrefages, 33, 99, 298

Rabaud, 36, 99, 305

Rahm, 35, 72, 73, 82, 99, 246. 249,

294, 315, 316, 317
Ramsden, 327
Reaumur, 76, 296
Regeimbal, 316

Rc.unard. S7. ll'.t, 2:;:i. 2!t4. 304

IJciii. L'.".:t. 1^!I5. 310

Rcusch, 171. TIS

Renter, 294

Reynolds. 307

Richniann. 110

Richter, 64, 99. 311

Rigaud, 138

Rivers, 16, 17, 18. 2 1. 99. 319

Robinson. 81, 99

Roedel, 74, 77, 78. 99, 302

Roth, 56. 299

Rowland. 21, 24. 26, 98, 247. 248,

294. 305, 306. 307

Rubentschik, 318

Rubner, 164

Rumbold, 63

Runnstrom. 115. IIS. 120, 226. 311

Russell, ;'>13

Ryckenl)oer, 197, 226

Sacharov, 80, 99, 321

Sachs. 140, 141, 142, 144. 228, 241,

268, 272, 274. 295. 29S. 299
Salimovskaja-Rodina. 25
Salvin-Moore, 18, 99, 310
Sanderson. 17, 24, 99, 318
Savellier, 71, 99, 314
Saxton, 162, 227, 314
Schacht. 262

Schaffnit, 286, 295, 311. 322
Schenk, 33, 35, 54, 55, 99, 237, 238,

295, 300
Schiff, 56

Schmidt. 71. 73, 90, 99, 239, 295,
314. 325

Schmidt-Nielsen, 61, 99

Schmiesing, 216

Schumacher, 26, 99, 301

Scott, 117, 228, 314. 317

Scott-Goetz, 221, 227. 326

Sedgwick, 306

Senebier, 262, 295

Shrivastava, 177

Simonin, 55, 99, 239, 295, 322

Simpson, 92, 93, 99, 100, 307

Smart. 19, 25. 38. 62, 100, 238, 295,

324
Smith, K.V.. 320
Smith. K.F.. 23. 24, 100, 308, 312

341

Sorauer, 270, 302, 307
Spallanzani. 33, 34, 100, 296
Stcliepkiiia, 73, 99, 314
Stiles, 263, 283, 295, 316, 321
Stockman, 18, 100, 318
Stone, 22, 98, 237, 293, 313
Strasl)urger, 37
Straub, 118

Strickland, 31, 100, 244, 295, 325
Stuckey, 263, 295, 327
Summers, 316
Swingle, 23, 24, 100, 308
Swithinbank, 248, 295

Tait, 93, 100, 317

Talyract, 14

Tammann, 101, 112, 113, 128. 129,

131, 167, 188, 203, 228, 263, 295
Tanner, 16, 19, 23, 26, 100, 319, 322
Taylor, C.V., 31, 100, 244, 295, 325
Taylor, G.F., 192, 194, 195, 210, 228,

316, 317

Teodoresco, 38, 100, 308
Tetsuda, 16, 17, 100, 312
Thiselton-Dyer, 41, 100, 245, 295,
305

Thoennes, 46, 221, 223, 227. 250,

252, 264, 294, 327
Thunberg, 15, 100, 315
Thwing, 170, 228
Torossian, 22, 98, 237, 293, 313
Torrey, 322
Tottingham, 289, 292
Trouton, 169, 228
Turner, 86
Turner, 171, 228
Turner, T.B., 271, 295, 327, 328

Uloth, 301

Uvarov, 34, 75, 81, 100, 231, 295,
322

Valentine, 116, 227, 316
Vass, 315
Vaughan, 315
Velten, 301
Vernon, 286
Verson, 300
Vipond, 170, 226
Vogel, 172, 228, 297

Voigtlander, 51, 100, 124, 127, 156,
159, 160, 161, 191, 198, 200, 201,
228, 239. 259, 278, 295, 310

Voyle, 302

Wallace, 16, 19, 100, 322

Walter, 50, 100, 122, 124, 156, 228,
324

Walther, 91, 100, 299

Waltner, 34

Walton, 131, 209, 228

Warburg, 28, 249, 295, 315

Wartenberg, 324

Wartman, 40, 100, 196, 228, 299

Washburn, 170, 228

Weber, H., 33

Weber, H.F., 169, 228

Weigmann, 75, 90, 100, 156, 201,

228, 240. 264, 295, 320, 321, 325
Weill, 14
Weimer, 321
Weinzirl, 248, 295, 323
Weiser, 248, 295, 323

Weismann, 50, 100, 122, 124, 156,

228, 324

West, G., 27, 66
West, W., 27, 66
Wettstein, 58

White, 24, 100, 247, 295, 305
Wiegand, 143, 228, 309
Wiesner, 308
Williams, 22, 24
Williamson, 23, 26, 100, 319
Winkler, A., 52, 100, 270, 295
Winkler, K.C., 320
Winslow, 306
Winternitz, 92, 100, 304
Wislouch, 27, 100, 311
Wolfson. 30, 100, 237, 238, 295, 324
Weight, 14, 192, 194, 195, 201, 228,
316. 317, 319

Young, 195, 228

Yung, 15, 18, 20, 26, 74, 100, 302

Zacharowa, 45, 51, 100, 124, 228

239, 295, 318
Zandbergen, 32, 100, 317
Zawadowski, 34
Zimpfer, 327

Zirpolo. 22, 100, 248, 295, 322, 323
Zopf, 25