The Leeuwenhoek Lecture: The Problem of Anabiosis or Latent Life: History and Current Concept Author(s): D.

Keilin Source: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 150, No. 939 (Mar. 17, 1959), pp. 149-191 Published by: The Royal Society Stable URL: http://www.jstor.org/stable/83251 . Accessed: 03/02/2011 05:49
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THE LEEUWENHOEK LECTURE

The problem of anabiosis or latent life: history and current concept

BY D. KEILIN, F.R.S.

Molteno Institute, University of Cambridge (Delivered 19 June 1958-Received 22 September 1958)

CONTENTS
PAGE

INTRODUCTION
I. EARLY WORK ON ANABIOSIS

1.50
150

1. 2. 3. 4.
II.

Leeuwenhoek's discovery and observations The work of Needham and Baker The work of Spallanzani, Roffredi and Fontana The work and views of Bauer, Ehrenberg and Doyere
OF INTEREST IN THE PROBLEM OF THE RESUSCITATION OF MICRO-

150 152 153 156

157

DECLINE

ORGANISMS III.
REVIVAL OF INTEREST IN THE PROBLEM OF THE RESITSCITATION OF DESICCATED

MICRO-ORGANISMS (1858-60)

158

1. Controversy between Doyere and Pouchet 2. Report of the Commission of the Biological Society of France (a) Conclusion of the report signed by all the members of the Commission (b) Critical examination of Broca's report 3. Reaction of French biologists to the Darwin-WVallace theory of evolution

158 159 160 160

(1858, 1859)
IV. V. PROBLEMS WH1ICH STIMULATED AND AND FURTHER WORK ON ANABIOSIS STATES OF ORGANISMS

163
164 165

NOMENCLATURE EXPERIMENTS

CLASSIFICATION

OF THE DIFFERENT FOLLOWING

VI.

VIEWS

ON ANABIOSIS

BROCA'S

REPORT

167

1. Anhydrobiosis (a) Experiments (b) Metabolism (c) Cytological 2. Anhydrobiosis 3. Anhydrobiosis 4. Anhydrobiosis 5. Anhydrobiosis
VII. VIII. IX. EFFECT EFFECT ANABIOSIS OF LIFE X. LATENT

in rotifers, tardigrades and nematodes on desiccation and resuscitation of desiccated tardigrades changes during anhydrobiosis in larger animals in bacteria in fungi in higher plants and the viability of seeds
ON THE LONGEVITY OF ORGANISMS

168 168 169 169 170 171 171 172

176 177

OF ANHYDROBIOSIS

OF LOW TEMPERATURES AND ITS BEARING ON THE EARTH AND THE STABILITY OF BIOLOGICAL MATERIALS ON PROBLEMS OF THE ORIGIN AND THE CONTINUITY

180 181

LIFE

1. Stability of haemoglobin and endoerythrocytic enzymes in vitro 2. Palaeobiochemical records of the stabilities of biological materials
IO

182 183

[ 149 ]

Vol. 150.

B.

(I7 March I959)

150
XI. XII.

D. Keilin
PAGE

IN MATERIALS VIVOAND IN VITRO AND ITS BEARING STABILITY OE BIOLOGICAL ON ANABIOSIS THEIR STRUCTUTRAL DYNAMIC AND STATICSTABILITYOF ORGANISMS, INTEGRITY AND ANABIOSIS REFERENCES INTRODUCTION

185 187 187

The eight previous Leeuwenhoek Lectures covered a great variety of problems in bacteriology and virology, and each of the lecturers paid an enthusiastic tribute to Antony van Leeuwenhoek as the founder of microbiology. When the Council honoured me by their invitation to deliver the ninth Leeuwenhoek Lecture I thought that it would be appropriate to devote it to the problem

of anabiosis or latent life. I selected this subject for three reasons: (1) This subject originated from one of Leeuwenhoek's important discoveries, and it is mainly in this association that his name appears in the modern biological
literature.

(2) It has a direct bearing on some aspects of ourwork on intracellular respiratory

catalysts of organisms.

(3) This subject is linked with a 'Discussion on viability of mammalian cells and tissues after freezing', which was recently held in this room under the leadership of Dr Parkes (1957) and with two symposia on 'Freezing and drying', organized by the Institute of Biology (June 1951 and April 1958). It is now well established that some micro-organisms can, under certain conditions, be deprived of all visible signs of life and yet these organisms are not dead, for, when their original conditions are restored, they can return to normal life and activity. This peculiar state of an organism is referred to in the literature as viable lifelessness, suspended animation, viability, latent life or the not very suitable, but widely used term anabiosis to which I shall return later on. In the meantime, the term anabiosis, or latent life, will be used in this lecture for the state of an organism when its metabolic activity is at its lowest ebb, sometimes reaching a hardly measurable value, and in some cases actually coming to a standstill, the physiological and biochemical processes being reversibly arrested for different periods of time.
I. EARLY WORK ON ANABIOSIS

1. Leeuwenhoek's discovery and observations The study of anabiosis originated in 1702 from Leeuwenhoek's observations recorded in his letter: 'On certain Animalcules found in the sediment in gutters of the roofs of houses.' In this letter, where he deals with the animalcules now known as rotifers, Leeuwenhoek writes: 'I have often placed the Animalcules I have before described out of the water, not leaving the quantity of a grain of sand adjoining to them, in order to see whether, when all the water about them was evaporated and they were exposed to the air, their bodies would burst, as I had often seen in other Animalcules. But now I found that when almost all the water was evaporated, so that the creature could no

The LeeuwenhoekLecture

151

longer be covered with water, nor move itself as usual, it then contracted itself into an oval figure, and in that state it remained, nor could I perceive that the moisture evaporated from its body, for it preserved its oval and round shape unhurt. 'In order more fully to satisfy myself in this respect, on the third of September, about seven in the morning, I took some of this dry sediment, which I had taken out of the leaden gutter and had stood almost two days in my study, and put a little of it into two separate glass tubes, wherein I poured some rain water which had been boiled and afterwards cooled.... 'As soon as I had poured on the water, I stirred the whole about, that the sediment which, by means of the hairs in it, seemed to adhere like a solid body, might be the sooner mixed with the water: and when it had settled to the bottom of the glass, I examined it, and perceived some of the Animalcules lying closely heaped together. In a short time afterwards they began to extend their bodies, and in half an hour at least a hundred of them were swimming about the glass, though the whole of the sediment which I had put into it did not, in my judgment, exceed the weight of two grains.... 'The preceding kinds of experiments I have many times repeated with the same success, and in particular with some of this sediment, which had been kept in my study for above five months, and upon pouring on it rain water, which had been boiled, and afterwards cooled, I saw in a few hours time many of the Animalcules before described. And if, after being so long in a dry state, these Animalcules, upon water being given to them, can unfold their bodies and move about in the usual manner, we may conclude, that in many places, where in summer time the waters stagnate, and at length dry up, there may be many kinds of Animalcules, which, though not originally in those waters, may be carried thither by water fowl, in the water or mud adhering to their feet or feathers.' This view on the distribution of freshwater organisms by birds is not unlike that developed by Darwin (i859) in The origin of species (pp. 386-9). It is important to note that Leeuwenhoek never suggested that his dried animalcule was completely desiccated, for its body 'preserved its oval and round shape unhurt'. Although he clearly demonstrated that these animalcules can be kept dry for several months without showing any visible sign of life, he never brought into the discussion of the phenomenon he discovered the concepts of life, latent life, resuscitation and death, which, as we shall see, occupied such a prominent place in the work which followed. At the time of these observations Leeuwenhoek (1632-1723)* was 70 years old, but his interest in the phenomenon he discovered never abated during the
* One can scarcely discuss Leeuwenhoek's work without referring to the scholarly book
by the late Clifford Dobell, F.R.S. (1932) on Antony van Leeuwenhoek and his 'little animals ',

the book which is universally recognized as one of the outstanding and lasting tributes to this great Dutchman. Dobell, a distinguished protistologist himself, dealt in this book mainly with Leeuwenhoek's discoveries of protozoa and bacteria. However, one must not forget that, great as these discoveries were, they covered but one aspect of his wide scientific interests and activities. I must also mention that Dobell's enthusiastic admiration of Leeuwenhoek and his little animals had occasionally led him astray. This is noticeable in his very striking underestimation of the scientific achievements of John Turberville Needham and Henry Baker.

152

D. Keilin

remaining 20 years of his life. Unfortunately, these remarkable observations failed to impress his contemporaries and were soon forgotten. This was not surprising considering that, while the world of the microscopic animalcules was to Leeuwenhoek as real as the world around him, to his contemporaries it was a very strange world indeed, where anything could happen, leaving no room for surprise. 2. The work of Needham and Baker Forty years later Leeuwenhoek's observations were followed by an important discovery made by John Turberville Needham (I743)* of microscopic eelworms (later recognized as a larval stage of Anguillulina tritici) which cause disease in wheat accompanied by the formation of galls known as 'cockles' or 'peppercorns', which replace the healthy grains. This discovery was announced in a letter dated 11 August 1743, read at the Royal Society and summarized in the Philosophical Transactions as follows: 'On opening such an infected black grain, he [Needham] perceived a soft white substance, a small portion of which he placed on the objectplate: it seemed to consist wholly of longitudinal fibres bundled together and without any least sign of life or motion. He dropped a globule of water on it, to try if the parts, when separated, might be viewed more conveniently; when, to his surprise, these imaginary fibres, as it were, instantly separated from each other, took life, moved irregularly, not with a progressive but twisting motion; and so continued for the space of 9 or 10 hours, when he threw them away. He satisfied they are species of aquatic animals, and may be denominated worms, eels or serpents, which they much resemble. He repeated the experiment several times, with the same success and gratified others with a sight of it.' A more detailed account of this phenomenon was given by Needham (1745) in his book New microscopical discoveries (pp. 85-9), where it is suggested that the blight in wheat 'is frequently occasioned by the sowing of Seed intermixed with blighted Grains'. As to the nature of these organisms, he writes: 'How these Eels subsist... whence they come, what they convert into, if they suffer any Change, or how they propagate, I could never learn.' Needham's observations were soon confirmed by Henry Baker (I753),' a distinguished naturalist, microscopist and later the founder of the Bakerian Lecture of the Royal Society. In chapter iv of part II of his Employment for the microscope, which deals with 'Eels in blighted Wheat', Baker writes: 'In a month of August 1743, a small Parcel of blighted Wheat was sent by Mr Needham to Martin Folkes, Esq; President of the Royal Society (with an Account of his new Discovery) which Parcel the President was pleased to give to me, desiring I would examine it carefully.' With this material he was very soon 'entertained with the pleasing sight of * John Turberville Needham (1713-81),Roman Catholicdivine, distinguishedfor scientific work, especially on microscopicorganisms. Elected F.R.S. 1747. Also famous for his controversy, on spontaneousgeneration,with Spallanzani(1769). t Henry Baker (1698-1774), distinguishedbiologist and author of well-knownbooks on microscopy,which contain all his scientific contributions. In 1741 he was elected Fellow of the Royal Society and in 1744 was awarded the Copley Medal. According to the Diet. Nat. Biog. (8, 9-10) he marriedthe youngest daughterof Daniel Defoe and made a fortune by inventing a method for educating deaf-mute children.

The Leeuwenhoek

Lecture

153

this wonderful Phenomenon'. He repeated these experiments at different times and even with grains kept dry for 4 years, always with the same success and demonstrated it to Mr Folkes and some other friends. 'We find [he writes] an Instance here, that Life may be suspended and seemingly destroyed; that by an Exhalation of the Fluids necessary to a living Animal, the Circulations may cease, all the Organs and Vessels of the Body may be shrunk up, dried, and hardened; and yet, after a long while, Life may begin anew to actuate the same Body; and all the animal Motions and Faculties may be restored, merely by replenishing the Organs and Vessels with a fresh supply of Fluid. Here is, I say, a Proof, that the Animalcules in the Grains of blighted Wheat can endure having their Bodies quite dried up for the Space of four Years together, without being thereby deprived of their living Power: and since, after they are become thus perfectly dry and hard, there seems little room for farther Alteration, unless their Organs should be broken or torn asunder; may they not possibly be restored to Life again, by the same Means, even after twenty, forty, an hundred, or any other Number of Years provided their Organs are preserved intire? This Question future Experiments alone can answer.' He further writes: 'What Life really is, seems as much too subtile for our Understanding to conceive or define, as for our senses to discern and examine.... 'Dr Butler, the late Bishop of Durham, in his ingenious Analogy of religion to the constitution and course of nature, gives it as his Opinion, pag. 21 that "We have no more Reason to think a Being endued with living Powers, ever loses them, during its whole existence, than to believe that Stone ever acquires them.... "The Capacity of exercising them for the present, as well as the actual Exercise of them, may be suspended (says he pag. 22), and yet the Powers themselves remain undestroyed."' Moreover, in a letter to Martin Folkes, dated 16 January 1744/5 and printed for the first time in Employment for the microscope 1753 (pp. 267-92) Baker confirmed the old observations by Leeuwenhoek on wheel-animals (rotifers) and gave a more detailed description of their structure. 'Mr Leeuwenhoek', he writes, 'kept some Dirt, taken out of a Leaden Gutter, and dried as hard as Clay, for twenty-one Months together; and yet when it was infused in Water, Multitudes of these Creatures soon appeared unfolding themselves, and quickly after began to put out their Wheels; and I myself have experienced the same with some that had been kept much longer.... I cannot conclude this Subject without doing all the Honour I am able to the Memory of Mr Leeuwenhoek, by repeating, that we are obliged to his indefatigable Industry for the first Discovery of this most surprising Insect' (pp. 291-2). 3. The work of Spallanzani, Roffredi and Fontana No biological phenomenon of any interest ever escaped the attention of Spallanzani (1767-69).* However, his first reaction to Needham's discovery was to
* Lazzaro Spallanzani (1729-99), Professor of Natural History of the University of Pavia. One of the foremost scientists of his time, Fellow of the Royal Society. Celebrated for his work on reproduction, digestion, circulation, tissue respiration and the controversy with J. T. Needham on spontaneous generation. For an account of his life and work see Senebier

(I803).

154

D. Keilin

deny the animal nature of the dried filaments present in blighted wheat grains. The phenomenon of reviviscence described by Needham and Baker he ascribed to the motion of elongated fibres brought about by penetration of fluid into them. Such a view, held also by some other workers, was not surprising considering: (1) that reviviscence of dried eel-worms appeared as a very strange and unexpected phenomenon even to a very experienced biologist; (2) that Leeuwenhoek's observations on wheel-animals were almost completely forgotten; (3) that there was still great confusion between rust- or ergot-invaded grain and Needham's blighted wheat grains; (4) that biologists were familiar with swelling and other movements exhibited by some dried vegetable fibres soaked in water; and (5) that nothing was known about the origin of eel-worms in blighted wheat and their further development. Spallanzani's view, backed by his great scientific reputation, prompted Needham to abandon his original view and to express in some notes to the French translation of Spallanzani's work (1769) that certain filaments or fibres elongated in the form of eels which he found in blighted wheat belong 'to a kind of purely vital being which does not manifest any spontaneity in its movements'. In the meantime l'Abbe Roffredi* (1775-76) and Felice Fontana (1776)'- each independently unravelled the main stages of the life-history of eel-worms found in blighted wheat. They found: (1) that wheat becomes infected with eel-worms when healthy and blighted grains are sown together; (2) that in the early stages of infection the blighted grain contains a few sexually mature eel-worms; (3) that the females, which are much larger than the males, produce a great number of eggs from which emerge the young eel-worms; (4) that while males and females die and disintegrate the larvae remain within the blighted grain, ultimately forming the white mass of dried eel-worms discovered by Needham; and (5) that the blighted grain, known also as purple, cockle or peppercorn, is not a true grain, but a gall (Fontana 1776) similar to plant galls produced by insects. Thus, the work of Roffredi and Fontana demonstrated, beyond any doubt, the animal nature of the material discovered by Needham in blighted wheat-grains. Moreover, Fontana (i776) repeated Needham's observations and showed that the lifeless eel-worms, although they were desiccated to such an extent that the mere touch of the point of a hair or a needle made them crumble to powder, they could yet revive within a few minutes when they were brought in contact with water. He demonstrated the phenomenon of reviviseence 'to professors of Tuscany, to many learned men from abroad and to numerous persons of distinction'. In the above papers both Roffredi (I 775 a) and Fontana (I776) attacked Needham, severely and with some sarcasm, for abandoning his original correct view as to the true
* D. Maurice Roffredi referred to himself as 'Abbe Regulier de l'Abbaye de Casanova, Ordre de Citeaux, en Piemont'. It may be mentioned that his contemporaries, Needham, Spallanzani, Fontana and others are often referred to as Abbe, without such a clear indication as that given by Roffredi. t Felice Fontana (1720-1805), versatile Italian scientist, physician to the Grand Duke of Tuscany and Director of the Natural History Museum of Florence; celebrated for his toxicological work on snake venom, prussic acid and opium and for his numerous contributions to other biological subjects. (See Nature, Lond. 175, 410, 1955.)

The Leeuwenhoek

Lecture

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animal nature of his desiccated and revived eel-worms. Needham ( I775) in a letter to Abbe Rozier (the editor of the Journal de Physique) pays a generous tribute to the remarkable achievement of Roffredi, but expresses some resentment at the spirit of criticism directed against his more recent view 'where the critic aims only to wound his adversary'. In addition to his work on eel-worms, Fontana (1776, I795) also corroborated Leeuwenhoek's and Baker's observation on the desiccation and reviviscence of wheel animals (rotifers) and recorded a similar phenomenon in the case of Gordius. The brief account of his observations was concluded as follows: 'I have since found a number of other small animals, either on the tops of houses, in earth or in water, which in the same way, alternately lose and recover the use of their organs, on being dried and afterwards returned again into water. I purpose speaking of these little prodigies in a work apart, to be entitled, On the Life and apparent Death of Animals' (Fontana 1795, Treatise on the venom of the viper, vol. 1, p. 113). However, this work was never published, but not, as we shall see, for the reason later suggested by Broca (I860). Following the work of Roffredi and Fontana on the life-history of the eel-worm. of blighted wheat, Spallanzani returned to the problem of organisms which can undergo reversible desiccation. The results of his extensive study on this subject were presented under the title of: 'Observations and experiments upon some singular animals which may be killed and revived', in two of his major and best known works, which appeared in Italian and were translated into French and English and published in several editions between 1776 and 1803.* Although the animalcula in infusions usually perish in the absence of water, 'there are other animalcula,' he writes, 'which, notwithstanding they inhabit infusions, are so much distinguished and privileged by nature, so to enjoy the advantage of real resurrection after death' (p. 119). His observations and experiments were carried out on micro-organisms found in the dry sand in roof gutters, comprising wheelanimalcules (rotifers), tardigrades, which he discovered and described for the first time, and free-living eel-worms; he also re-investigated the eel-worms of blighted wheat. On this material he confirmed and greatly extended the work of Leeuwenhoek, Needham and Baker. The comparative rarity of these animalcules 'arises', he writes, 'less from the scarcity of such beings than the rareness of the philosophers who have entered on this branch of natural philosophy. Leeuwenhoek was the first who, by his reviving of the wheel-animal, drew the curtain from before these objects; and seems surprised of a fact which was unique and unexampled in nature: and indeed, no one thought there was another creature in all the animated world possessed of this prodigy. But since the profound research of more modern naturalists has discovered others, I doubt not that the number of these wonderful beings will increase as the study is cultivated' (p. 196). In another place referring to wheel-animals he writes: 'They are not the only animals, however, that enjoy the privilege of resurrection; others also possess it: among these, the celebrated eels of blighted corn deserve to be particularly mentioned. All the world knows
* The quotations which follow were taken from the second edition of the English translation of his Tracts on the natural history of animals and vegetables, published in 1803.

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that Mr Needham is the author of this famous discovery.' 'The fact was too wonderful for others not to endeavour to ascertain it' (p. 169), and further, 'In short, there is not at this day any professor, any amateur of natural history, particularly in Italy, who does not take pleasure in amusing himself, and gratifying the curiosity of his learned friends with these admirable resurrections' (p. 170). About the general philosophical significance of this work Spallanzani wrote: 'An animal, which revives after death, and which within certain limits, revives as often as we please, is a phenomenon, as incredible as it seems improbable and paradoxical. It confounds the most accepted ideas of animality; it creates new ideas, and becomes an object no less interesting to the researches of the naturalist than to the speculation of the profound metaphysician' (p. 160). Spallanzani, unlike his predecessors, was not satisfied with the mere observations of these animalcules during desiccation and reviviscence. He submitted them to different temperatures, to vacuum, to electric shocks, and to treatment by other agents. He found, for instance, that while active rotifers were killed at about 45 ?C desiccated forms withstood temperatures up to 73 ?C. He also found that active as well as desiccated rotifers withstood cooling to - 24 ?C, obtained by freezing mixtures. Vacuum did not prevent but delayed the reviviscence. Eel-worms of blighted wheat were found by him less resistant to higher temperatures, and on successive drying the proportion of eel-worms which could be revived gradually diminished. 4. The work and views of Bauer, Ehrenberg and Doyere The view of Spallanzani was not generally accepted and the study of the problem of resuscitation of micro-organisms was continued by his contemporaries and a generation of biologists who followed. While some among these workers confirmed his results, others rejected them. Of the work belonging to this period we shall examine only that of Bauer (1823), Ehrenberg (1838) and Doyere
(I842).

Bauer,* who studied the eel-worms (Anguillulina tritici) of blighted wheat, confirmed in all details the results of previous work on the resurrection of desiccated larval forms. On the other hand, his study of the life-history of eel-worms was not as complete and correct as the work of Roffredi (1775-76) and Fontana (1776). Unlike these two biologists, he overlooked the existence of males, assuming that the adult eel-worms are hermaphrodites, and he failed to recognize the tumour or gall nature of blighted grain. However, as his work formed the subject of the Croonian Lecture of the Royal Society (1823) and was published in the Philosophical Transactions, it became more widely known than the excellent work of his predecessors. Even the recent work on anabiosis by Schmidt (1948) not only refers to Bauer's work but reproduces his incorrect illustrations of the adult eel-worm.
* Francis Bauer (1758-1841), scientific draughtsman of the first rank. He was employed in that capacity at the Royal Botanical Gardens (Kew) by Sir Joseph Banks whom he later joined in his study of diseases of corn, blight caused by eelworms and ergot. The illustrations of his Croonian Lecture (1823) reveal his professional skill but no knowledge of helminths.

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Ehrenberg (1838),* on the other hand, was one of the strongest opponents of the concept of resuscitation of lifeless desiccated micro-organisms. The life processes of these organisms, according to him, are not arrested but only greatly slowed down and their state is that of apparent death ('Scheintod'). Since it was impossible to ascertain that these organisms are deprived of all traces of water no serious objection could be raised against this view. However, Ehrenberg was completely wrong when he suggested that rotifers in dry sand continue to feed and even to lay eggs which give rise to a new generation of rotifers, so that the organisms claimed to be resuscitated were not the specimens originally present in the sand, but the forms belonging to one of the subsequent generations. Although this view contradicted numerous observations and experiments which had been carefully carried out, Ehrenberg's authority was so great, that his view impressed his contemporaries and was widely accepted. Four years later the problem was re-investigated by Doyere (1842) in his extensive work on tardigrades which he concluded with a chapter on 'The faculty which possess tardigrades, rotifers, anguillules of the roofs, and certain other animalcules, of returning to life after being completely desiccated'. Doyere greatly improved the experimental method by drying the material (moss or sand) containing the micro-organisms, first of all in air and then in vacuum over sulphuric acid, or calcium chloride. He found that, while active forms can withstand warming to only about 48 ?C and perished at 50 ?C, the desiccated forms withstood for a few minutes temperatures up to 120 to 125 ?C. The differences in the resistance of active and desiccated micro-organisms at different temperatures he compared with those of albumin (protein) in solution and in the dry form. He was also successful in demonstrating that by graded drying, micro-organisms such as rotifers can be revived after they have been desiccated in distilled water on a microscope slide. What is left of the organisms, according to him, is the molecular composition of their tissues and their organic arrangement, or organized material endowed with potential life. Expressed in modern language, the desiccated microorganisms which can return to life have the composition and the intimate structure of their tissues intact. Some of his experiments on the resuscitation of dried micro-organisms, which had been warmed to 122 to 125 ?C, were carried out under the observation of de Jussieu, Dumas, Milne-Edwards and de Quatrefages (see Milne-Edwards 1842). Although Doyere strongly criticized the views of Ehrenberg, his work remained practically unnoticed for almost 20 years.
II.
DECLINE OF INTEREST IN THE PROBLEM MICRO-ORGANISMS OF THE RESUSCITATION OF

Doyere's work was not followed by further experimental investigation or discussion of the problem of reviviscence for at least 17 years. The gradual decline
* Christian Gottfried Ehrenberg (1795-1876), one of the distinguished scientific figures of his time. He made fundamental contributions to the study of protozoology as well as to other branches of biology. He was elected Foreign Member of the Royal Society in 1837 and was the first recipient in 1875 of the Leeuwenhoek Medal of the Royal Academy of Sciences of Amsterdam (Dobell I923, I932).

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of the interest in the problem of reviviscence was due, not only to Ehrenberg's adverse criticism, but also to several other factors, which were manifested long before Ehrenberg's work and which were: (1) The discrepancy in the results obtained by different workers, which in some cases could be traced to differences in the species of micro-organisms used in their experiments. This was not surprising considering how little at that time was known of the structure, taxonomy and life histories of these micro-organisms. (2) The inadequacy of methods used in the desiccation of micro-organisms and in the proper assessment of the degree of their dehydration. In this respect Doyere's work marks a great advance in the study of the problem of resuscitation. (3) The loss of confidence in work depending on microscopical observations. The microscope, on becoming a fashionable source of amusement in the hands of some people with more imagination than knowledge or experimental skill, was gradually discredited as a tool for serious research. Some workers even began to doubt the correctness of early observations and discoveries made with the use of the microscope, including those of Leeuwenhoek. One of the early warnings against the misuse of the microscope was given by Fontana in the chapter 'On microscopical errors; and Consequences deduced from Microscopical Observations' of his Treatise on the venom of the viper; etc. (i795, vol. 2, p. 284). The feeling of mistrust of some of the work carried out with the microscope was also shown by Prevost & Dumas (I824) in their study of seminal fluid. They expressed it as follows: 'L'abus etrange que les amis des hypotheses hasardeuses ont fait du microscope a cette occasion, justifie assez la repugnance que les esprits positifs eprouvent a discuter des observations faites avec un instrument decevant pour tous ceux qu'une longue habitude n'a pas rendus maitres de son emploi' (p. 15). According to Geoffroy Saint-Hilaire & Milne-Edwards (1838), it is to the work of Prevost & Dumas that we owe the rehabilitation of the microscope as an instrument for research. It may be interesting to note that the young Dumas, who began his scientific apprenticeship as a biologist under Dr J. L. Prevost in Geneva, became later the well-known Jean Baptist Andre Dumas (1800-84), one of the greatest chemists of his time, who taught Louis Pasteur, encouraged him in his work and supported him in several of his scientific controversies, including that with F. A. Pouchet to which I shall return later on.

III.

REVIVAL

OF INTEREST

IN THE PROBLEM

OF THE RESUSCITATION

OF DESICCATED MICRO-ORGANISMS

(1858-60)

1. Controversy between Doyere and Pouchet The work on the resuscitation of desiccated micro-organisms was, after a long period of stagnation, suddenly revived in 1858-59 by a sharp discussion between two French biologists, Doyere and Pouchet, who carried out their experiments on micro-organisms (rotifers and tardigrades), but with very different results.

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According to Doyere, who began his investigations in 1842 and whose early work was practically forgotten, these micro-organisms can be revived after complete desiccation and the cessation of their life processes (cf. p. 157). According to Pouchet (1859a, b, c) no organism can survive complete desiccation, or return to life, once all life processes have been arrested. Between 1858 and 1859 members of learned societies and the lay press of Paris were, according to Broca, divided into two hostile groups: the resurrectionists and the anti-resurrectionists. One may ask what provoked the controversy between Doyere and Pouchet, and why it revived so much general interest in the problem of resuscitation considering that no new facts on this subject had come to light ? The only answer that can now be given is that the discussion of the problem of resuscitation crossed the path of the controversy on spontaneous generation which agitated the Academie des Sciences and the press of Paris. Pouchet,* who was interested in the problem of the origin of life, carried out with his co-workers experiments which he thought had demonstrated the spontaneous generation of certain micro-organisms. The results of this work, which he communicated on 20 December 1858 to the Academie des Sciences, were severely criticized at the meeting of the Academie (held on 3 January 1859) by MilneEdwards, Claude Bernard, Dumas, Boussignault, de Quatrefages and Payen.f It is in the course of this discussion that Milne-Edwards referred to Doyere's (I842) demonstration of the thermostability of desiccated rotifers and tardigrades (cf. p. 157). This compelled Pouchet to carry out experiments on the desiccation, stability and resuscitation of rotifers and tardigrades with results very different from those obtained by Doyere. 2. Report of the Commission of the Biological Society of France Having failed to reach any agreement in their comparatively simple experiments, Doyere and Pouchet approached in 1859 the Biological Society of France for an impartial examination of their claims. The Society agreed to their request and appointed for this purpose a special 'Commission' composed of the following members: Balbiani (protistologist), Berthelot (chemist), Broca (anatomist and anthropologist), Brown-Sequard (physiologist), Darest (embryologist), Guillemin (biophysicist) and Ch. Robin (anatomist and embryologist).
* Plouchet, Felix Archimide (1800-72), a distinguished biologist, director of the Natural History Museum and of the Botanical Garden of Rouen. Elected 'Membre Correspondant de l'Academie des Sciences', Paris, in 1849. t This first stage of the controversy on spontaneous generation between Pouchet and the distinguished members of the Academie des Sciences, a year before the discussion was joined and dominated by Pasteur, is seldom referred to in the literature dealing either with the history of this problem, or with the life of Pasteur. The controversy on the resuscitation of desiccated organisms which emerged from that on spontaneous generation can be followed in vol. 1.4 (1859) of Cosmos edited by M. 1'abbe Moigno, who not only recorded its successive steps but also kept the fire of the discussion going by harassing the adversaries with his questions and inviting them to express their views in his journal. The following pages of this volume of Cosmos contain some of the records of these discussions: pp. 22-3, 33-6, 65-6, 70-2, 380-2, 426-35, 591-3, 635-40.

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The commission spent a few weeks each with Doyere and Pouchet carefully recording their experiments. They then carried out their own experiments. In less than a year they had about forty-two meetings and in March 1860 they presented an extensive report of more than 60000 words, which was published as a Memoire of the Biological Society. The report and its summary were written by Broca himself and the conclusion only was drawn up and signed by all the seven members of the Commission. Since this report is considered as one of the outstanding contributions to the knowledge of anabiosis in micro-organisms (Schmidt 1948), I shall examine it in some detail and I shall begin with the conclusion signed by all the members of the Commission. (a) The conclusion of the report signed by all the members of the Commission It reads as follows: 'The resistance of tardigrades and rotifers to high temperatures appears to increase in proportion to the degree of desiccation previously attained. Rotifers can be revived after being preserved in the dry state for 82 days in vacuum and submitted immediately afterwards to a temperature of 100 ?C for 30 minutes. That is to say, animals which have been successively dried in vacuum at ordinary temperatures and then heated at 100 ?C at atmospheric pressure, so reaching the most complete degree of desiccation that can be realized under these conditions and in the present state of scientific achievement, may yet retain the ability to revive in contact with water.' Although this conclusion corroborated the main experimental results obtained by Doyere, it did not appear to follow him as far as to say that these microorganisms could be revived after their complete desiccation, or after the cessation of all processes of life. (b) Critical examination of Broca's report The report, which was the work of Broca, * was written with great eloquence and a tendency to dramatization, which is already reflected in the motto: 'To be or not to be, that is the question.' It is difficult to understand the omission from the report of the fact that the discussion between Doyere and Pouchet and the general revival of interest in the resuscitation of desiccated organisms originated from and was intimately linked with the problem of spontaneous generation. The report gives a detailed account of the experimental procedures used and the results obtained by all the participants in this joint investigation, and the Commission accepted full responsibility for the accuracy of these experimental records. It also contains an excellent discussion of different aspects of the problem and an extensive review of the work on this subject between 1702 and 1860. For almost a century this report was the chief source of information on the history of the development of the subject, and it is still frequently used for this purpose, even in recent works including the important book on Anabiosis by Professor Schmidt (1948), the distinguished Russian biologist well known for his contributions to this
* Broca, P. P. (1824-80). French anatomist, Distinguished founder of the French Society of Anthropology (1850). surgeon and anthropologist,

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subject since 1916. Unfortunately, the historical treatment of the subject is not free from serious inaccuracies. The conclusion reached by Broca in his report differed from that which he had drawn up jointly with the other members of the Commission in that he rightly and more openly supported the original view of Doyere (1842), namely, that desiccated micro-organisms can be revived after all their life processes have been suspended. On the other hand, he failed to appreciate the natural and general reluctance of workers to accept such a view and wrongly suspected this to be due to religious prejudice. He even made an elaborate attempt to demonstrate that the Church kept a vigilant eye on works dealing with the problem of resuscitation, and that early workers like Needham, Baker and Fontana were greatly inhibited by the fear of being censured, or even penalized by, the Church. Thus, in reviewing the work of Needham, Broca wrote: 'It did not take him [Needham] long to realize that he had clashed with generally accepted ideas. Subjected to condemnation and to sarcasm, now censured as a reprobate, now derided as an absurd visionary, the unfortunate Needham failed to re-establish his reputation even at the cost of the repeated sacrifice of his ideas to the capricious demands of the age.' Having studied the work of Needham and of his contemporaries, I have failed to find in them any indication that he ever faced the difficulties so dramatically depicted by Broca. On the contrary, his work on blighted wheat was accepted by the Royal Society and was very early confirmed by Henry Baker, Fontana, Spallanzani and others. In fact, it was not Needham's work on the resuscitation of desiccated eelworms, but his work on spontaneous generation that was attacked on religious grounds. It was attacked not by Spallanzani who, while he contested Needham's view, never failed to show his deep admiration for him and for his work, nor was it attacked by the Church; it was, however, mischievously attacked by Voltaire* in articles, letters and even verses describing Needham as an Irish Jesuit disguised as a layman who by stupid experiments provided weapons for atheistic philosophy. In referring to some of these attacks Needham mildly complained of being accused of atheism, adding that Voltaire 'omits nothing to amuse the public by ingenious jokes at my expense' (see Needham's notes in the book by Spallanzani, 1769). Baker's use of a quotation from the work of Dr Butler, the Bishop of Durham (cf. p. 153), was, according to Broca (i86o), nothing but a desire to protect himself from attacks on religious grounds. This view was accepted and further elaborated by Schmidt (I948), who assumed that Baker was able to publish his observations on the resuscitation of eel-worms only after soliciting the permission of the Bishop of Durham. In making such allusions Broca and Schmidt revealed only that they were not aware of the fact that Butler's Analogy of religion to the constitution and course of nature was very widely read by successive generations of men of learning. We find, for instance, that in the fourth and subsequent editions of Darwin's Origin of species (i866-72), of the three quotations facing the title page, the second is from
* Voltaire, F. M. Arouet de Every more or less complete edition of his work (1694-1778). and letters contains several somewhat crude sarcastic remarks about Needham. In the edition given in the references to this lecture, these remarks are found in vol. 18, p. 372; vol. 27 pp. 159, 219; and especially vol. 46, pp. 105, 106.

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Butler's Analogy of religion and it concerns the definition of the word 'natural'. Later John Tyndall (I874), in his presidential address to the British Association for the Advancement of Science given at Belfast, before a lengthy discussion of Butler's work, remarked: 'The Bishop still influences superior minds, and it will repay to dwell for a moment on his views.' ' Finally, in referring to Fontana's promised work: On the life and apparent death of animals' which was never published (cf. p. 155), Broca (I860) uncritically accepted Dupaty's* (I789) statement that it was the fear of being excommunicated by the Church that prevented him from publishing this work. It was while staying in Florence in 1785 that Dupaty visited the then celebrated Cabinet of Natural History of the Grand Duke of Tuscany which was under the direction of Fontana. In his account of this visit (Letter xxxiii, pp. 126-9) he described the experiment on the resuscitation of the desiccated eel-worms of blighted wheat grain which Fontana demonstrated. Dupaty wrote: 'He only makes use of a drop of water. He takes care not to let it fall over these dustanimals, for it might crush them in falling: he approaches by degrees the drop of water on the head of a needle, and by degrees the little animal is penetrated by its freshness. All the atoms that compose it draw nearer and nearer, join themselves, and form but one; motion already exists; it gains ground, it advances, it circulates, and the animal receives life. The consequences resulting from this experiment are of the greatest importance; they throw much light on the life and death of matter. M. Fontana dares not write on this subject; he is afraid of being excommunicated. All the Grand Duke's power could not save him from the ill-consequence of excommunication. .. his great occupations, and above all, the proximity of Rome, prevent him from writing, and discouraged him often from thinking.' This naive account of resuscitation only confirms Spallanzani's statement that in Italy professors and amateur naturalists amused themselves and gratified the curiosity of their friends 'with these admirable resurrections'. As to Dupaty's statement that it was the fear of being excommunicated that prevented Fontana from publishing his work, this was in complete disagreement with the facts: (1) that about 10 years before Dupaty's visit, Fontana (1776) had already published his observations on the resuscitation of desiccated organisms; and (2) that the same year there appeared Spallanzani's (1776,t 1787, 1803) important study of 'animals that can be killed and revived', in which he freely used the expression 'the resurrection of killed animals'. The only explanation of Fontana's failure to publish his promised work is that he had nothing to add to his previous work, especially after the appearance of the works of Roffredi (I775-76) and Spallanzani (1776--1i803). I examined Broca's report in some detail, not only because it is still considered to be one of the outstanding contributions to our knowledge of anabiosis, but also
* French liberally minded magistrate who was associated with Dupaty, C. (1746-88). intellectuals and philosophers of his time, especially Diderot. Author of well-known Lettres sur l'Italy, translated into English under the title: Sentimental letters on Italy. t The year 1776 refers to the original Italian edition.

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because I found it important to correct some of its errors, which have remained unchallenged for almost a century. Moreover, I hope that the critical examination of this report will dispel, from further discussion of the problem of the possible discontinuity of life processes, all suspicion that workers who failed to accept such a possibility were in any way influenced by religious prejudices. During the same period there appeared two important papers on this subject which deserve special consideration. The first paper is that by Davaine (I856) on the eel-worm (Anguillulina tritici) of blighted wheat grains. It contains the first complete and correct account of the anatomy, life-history and biology of this organism. He confirmed and extended the work of Roffredi and Fontana by following the successive steps of the invasion of wheat seedlings by larvae liberated from blighted grains, and the fornation of galls from the parenchyma of different portions of young flowers. Davaine also carried out careful experiments on the resuscitation of desiccated larvae and confirmed the main results obtained by Needham, Baker, Spallanzani and Fontana. The second paper was on rotifers and tardigrades by Gavaret (1859), who found that after drying these organisms for 50 days in vacuum (4 mm Hg) over sulphuric acid, they withstood warming to 110 ?C, whereas active forms were rapidly killed at 50 ?C. Broca's report temporarily closed, at least in France, the controversy (1858-60), on the resuscitation of desiccated organisms, whereas the discussion on spontaneous generation was concluded only in 1864 by Pasteur's brilliant victory (see ValeryRadot
900o).

3. Reaction of French biologists to the Darwin-Wallace theoryof evolution (1858, 1859) As the years 1858 and 1859 are linked in our minds with another and certainly more important event in the history of biology, an event which will shortly be celebrated here, one may ask whether the intensity of the discussions which I have summarized was not somewhat exaggerated. I should like therefore to digress for a while from my subject and to remind you, that while the problems of resuscitation and of spontaneous generation provoked, between 1858 and 1864, stormy and often dramatic discussions in France, the joint announcement in 1858 by Darwin and Wallace of their theory of evolution by natural selection and the appearance in 1859 of the Origin of species produced scarcely a ripple in the Academie des Sciences and in other learned societies of France. It took more than 20 years before the new idea of evolution received even partial recognition in France. Two factors contributed to this delay: (1) the French Academie des Sciences was still strongly dominated by the spirit of Cuvier, and (2) the rise of a purely experimental approach to biology which was inaugurated by the brilliant investigations of Claude Bernard and Pasteur. It was, indeed, significant that Darwin's candidature as a 'Membre Correspondant de l'Academie des Sciences', proposed by zoologists in 1872, was strongly opposed and defeated. He was elected only in 1878 and not in the zoological but in the botanical section (see Life and letters, 1, 223-4 and Caullery 1924, p. 255). However, this lack of recognition of the Origin of species by the French 'Academie

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des Sciences' was to some extent shared by the Royal Society. Thus, when Darwin received the award of the Copley Medal (1865), the President (then General Sabine) in his Anniversary address praised Darwin's contributions to zoology, botany and geology, referring to the Origin of species only in so far as it contained numerous interesting, detailed and patient observations. He even stated that 'Speaking generally and collectively we have expressly omitted it from the ground of our award' (Life and letters, vol. 3, p. 29). It is, therefore, not surprising that, between 1858 and 1864, the attention of French biologists was not diverted, by the appearance of the Origin of species (I859), from their exciting polemical discussions of spontaneous generation and the resuscitation of desiccated micro-organisms.
IV. PROBLEMS WHICH STIMULATED FURTHER WVORK ON ANABIOSIS

The problem as to whether an organism can be revived after the complete cessation of all life processes, that is, whether life under certain conditions may be a discontinuous process, has, apart from its great scientific interest, a fascination of its own. In fact it is one of the oldest problems which preoccupied man when he began to think about life, death and immortality. It is reflected in almost all religions, in some legends and even in fairy stories. The desire to prolong life, or to cheat death whenever possible, was gratified even when it was achieved only with respect to small animalcules or the seeds of plants. This explains the relish with which Spallanzani repeatedly referred to 'resurrections of killed animals', it explains the great popularity of Needham's experiments on the resuscitation of eel-worms and the excitement of the popular press provoked by every new record on the longevity of seeds and by experiments on the resuscitation of cooled animals, which show no breathing and no heart beats. This yearning to prolong life was expressed in a somewhat frivolous manner by John Hunter in one of his lectures 'On the heat of animals'. Having failed in 1776 to revive some frozen carp, Hunter remarked: 'Till this time I had imagined that it might be possible to prolong life to any period by freezing a person in the frigid zone, as I thought all action and waste would cease until the body was thawed. I thought that if a man would give up the last ten years of his life to this kind of alternate oblivion and action, it might be prolonged to a thousand years; and by getting himself thawed every hundred years, he might learn what happened during his frozen condition.* Like other schemers, I thought I should make my fortune by it; but this experiment undeceived me' (Work of Hunter 1835, vol. 1, p. 284). Following Broca's report the interest in anabiosis rapidly developed in different
* Lucretius in his great poem on 'The nature of the Universe' showed no such curiosity as to what might happen long after his time. He was satisfied that all the knowledge that man can acquire is found in the teaching of the great Master, Epicurus. Meditating on life and death, he exclaimed: 'What is this deplorable lust of life that holds us trembling in bondage to such uncertainties and danger? A fixed term is set to the life of mortals, and there is no way of dodging death.... However many generations you may add to your store of living, there waits for you none the less the same eternal death' (Book III, pp. 128-9 [1038-94]).

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fields of biology, being stimulated by a great variety of problems of fundamental and practical importance. These problems can be grouped as follows: (1) The discontinuity of physiological and biochemical processes. The problem as to whether all processes of life can come reversibly to a standstill is of fundamental importance and is intimately linked with all other aspects of the work on anabiosis. (2) The effects upon organisms of great seasonal variations and of extreme climatic conditions such as life in deserts and in polar regions. Outstanding in this respect is the extensive monograph by Uvarov (I93 I) on 'Insects and climate' which contains a wealth of information on insects under a great variety of natural and experimental conditions. It also deals with the problems of hibernation, aestivation, anhydrobiosis and diapause. (3) The resistance of micro-organisms, of their spores and cysts to desiccation, their longevity and their dissemination in nature, which is of great medical and agricultural importance. (4) The preservation of biological materials, such as tissues for transplantations, blood for transfusion, semen for artificial insemination, micro-organisms for type cultures, seeds for germination and certain articles of food. All these problems were extensively discussed in the three symposia referred to at the beginning of this lecture. (5) Hypothermia, or lowering the temperature and therefore the metabolism of mammals and man, which is of great biological interest and of medical as well as surgical importance. The extensive work in this comparatively new field of research was summarized by Dr Audrey Smith (I958) in her excellent review 'On the resistance of animals to cooling and freezing', which appeared in the last number of Biological Reviews. (6) The origin of life, especially the early, now-abandoned, speculations on the interplanetary or interstellar dissemination of living organisms or 'panspermy' through cosmic dust and meteorites. Such speculations, which were at different times favoured by distinguished scientists like Lord Kelvip, Helmholtz, Arrhenius and others (see Oparin 1957), stimulated a number of experiments on anabiosis which I shall examine later on.
V. NOMENCLATURE AND CLASSIFICATION OF DIFFERENT STATES OF ORGANISMS

Before dealing with some specific cases of anabiosis I shall define some of the terms which are already used and a few new terms which may be helpful in further work on this subject. The term anabiosis or return to life ('Wiederbelebung') was introduced by Preyer* (i872, I891) for the phenomenon of resuscitation, or resurrection of completely lifeless but viable organisms. This term was, however, extended by him and by other workers to the state of viable lifelessness itself, which clearly
* Wilhelm Preyer (1841-97), Professor of Physiology blood gases, haemoglobin and its crystalline forms.
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emerges from the following distinction made by Preyer (I89I) of the two possible states of an organism:* 'lifeless and viable= anabiotic, lifeless and not viable = dead.' To avoid this confusion Schmidt (1948) proposed for the state of viable lifelessness the term abiosis. Unfortunately, this term is too closely linked with the terms abiotic and abiogenesis which are used in a very different sense in the literature dealing with the problem of the origin of life. I should like to propose the term cryptobiosis, that is, latent life, for the state of an organism when it shows no visible signs of life and when its metabolic activity becomes hardly measurable, or comes reversibly to a standstill. In proposing this term I am aware of the reluctance shown by Broca (I86o), Preyer (1891) and Schmidt (1948) in applying the term latent life to an organism which shows no visible or measurable signs of life. However, Claude Bernard (1878), who accepted Broca's report, defined latent life as a state of an absolute chemical indifference characterized by the suppression of all interrelationships between organisms and their media. Such a state, according to him, was experimentally demonstrated for seeds of plants and for certain resuscitating animals, such as eel-worms, tardigrades and rotifers (see vol. 1, pp. 67-9). In nature and under experimental conditions the same organism may show a complete gradation between the states of high biological and metabolic activity, a more or less deep dormancy, or torpor due to hibernation, aestivation, diapause or quiescence associated with a lower but still measurable metabolism (hypometabolism), and the state of ametabolic latent life, or cryptobiosis. All these states of an organism are well covered by the term hbypobiosisintroduced, in a somewhat different sense, by Monterosso (I934). The relationships between these different states of organisms are summarized in table 1.
TABLE 1

active life = normal metabolism

dormancy (hypometabolism) hypobiosis hibernation aestivation diapause ] quiescence

(Monterosso 1934) (ametabolism)

latent life = cryptobiosis (D.K.) = anabiosis (Preyer 1891) -abiosis (Schmidt 1948)

* Preyer (i891) expresses this as follows: 'Die Thatsache der Anabiose, d.h. der Wiederbelebung volkommen lebloser Organismrnen und ihrer Theile, deren Zustand sich von dem untersdmtlicher Lebensvorgange gew6hnlichen Scheintod durch die totale Unterbrechung scheidet, habe ich vor mehr als 25 Jahren experimentell festgestellt und seitdem in meinen Vorlesungen und mehreren Schriften begriindet, auch ihre grosse theoretische Bedeutung Namentlich lege ich Gewicht auf den von mir gelieferten Bewies fuir die hervorgehoben. der beiden Gegensatze des Lebens, namlich: Verschiedenheit (1) Leblos und lebensfahig = anabiotisch. (2) Leblos und lebensunfahig = tot.'

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As to the conditions which can bring about different states of hypobiosis, including latent life or cryptobiosis, they are: loss of water, lowering of temperature, absence of oxygen and high salt concentration, or any combination of these factors. The corresponding states of hypobiosis can therefore be termed anhydrobiosis (introduced by Giard I895), cryobiosis, anoxybiosis and osmobiosis or their combinations as shown in table 2.
TABLE 2 states*
(1) anhydrobiosis (Giard I894) (2) cryobiosis

conditions
(1) dehydration (2) cooling

(D.K.)
hypobiosis (Monterosso 1934) (3) anoxybiosis (3) lack of oxygen (D.K.) (4) high salt concen(4) osmobiosis tration (D.K.) (5) combinations of (1) to (4) D,K.

* New terms in tables 1 and 2 are initialled

The dehydration of organisms is invariably accompanied by a rise in salt concentration. This, however, can also be brought about by the evaporation of their medium, which may be rich in salt, e.g. sea water. Lowering the temperature, which leads to freezing, is usually accompanied by the loss of water and a consequent rise in salt concentration. The loss of oxygen under experimental conditions is obtained by the high vacuum often used for the desiccation of organisms. Under natural conditions anoxybiosis is brought about by micro-organisms which use oxygen for their metabolism and thus remove it from the surrounding medium, as, for instance, in the case of plant seeds deeply buried in soil or in peat. Although I hope that in all future work on this subject the term cryptobiosis will replace the old and awkward term anabiosis, I shall continue to use this old term until the end of this lecture mainly because the historical background of this field of research is one of the main objects of this lecture.
VI.
EXPERIMENTS AND VIEWS ON ANABIOSIS FOLLOWING BROCA'S REPORT

Broca's conclusions failed to satisfy all biologists and the work on the resuscitation of desiccated organisms, as well as the controversy on the true interpretations of experimental results, continued until the present time. From the rich literature on this subject containing numerous interesting records I selected only a few examples dealing in the first instance with cases of anhydrobiosis and anoxybiosis. These examples can be divided into five groups: (1) rotifers, tardigrades and nematodes; (2) larger animals; (3) bacteria; (4) fungi; and (5) seeds of plants.
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1. Anhydrobiosis in rotifers, tardigrades and nematodes

(a) Experiments on desiccation and resuscitation Davis (i 873), who worked on a rotifer, Philodina roseola, concluded that successful resuscitation can be observed only after slow drying in the presence of sand when the organism had had time to secrete a mucus envelope which protected it from complete desiccation. This observation prompted Delage & Herouard (I897) to make a sweeping statement that 'the words reviviscence and resistance to desiccation must disappear from the zoological vocabulary in so far as they concern rotifers' (pp. 206-7). However, Davis's results were completely disproved by Jacobs (i909) and Hickernell (I917), who, by direct examination and histological study of the same organism, failed to detect the secretion of any protective mucus envelope. Jacobs also found that while active rotifers stained with neutral red turn yellow when exposed to ammonia vapour, stained rotifers after desiccation remained red when they were exposed to dry ammonia, which suggested the absence of free water. On the other hand, the facts that properly fed specimens withstand better the effect of desiccation and that on repeated dehydration and hydration the number of rotifers revived rapidly diminished, indicate, according to Jacobs, the possibility that desiccated organisms may still contain sufficient water for their tissue to have a continuous although very slow metabolism. A similar conclusion was reached by Baumann (1922), as a result of his extensive study of anabiosis in a tardigrade (Macrobiotus hufelandi). One must not, according to him, consider desiccated tardigrades as examples illustrating the interruption of life processes, or of metabolism, as was postulated by Preyer, Pfliiger and Verworn. On the contrary, he found that tardigrades which survive are not completely deprived of water, so that their metabolism although imperceptible, owing to their small size, must proceed. Schmidt (I948), in reviewing his early experiments on rotifers, tardigrades and nematodes desiccated with moss, or free on slides over calcium chloride in vacuum (0-2 mm Hg), found that, after keeping them dry for 3 months, only a certain small proportion of rotifers could be revived. He concluded that the experiment carried out even under these conditions cannot completely exclude the possibility of the persistence in the desiccated rotifers of certain 'minimal life'. Moreover, even the workers who believed to have demonstrated the resuscitation of desiccated organisms after the complete cessation of life processes were puzzled by the following three facts: (1) The slowing down of resuscitation with the increase in the time the organisms are kept in a dry state. (2) The rapid decrease in the number of successful resuscitations after repeated desiccations and hydrations. (3) The gradual decrease in the number of organisms resuscitated with the increase in the time they are kept dry. Thus the maximum life span of desiccated eel-worms (Anguilhdlina tritici) in wheat grains determined by Goodey was about 10 years. A similar life span was found for desiccated rotifers and tardigrades belonging to several moss-inhabiting species.

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(b) Metabolism of desiccated tardigrades One of the important problems which arose from the study of anabiosis in micro-organisms was to determine the effect of desiccation on their metabolism. This was only recently achieved by Pigon & Weglarska (I955) who measured the rates of oxygen uptake during anabiosis and active life of two species of tardigrades: Macrobiotus dispar, a freshwater form capable of forming cysts, and Macrobiotus hufelandi, which can withstand desiccation. Oxygen uptake was measured by a somewhat modified Cartesian diver microgasometer. While the respiratory activity of M. dispar cysts was much higher than that of desiccated M. hufelandi, their active forms showed very little difference in this respect (table 3).
TABLE 3. THE RATES OF 02 UPTAKE OF MARCROBIOTUS DISP4AR AND M.i HUFELANDI species and stage MV.dispar active cysts ML.hufelandi active dried 02 uptake (,1l. x 10-6)/h/individual 1000 240 980 1-6

On measuring the 02 uptake of -l. hufelandi equilibrated with different relative humidities of air, they found that at the lowest humidities (25 to 48 %) the rate was very slow and not reliable, that at 59 % the 02 uptake represents less than 0 1 % of that of the fully active tardigrade in water and that there is a logarithmic increase in the metabolic rate with the increase of humidity. They, nevertheless, believe that even in the dry state the tardigrades may have perceptible metabolism. It is, however, possible that the very low 02 uptake by desiccated tardigrades at low humidities (25 to 48 %) is brought about not by the complex respiratory catalytic systems of cells, but by some oxidative reactions not occurring normally and therefore more harmful to the organism than the normal but slow utilization of reserve substances which may lead to starvation. (c) Cytological changes during anhydrobiosis The cytological structure of an organism in the state of anhydrobiosis and during resuscitation was studied by Hickernell (I917) on a rotifer, Philodina roseola, and by May (1949) on the intestinal cells of a moss-inhabiting anguillule, Plectus parientinus, and the body cavity cells of a tardigrade, Macrobiotus hufelandi. These investigations were carried out by the usual cytological techniques and optical microscopy. Cells of the intestinal epithelium in an active anguillule show distinct nuclei provided with a darkly stained nueleolus, while .numerous mitochondria surround the nucleus and are dispersed in the cytoplasm. On desiccation all these structures become scarcely recognizable; the intensely basophilic cytoplasm forms a condensed mass, the mitochondria are fused into compact structures and the nucleus,

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which can only be revealed by the silver impregnation method, is hyaline with chromatin forming a peripheral ring. On placing desiccated anguillules in drops of water and fixing them every 30 s, drastic changes become noticeable already after the first 30 s; the cytoplasm becomes less basophilic and there emerge in it the nucleus and mitochondria. Within 2 min the cells acquire normal cytological structures indistinguishable from those of normal active anguillules. However, it is only with the more modern electron microscope technique that the fine structural changes accompanying anhydrobiosis will be revealed. 2. Anhydrobiosis in larger animals Although larger animals cannot withstand as complete desiccation as can some micro-organisms, they are more suitable for the precise estimation of the loss of water due to drying. From the data obtained by Schmidt (1920, I948) and Hall (1922) I have selected only a few examples showing the maximum loss of water compatible either with life or with reviviscence. The loss of water was expressed as a percentage of the total body weight of the organism or of its total water content. As shown in table 4, the earthworm (Allolobophora chlorotica) and the leech (Placobolella parasitica) can lose a very great proportion of their water; they contract, lose their elasticity and give the impression of mummified organisms. Even Amphibia and mammals can lose an appreciable amount of water, while, in the case of man, a loss of water representing 10 % of the body weight or 14 % of the total water content is already dangerous.
TABLE 4. MAXIMAL LOSS OF WATER BY DIFFERENT ANIMALS COMPATIBLE WITH VIABILITY maximal % of body weight 70 70 41 24 loss of water % of total water 83 92 50 34

earthworm leech frog mouse

An interesting case of anhydrobiosis was described by Hinton (1951, 1953). It concerns the larvae of a chironomid, Polypedilum vanderplanki Hint. which breeds in shallow rock pools in Northern Nigeria. They can survive drying at 65 ?C for 20 h, and their moisture content can be reduced below 1 %. When dry, about 30 % of the larvae survived the temperature of about 68-5 ?Cfor 11 h. The respiratory activity of a dried larva is about 170 times lower than that of an active larva. Hinton also cited a few interesting examples which show that the deficiency of water in the surrounding medium, even if it does not suppress the metabolism, prolongs the life of an insect and delays its development. Thus the larva of the South American coccid Margarodes vitium, known as 'ground pearl', was found to be alive after it had been kept about 17 years in a museum. The development of the wood-boring larvae of Eburia quadrigeminata in dry wood may be delayed up to 40 years and that of Buprestis aurulenta up to 26 years.

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3. Anhydrobiosis in bacteria The stability of certain micro-organisms when their growth and proliferation are abolished is of great practical importance; since it gives valuable information as to the time during which pathogenic organisms and their spores may remain viable in soil and different objects which harbour them. According to Bulloch (I928), who obtained numerous records on this subject, anthrax spores upon silk threads remained viable up to 31 years and spores of Clostridium sporogenes were found to survive about 46 years in pathological specimens kept in alcohol in sealed museum jars. The great stability of bacteria in the desiccated state was applied during the last 20 years to their preservation in different collections. About 2700 strains of bacteria in the National Collection of Type Cultures, dried over phosphorus pentoxide, were examined at different intervals of time up to 14 years, and 83 % of cultures were found to be viable (Rhodes I950). Some of the factors affecting the viability of bacteria in the type collection of the Wellcome laboratories were discussed by Proom (I95i) at the symposium of freezing and drying organized by the Institute of Biology. The stability of certain bacteria and their spores was responsible for the extravagant claims made by some workers as to their remarkable longevity. Thus, Gallipe and his co-workers (1920, 192I) claimed that they were able to isolate living 'organites' from crushed amber, meteorites, quartz, basalts, etc., while Lipman (1928, 193I) believed he had discovered living micro-organisms in preCambrian and other rocks. There is very little doubt that these claims were due to faulty bacteriological technique. It was also claimed by Lipman (I93I, 1934) that bacteria he was able to isolate from anthracite were probably the microorganisms originally present in the peat and remaining from earliest times in a quiescent state. This view was, however, opposed by Lieske (1932), who considers them as anaerobic bacteria which have a very low metabolic activity and a low rate of multiplication, adapted to life in media containing very little nutrient material, a low water content and practically no oxygen. It is surprising that no attempt was made by other bacteriologists to repeat the experiments carried out by Gallipe and Lipman, with the result that their claims are still referred to in the literature as the most remarkable examples of the stability and longevity of a bacterial cell. 4. Anhydrobiosis in fungi One of the most interesting cases of anhydrobiosis is that of the xerophytic agaric, Schizophyllum commune, investigated by Bisby (I945). Portions of a fruit body of this fungus were found by him to be capable of producing active spores after being kept dry in sealed tubes in vacuum (0-1 mm Hg) for 35 years. The pilei they contained, when moistened and placed in a damp chamber, expanded and shed spores in different stages of development. Thus each of the tubes opened contained specimens of agaric still living more than 35 years after they developed, 34 years of which were spent in high vacuum and in one case 3 weeks having been spent in liquid air (- 190 ?C).

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5. Anhydrobiosis in higher plants and the viability of seeds The problem of the viability of seeds has always appealed to the imagination and has attracted the attention of general biologists, botanists and agriculturalists, with the result that numerous records have been accumulated on this subject, especially during the last 50 years. While some of these records were based upon observations and circumstantial evidence, others were based upon well-planned experiments on the germination of seeds properly dated and put aside for this purpose, or of seeds obtained from plants dated and preserved in well-known herbaria. Extensive observations, experiments and critical reviews of the literature on this problem can be found in papers by Becquerel (1907), Ewart (I908), Turner (i933) and Crocker (1938). It was known that many seeds are better preserved when they are buried 3 ft. or more below the surface where the temperature and the oxygen supply are much reduced and the amount of moisture is more uniform. It was mentioned by Turner (1933) 'that on the soil of old pastures and woodlands freshly turned up, or on the mud of drained ponds, plants previously unknown in that locality appear...'. Turner writes further: 'Hill, in the Flora of the Somme Battlefields, says "In July poppies (Papaver rhoeas L.) predominated, and the sheet of colour as far as the eye could see was superb; a blaze of scarlet unbroken by tree or hedgerow. Here and there long stretches of chamomile (Xllatricaria chamomilla L.) broke into the prevailing red and monopolised some acres; and large patches of yellow charlock were also conspicuous, but in the general effect no other plants were noticeable.... ." ... No doubt in the ordinary operation of ploughing and tilling of the ground in years before the war much seed was buried which has been brought to the surface by the shelling of the ground and subsequent weathering.' The appearance of charlock (Sinapis arvensis) in shell craters while it was absent on the undisturbed ground was recorded by other botanists during the War. This also explains why farmers often object to deep ploughing. These observations corroborated the experiments on the viability of seeds buried 6 to 42 in. below the surface of the soil, which showed that the seeds buried most deeply retained their vitality the longest (Turner I933). On testing the germination of seeds preserved in the herbarium of the Natural History Museum of Paris, Becquerel (I907) found that several species were viable for 56 to 87 years, while Turner (1933) found seeds of a number of leguminous plants viable up to 90 years. Ewart (I908) from personal experience and the study of the literature, concluded that 'However dry the seeds may be they cannot indefinitely prolong their vitality. Even the most resistant seeds after 50 to 100 years show a pronounced decrease in the percentage germination, and the general trend of the curves is such as to show that the probable extreme duration of vitality for any known seed may be set between 150 and 250 years (Leguminosae).' According to Ewart the minimum water content of seeds compatible with their viability is 2 to 3 %. However, the most remarkable example of the longevity of plant 'seeds' or fruits which excited the daily press, is the well-known case of the water lily or

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sacred lotus, Nelumbo nucifera Gaertner (= Nelumbium speciosum Willd),* which grows in shallow water. The first experiments on the germination of Nelumbium seeds were carried out by Robert Brown in 1850 with seeds from the 150-year-old collection of Sir Hans Sloane in the British Museum. He found that 85 % of seeds retained their viability. However, a further attempt by the Japanese botanist Ichiro Ohga, made in I926, to germinate the seeds from this collection, then 226 years old, were unsuccessful probably because they showed some invasion by moulds. Thus Brown's demonstration of the germination of Nelumbo seeds 150 years old was the record of viability of plant seeds, or fruits (Turner, I933). On the other hand, the seeds of Nelumbo nucifera were found by Ohga (i923) in the peat of an old dried-up lake in the Pulantien Basin of southern Manchuria, and from these seeds he obtained 100 % germination. In 1926 Ohga sent thirty of these seeds to the British Museum so that their viability could be tested at different intervals. To induce the germination of these seeds the hardened and impermeable envelope or pericarp had to be removed by soaking the seeds for several hours in concentrated sulphuric acid followed by washing and soaking in water. Two of these seeds were germinated in 1931, the seedlings were potted and planted in the Victoria Regia tank at Kew Gardens where they flowered in 1932. Nelumbo seeds of the same origin were successfully germinated and produced flowering plants in 1951 in Washington and at Berkeley, California. From the present level of the Pulantien river and the age of the trees Salix babylonica growing on the peat containing Nelumbo seeds, Ohga estimated their age to be approximately 400 years. However, the proper estimation of their age requires the knowledge of the age of the peat which contains them and this can be obtained from the geological study of the region. In this respect, the article: 'How old are the Manchurian Lotus seeds ? by Professor R. W. Chaney (1951 )t- of the University of California is of special interest. He re-examined this problem in the light of his personal geological observations in this part of the world and those of the Japanese geologist Dr Seido Endo, an authority on north-eastern Asia. The important facts which, according to Chaney, should be considered in determining the age of Nelumbo seeds are: (1) That the peat was gradually formed in the lake basin, about a mile across. (2) That at some later date the lake was drained and the layer of peat with Nelumbium seeds, thus exposed to the atmosphere, was covered by several feet of loess composed of a fine wind-borne clay dust from the Gobi Desert and other northern arid regions. (3) That the disappearance of the lake was probably due to the uplifting of the coastal plain on which lay the peat. This made it possible for a river to erode back until its valley cut below the
* It is a sacred plant in China, Tibet and generally in Asia. Although it is represented on ancient Egyptian monuments it is not found any more in the Nile region. It is also known as the sacred bean in India, where nuts and rhizomes are used as food. The leaves and especially the flower stalks are rich in spiral tubes, which can be extracted with care by gently breaking the stems and slowly drawing them apart. From these filaments are prepared the wicks which are burnt in lamps before the shrines on solemn religious occasions. This information and the coloured slides [shown at the lecture] representing these plants are taken from Curtis's Botanical magazine or flower garden displayed, 1842, vol. 68. t For the photostatic copy of this article I am very grateful to Dr G. Taylor, the Director of the Royal Botanic Gardens, Kew.

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level of the lake basin, so that the Pulantien river in its present course has cut down through the layers of loess and the underlying peat, and to a depth of more than 40 ft. into the bedrock. (4) That the Nelumbo plant is no longer native in northern Asia and does not grow within 1000 miles of the peat containing the seeds. (5) That the modern Indian lotus seeds differ in size, shape and certain structural features from the Pulantien seeds. Endo believed that the loess deposits overlying the peat are of the Pleistocene age, and that the peat was deposited about 50000 years ago. If this were so, the lotus seeds would be contemporary with our oldest human ancestor, Peking Man. 'It would be interesting to know', writes Chaney, 'whether this First Family (anthropologically) of China ever admired the flowers of our Pleistocene lotus, if Pleistocene it proves to be, and whether its roots and seeds were used for food in this oldest known human home.' The view that the Pulantien peat is of the Pleistocene age would scarcely surprise geologists were it not for the viable Nellumbo seeds it contains. Chaney rightly concluded that the problem of the true age of these seeds could only be solved by determining the residual 14C isotope they contain, the half-life of this isotope being about 5550 years. This was done by Dr W. T. Libby (I95I), who in a preliminary report on the estimation of the residual 14Cisotope of a few Nelumbo seeds from Pulantien peat puts their age at about 1040 (? 200) years. Chaney (I941), referring to this report in a footnote to his article, remarked: 'It is hoped that more seeds can be secured so that a test can be run using only their shells, and that some of the peat in which they were buried may also be tested. With this additional information, the maximum age of the seeds from Pulantien may be established.' Such work may clarify the discrepancy in the age of Manchurian lotus seeds obtained from geological considerations and the estimation of 14C. However, pending further work on this subject, one cannot exclude the possibility that one or two thousand years ago some more or less deep crevasses have been formed in the Pulantien loess and peat bed forming temporary water reservoirs into which lotus seeds may have been introduced by birds. Of special interest in this connexion are some of the remarks made by Darwin (1859) in the course of his discussion of the distribution of water plants: 'I have stated', he wrote, 'that fresh-water fish eat some kind of seeds, though they reject many other kinds after having swallowed them; even small fish swallow seeds of moderate size, as the yellow water-lily and Potamogeton. Herons and other birds, century after century, have gone on daily devouring fish; they then take flight and go to other waters, or are blown across the sea; and we have seen that seeds retain their power of germination, when rejected in pellets or in excrements, many hours afterwards. When I saw the great size of the seeds of that fine water-lily, the Nelumbium, and remembered Alph. de Candolle's remarks on this plant, I thought that its distribution must remain quite inexplicable, but Audubon states that he found the seeds of the great southern water-lily (probably according to Dr Hooker, the Nelumbium luteum) in a heron's stomach.. .the wide distribution of fresh-water plants and of the lower animals, whether retaining the same identical form or in some degree modified, I believe mainly depends on the wide dispersal of their

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seeds and eggs by animals, more especially by fresh-water birds, which have large powers of flight, and naturally travel from one to another and often distant piece of water' (Origin of species, 1st edition, pp. 386-9). The germination of Pulantien lotus seeds at Washington (United States) in March 1951 aroused great interest mainly because of their possible age estimated by Endo at about 50000 years. The daily press on both sides of the Atlantic eagerly recorded step by step their development, informing the readers that the lotus plants which produced these seeds flowered when probably Neanderthal man roamed Europe. However, this excitement soon subsided when a few months later Libby and Chaney announced that these seeds may have been only 1040 (? 200) years old. Yet, even if we take the lowest value, that is about 800 years, as the true age of these seeds it still appears to me as a very remarkable age, especially if we think of the events, in practically every aspect of human history, which have taken place during the dormancy of these seeds.* The longevity of Nelumbo seeds is greatly enhanced by their thick, impermeable envelope or pericarp, which protects the embryo, and the exceptionally favourable conditions which are found in the peat, such as lack of oxygen and the presence of humic substances with their acidic and tannic properties (Waksman I936; Erdtman I943). Yet, when the hard envelope of the seed is removed, its germination in contact with water and oxygen is a very rapid process. In order to germinate the embryo must consist of intact cells provided with a complete equipment of enzymes, coenzymes and substrates organized into active and complex catalytic systems supported by the intracellular structural elements which assure their proper spatial distribution and their mutual accessibility. The physiological state of seeds during their more or less protracted period of quiescence under natural and experimental conditions was the subject of much work and discussion. Of special interest in this respect is Becquerel's (1907) extensive study of the latent life of seeds. He found that the tegument or envelope of seeds, especially in leguminous plants, when dry, is impermeable to gases, alcohol and chloroform. When exposed to air containing some moisture the seeds show measurable gas exchange which gradually slows down. This gas exchange appears to be localized in the tegument, which is devoid of living tissue, and is greatly stimulated by light of short wave lengths (blue to ultraviolet). Dried seeds deprived of their tegument or pericarp and kept in the dark in pure N2 produced no C02, and yet they were viable; they also remained viable after being kept for 1 or 2 years in pure C02, or in high vacuum in a sealed glass tube or under mercury. According to Becquerel the gas exchange detected in air-dried
* As to the supposed viability of wheat grain found in ancient Egyptian, Indian or Graeco-Roman tombs, it was completely disproved by repeated germination tests and microscopic study of the embryo of grain obtained from undisturbed tombs (Gain 1900; Becquerel 1907; Turner 1933). This was not surprising considering that the viability of cereals was found not to exceed 15 to 20 years. Yet, the credulity of even intelligent people, eager to find something bearing upon immortality, is so great that it was frequently exploited by some of the guides who kept supplies of modern grain carefully hidden in tombs for visitors who are willing to buy them. as viable 'mummy wheat' or 'mummy barley' (Turner I933).

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seeds is brought about, not by a true respiratory activity of their living tissues, but by some oxidation reactions taking place in their tegument. This was more recently confirmed by several workers who found that while the rate of respiration of air-dried wheat grains deprived of their embryos by drilling (Leach 1944), or by the attack of a moth larva (Oxley & Jones I944) was practically the same as that of intact grains, the rate of respiration of grains deprived of their pericarp (by abrasion with coarse carborundum), but which nevertheless remained viable, was reduced to about 5 % of its original value. Much of the recent work on seed metabolism was stimulated by certain practical problems which arose from the need to store grain on a large scale. It was shown moisture content, and that the very low rate of respiration of grain, at 8 to 14 %0 which is localized in the pericarp, is almost entirely due to the mycelium of moulds present under the epidermis of the pericarp (Oxley & Jones I944). On the other hand, the rapid increase in the rate of respiration of non-germinating seeds, at moisture contents of 18 % and above, is due to the development of external moulds such as Aspergillus or Penicillium (Hyde I954).* The results of extensive investigations on various aspects of seed metabolism, under different conditions, including dormancy, were recently critically examined by Crocker & Barton (I953) in their excellent monograph the Physiology of seeds. The fact that the gas exchange of dried non-germinating seeds is localized in the tegument and is probably due to moulds clearly shows that the respiratory activity of seeds and their metabolism is at a standstill, which is in agreement with the view concerning the latent life of seeds put forward by Claude Bernard (1878) in his famous lectures on the phenomena of life common to animals and plants.
VII. EFFECT OF ANHYDROBIOSIS ON THE LONGEVITY OF ORGANISMS

One of the effects of anhydrobiosis, which is usually accompanied by anoxyand osmobiosis, upon organisms is the marked increase of their life span. This can be illustrated by a few examples taken from cases of anhydrobiosis I have already described in some detail. Thus (1) rotifers, nematodes and tardigrades which have a comparatively short span of life, of a couple of months only, can return to life after being kept dry for 7 to 13 years; (2) spores of bacteria (Clostridium sporogenes) survived for 46 years in pathological specimens kept in alcohol in sealed museum. jars; (3) the fruit body of a fungus (Schizophyllum commune) retained the ability to produce viable spores after having been kept in vacuo (0-1 mm Hg) for over 35 years, and (4) seeds of different plants kept dry remained viable from 50 to about 1000 years. That certain organisms can withstand better than others long periods of extreme dehydration may be due partly to their smaller size and partly to a more favourable distribution of water within their cells, so that its withdrawal does not cause the disruption of their intimate structure. While some workers, as we have seen, strongly supported the view that in desiccated organisms all life processes can be reversibly suspended for considerable
* For this information and for references to some recent work on grain respiration grateful to Miss Mary B. Hyde of the Pest Infestation Laboratory. I am

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periods of time, others on the contrary were reluctant to consider even the possibility of a temporary discontinuity of metabolic processes. They were inclined to believe that, however slow these processes may be, they are, nevertheless, proceeding continuously in order to supply the energy necessary for the upkeep of the complex structure of the organism. This view was clearly formulated by Kalabuchow (i935) in his paper on anabiosis of mammals and insects cooled to temperatures below 0 ?C. Although the results of this work are very important inasmuch as they demonstrated the re-animation of mammals after their body temperature had dropped below 0 ?C and. their breathing and heart beats had stopped, they had no direct bearing upon the problem of the discontinuity of life processes, considering that all the cells and tissues of his cooled animals still had an active metabolism. Kalabuchow, nevertheless, made a sweeping statement which he underlined in his paper, namely, that 'the theory of anabiosis, that is the supposition of the possibility of resuscitation of an organism after the complete cessation of its life processes, is not correct. Life is an uninterrupted process and its cessation even for a, short time leads to death' (p. 63). The concept that the continuity of metabolic processes is inevitable, which in my opinion can scarcely be accepted for the extreme cases of anhydrobiosis I described, breaks down completely when we consider the effect upon organisms of very low temperatures.

VIII.

EFFECT OF LOW TEMPERATURES

The first experiments on the effect of freezing upon microscopic animals were recorded between 1661 and 1664 by Henry Power, M.D. and Fellow of the Royal Society, in his work Experimental philosophy in three books: containing new experiments: microscopical, mercurial, magnetical. His experiments, which were carried out on eel-worms (Turbatrix aceti), were recorded in a chapter under the title 'Of the little white Eels or Snigs, in Vinegar or Aleger' (pp. 32-3). 'Now', wrote Power, 'though heat hath that killing property, yet it seems that cold hath not; for I have taken a jar-glass full of the said Vineger, and by applying Snow and Salt to it, I have artificially frozen all the said Liquor into a mass of Ice, (wherein all these Animals it seemed lay incrystalled) though I could discover none of them in it (though I have taken the Icy-mass out on purpose to look at it) so that now I gave them gone for ever; yet when I came again (about two or three hours later) to uncongeal the Liquor, by keeping the glass in my warm hand, when the Vineger was again returned to its former liquidity, all my little Animals made their re-appearance, and danced and frisked about as lively as ever. Nay I exposed a jar-glass full of this Vineger all night to a keen Frost, and in the morning have thaw'd the Ice again and these little vermine have appeared again and endured again that strong long Conglaciation without any manifest injury done to them; which is both a pretty and strange Experiment.' At about the same time Robert Boyle (I665, 1683) published the results of his important observations and experiments upon the effect of cold on animals (see

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Parkes i957; Smith 1958). To the early period in the development of this subject also belong the experiments on cooling and freezing of caterpillars of different insects and of their blood by R6aumur (1736) and of rotifers, tardigrades and eelworms inhabiting moss by Spallanzani (1787) as well as Hunter's study: 'On the heat of animals' to which I have already referred (see p. 164). Much work on the effect of cold and freezing has been done, since these early observations and experiments and the results of these investigations were carefully collected and critically examined by Luyet & Gehenio (1940) in their important book Life and death at low temperatures. More recently, theoretical, medical and industrial aspects of freezing and drying of biological materials were discussed by several workers at the three important symposia to which I referred at the beginning of this lecture. The problem of the physiology of frozen plants and animals in the arctic regions was the subject of important investigations carried out by Scholander, Flagg, Hock & Irving (1953) at the Arctic Research Laboratory (Alaska, U.S.). I scarcely need to refer in this lecture to the elegant investigations recently carried out by Dr Parkes, Dr Audrey Smith, Dr Andjus and Dr Lovelock at Mill Hill on reanimation of mammals (mice, rats and hamsters) cooled to the body temperature a few degrees below 0 ?C, which abolishes their breathing and heart beats, and kept in that state for about 1 h. These investigations, as well as their historical background, have already been discussed in some detail at the Royal Society Symposium (Parkes I957) and in an excellent review by Dr Audrey Smith (1958) on the 'Resistance of animals to cooling and freezing' which also deals with the problem of the experimental hypothermia. It was shown by several workers that representatives of a great variety of organisms revive after being kept for several weeks at the temperature of liquid gases, and numerous records on this subject were brought together by Luyet & Gehenio (I940, see their tables 2 to 4, pp. 243-50). Of special interest in this respect are the investigations carried out by Rahm (19I9-26)* and by Becquerel (1905-51 ), who demonstrated that desiccated moss-inhabiting and other organisms can withstand long periods of cooling in liquid air (- 190 ?C), hydrogen (- 253 ?C) and helium (-269 to -271 ?C). Moreover, in collaboration with the Kamerlingh Onnes Cryogenic laboratory at Leiden, Becquerel (95o a, b, I951 a,b) carried out experiments on the effect of temperatures between 0.008 and 0-047 ?K, obtained by adiabatic demagnetization of iron alum in liquid helium, on different species of rotifers, tardigrades, algae, spores of bacteria and moulds, fragments of lichens and mosses and seeds of different plants. The organisms were previously desiccated and incorporated within the compressed powder of iron alum either directly or
* It was stated by Schmidt (1948, p. 115) that when Rahm, a Roman Catholic priest and a member of the Order of Jesuits, had demonstrated the resuscitation of lifeless rotifers and tardigrades, he was penalized by being sent as a missionary to Peru and Chile so that he was unable to complete this work. This statement, which is reminiscent of those made by Broca (i86o) in reference to Needham, Baker and Fontana, is not supported by the available evidence. In fact P. Gilbert Rahm, O.S.B. (Order of St Benedict) published between 1919 and 1926 about twenty papers on different aspects of this subject and it is most unlikely that he could add anything valuable to the important results he had already obtained and which were incorporated in his excellent papers.

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protected by Cellophane. All organisms revived after being kept for about 2 h at a temperature a fraction of a degree above absolute zero. I shall now enumerate certain features of organisms kept at very low temperatures between -190 and -272 ?C. (1) All cell constituents are solidified by vitrification to glass (Luyet & Gehenio) or by syneresis or contraction and solidification of the nucleo-cytoplasm after the separation of water which crystallizes out (Becquerel 195 a, b).* (2) All gases: N2, 02 and C02, if still present, undergo liquefaction or solidification. Of 02, which is paramagnetic, about 50 % will appear as diamagnetic 04 which is responsible for the bluish colour and strong absorption bands of highly compressed or liquid oxygen. However, very little is known of the chemical properties of this compound. (3) The dissociation and ionization of molecules are completely suppressed, and this already takes place at moderately low temperatures. (4) All reactions are slowed down to a vanishing point, that is, reactions at - 200 ?C, if at all posrible in the solid state of the matter, would be about eight million times slower than at 20 ?C. This was calculated for very simple reactions (Becquerel I95I). The reactions in which complex catalytic systems are involved will certainly be more strongly affected by cold. (5) Marked changes in the molecular states of certain substances are reflected in the sharpening, displacement and often splitting of their absorption bands. This can be observed in the components of the cytochrome system, peroxidase, catalase, haemoglobin and other light-absorbing molecules. The effect of low temperatures upon the absorption spectra of pigments was of great help in our study of intracellular haemoprotein compounds. The phenomena of the sharpening, displacement and often splitting of absorption bands, which was known to physicists, is purely a temperature effect and depends upon the molecular structure of the substance. It can be observed in the material dissolved or suspended in 50 % glycerol and rapidly cooled to glass in liquid air. In addition to this phenomenon, Dr Hartree and I observed a very marked (about 20-fold) intensification in colour and absorption bands, which takes place only when the cooled glass-like material, on warming, undergoes devitrification to a microcrystalline mass, which is then rapidly recooled in liquid air. There is no doubt that the striking intensification of the absorption bands, which is equivalent to a corresponding increase in the effective optical depth of the coloured substance, is a purely optical effect. It can be explained by an interplay between the lightabsorbing substance and light-reflecting surfaces of the microcrystalline mass of the solvent (Keilin & Hartree I949b, I950). Both phenomena, the sharpening and the intensification of the absorption bands, were of very great help in our study of cytochrome and other haemoproteins,
* In the last few letters which I received from the late Professor Paul Becquerel (Iv, v, 1954) he strongly supported his view that the true mechanism of rapid freezing of an organism at the very low temperatures of liquid gases consists in a reversible cyto-nucleoplasmic cryosyneresis and not vitrification for which no X-ray evidence was available.

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especially in cells and cell-free preparations. This can be illustrated by the study which Dr Hartree and I carried out on spores of Bacillus subtilis. Spectroscopic examination of these spores revealed no absorption bands of cytochrome, which was not surprising since their respiratory activity was less than 4 % of that of the vegetative stages. However, by applying the low-temperature technique we were able, not only to observe, but also to estimate the cytochrome content of spores, which was found to be about 6 % of that of the vegetative forms (Keilin & Hartree I947).* The low-temperature technique also enabled us to identify additional components of the cytochrome system, the absorption bands of which at room temperature are fused with and masked by the bands of other components (Keilin & Hartree I949b, I955). The spectroscopic study of haemoproteins at low temperatures was further developed by Estabrook (1956), who devised a more quantitative method, which enabled him to plot the spectrophotometric curves of cytochromes and some other haemoproteins at the temperature of liquid nitrogen. Returning now to the state of an organism submitted to a very low temperature, we can see that its cells are affected not only on the colloidal but also on the molecular level. Moreover, the effects of low temperatures are usually accompanied by extreme desiccation, anoxia and an increase in the salt concentration. Yet, when the organisms are brought back to normal conditions they rapidly regain their normal appearance and metabolic activity. There is therefore no doubt that, at very low temperatures, all biochemical and physiological reactions, in other words, all processes of life, can be reversibly suspended for considerable periods of time.
IX. ANABIOSIS AND ITS BEARING ON PROBLEMIS OF THE ORIGIN AND THE CONTINUITY OF LIFE ON THE EARTH

I have already mentioned that some of the experiments on anabiosis were stimulated by some early speculations on the problem of the origin of life (cf. p. 165). Several generations of workers, failing to find a fruitful method of approach to this problem, obtained a certain feeling of relief in relegating the origin of life to some remote part of the universe. In a paper dealing with the resistance of dried non-sporulating bacteria, Shattock & Dudgeon (1912) wrote: 'The most fascinating problem in connection with the vitality of bacteria in vacuo is the possibility of their interplanetary life.' However, they found that dried bacteria are rapidly killed by ultra-violet radiation; they also mentioned that, according to Sir James Dewar (who presented their paper to the Royal Society), ultra-violet rays kill undried bacteria frozen at the temperature of liquid air ( -- 190 ?C). They concluded that their results militate against the view 'that free particulate life has entered the earth's atmosphere as a result of light propulsion, from extramondane space'. This conclusion was in complete agreement with the view expressed by Becquerel in I9IO and further developed in his recent work (Becquerel I95ob).
* This example was discussed in our paper: 'Comparative study of spores and vegetative forms of Bacillus subtilis', which appeared in the Jubilee volume of the journal Antonie van Leeuwenhoek dedicated to the late Professor Albert J. Kluyver, For.Mem.R.S., of Delft, who in 1952 delivered the third Leeuwenhoek Lecture (Kluyver I953).

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On the other hand, the ability of some organisms to withstand desiccation and very low temperatures enables them to survive in polar regions or at very high altitudes (Becquerel I951 a). Such organisms, according to Becquerel, may even assure the preservation of life under the most adverse hypothetical conditions. It is interesting in this respect to recall a remark made by Darwin in his autobiography: 'With respect to immortality, nothing shows me [so clearly] how strong and almost instinctive a belief it is, as the consideration of the view now held by most physicists, namely that the sun with all the planets will in time grow too cold for life, unless indeed some great body dashes into the sun and thus gives it fresh life. Believing as I do that man in the distant future will be a far more perfect creature than he now is, it is an intolerable thought that he and all other sentient beings are doomed to complete annihilation after such long-continued slow progress. To those who fully admit the immortality of the human soul, the destruction of our world will not appear so dreadful' (see Life and letters, vol. 1, p. 312). Although no answer to the speculations of physicists can be given which would have satisfied Darwin in so far as it concerns the fate of :man and some other higher organisms, 'the latent life of germs [wrote Becquerel (I95ob)], at very low temperatures is still the most efficient way for the preservation of life on earth as long as possible. In fact were cold to overcome our planet, either by its moving farther away from the sun, or by absorption of solar radiation or their extinction caused by some astronomical event, our atmosphere would be liquefied as this has already taken place in the case of Jupiter, Uranus, Neptune and Pluto. The animals and plants would disappear. Only some spores, some eggs and resuscitating animalcules would subsist ready for a new evolution if favourable cosmic conditions return.' However, according to the more recent speculations of astronomers, the extinction of life on earth will be brought about not by a decrease, but by an increase in the intensity of solar radiation, which will gradually raise the temperature of the earth and ultimately vaporize it (see Hoyle 1955, pp. 141-3). All forms of life, including spores, eggs and the resuscitating, or anabiotic organisms will already be destroyed when the temperature of the earth approaches 100 ?C. On the other hand, astronomers give us many million years of respite before the earth will reach even a moderately high teniperature: one which will already be lethal to man and to some other higher forms of life. Man thus has ample time to develop into what Darwin earnestly believed to be 'a far more perfect creature than he now is' and probably to find some way of escaping the annihilation now predicted by astronomers. Unfortunately, a far greater and more imminent danger to man may come not from any hypothetical cosmic event, but from man himself, if some of his great scientific achievements, which provide him with unimaginable power of destruction, should happen to serve the end of his still greater folly.
X. LATENT LIFE AND THE STABILITY OF BIOLOGICAL MATERIALS

We have seen that anhydrobiosis greatly enhances the longevity of certain organisms. One may now ask to what extent the life-span of an organism and therefore the integrity of its structure, during latent life (==cryptobiosis) or
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anabiosis, is limited by the stability of its constituents such as proteins and other complex molecules That there is a certain parallelism between the increased thermostability of desiccated micro-organisms and desiccated albumin had already been noticed by
Doyere (1842).

1. Stability of haemoglobin and endoerythrocytic enzymes in vitro Of all biological materials proteins exhibit the greatest diversity in their properties and functions and, since their structure is very complex, they are usually more fragile than are other cell constituents. The best material for the study of protein stability is mammalian blood, which can easily be obtained aseptically, and which contains a number of well-recognizable, biologically active substances, such as haemoglobin and different enzymes. The smallest changes in the structure of these substances are reflected and can be very precisely measured by their specific biological properties, which may be greatly affected, or even abolished, before any alteration in their physical and chemical properties can be detected. The study of protein stability, or its life-span, which can be expressed in terms of the time during which it retains its specific biological properties, has to depend upon the chance of finding suitable material, collected and preserved for entirely different purposes, and which has been kept for a considerable period of time. Dr Wang and I were fortunate in finding some samples of whole blood in the rich collection of specimens obtained aseptically and preserved by the late Professor G. H. F. Nuttall between 1901 and 1904. They were left over from his extensive serological investigations, the results of which were incorporated in his classical monograph on Blood immunity and blood relationship published in 1904. All these specimens were in glass tubes which had been kept in boxes at room temperature, but protected from light. In the samples of blood which we examined (Keilin & Wang I947), the red blood corpuscles were laked and the haemoglobin was completely deoxygenated. Yet, after 44 years, all the properties of the haemoglobins such as their oxygen capacity, oxygen dissociation curves, absorption spectra, ease of crystallization and crystalline forms were indistinguishable from those of the corresponding haemoglobins in freshly collected blood samples. Moreover, the endoerythrocytic enzymes such as carbonic anhydrase, catalase, glyoxalase and choline esterase retained up to 85 % of their activities, when compared with those of fresh blood samples. The factors which enhanced the preservation of haemoglobin and the endoerythrocytic enzymes in these samples were: (1) protection from bacterial infection; (2) absence of oxygen; (3) protection from light; and (4) the high protein
concentration (Keilin & Wang I947).

Among the records of protein stability available in the literature one can mention: (1) a Haldane standard tube of carbon monoxide haemoglobin which for 30 years showed practically no change (Donaldson, Harding & Wright 1943) and (2) tobacco mosaic virus in cured, dried tobacco, which retained some of its virulence for 52 years (Thornberry, Valleau & Johnson 1938). If we consider viruses as living organisms, this example also illustrates a state of perfect anabiosis.

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2. Palaeobiochemical records of the stability of biological materials The highest value so far recorded of the life span of proteins in vitro is only about 50 years, which is not surprising, considering that these records were based on the study of materials obtained accidentally and preserved in solutions and at room temperature. Much higher life span values were recorded for certain other biologically active compounds synthesized by organisms. Thus, Boyd & Boyd (I937) tested serologically the tissues of more than 300 Egyptian and American mummies and in several cases, especially those of pre-dynastic Egyptian mummies about 5000 years old, they obtained distinct serological reactions of blood groupings. More recently, successful identification of blood group antigens in tissues of South American and pre-Dynastic Egyptian mummies were obtained by Gilbey & Lubran
(1952, I953)-

A spasmogenic substance, probably of a histamine-like nature, was obtained by Graf (I949) from different tissues of an Egyptian mummy about 3000 years old. Haematin in mammoth blood. From dried blood mixed with sand, found within the remains of a frozen mammoth (Siberia), crystals of haemin were obtained which were indistinguishable from those given by fresh blood (Sansky I904). Thus, the haematin nucleus of haemoglobin remained unchanged for a period of 30000 to 100000 years. A positive precipitin reaction was easily obtained with this mammoth and elephant (Friedenthal 1904) because of a very good preservation of mammoth tissues by cold (see also W. A. Schmidt I908). Fossil porphyrins. It was shown by Treibs (I934, 1935) that porphyrins derived from chlorophyll and haemoglobin can be extracted from crude mineral oils, bituminous shales, asphalt and coal, the oldest deposits belonging to the lower Silurian period. Thus the porphyrins synthesized by plants and animals living 400000000 years ago have withstood destruction. Such exceptional instances of the preservation of complex organic molecules must be due to the great stability of these compounds when they are protected from oxygen and light. In fact, solutions of porphyrins freshly prepared from haemoglobin and exposed to oxygen and light are rapidly degraded. Succinic acid in amber. One of the interesting organic compounds present in amber (a fossil resin of the Eocene and Oligocene periods) is succinie acid, which may constitute from 3 to 8 % of the resin, hence the name succinite used for this resin (i.e. Baltic amber). The preservation of suceinic acid in amber about 40000000 years old is due to its great stability and resistance to oxidation by molecular oxygen. This stability is in sharp contrast to the remarkable ease with which it is oxidized by the succinic dehydrogenase-cytochrome system, which is one of the most powerful and widely distributed catalytic systems of cells (Keilin & Hartree 1940). The biological oxidation of succinic acid is one of the best examples of the remarkable activity shown by the catalytic system of the cell. Organic compounds in peat. Peat of different ages containing the remains of plants and animals in different states of preservation is an exceptionally rich source of material for the study of the stability of a great variety of organic

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substances synthesized by organisms. The preservation of biological materials in peat from the beginning of its formation is greatly favoured by the very low oxygen content in undrained peat bogs, an acid medium and protection from light. Much valuable knowledge on the chemical development of peat is already available (Waksman i936; Erdtman 1943). The presence in certain peats of 'proteins' and .amino acids is of great interest, especially as the modern methods of analysis and estimation of these compounds, when applied to certain forms of peat, may yield results of general biological importance. The examination of these few records of the stability of biological materials convinced me that 'The study of substances synthesized by organisms living during past geological periods is of great biological importance. The scattered observations which are already available, when brought together under the heading of palaeochemistry, or palaeobiochemistry, cannot fail to stimulate more work in this interesting and hardly explored field of research' (Keilin 1953). Proteins and amino acids in fossil shells and bones. It was indeed gratifying to find that already within the last 4 years palaeobiochemistry has been recognized as a fertile and rapidly developing field of research. The recent progress in palaeobiochemistry was chiefly due to the important work by Dr P. H. Abelson (Director of the Geophysical Laboratory, Washington, U.S.A.) who investigated the state and the distribution of proteins and amino acids in fossil shells of molluscs and
bones of vertebrates (Abelson 1955, 1956, 1957).

Of special interest are the results of his study of the edible clam Merceneria mercenaria, which has been common for more than 25000000 years, so that the fossil shells of the Pleistocene, Pliocene and Miocene ages could be compared with those of the modern clam. The laminated, colourless sheets of protein in the shells of the present-day clam have a certain mechanical resistance and strength, and are composed of some fifteen different amino acids. The protein in shells 1000 years old was brown and had lost its mechanical strength, although it still had its peptide bonds intact. In shells 100000 to 500000 years old, the protein was replaced by a black tar-like substance containing free amino acids mixed with short peptide chains, the total content of amino acids being one-tenth of that of the modern clam. Finally, Miocene shells 25000000 years old contained only a small amount of free amino acids: alanine, glycine, glutamic. acid, leucine, isoleucine, valine and proline. The amino acid constituents of some fossil shells and bones shown in table 5 indicate that the general chemical structure of proteins in animals living 400 000 000 years ago differed little from that of animals living at present. The preservation of amino acids in fossils depends not only on their stability, but also upon the conditions, especially the temperature, to which the material was submitted during past geological periods. Although the palaeobiochemical records revealed that many substances synthesized by living organisms have a practically unlimited span of life, so far they gave little valuable information as to the life span of native proteins. In fact, since the stability of the protein in fossil clams was assessed mainly by the integrity of its peptide bonds, it could only throw light upon the chemical stability of denatured,

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but not of biologically active native proteins. The latter, on undergoing denaturation, lose their specific biological properties, while their chemical composition, including the sequence of amino acids in their peptide chains, may remain undisturbed.
TABLE 5. AMINO ACIDS EXTRACTED FROM DIFFERENT FOSSILS A somewhat fossil material (1) prehistoric tooth (2) snail (3) dinosaur (4) fish (5) brachiopod horse simplified table after Abelson (I957). amino acids (1) gly, ala, len, val, glu (2) as (l)+pro (3) as (1) +asp (4) as (1)+asp (5) as (1)

approximate age (years) Later Pliocene 5 x 106 Miocene 25 x 106 Cretaceous 100 x 106 Devonian 360 x 106 Ordovician 430 x 106

Abbreviations: ala, alanine; asp, aspartic acid; glu, glutamic acid; gly, glycine; leu, leucine or isoleucine; pro, proline; val, valine.

XI.

STABILITY OF BIOLOGICAL MATERIALS IN VIVO AND IN VITRO AND ITS BEARING UPON ANABIOSIS

The only record of the life span of native proteins in vitro so far available is that of the blood sample, to which I have already referred, in which haemoglobin and four endoerythrocytic enzymes, carbonic anhydrase, catalase, glyoxalase and choline esterase, retained for almost 50 years most of their original activity (Keilin & Wang 1947). This great stability of haemoglobin and of endoerythrocytic enzymes in vitro contrasts with their rapid degradation in vivo. Thus, the life span of our red blood corpuscles, determined by different methods, is about 120 days. Our body loses every 24 h about 25 ml. of old erythrocytes, which are destroyed and replaced by new cells. Yet neither haemoglobin nor its derivatives, nor the erythrocytic enzymes are found in the circulating blood. However, this is due not to their instability, but to well-regulated mechanisms for their degradation in vivo, involving a series of catalyzed reactions localized in certain organs and tissues of the body. The life span of haemoglobin and enzymes in vitro (about 50 years), which was so much longer than their life span in vivo within the body during the normal active metabolism, is, however, much shorter than the life span of enzymes and other complex molecules incorporated within the cells of an organism during its ametabolic latent life (=cryptobiosis) or anabiosis which, as in the Manchurian lotus seeds, may last 1000 years. This greatly increased life span of complex biological molecules, especially of enzymes during anabiosis, is primarily due to the very marked slowing down, or the complete suppression, of metabolic processes and other chemical reactions in

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which these molecules could be involved and degraded. It is also due to the fact that the enzymes are not free within the cells which contain them, but are linked by more or less stable bonds to other components of catalytic systems of which they form integral parts. That an enzyme incorporated within a complex catalytic system is much more stable than when it is isolated and kept in vitro, was recently demonstrated in our experiments on the reconstitution of the succinic dehydrogenase cytochrome system from its components (Keilin & King I958). This widely distributed and very active system, which catalyzes the aerobic oxidation of succinic to fumaric acid, can be obtained from heart muscle and other tissues as a cell-free colloidal, or particulate preparation. The minute particles of this preparation, which derive from physically disintegrated mitochondria or sarcosomes, contain a typical cytochrome system comprising cytochromes a3, a, c, c1 and b, a powerful and very stable succinic dehydrogenase and certain other components of the respiratory chain (Keilin I929; Keilin & Hartree 1940, 949 a; Keilin & Slater I953). Succinic dehydrogenase has recently been isolated and purified in a soluble form by Singer (1956, I957) and Wang (I956) with their co-workers. The soluble succinic dehydrogenase, unlike its insoluble endogenous form of the particulate preparation, is very unstable and does not react with cytochrome c. However, when the soluble form is re-incorporated within the particulate heart-muscle preparation deprived of this enzyme, it re-acquires all the properties of the endogenous form, namely, its stability and reactivity towards cytochrome c. Spectroscopic and manometric studies of the succinic dehydrogenase cytochrome system of the particulate heart-muscle preparation show that the activity of this system depends not only on the integrity of each of its components, but also on the structural integrity of the particle which supports them and assures their proper spatial orientation and their mutual accessibility (Keilin & Hartree 1940). The structural change of the particle, even if it brings about a slight displacement of one of the components of the system may disrupt the activity of the whole respiratory chain involved in the hydrogen or electron transfer from the molecules of substrate to oxygen. During anabiosis of an organism caused by desiccation or solidification at low temperatures accompanied by the rise in salt concentration, the structural elements of cells such as mitochondria, which harbour the components of the catalytic systems involved in respiration, may undergo certain marked although reversible changes. It is conceivable that these changes may cause the reversible loss of accessibility between the components of the respiratory chain, with the result that all the metabolic processes come to a standstill. It is also possible that this state of an ametabolic latent life can be brought about by comparatively mild desiccation such as occurs in stored non-germinating seeds, or by low temperatures still well above that of liquid gases. The attempts by several generations of workers to submit an organism to complete desiccation, or to temperatures very near the absolute zero, although they were of great biological interest, they were probably not essential for the demonstration that life may be a discontinuous process.

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187

I should like to conclude this lecture by comparing the stability of an organism in its two states: (1) normal activity and (2) extreme anabiosis. In a living active organism the state of many of its constituents is the result of a dynamic equilibrium between the reactions involved in their constant degradations and regenerations. The organism must constantly provide the energy for the upkeep of its complex structure, which has a tendency to collapse. The stability of such an organism is of a dynamic nature. During anabiosis no energy can be supplied by the organism for the upkeep of its complex structure which, nevertheless, remains intact. This is not surprising, since reactions involved in the degradation of the structure also require water, heat and oxygen. The stability of such an organism is of a purely static nature, resembling the stability of biological material in vitro obtained experimentally or from palaeobiochemical data; and as long as the structure of these organisms remains intact they retain the ability to return to normal active life. The concept of life as applied to an organism in the state of anabiosis (cryptobiosis) becomes synonymous with that of the structure, which supports all the components of its catalytic systems. Only when the structure is damaged or destroyed does the organism pass from the state of anabiosis or latent life to that of death.
REFERENCES Abelson, P. H. 1955 Annual report of the director of the geophysical laboratory (1954-55). Yearb. Carneg. Instn, no. 54. Sci. Amer. 195, 83. Abelson, P. H. 1956 Paleobiochemistry. Ann. N.Y. Acad. Sci. 69, 276. Abelson, P. H. I957 Some aspects of paleobiochemistry. Baker, H. 1753 Employmentfor the microscope (see part II, chapter iv, Eels in blighted wheat, 250-60). London. Baker, H. I754 The microscope made easy (4th ed.). London. Bauer, F. I823 Microscopical observations on the suspension of the muscular motion of the Vibrio tritici (Croonian Lecture). Phil. Trans. 113, 1. Z,ool. Jb. 45, 501. Baumann, H. I922 Die Anabiose der Tardigraden. Becquerel, P. 1907 Recherches sur la vie latente des graines. Ann. Sci. nat. (9e s. Bot.), 5, 193-311. Becquerel, P. I95oa La suspension de la vie au-dessous de 1/20 ?K absolu par demagnetisation adiabatique de l'alun de fer dans le vide le plus eleve. C.R. Acad. Sci., Paris, 231, 261. Becquerel, P. 195ob La suspension de la vie des spores des bacteries et de moisissures dessechees dans le vide vers le zero absolu. Ses consequences pour la dissemination et la conservation de la vie dans l'Univers. C.R. Acad. Sci., Paris, 231, 1392. Becquerel, P. 195i a La suspension de la vie des algues, lichens, mousses, aux zero absolu et rSle de la synerese reversible pour 1'existence de la flore polaire et des hautes altitudes. C.R. Acad. Sci., Paris, 232, 22. Becquerel, P. 1951 b La suspension de la vie au confins du zero absolu entre 0,0075 ?K Proc. 8th Internat. et 0,047 ?K. Role de la synerese reversible cytonucleoplasmique. Congr. Refrig. p. 326. Bernard, Claude 1878 Lecons sur les phenomenes de la vie communs aux animaux et vegetaux. Paris. Bisby, G. R. I945 Longevity of Schizophyllum commune. Nature, Lond. 155, 732.

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Boyd, W. C. & Boyd, L. G. 1937 Blood grouping tests on 300 mummies. With notes on the precipitin-test. J. Immunol. 32, 307. Boyle, R. I683 New experiments and observations touching cold (2nd ed.). London. Broca, P. I86o Rapport sur la question soumise A la Societe de Biologie au sujet de la reviviscence des animaux desseches. Mem. Soc. Biol., Paris, 2, 1-140. Bulloch, W. 1928 The viability of bacteria in antiseptic solutions. Zbl. Bakt. 106, 21. Butler, J. I736 The analogy of religion natural and revealed to the constitution and course of nature. London. Caullery, M. 1924 Histoire des sciences biologiques in G. Hanotaux: Histoire de la nation
Frangaise 15, 1-296. (4?) Paris: Plon-Nourrit.

Chaney, R. W. 1951 How old are Manchurian lotus seeds ? The GardenJ. N. Y. Bot. Gdn, 1,137. Crocker, W. 1938 Life span of seeds. Bot. Rev. 4, 235. Crocker, W. & Barton, L. V. 1953 Physiology of seeds. Mass., U.S.A.: Wattham.
Curtis's bot. Mag.
I842

68.

Darwin, C. I859 Origin of species (1st ed.). London: John Murray. See also: Reprint of the 6th edition in the World Classics, 1956. Oxford University Press. Darwin, C. I887 The life and letters of, including an autobiographical chapter, edited by Francis Darwin. 3 vols. London: John Murray. Davaine, C. I856 Recherches sur l'anguillule de ble nielle consideree au point de vue de l'histoire naturelle et de l'agriculture. Mem. Soc. Biol., Paris (2e s.), 3, 201-271. Davis, H. I873 A new Callidina: with the results of experiments on the desiccation of rotifers. Mon. Micr. J. (now J. R. Micr. Soc.) 9, 201. Delage, Y. & Herouard, E. 1897 Traite de Zoologie Concrete, vol. 5. Les Vermidiens. (See pp. 206-207.) Paris: Schleicher. Dobell, C. I923 C. G. Ehrenberg (1795-1876). A biographical note. Parasitology, 15, 320. Dobell, C. 1932 Antony van Leeuwenhoek and his 'Little Animals'. London: John Bale and Danielson. Donaldson, R., Harding, H. G. W. & Wright, G. P. I943 Standardisation of the Haldane haemoglobinometer. J. Path. Bact. 55, 205. Doyere [P. L. N.] 1842 Memoirs sur les Tardigrades. Sur la faculte que possedent les Tardigrades, les Rotifbres, les Anguillules des toits et quelques autres animalcules, de revenir A la vie apres avoir ete completement desseches. Ann. Sci. nat. (2e s.), 18, 5. (See also footnote p. 159.) Dupaty, President I789 Sentimental letters on Italy in 1785, translated from French by J. Povoleri. London: J. Crowder. Estabrook, R. W. I956 The low temperature spectra of hemoproteins. I. Apparatus and its application to a study of cytochrome c. J. Biol. Chem. 223, 781. Ehrenberg, C. G. I838 Die Infusionsthierchen als vollkomene Organismen. Ein Blick in das tiefere organische Leben der Natur. Leipzig. Erdtman, G. I943 An introduction to pollen analysis. Mass., U.S.A.: Waltham.
Ewart, A. J. 1908 On longevity of seeds. Proc. Roy. Soc. Vict. (N.S.) 21, 1-210.

Fontana, F. I776 Lettre a un de ses amis sur l'ergot et le Tremella. J. de Physique de l'Abbe Rozier, 7, 42. Fontana, F. I795 Treatise on the venom of the viper, etc. in 2 vols. translated from the original French (2nd ed.). London. Freezing and drying 195I First international symposium under Institute of Biology, edited by R. J. C. Harris. Freezing and drying 1958 Second international symposium under Institute of Biology, edited by A. S. Parkes. Friedenthal I904 [On Precipitin reaction of Siberian Mammoth and elephant, see Schmidt,
W. A. (I908).]

Gain, E.
1643.

I900

Sur les embryons du ble et de l'orge pharaonique. C.R. Acad. Sci., Paris, 130,

Galippe, V. i92o Recherches sur la resistance de microzymes a l'action du temps et sur leur survivance dans l'ambre. C.R. Acad. Sci., Paris, 170, 856. Gallipe, V. & Souffland, G. 1921 Recherches sur la presence dans les meteorites, etc., les cendres et les laves volcaniques, d'organites susceptibles de reviviscence et sur leur resistance aux hautes temperatures. C.R. Acad. Sci., Paris, 172, 1252.

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Gavaret [J.] I859 Quelques experiences sur les rotiferes, les tardigrades et les anguillules des mousses des toits. Ann. Sci. nat. (4e s.) Zool. 11, 315. Geoffroy Saint-Hilaire, I. & Milne-Edwards, H. I838 Rapport sur une note de M. Mandl C.R. Acad. Sci., relative A la forme des globules du sang chez quelques mammiferes. Paris, 7, 1136. Giard, A. I894 L'anhydrobiose ou ralentissement des phenomenes vitaux. C.R. Soc. Biol., Paris, 46, 497. Gilbey, B. E. & Lubran, M. 1952 Blood groups of South American and Indian mummies. Man, 52, 115. Gilbey, B. E. & Lubran, M. 1953 The ABO and Rh blood group antigens in pre-Dynastic Egyptian mummies. Man, 53, 23. Goodey, T. 1923 Quiescence and reviviscence in Nematodes with special reference to Tylenchus tritici and Tylenchus dispar. J. Helminth. 1, 47. Graf, W. 1949 Presence of a spasmogenic substance, presumably histamine, in extracts of mummy tissue. Nature, Lond. 164, 701. Graham-Smith, G. S. & Sanger, F. I903 The biological precipitin test for blood considered mainly from its medico-legal aspect. J. Hyg., Camb., 3, 258. Hall, F. G. I922 The vital limit of exsiccation of certain animals. Biol. Bull., Woods Hole, 41, 31. Hickernell, L. M. 1917 A study of desiccation in the rotifer, Philodina roseola, with special Biol. Bull., Woods Hole, 32, reference to cytological changes accompanying desiccation. 343-397. Hinton, H. E. 195I A new chironomid from Africa, the larva of which can be dehydrated without injury. Proc. Zool. Soc. Lond. 121, 371. Hinton, H. E. I953 Some adaptations of insects to environments that are alternately dry and Trans. Soc. Brit. Ent. 11, 209. flooded, with some notes on the habits of the Stratiomyidae. Hoyle, F. 1955 Frontiers of astronomy. London: W. Heineman. Phil. Trans. 68, 7-49. Hunter, J. I778 Of the heat of animals and vegetables. Hunter, J. i835 The Works of, in 4 vols. edited by J. F. Palmer. See vol. 1, chapter viiI. On the heat of animals, pp. 278-298. London: Longman. Hyde, M. B. 1954 The respiration of mature wheat grains in relation to moisture content and 8th Int. Bot. Congr., Paris. fungal contamination. Jacobs, M. H. 1909 The effects of desiccation on the rotifera Philodina roseola. J. Exp. Zool. 6, 207-263. Kalabuchow, N. J. 1935 Anabiose bei Wirbeltieren und Insekten bei Temperaturen unter 0?. Zool. Jb. (Abt. 3) Allg. Zool. Physiol. 55, 47. Keilin, D. I929 Cytochrome and respiratory enzymes. Proc. Roy. Soc. B, 104, 206. Keilin, D. 1953 Stability of biological materials and its bearing upon the problem of anabiosis. Sci. Progr. Twent. Cent. 41, 577. Keilin, D. & Hartree, E. F. 1940 Succinic dehydrogenase-cytochrome system of cells. Proc. Roy. Soc. B, 129, 277. Keilin, D. & Hartree, E. F. 1947 Comparative study of spores and vegetative forms of Bacillus subtilis. Leeuwenhoek ned. Tijdschr. 12, 115. Keilin, D. & Hartree, E. F. 1949a Activity of succinic dehydrogenase-cytochrome system in different tissue preparations. Biochem. J. 44, 205. Keilin, D. & Hartree, E. F. I949b Effect of low temperature on the absorption spectra of haemoproteins; with observation on the absorption spectrum of oxygen. Nature, Lond. 164, 254. Keilin, D. & Hartree, E. F. 1950 Further observations on absorption spectra at low temperatures. Nature, Lond. 165, 504. between certain components of the Keilin, D. & Hartree, E. F. I955 Relationship cytochrome system. Nature, Lond. 176, 200. of the succinic oxidase system from soluble Keilin, D. & King, Tsoo E. 1958 Reconstitution succinic dehydrogenase and a particulate cytochrome system preparation. Nature, Lond. 181, 1520. Brit. Med. Bull. 9, 89. Keilin, D. & Slater, E. C. I953 Cytochrome. Keilin, D. & Wang, Y. L. I947 Stability of haemoglobin and of certain endoerythrocytic enzymes. Biochem. J. 41, 491.

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Kluyver, A. J. 1953 Leeuwenhoek Lecture. The changing appraisal of the microbe. Proc. Roy. Soc. B, 141, 147. Leach, W. I944 Studies on the metabolism of cereal grains. Canad. J. Res. C, 22, 150. Leeuwenhoek, A. van 1702 On certain animalcules found in the sediment in gutters of the roofs of houses. Letter 144 in The Select works of Antony van Leeuwenhoek, translated by Samuel Hoole, 1798, 1807, 4?, 2 vols. London. (See vol. 2, pp. 207-213.) Leeuwenhoek, A. van I705 A letter to the Royal Society concerning animalcules on the roots of duck-weed. Phil. Trans. 24, no. 296. Radiocarbon dates. II. Science, 114, 291. Libby, W. F. I95I Biochem. Z. 250, 339. Lieske, R. 1932 Uber das Vorkommen von Bakterien in Kohlenfl6zen. The discovery of living micro-organisms in ancient rocks. Science, 68, Lipman, C. B. I928

272.
Lipman, C. B. 1931 Living micro-organisms in ancient rocks. J. Bact. 22, 183. Lipman, C. B. 1934 Further evidence on the amazing longevity of bacteria. Science, 79, 230. Lucretius The nature of the universe. A new translation by R. E. Latham 195 . The Penguin Classics. Luyet, B. J. & Gehenio, P. M. 1940 Life and death at low temperatures. Normandy, Missouri: Biodynamica. Proc. 6th Int. Congr. Exp. Cytol. suppl. 1, 390. Cytologie de la reviviscence. May, R. M. I949 Milne-Edwards, [H.] i842 Rapport sur un memoire de M. Doyere. C.R. Acad. Sci., Paris, 15, 320. L'anabiosi nei Cirripedi e il problema della vita latente (ipobiosi). Monterosso, B. I934 Arch. zool. (ital.), Napoli, 19, 17-379 [see Schmidt, P. 1948 Anabiosis, p. 339]. Needham, J. T. I743 Concerning certain chalky concretions, called malm; with some microscopical observations on the farina of Red Lilly, and worms discovered in Smuthy Corn.

Phil. Trans. 42, 634.

Needham, J. T. 1745 New microscopical discoveries. [Of eels in blighted wheat pp. 85-89.] London. Needham, J. T. 1775 Lettre ecrite a l'auteur de ce Recueil. J. de Physique, l'abbe Rozier, 5,226. Nuttall, G. H. F. 1904 Blood immunity and blood relationship. Cambridge University Press. On the longevity of the fruits of Nelumbo nucifera. Bot. Mag., Tokyo, 37, 87. Ohga, I. I923 The germination of century-old and recently harvested Indian lotus fruits, I. I926 Ohga, with special reference to the effect of oxygen supply. Amer. J. Bot. 13, 754. Oliver and Boyd. The origin of life on the earth. Edinburgh: Oparin, A. I. I957 Oxley, T. A. & Jones, J. D. 1944 Apparent respiration of wheat grains and its relation to a fungal mycelium beneath the epidermis. Nature, Lond. 154, 826. Parkes, A. S. 1957 A discussion on viability of mammalian cells and tissues after freezing, under the leadership of, held 31 January 1957. Proc. Roy. Soc. B, 147, 423-553. Pigoni, A. & Weglarska, B. 1955 Rate of metabolism in tardigrades during active life and anabiosis. Nature, Lond. 176, 121. C.R. Acad. Pouchet, F. i859a Nouvelles experiences sur les animaux pseudo-ressuscitants.

492. Sci., Paris, 49,

F. i859b

Pouchet,

Experiences

sur la resistance vitale des animalcules

C.R. Acad. Sci., Paris, 49, 886.

pseudo-ressuscitants.

Generation spontanee et resurrection des Rotifbres. Cosmos, Paris, 14, Pouchet, F. 1859 380. (See also footnote p. 159.) Power, H.I663-4 Experimental philosophy in threebooks: containing new experiments: Micro-

scopical, mercurial, magnetical. London.

Prevost [J. L.] & Dumas [J. B. A.] 1824 Nouvelle theorie de la generation. Ann. Sci. nat. 1, 1-29. Preyer, W. I872 Mittheilung aus dem Vortrag des Herrn Prof. Dr Preyer. Tagebl. 45

Versamml. dtsch. Naturf. Aertze, Leipzig, 18, 46.

Preyer, W.I891 Proom, H.I95I

Ueber die Anabiose. Biol. Zbl. 11, 1. Some of the factors affecting the viability

of bacterial

cultures subjected

to the freeze-drying process. Symposium on freezing and drying, Institute of Biology. (See
und physiologische Beitrage zur Kenntnis der Moosfauna.

pp. 117-126.) Rahm, P. G. 1I922 Biologische

Z. allg. Physiol. 20, 1-34.

The LeeuwenhoekLecture

191

Versuche mit niederen Temperaturen. Ein Rahim, P. G. I924 Weitere physiologische Verh. dtsch. zool. Ges. p. 106. Beitrag zur L6sung des Kalteprobleme. Rahm, P. G. I 926 Die Trockenstarre (Anabiose) der Moostierwelt (Ihr Verlauf, ihre Bedeutung Biol. Zbl. 46, 452. und ihr Unterschied der Cystenbildung). Reaumur, R. A. F. de I736 M~emoire pour servir a l'histoire des Insectes, Paris, Imprimerie Royal. (See vol. 2, pp. 141-147.) Rhodes, M. 1950 Viability of dried bacterial cultures. J. gen. Microb. 4, 450. Roffredi, M. I775 a Sur l'origine des petits vers ou Anguilles du Bled rachitique. J. Physique, l'Abb6 Rozier, Paris 5, 1. Roffredi, M. 1775b Seconde lettre ou suite d'observations sur le rachitism du Bled, sur les Anguilles de la colle de farine et sur le grain charbonne. J. Physique, l'Abbe Rozier, 5, 197. Roffredi, M. 1776 Memoire pour servir de supplement et d'eclaircissement aux m6emoires sur les Anguilles du Bled et de la colle de farine. J. Physique, l'Abbe Rozier, 7, 369. der Erforschung des im Jahre 1901 am Ufer Sansky, W. I904 Ueber due Hauptresultate 6s Congr. Int. Zool. Berne, der Beresowka endeckten maenlichen Mammuthcadavers. C.R. Seances, p. 67. Schmidt, P. 1920 L'anabiose des vers de terre. Trav. Soc. Nat. Petrograd, 50, 1 [in Russian]. Schmidt, P. 1948 Anabiosis, S.S.S.R. Academy of Science, Moscow and Leningrad (in Russian). von agyptischen MumSchmidt, W. A. 1908 Chemische und biologische Untersuchungen der alten Agypter. mienmaterial, nebst Betrachtungen fiber Einbalsamierungsverfahren Z. allg. Physiol. 7, 369. Scholander, P. F., Flagg, W., Hock, R. J. & Irving, L. I953 Studies on the physiology of frozen plants and animals in the arctic. J. Cell. Comp. Physiol. 42, suppl. 1, 1-56. Senebier, J. i8o3 Notice historique sur la vie les ecrits de L. Spallanzani, in Me&moires sur Gen6ve. la respiration par Lazzare Spallanzani. Shattock, S. G. & Dudgeon, L. S. 1912 Certain results of drying non-sporulating bacteria in a charcoal liquid air vacuum. Proc. Roy. Soc. B, 85, 127. Singer, T. P., Kearney, E. B. & Massey, V. 1957 Newer knowledge of succinic dehydrogenase. Advanc. Enzym. 18, 65. Smith, A. U. I958 The resistance of animals to cooling and freezing. Biol. Rev. 33, 197-253. Spallanzani, L. 1769 Nouvelles recherches sur les decouvertes microscopiques et sur la generation des corps organises. 2 vols., translation from Italian book (1767) with notes by Needham. See vol. 1, p. 25. Spallanzani, M. l'Abbe 1787 Opuscules de physique animale et vegetale (translated from Italian, 1776, by J. Senebier), 2 vols. Paris. [Vol. 2 contains a chapter: Observations et experiences sur quelques animaux surprenans que l'observateur peut a son gre faire passer de la mort a la vie, pp. 203-285.] Spallanzani, The Abbe 1803 Tracts on the natural history of animals and vegetables, 2 vols., translated from Italian by J. G. Dalyell. Creech and Constable, Edinburgh. Second edition, see pp. 119-194: 'Observations and experiments on some singular animals which may be killed and revived.' An abridged edition in 1 vol. appeared in 1799. Thornberry, H. H., Valleau, W. D. & Johnson, E. M. 1938 Inactivation of tobacco-mosaic virus in cured tobacco-leaves by dry heat. Phytopathology, 28, 129. Treibs, A. 1934 Uber das Vorkommen von Chlorophyll Derivaten in einem Olschiefer aus der oberen Trias. Liebigs Ann. 509, 103. in bitumin6sen Gesteinen, Erdolen, Treibs, A. I935 Chlorophyll- und Hamin-Derivate Kohlen, Phosphoriben. Liebigs Ann. 517, 172. Turner, J. H. 1933 The viability of seeds. Kew Bull. no. 6, 251. Tyndall, J. 1874 Presidential address to the British Association for advancement of Science, Belfast, pp. 66-97. (See p. 79.) Uvarov, B. P. I931 Insects and climate. Trans. R. Ent. Soc. Lond. 79, 1-247. Valery-Radot, R. I900 La vie de Pasteur. Paris: Hachette. Voltaire, F. M. A. I882 (Euvres completes. Paris: Garnier Freres. London: Waksman, S. A. 1936 Humus: origin, chemical composition and importance. Bailliere, Tindall and Co. Wang, T. Y., Tsou, C. L. & Wang, Y. L. 1956 Studies on succinic dehydrogenase. Scientia Sinica, 5, 73.